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ndustrial biotechnology from fundamentalso practice (ACIB Session)

CIB-1

wo promising biocatalytic tools: regioselective car-oxylation of aromatics and asymmetric hydration oflkenes

ilvia M. Glueck1,∗ , Christiane Wuensch1, Tamara Reiter1,ohannes Gross1, Georg Steinkellner1, Andrzej Lyskowski1, Karlruber2, Kurt Faber2

ACIB GmbH c/o University of Graz, Department of Chemistry, AustriaUniversity of Graz, Austria

Due to the growing demand for alternative carbon sources,he development of CO2-fixation strategies for the productionf valuable chemicals is a current challenge in synthetic organichemistry [1]. The enzymatic carboxylation of aromatic com-ounds [2,3] catalyzed by various decarboxylases represents aromising ‘green’ alternative to the classic Kolbe-Schmitt reaction4] which requires harsh reaction conditions.

Depending on the type of enzyme, the carboxylation proceededn a highly regio-complementary fashion: Benzoic acid decarboxy-ases selectively catalyzed the ortho-carboxylation of phenolicubstrates (conv. up to 80%) whereas phenolic acid decarboxylasesxclusively catalyzed the �-carboxylation of styrenes (conv. up to0%) [5].

During these studies a promiscuous ‘hydratase-activity’ of phe-olic acid decarboxylases was discovered, which leads to thetereoselective hydration of hydroxystyrenes to yield the corre-ponding sec-alcohols with high conversion (up to 82%) and e.e.’s
f up to 71% [6].
Acknowledgements: This work has been supported by theustrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT

hrough the Austrian FFG-COMET-Funding Program.

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eferences

].Tsuji Y, Fujihara T. Chem Commun 2012;48:2365.].Glueck SM, Gümüs S, Fabian WMF, Faber K. Chem Soc Rev2010;39:313.

].Matsui T, Yoshida T, Yoshimura T, Nagasawa T. Appl Microbiol Bio-technol 2006;73:95.

].Lindsey AS, Jeskey H. Chem Rev 1957;57:583.].Wuensch C, Gross J, Steinkellner G, Lyskowski A, Gruber K, GlueckSM, Faber K. RSC Adv 2014;4:9673.

].Wuensch C, Gross J, Steinkellner G, Gruber K, Glueck SM, Faber K.Angew Chem Int Ed 2013;52:2293.

ttp://dx.doi.org/10.1016/j.nbt.2014.05.1615

CIB-2

nconventional substrates for enzymatic reduction: car-oxylates and nitriles

. Winkler1,∗ , K. Napora-Wijata1, B. Wilding1, N. Klempier2

ACIB GmbH, Petersgasse 14/III, 8010 Graz, AustriaInstitute of Organic Chemistry, Graz University of Technology, Stremayrgasse, 8010 Graz, Austria

eywords: Biocatalysis; Nitrile Reductase; Carboxylate reductase;eduction; Enzyme

The reduction of carbonyls by enzymes has been extensivelytudied and is now widely applied on industrial scale. In com-arison, enzyme catalyzed carboxylic acid/carboxylate and nitrileeduction are relatively young fields. Their full potential is cur-ently being explored. The chemical reduction of nitriles andarboxylic acids requires strong reducing agents due the low reac-ivity of these moieties. Consequently, mild alternatives would be

valuable addition to the current toolbox of biocatalysis. Theroducts of nitrile reduction are primary amines and we have
ecently expressed nitrile reductase enzymes (NReds), in order topply these enzymes as biocatalysts. The substrate scopes of wildype and mutant NReds as well as their biochemical characteristicsere studied [1]. The reaction mechanism of NReds involves the
www.elsevier.com/locate/nbt S1

SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACI

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igure 1 Whole cell biotransformation approach towards 3-ydroxytyrosol – an antioxidant from olives.

eaction of the substrate with an active site thiol and subsequentADPH mediated reduction of the intermediate [2], which is simi-

ar to the mechanism proposed for carboxylate reductase enzymesCARs) [3]. The latter enzyme was in focus of the development of ahole cell system (Figure 1) for the production of the antioxidant-hydroxytyrosol [4].

eferences

].(a) Wilding B, et al. Adv Syn Catal 2012;354(11–12):2191–8;(b) Wilding B, et al. Chem Eur J 2013;19(22):7007–12.

].Chikwana VM, et al. J Biol Chem 2012;387:30560–70.].Venkitasubramanian P, et al. J Biol Chem 2007;282:478–85.].Napora-Wijata K, et al. ChemCatChem, Patent application pending[in print].


CIB-3

ngineering of cupin hydroxynitrile lyases

erstin Steiner1,∗ , Romana Wiedner1, Bettina Kothbauer1, Man-ana Gruber-Khadjawi1, Helmut Schwab2

ACIB GmbH, AustriaTU Graz, Austria

Enantiopure cyanohydrins serve as versatile building blocks forbroad range of chemical and enzymatic reactions resulting inighly valuable products for many applications. Hydroxynitrile

yases comprise a diverse group of enzymes which catalyze theeversible cleavage of cyanohydrins to carbonyl compounds andydrogen cyanide [1]. For a long time HNLs were only known toxist in plants, but recently (R)-selective HNL activity has beendentified in several bacterial proteins [2]. The structure of onearget protein from Granulicella tundricola (GtHNL) was solved andhowed high similarity to proteins of the cupin superfamily, a fold,hich has not been reported for HNLs before. Cupins are ubiqui-

ous small beta-barrel proteins and ideal candidates for applicationn industrial processes and as scaffold for enzyme engineerings they are easily expressed as soluble proteins in exceptionallyigh yield (> 50% of total protein) in E. coli. A detailed bio-hemical characterisation of GtHNL revealed that the enzyme isetal-dependent. Several amino acids were identified, which are

mportant for activity. The initial specific activity of 1.74 U/mg atH 6 with (R)-mandelonitrile, and the 80% conversion of benzal-
ehyde to (R)-mandelonitrile with 90% enantiomeric excess wereignificantly improved by applying random and rational proteinngineering approaches.
2 www.elsevier.com/locate/nbt

B SESSION) New Biotechnology · Volume 31S · July 2014

Acknowledgement: This project is financed by the Europeannion’s Seventh Framework Programme FP7/2007-2013 underrant agreement no. 289646 (Kyrobio).

eferences

].Winkler M, et al. Comprehensive chirality. Synthetic methods VI, vol. 7.Amsterdam: Elsevier; 2012. p. 350–71.

].Hajnal I, et al. FEBS J 2013;280:5815–28.


CIB-4

nzyme responsive polymers

lexandra Rollett 1,∗ , Andrea Heinzle2, Konstantin Schneider2,oris Schiffer2, Gregor Tegl3, Eva Sigl 2, Georg Guebitz1

BOKU-Vienna/ACIB GmbH, AustriaACIB-Austrian Centre of Industrial Biotechnology GmbH, AustriaBOKU-Vienna, Austria

Smart polymers can change their properties depending onertain stimuli like pH, temperature, light or electrical fields. Espe-ially in biomedical materials, but also in technical applications,ike the packaging industry, enzymes are useful biomarkers forensoring purposes. Here we present different strategies towardsnzyme responsive polymer systems for the detection of microbialontaminations in distinctive fields.

Enzyme responsive hydrogel based systems were constructedsing methacrylated biopolymers like polygalacturonic acid1], alginic acid [2], carboxymethylcellulose [3], peptidoglycan,elatin, collagen, alginate and agarose [4–6] while both stabil-ty and sensitivity were tuned via the degree of UV crosslinking.heses systems were successfully used to detect contaminatingicroorganisms based on their extracellular enzymes, and infec-

ion of wounds based on enzymes from the human immuneystem. The latter included myeloperoxidase, cathepsin G, elas-ase and lysozyme, which were found to show an elevated activityn infected wounds.

eferences

].Schneider, et al. N Biotechnol 2012;29:502–9.].Schneider, et al. Enzyme Microbial Technol 2011;48:312–8.].Schneider, et al. Process Biochem 2012;47:305–11.].Hasmann, et al. Exp Dermatol 2011;20:508–13.].Heinzle, et al. Wound Repair Regen 2013;21:482–9.

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ew Biotechnology · Volume 31S · July 2014 SUNDAY 13 J

CIB-5

sterases from Clostridium are involved in anaerobicegradation of synthetic polyester

eronika Perz1,∗ , Veronika Perz2, Armin Baumschlager2, Klausleymaier2, Andrzej Łyskowski2, Altijana Hromic2, Karl Gruber3,arsten Sinkel4, Ulf Küper4, Melanie Bonnekessel 4, Doris Ribitsch2,eorg Guebitz5

ACIB GmbH, AustriaACIB GmbH, Petersgasse 14, 8010 Graz, AustriaInstitute of Molecular Biosciences, University of Graz, AustriaBASF SE, Carl-Bosch-Straße 38, 67056 Ludwigshafen, GermanyInstitute of Environmental Biotechnology, University of Natural Resources andife Sciences, Austria

The anaerobic degradation of synthetic aliphatic–aromaticolyesters used in food packaging is of great importance duringnaerobic digestion. Several studies have proven the biodegrad-bility of PBAT (poly(butylene adipate-co-butylene terephthalate))nder aerobic conditions while there is only little information onnaerobic degradation.

In this study, imaging analysis (CLSM, SEM) and quantifica-ion of degradation products indicated anaerobic hydrolysis ofBAT in biogas sludge. However, the detected hydrolysis ratesre still too low for efficient PBAT degradation in industrial bio-as plants. Consequently, hydrolysis of PBAT by enzymes fromifferent anaerobic organisms (Clostridium species) was investi-ated. Therefore, various hydrolases from these organisms wereuccessfully heterologously expressed in E. coli BL21-Gold(DE3).he kinetic parameters on the standard substrate p-nitrophenylcetate were determined and revealed high activity of up to 700/mg (vmax). Analysis of the crystal structure of one esterase from. botulinum disclosed the presence of a Zn2+ metal ion that lieseep beneath the protein surface.

The degradation of synthesized oligomeric and polymericodel substrates was studied in order to get a deeper insight into

he reaction mechanisms of these new hydrolases.Esterases from C. hathewayi and C. botulinum were indeed

ble to hydrolyze aliphatic-aromatic polyesters like PBATnd different model substrates as indicated by HPLC/MSuantification of the hydrolysis products terephthalic acid,dipic acid, mono(4-hydroyxbutyl)terephthalate and bis(4-ydroxybutyl)terephthalate. We could demonstrate that thesterases show activity under mild conditions as well as in biogas
ludge.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1619aatmt

opatide

NDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)

CIB-6

esigning robust Saccharomyces cerevisiae strainsgainst stresses encountered during bioethanol fermen-ations from lignocellulosic biomass

inod Kumar1,∗ , Darren Greetham1, Tithira Wimalasena2

The University of Nottingham, United KingdomKingston Research Limited (BP and DuPont JV), United Kingdom

During the pre-treatment of lignocellulosic biomass inhibitoryompounds are released which can exert adverse effects on cellularrowth, metabolism and ethanol production. In addition, osmotictress caused by the high concentrations of available sugars andnd-stage ethanol toxicity reduce overall rates of bioethanol fer-entation. In previous studies, F1 hybrid segregants derived from

lean lineage Saccharomyces cerevisiae strains were assessed forolerance to a range of stresses encountered during industrialioethanol fermentations. Using a systems biology approach (QTL-uantitative Trait Locus) chromosomal loci conferring resistance

gainst weak acid and osmotic stress were identified, and withinhose loci genes COX20 (acetic acid) and RCK2 (osmotic) wereetermined as important for stress response.

In the present study, strains in which COX20 or RCK2 haveither been deleted or inserted into on tetracycline induced vec-ors. Phenotypic microarray assessment (Biolog, US) of thesetrains under acetic acid, formic acid, furfural, HMF, vanillin andorbitol stress have been determined and compared against appro-riate controls. Results have highlighted that presence of COX20

mproves tolerance to weak acids while RCK2 gene conferred resis-ance to osmotic stress. The presence of either gene also enhancedhe tolerance against furanic compounds such as furfural and HMF.


CIB-7

ystems biology of Pichia pastoris

rigitte Gasser

Dep. of Biotechnology, BOKU University of Natural Resources and Life Sciencesienna, Austria

Pichia pastoris is the most frequently used yeast system foreterologous protein production today, however, the toolbox ofvailable genetic elements is rather limited. To enable more robustnd cost-effective production processes for biopharmaceutical pro-eins and for industrial enzymes, it is crucial to understand the

olecular physiology of the host, and the specific limitations thathe product may exert on expression.

Instead of classical genetic approaches, we applied systems biol-gy tools to improve several aspects of the P. pastoris productionlatform. Combined transcriptomics, proteomics, metabolomicsnd flux data were used to investigate the interplay between pro-ein production and cell metabolism. Thereby we gained insight
n key regulatory effects exerted during heterologous protein pro-uction processes. These genome scale data were then furtherxploited for the identification of novel regulatory elements and

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UNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTIC

he prediction of cell engineering targets for improved productiv-ty and robustness. Additionally, the endowment of genes involvedn the protein secretory pathway was analysed in a comparativeenomics approach, as productivities are often limited at the levelf protein folding and secretion.


CIB-8

ross-species comparison of recombinant protein secre-ion in CHO cells and Pichia pastoris

ils Landes1,∗ , Andreas Maccani2, Christian Leitner3, Michaelaurer4, Alexandra B. Graf4, Minoska Valli 2, Clemens Gruber5,erda Modarres4, Friedrich Altmann5, Brigitte Gasser3, Wolfgangrnst3, Renate Kunert3, DiethardMattanovich3

ACIB, AustriaAustrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, AustriaDepartment of Biotechnology, University of Natural Resources and Life Sci-nces, Vienna, AustriaSchool of Bioengineering, University of Applied Sciences FH Campus Wien,ienna, AustriaDepartment of Chemistry, University of Natural Resources and Life Sciencesienna, Austria

Chinese hamster ovary (CHO) cells are currently theorkhorses in biopharmaceutical industry. However, yeasts such asichia pastoris are about to enter this field. Although a vast numberf research studies has focused on characterization of each of thendividual expression system, information on direct cross-speciesomparative studies are limited. Here we present a comprehensiveomparison of recombinant P. pastoris strains and CHO cell lines,ncluding bioprocess engineering aspects as well as systems biologypproaches.

Two model proteins of different complexity were chosen:onomeric and non-glycosylated human serum albumin and aore complex 3D6 single-chain Fv-Fc fusion antibody (3D6scFv-

c), which is secreted as a homodimer and contains the Fc-specificlycosylation sites.

High and low producing strains or cell lines of the two modelroteins were established and characterized in lab-scale bioreac-or cultivations. To evaluate the performance of each expressionystem, fed batch cultivations were performed. The productionrocesses were characterized by monitoring biomass and prod-ct formation as well as product quality. The two host systemsere then compared regarding their biomass specific secretion

ates and space-time yields of the processes. Obtained results showhat P. pastoris is the preferred host system for production of HSA,hereas CHO cells are more suited for the production of a complexolecule like the 3D6 scFv-Fc fusion protein.For transcriptomics and proteomics analysis steady state

amples from chemostat cultures were taken. Similarities and dif-erences between the investigated host systems as well as proteinpecific effects will be presented.

ttp://dx.doi.org/10.1016/j.nbt.2014.05.1622 ria


B SESSION) New Biotechnology · Volume 31S · July 2014

CIB-9

tilization of recombinase mediated cassette exchangeRMCE) for the generation of recombinant CHO cell linesith defined expression properties

artina Baumann1,∗ , Elisabeth Gludovacz2, Sabine Vcelar1, Nicoleorth3

ACIB, AustriaBOKU, AustriaBOKU/ACIB, Austria

For diverse applications, including generation of recombinantroduction cell lines, stable integration of transgenes into theenome of the host cell is a pre-requisite. The precise positionf the integrated transgene is a major determining factor, bothor gene expression level and long-term stability, which makeshe screening for a suitable cell clone very time-and labour inten-ive. The utilization of site-specific Recombinase Mediated Cassettexchange (RMCE) opened up the possibility to transfer the genef interest (GOI) into pre-selected genomic locations with definedxpression properties. In this study we developed a strategy sup-orting the identification of recombinant Chinese Hamster OvaryCHO) cells with integration sites favourable to persistent highransgene expression and good gene-exchangeability. A sortableeporter gene (CD4) with a leaky start codon, flanked by het-rospecific FRT (Flippase recognition targets) sequences was stablyransfected into CHO cells. The leaky start codon reduces trans-ation efficiency and allows sorting for the highest transcriptionates, while the FRT sites mediate subsequent exchange of thexpression cassette. Two rounds of RMCE followed by FACS sor-ing for top producers were performed to select for sites that alloweliable cassette exchange besides high transgene expression. Theesulting master cell line can be used repeatedly for insertion of theOI without major genetic alterations. A combination of chemi-

al selection using alternative selection markers and FACS sortingor absence of CD4 expression as criterion for successful genexchange enables fast establishment of recombinant cell lines withredictable expression properties.


CIB-10

ntegrated continuous refolding and precipitation ofroteins in a tubular reactor

iqi Pan ∗ , Monika Zelger, Rainer Hahn

Austrian Centre of Industrial Biotechnology, Austria

Large amounts of therapeutic proteins expressed in Escherichiaoli as inclusion bodies have created a bottleneck in downstreamrocessing. Process integration of continuous processes is one wayo alleviate this bottleneck. In view of this consideration, lab-ratory scale tubular reactors for the continuous processing of
ecombinant proteins were developed. Benefits include faster mix-ng, high design flexibility and efficient heat transfer. With thesedvantages in mind, refolding strategies like pulsed refolding and

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ew Biotechnology · Volume 31S · July 2014 SUNDAY 13 J

emperature leap refolding in tubular reactors were successfullyerformed. Additionally, the tubular reactor offer better integra-ion between processing steps while the ability to reach steadytate ensured consistent product quality. In an exemplary exam-le, a fully continuous process for an autoprotease fusion proteinas established. This includes inclusion body dissolution, refol-ing and autocleavage, followed by purification by precipitatingut impurities. Process runs were robust over extended periodshile reaction rates, product quality and yields were equal to batchrocessing. In an advanced setup, oxidative refolding by aeration

n loop configuration, equipped with static mixers, sparging ele-ents and air-traps was successfully performed. The integration

f inline sensors also allowed the monitoring of critical processarameters. Process runs confirmed that productivity with theubular reactor were more than twice of the batch reactor. With arowing interest in integrated continuous biomanufacturing, thelear advantages of the tubular reactor would be essential to thiseld.


CIB-11

rom multi-sequence alignment to metabolic engineer-ng to minimal drug cocktails: a new hitting setalculator

uergen Zanghellini ∗ , Christian Jungreuthmayer

University of Natural Resources and Life Sciences, Austria

Suppose you are given a set of drugs for the treatment of malig-ant cell lines. Typically a single drug does not treat all malignantells. So what is the (smallest) set of drugs killing all malignant

NDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)

ells? Such a problem is called a (minimal) hit-ting set [(M)HS]roblem–a classical problem in combinatorial optimization. Theroblem of calculating MHSs is often encountered in computa-ional biology with various applications in shotgun proteomics,ene expression analysis, multi-genome alignment and metabolicngineering.

Here we present MHScalculator, which allows for fast calcula-ion of MHSs in large systems. MHScalculator was developed at acibnd is freely available. We illustrate the power of MHScalculatoror genomics, systems pharmacology, and systems biotechnologyy aligning multiple genomes, by predicting minimal drug cock-ails, and by finding minimal metabolic intervention strategies tourn microorganism into optimal cell-factories for bio-productionf chemical commodities. MHScalculator is able to process largenput data sets typically within a minute and allows for a fastnd comprehensive analysis on standard computer infrastructure


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S

IOMOLECULAR TECHNOLOGYOF PROTEINS

iomolecular technology of proteins

IOTOP-1

esign and engineering of next generation mammalianell factories

avid James

The University of Sheffield, United Kingdom

The majority of new biopharmaceuticals are made in Chineseamster ovary cells, a transformed cell type originally isolated

n the 1950s. The adaptability and utility of CHO cell factorieserives from our exploitation of their acquired genetic/functionalariation using high-throughput functional screening and selec-ion processes which enable industry to isolate and maintain cellactories with unusual and desirable properties.

However, we still have a limited understanding of enablingellular mechanisms that underpin the ideal manufacturing phe-otype. This knowledge would permit design and construction of

ntrinsically better cell factories using the new concepts and toolsf systems and synthetic biology.

We now have the potential to provide disruptive new solutionsor cell and process engineering, where we will be able to createespoke cell factories with a predictable ability to manufactureomplex biopharmaceutical proteins.


IOTOP-2

haracterization of a novel cell penetrating peptideerived from human Oct4

va Harreither1,∗ , Hanna Rydberg2, Helene Amand2, Vaibhavadhav1, Lukas Fliedl 3, Christina Benda4, Miguel Esteban5,uanqing Pei5, Nicole Borth1, Regina Grillari-Voglauer1, Oliverommerding6, Frank Edenhofer7, Bengt Nordén2, Johannesrillari 1

Department of Biotechnology, BOKU University Vienna, AustriaDepartment of Chemical and Biological Engineering/Physical Chemistry,halmers University of Technology, Gothenburg, SwedenACIB GmbH, Austrian Center of Industrial Biotechnology, AustriaKey Laboratory of Regenerative Biology, Chinese Academy of Sciences,uangzhou, ChinaChinese Academy of Sciences, Guangzhou, ChinaStem Cell Engineering Group, Institute of Reconstructive Neurobiology, Uni-ersity of Bonn, GermanyStem Cell and Regenerative Medicine Group, Institute of Anatomy and Celliology, Julius-Maximilians-University Würzburg, Germany

Oct4 is a transcription factor that plays a major role for thereservation of the pluripotent state in embryonic stem cells asell as for efficient reprogramming of somatic cells to inducedluripotent stem cells (iPSC) or other progenitors. We here reporthat a 16 amino acid peptide representing the third helix of the
uman Oct4 homeodomain can internalize in mammalian cellspon conjugation to a fluorescence moiety thereby acting as a cellenetrating peptide (CPP). This Oct4-protein transduction domainPTD) was characterized by comparison to the well studied CPP

New Biotechnology · Volume 31S · July 2014

enetratin. Flow cytometry measurements and confocal imaginghow that human Oct4-PTD internalizes into live cells slightlyore efficiently than penetratin. However, the cellular distribu-

ion differs greatly, with Oct4-PTD showing diffuse cytosolic anduclear staining, whereas penetratin is strictly localized to a punc-

uate pattern in the cytoplasm. By using a Cre/loxP-based reporterystem, we further show that this peptide also drives translocationf a functionally active Oct4-PTD-Cre-fusion protein into reporterells. Finally, recombinant full length Oct4 is shown to translo-ate into human and mouse fibroblasts even without addition ofny kind of cationic fusion tag. Our data therefore support the ideahat transcription factors might be part of an alternative signallingathway, where the proteins are transferred to neighbouring cellso actively change the behaviour of the recipient cell.


IOTOP-3

rom slow to fast: effects of growth rate on global genexpression and recombinant protein secretion in Pichiaastoris

orinna Rebnegger1,∗ , Alexandra B. Graf2, Minoska Valli 3, Brigitteasser3, Michael Maurer2, DiethardMattanovich1

Department of Biotechnology, University of Natural Resources and Life Sci-nces, Vienna, AustriaSchool of Bioengineering, University of Applied Sciences FH-Campus Vienna,ienna, AustriaAustrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria

In Pichia pastoris recombinant protein production is related tohe specific growth rate �. To investigate the impact of varying

on productivity in more detail, we studied secretion rates andhe transcriptome of a recombinant P. pastoris strain producinguman serum albumin (HSA) at � ranging from 0.015 to 0.15 h−1

n glucose-limited chemostat cultures. Production rates of HSAorrelated positively with � while product quality remained unaf-ected. Analysis of global gene expression showed that ribosomalenes and other genes involved in gene expression and translations well as mitochondrial genes were upregulated with increasingrowth rate, while many transcriptional regulators, carbon sourceesponsive genes, autophagy and other proteolytic processes wereownregulated at higher �. Expression of mating and sporula-ion genes peaked at intermediate � of 0.05 and 0.075 h−1, andt very slow growth (� = 0.015 h−1) many transporter genes wereifferentially expressed. Analysis of a subset of genes related to pro-ein folding and secretion revealed that unfolded protein responseargets such as translocation, endoplasmic reticulum genes, andytosolic chaperones were upregulated with increasing � whileroteolytic degradation of secretory proteins was downregulated.e therefore conclude that a high � positively affects specific pro-

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ew Biotechnology · Volume 31S · July 2014

IOTOP-4

he human anti-HIV antibodies 2F5, PG9 and 2G12 differn their proteolytic susceptibility

elanie Niemer1,∗ , Melanie Niemer2, Ulrich Mehofer2, Juan Anto-io Torres Acosta2, Maria Verdianz2, Theresa Henkel2, Andreasoos2, Richard Strasser2, Daniel Maresch2, Thomas Rademacher3,erta Steinkellner2, Lukas Mach2

University of Natural Resources and Life Sciences Vienna, AustriaUniversity of Natural Resources and Life Sciences, AustriaInstitute of Molecular Biotechnology, RWTH Aachen University, Germany

The tobacco-related species Nicotiana benthamiana has recentlymerged as a promising host for the manufacturing of proteinherapeutics. However, the production of recombinant proteinsn N. benthamiana is frequently hampered by undesired proteo-ysis. Here we show that the expression of the human anti-HIVntibodies 2F5, PG9 and 2G12 in N. benthamiana leaves leadso the accumulation of discrete heavy-chain derived degradationroducts of 30–40 kDa. Incubation of purified 2F5 with N. ben-hamiana intercellular fluid resulted in rapid conversion into the0-kDa fragment, whereas 2G12 proved largely resistant to degra-ation. Such a differential susceptibility to proteolytic attack waslso observed when these two antibodies were exposed to variousypes of proteinases in vitro. While serine and cysteine proteinasesre both capable of generating the 40-kDa 2F5 fragment, the0-kDa polypeptide is most readily obtained by treatment withhe latter class of enzymes. The principal cleavage sites resideithin the antigen-binding domain, the VH-CH1 linker segmentnd the hinge region of the antibodies. Collectively, these resultsndicate that down-regulation of endogenous serine and cysteineroteinase activities could be used to improve the performance oflant-based expression platforms destined for the production ofiopharmaceuticals.


IOTOP-5

hlorite dismutases and dye-decolorizing peroxidases –imilarities and differences within a structural super-amily of heme proteins

rene Schaffner ∗ , Stefan Hofbauer, Paul Furtmüller, Christianbinger

BOKU Vienna, Austria

Chlorite dismutases (Clds) are heme b containing enzymeshich were discovered in chlorate- and perchlorate-reducingacteria (PCRBs) [1] but are found in many other bacterial andrchaeal phyla [2]. Cld protects PCRBs from the accumulation ofhe harmful metabolic intermediate chlorite by degrading it tohloride and dioxygen [1]. This catalytic function turns Cld into aighly interesting enzyme for bioremediation as the environmen-

al pollutants chlorate and chlorite are used as bleaching agents, asisinfectants, in pesticides etc. Additionally, Cld is extremely inter-sting from a biochemical point of view as it is the only known

BIOMOLECULAR TECHNOLOGYOF PROTEINS

nzyme system which efficiently catalyzes O-O bond formationeside photosystem II. Moreover, Clds are phylogenetically relatedith a recently discovered new heme peroxidase family calledye-decolorizing peroxidases (Dyps). They were shown to have anique tertiary structure with a distal heme region that is differ-nt from conventional peroxidases from plants and animals [3] butimilar to Cld. Here, we show an up-to-date phylogenetic analysisf Clds and Dyps and point out their structural relationship as wells their functional differences [4].

eferences

].van Ginkel CG, Rikken GB, Kroon AG, Kengen SW. Arch Microbiol1996;166:321–6.

].Maixner F, Wagner M, Lücker S, Pelletier E, Schmitz-Esser S, Hace K,et al. Environ Microbiol 2008;10:3043–56.

].Sugano Y, Muramatsu R, Ichiyanagi A, Sato T, Shoda M. J Biol Chem2007;282:36652–8.

].Hofbauer S, Schaffner I, Furtmüller PG, Obinger C. Biotechnol J 2014[in press].


IOTOP-6

nzymatic oxidation of plant polysaccharides adsorbedo cellulose surfaces

ilip Mollerup1,∗ , Matti Häärä2, Kirsti Parikka3, Chunlin Xu2,aija Tenkanen3, EmmaMaster4

Department of Biotechnology and Chemical Technology, Aalto University,inlandLaboratory of Wood and Paper Chemistry, Åbo Akademi University, FinlandDepartment of Food and Environmental Science, University of Helsinki,inlandDepartment of Chemical Engineering and Applied Chemistry, University oforonto, Canada

The development of renewable energy and materials dependsn intelligent utilization of abundant plant resources. Whileiotechnology for biofuels has received considerable attention, theotential of enzymes to tailor plant polymers for novel biopolymerynthesis is comparatively unexplored.

In particular, enzymes that catalyze selective oxidation ofpecific hydroxyls to carbonyl and carboxyl groups can facili-ate targeted chemical derivatizations of polysaccharides to createenewable polymers with preferred physical and chemical proper-ies [1]. In this regard, galactose oxidase (GaOx) continues to bepromising catalyst for specific delivery of new functionality tolant polysaccharides. Current chemo-enzymatic protocols withaOx begin by oxidizing galactose substituents in polysaccharides

e.g. galactoglucomannan, xyloglucan) that are suspended in solu-ion. Oxidized polysaccharides can then be adsorbed to celluloseurfaces to modify the surface reactivity and/or barrier properties


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IOMOLECULAR TECHNOLOGYOF PROTEINS

Accordingly, the aim of this study was to investigate the poten-ial to oxidize xyloglucan and galactoglucomannan followingdsorption of these polysaccharides to cellulose (Whatmann filter
aper no. 1). Wild-type GaOx along with GaOx fused to a family9 CBM from Piromyce equi were used in the analysis. Recent char-cterization of the GaOx RQW mutant fused to a cellulose-bindBM3 will also be presented.
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eference

].Xu C, Spadiut O, Araújo AC, Nakhai A, Brumer H. Chemo-enzymaticassembly of clickable cellulose surfaces via multivalent polysaccha-
rides. ChemSusChem 2012;5:661–5.

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pening Ceremony

L1 – 1

ow to get biotechnology to work for us?

nne Glover (CBE)

CSA to President Barroso, European Commission

One thing scientists are not short of is imagination and ourndeavours in biotechnology are an example of that. At thisonference we will hear about synthetic polymers, plants asio-factories, stem cell therapies, climate change mitigation andiofuels to mention just some of the topics. This harnessing ofellular and biomolecular processes to develop new products androcesses and safeguard our planet requires more than just sci-ntists to make sure it is the success that we hope for. We needommunicative scientists who can talk about the opportunities asell as the threats of new technologies; we need citizens to engagend question what we do; we need honest and transparent busi-esses to translate knowledge into the economy and offer newossibilities; we need imaginative policymakers to develop smartptions to allow exploitation of the knowledge that we producend we need effective and transparent politicians who make surehat citizens realise the maximum benefit of the research that theyave funded. We are living in an era dominated by biology and theioeconomy has never held out so many possibilities. Only if well work together will we be able to achieve the best for citizens,ur lifestyle and our planet.


L1 – 2

ynthetic biology for synthetic chemistry

ay Keaslinga,b,c

Departments of Chemical Engineering and Bioengineering, University ofalifornia, Berkeley, CA 94720, United StatesSynthetic Biology Department, Physical Biosciences Division, Lawrenceerkeley National Laboratory, Berkeley, CA 94720, United StatesJoint BioEnergy Institute, Emeryville, CA 94608, United States

Synthetic biology is the design and construction of new bio-ogical entities such as enzymes, genetic circuits, and cells or theedesign of existing biological systems. Synthetic biology buildsn the advances in molecular, cell, and systems biology and seekso transform biology in the same way that synthesis transformedhemistry and integrated circuit design transformed computing.he element that distinguishes synthetic biology from traditionalolecular and cellular biology is the focus on the design and con-

truction of core components (parts of enzymes, genetic circuits,etabolic pathways, etc.) that can be modeled, understood, and

uned to meet specific performance criteria, and the assembly ofhese smaller parts and devices into larger integrated systems that
olve specific problems. Just as engineers now design integratedircuits based on the known physical properties of materials andhen fabricate functioning circuits and entire processors (with rel-
OPENING CEREMONY

tively high reliability), synthetic biologists will soon design anduild engineered biological systems.

We have used synthetic biology to create inexpensive, effective,nti-malarial drugs. Currently, malaria infects 300–500 millioneople and causes 1-2 million deaths each year, primarily children

n Africa and Asia. One of the principal obstacles to addressinghis global health threat is a lack of effective, affordable drugs.he chloroquine-based drugs that were used widely in the pastave lost effectiveness because the Plasmodium parasite whichauses malaria has become resistant to them. The faster-acting,ore effective artemisinin-based drugs – as currently produced

rom plant sources – are too expensive for large-scale use in theountries where they are needed most. The development of thisechnology will eventually reduce the cost of artemisinin-basedombination therapies significantly below their current price. Toeduce the cost of these drugs and make them more widely avail-ble, we have used synthetic biology to engineer microorganismso produce artemisinin from renewable resources.

Having successfully completed the artemisinin work, we areow engineering the metabolism of the same microorganisms

Escherichia coli and Saccharomyces cerevisiae) for production ofdvanced biofuels and chemicals that might otherwise be pro-uced from petroleum. Unlike ethanol, the advanced biofuelsave the full fuel value of petroleum-based biofuels, will be trans-ortable using existing infrastructure, and can be used in existingutomobiles and airplanes. Similarly, the microbially sourcedhemicals can be dropped into existing processes used to pro-uce existing materials. These chemicals will be produced fromatural biosynthetic pathways that exist in plants and a varietyf microorganisms as well as from pathways that have no rep-esentation in the natural world. Large-scale production of thesehemicals and fuels will reduce our dependence on petroleum andeduce the amount of carbon dioxide released into the atmosphere,hile allowing us to take advantage of our current transportation


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ONDAY 14 JULY BIOCHEMICAL ENGINEERING JOURNAL YOUNG INVESTIGATOR AW

onday 14 July

iochemical Engineering Journal Youngnvestigator Award Lecture

I – 1

glimpse into the future of mammalian cell culture pro-ess development: innovative approaches to impact timeo clinic, product quality, and cost of process develop-ent and commercial manufacturing

hetan Goudar

Amgen Inc., United States

There has been remarkable progress in cell line generation andell culture processes over the past 2 decades. Expression levelsave increased by orders of magnitude and a broad spectrum ofomplex biotherapeutics have been successfully manufacturedt commercial scale. Despite these advances, it is important to
ecognize that the future poses fundamentally different chal-enges. While development costs and speed to in vivo validationf biology will face increasing pressure, decentralized manufac-uring is increasingly becoming a necessity for global reach of a
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ECTURE New Biotechnology · Volume 31S · July 2014

iotherapeutic, both from regulatory and economic standpoints,hich has implications for commercial manufacturing. Increased

egulatory clarity around biosimilars has intensified efforts inhis space and matching product quality of molecules developedecades ago with current processes is most effectively done withobust mechanistic understanding of cellular processes ratherhan by purely empirical approaches. In this changing landscape,ecent advances in systems biology and a renewed interest inontinuous perfusion cultivation offer avenues to bring aboutaradigm shifts in cell culture process development and commer-ial manufacturing. An approach for streamlined cell line andarly-stage cell culture process development will be presentedhich can accelerate time to human clinical testing by as much asmonths. Application of a poly-omics systems biology approach

or identifying modulators for a critical product quality attributeill be presented followed by validation of these targets using cell

ine engineering. Recent dramatic productivity enhancementsrom continuous perfusion cultures will be presented and themplications of these advances on next-generation commercial


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ymposium 1: Stem cell applications and geneherapies: where are we?

1-1

ngineering synthetic stem cell niches

atthias P. Lutolf

Laboratory of Stem Cell Bioengineering and Institute of Bioengineering, Schoolf Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne,witzerland

In vivo, the behavior of stem cells in a tissue is governedy the three-dimensional (3D) microenvironment, or ‘niche’,hich involves a dynamic interplay between biochemical andechanical signals provided by the extra-cellular matrix (ECM),

ell-cell interactions and soluble factors. The complexity of nichesnd the context-dependent cell-responses that arise from thesenteractions have posed a major challenge to understanding thenderlying regulatory mechanisms and to identify artificial nichesontrolling the behaviour of specific stem cell types. To system-tically dissect the role of the various interacting factors thatetermine stem cell fate in a 3D context, we have developedxperimental paradigms to simultaneously generate hundreds tohousands of unique microenvironments and probe their effectsn cell fate. In this talk I will discuss our approach by way of sev-ral experiments in which we have measured the combined effectsf microenvironment stiffness, proteolytic degradation, and threelasses of signaling proteins on pluripotent stem cell fate, unveil-ng a comprehensive map of the interactive involvement of thesearameters in regulating self-renewal and early differentiation.ur approach is broadly applicable to gain a systems-level under-

tanding of multifactorial 3D cell-matrix interactions and openshe door for discovering unique artificial niches that control theehavior of difficult-to-culture mammalian stem cells.


1-2

quick potency assay for osteogenic and chondrogenicifferentiation of adipose derived stem cells

leni Oberbauer1,∗ , Florian Hildner2, Ara Hacobian1, Susanneolbank1, Carolin Steffenhagen1, Georg Feichtinger1, Anjaeterbauer2, Christian Gabriel 2, Heinz Redl1

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology,ustriaRed Cross Blood Transfusion Service of Upper Austria, Austria

The field of bone- and cartilage regeneration has evoked strongnterest in tissue engineering. Osteogenic and chondrogenic dif-erentiation potential of adipose derived stem cells (ASC) arehallenging and promising for bone and cartilage repair. Currentn vitro methods to analyze the differentiation capacity are time
onsuming and thus we designed and established novel enhancernd tissue-specific promoter for osteogenic and chondrogenicifferentiation of ASC together with a quick potency bioassay.or this, osteocalcin or collagen type II promoter was coupled
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SYMPOSIUM 1: STEM CELL APPLICATIONS ANDGENE THERAPIES: WHERE AREWE?

o a strong viral enhancer for signal amplification. Human pri-ary ASC were co-transfected with luciferase based reportergenes

CMVE/mOCP-MetLuc (osteocalcin) or pCMVE/mACDC-MetLuccollagen type II) together with renilla control plasmid. For trans-ection, a mild lipofection was applied and a green fluorescencerotein (GFP) control was included to assess transfection effi-iency. Secreted metridia luciferase allows to measure promoterctivation in the supernatant without need of cell lysing. The activ-ty of the reportergenes was enhanced in transfected ASC treatedith osteogenic or chondrogenic differentiation media compared

o control condition. Osteogenic differentiation was confirmedith Alizarin red quantitative and histological stainings whereas

hondrogenic differentiation was confirmed with collagen typeI staining. These findings were further verified by qRT-PCR.lthough osteocalcin and collagen type II are late markers, thectivation of the signal-amplified and sensitive reportergenes canlready be determined after 3 days of differentiation in vitro.


1-3

epeated systemic administration of human adipose-erived stem cells attenuate diabetic nephropathy in theats

ue-Yuan Bai ∗ , YuxiangMa, Xiangmei Chen

Department of Nephrology, Chinese PLA General Hospital, China

Diabetic nephropathy (DN) is a major complication of diabetesnd represents the leading cause of end-stage renal disease world-ide. Unfortunately, once the patients develop overt proteinuria,

here is no cure for DN. Therefore, the availability of a strategyimed to delay or revert DN would be highly desirable.

To investigate the role of human adipose-derived stem cellsASCs) in treatment of diabetic nephropathy, Sparague-Dawleyats were made diabetes by intraperitoneal injection of strepto-otocin after uninephrectomy. After 12 weeks proteinuria wasell-established. The rats received injection of human ASCs via tailein at 5 × 106 every 4 weeks for five times. Reduction of protein-ria was not observed in diabetic rats until 24 weeks after threeoses of ASCs. However, since 28 weeks, urinary protein excre-ion was significantly suppressed, and persisted up to 32 weeksfter streptozotocin. Hypoalbuminemia and hyperlipidemia werelso improved in ASCs treated group. ASCs significantly attenu-ted glomerulus hypertrophy and renal tubular interstitial injurys well as downregulation of podocyte markers, WT-1 and snap-opodin. Hoechst labeled ASCs were injected into DN rat via tailein. Cells were detected in lung and spleen, and peritubularegions, but rarely in glomeruli and pancreas within 48 hours afternjection. Gene expression of human Alu could be detected inat lung and spleen till 4 weeks after injection. The ASCs did notmprove hyperglycemia and pancreatic damage.

These findings indicate that repeated intravenous ASCs can
educe diabetic kidney damage in rats even at the progressive stage.


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1-4

roducing and harvesting culture-derived platelets withunctional activity from blood stem cells

illiam Miller1,∗ , Alaina Schlinker1, Katherine Radwanski2,hristopherWegener2, K. Augustine Min2

Northwestern University, United StatesFresenius Kabi, USA

Platelet production in culture by megakaryocytic cells (Mks)erived from hematopoietic stem and progenitor cells (HSPCs)ould supplement donations, but it is very challenging to purify

he platelets. As an alternative to multi-step centrifugation, wesed a polycarbonate spinning membrane with 4-�m cylindri-al pores. Because platelet release is asynchronous, we exploredhether we could harvest platelets generated early in culture and

eseed immature Mks to generate platelets later in culture. Exper-ments with immortalized Mks and apheresis platelets showedfficient platelet recovery and the exclusion of Mks. Recoveredlatelets had little pre-activation and the Mks retained viabil-

ty and proplatelet formation. Next, primary CD34+ HSPCS wereifferentiated to Mks that released platelets. As for the Mk line,rimary Mks were excluded from the platelet fraction and the via-ility and ploidy distribution of recovered Mks were similar tohose of input Mks. Recovered platelets expressed surface mark-rs and spread after activation by thrombin in a similar manner tonprocessed platelets. However, culture-derived platelet recoveryas lower than for apheresis platelets (70 vs. 90% at 3000 rpm).his is likely due to the larger size of culture-derived plateletsnd preplatelets (5.6 vs. 3.3 �m). After screening experimentsith even larger red blood cells (7.3 �m), we increased culture-erived platelet recovery with essentially no Mk contaminationy decreasing the rotation rate to 2000 rpm. Recovered plateletshowed minimal pre-activation and were activated by thrombin.mportantly, Mks in the cell fraction that were returned to cultureeleased platelets that were harvested two days later.


1-5

dipose derived stem cells respond to in vitro extracorpo-eal shockwave treatment with increased stemness andultipotency

hristina Schuh1,∗ , Philipp Heher1, Anna Weihs2, Asmitaanerjee1, Susanne Wolbank1, Rainer Mittermayr1, Heinz Redl1,ominik Rünzler2, Andreas Teuschl2

LBI for Experimental and Clinical Traumatology, AustriaUniversity of Applied Sciences Technikum Wien – Department of Biochemicalngineering, Austria

Tissue resident adipose derived progenitor/stem cells (ASCs) arepromising tool for tissue engineering, addressing the problem of

issue and organ shortage. Limiting factors for the use of ASCs areonor variation and senescence, loss of differentiation capacity asconsequence to loss of multipotency. For several decades extra-

orporeal shockwave treatment (ESWT) has been proven to be a

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uitable clinical tool to improve regeneration of a variety of tis-ues, however, the mechanisms underlying these beneficial effectstill remain widely unknown. In this study we address the effectsf ESWT onto ASCs and their stemness as well as differentiationotential in high passages.

In our study we show that human and rat ASCs respondtrongly to repetitive shockwave treatment in vitro, resulting inaintenance and significant elevation of mesenchymal mark-

rs (flow cytometry: CD73, CD90, CD105), while cell viabilitynd proliferation remain at a comparable level to control group.nother effect observed was a significant increase in differ-ntiation capacity into osteogenic (von Kossa staining; PCR:steocalcin, biglycan) and adipogenic lineage (Oilred O staining)s well as into Schwann like cells (flow cytometry: P75, S100, P0)n high passages.

Our results indicate that ESWT preserves stemness and multipo-ency of ASCs even in higher passages after extensive expansion.ence, ESWT might be a promising tool to improve the quality

nd consistency of ASCs for cell therapy in tissue engineering andegenerative medicine.

Acknowledgement: Financial support from FFG (#818412)nd the “City of Vienna Competence Team Tissue Engineeringioreactors” is gratefully acknowledged.


1-6

ngineering bacteria for the discovery of potential ther-peutic compounds against protein misfolding diseases

eorgios Skretas1,∗ , Dafni Delivoria2, Ilias Matis2, Nikolettaapaevgeniou2, Niki Chondrogianni2

National Hellenic Research Foundation, GreeceInstitute of Biology, Medicinal Chemistry & Biotechnology – National Hellenicesearch Foundation, Greece

It has now been widely recognized that many incurable dis-ases with enormous socioeconomic impact, such as Alzheimer’sisease, Parkinson’s disease, type 2 diabetes, etc., are initiated bycommon mechanism: the misfolding of specific proteins. Here,e describe the use of engineered bacterial cells as a platform for

he discovery of potential therapeutics against such protein mis-olding diseases (PMDs). The topic of the described research is thepplication of molecular evolution approaches for the discoveryf compounds that rescue the misfolding of PMD-associated pro-eins. To achieve this, Escherichia coli cells are first engineered toiosynthesize large libraries of test compounds with high struc-ural diversity. Then, the same cells are modified further so thathey allow the identification of the rare molecules that correct theolding of particular misfolding-prone and PMD-associated pro-eins (MisPs) with the use of a genetic screen. Lead compoundsdentified by this initial screen, are then subjected to more detailedvaluation by biochemical and biophysical methods of proteinnalysis, and their ability to inhibit MisP-induced pathogenicity
s tested using appropriate human cell assays or in vivo modelsf the disease of interest. The molecules capable of rescuing theisfolding of the target MisP and of antagonizing its associated

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athogenicity become drug candidates against the specific dis-ase. We will describe our efforts to identify such “pharmacologicalhaperones” against the misfolding of the amyloid � (A�) peptidend of certain carcinogenic misfolded variants of human p53, withhe aim of developing potentially therapeutic compounds againstlzheimer’s disease and cancer, respectively.


1-7

omputational prediction of associations between psori-sis, rheumatoid arthritis and osteoarthritis

uba Sevimoglu ∗ , Kazim Yalcin Arga

Marmara University, Turkey

Microarray technologies have enabled rapid and efficientxpression profiling of thousands of genes simultaneously.icroarray studies on disease datasets have already revealed genes

hat could be implicated in the pathogenesis of that disease thus
pening the path to accurate diagnosis and potential biomarkers.his study aims at comparative analysis of three common diseasesith important genetic origin: Osteoarthritis (OA), rheumatoidrthritis (RA) and psoriasis.
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SYMPOSIUM 1: STEM CELL APPLICATIONS ANDGENE THERAPIES: WHERE AREWE?

A comparison of gene expression data from five independenttudies, obtained from Gene Expression Omnibus has been per-ormed. Each dataset was statistically analyzed in order to identifyifferentially expressed genes (DEGs). Proteins encoded by DEGsere determined and integrated with protein-protein interaction

ppi) data for further analyses and to identify hub proteins. Enrich-ent analyses were performed to map the interconnectivities

etween diseases and biological pathways.Comparative analyses indicated that six DEGs were common

etween all three diseases, which are immune related and havessociations to cardiovascular diseases. Enrichment analyses leado a significant association with PPAR signaling pathway. There are4 Transcriptional Factors regulating these DEGs. The hub proteinsn the disease ppi network are: CEBPD and MMP1.

This study suggests a genetic cause common between psoriasis,A and RA. The commonality between psoriasis and RA comes

rom the fact that they are both autoimmune diseases. In thease of OA and psoriasis, Psoriatic Arthritis might be the link thatonnects psoriasis to OA. This study will elucidate molecular andellular mechanisms which will lead to a better understanding and



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YMPOSIUM 2: PLANTS FOR THE PRODUCTIONOF HIGH VALUE CHEMICALS

ymposium 2: Plants for the production ofigh value chemicals

2-1

ultured plant cambial meristematic cells as a chassis forhe production of pharmaceuticals

usan Howat1 , Marisol Ochoa Villarrea1, Rabia Amir1, Eunjungwon1, Zejun Yan1, Young-Woo Jin2, Eun-Kyong Lee2, Gary J.

oake1,∗

Institute of Molecular Plant Sciences, School of Biological Sciences, Universityf Edinburgh, King’s Buildings, Edinburgh EH9 3JR, UKUnhwa Corp., 874-1, 2Ga, Wooah-Dong, Dukjin-gu, Jeonju, South Korea

A plethora of important, chemically diverse natural productsre derived from plants. In principle, plant cell culture offersn attractive production platform for some natural products butften is not a commercially viable strategy because of difficul-ies associated with culturing dedifferentiated plant cells (DDCs)n an industrial scale. To address this issue we have isolatednd cultured innately undifferentiated cambial meristematic cellsCMCs). Utilizing a combination of deep sequencing technologies,e identified marker genes and transcriptional programs consis-

ent with a stem cell identity.CMCs derived from Taxus cuspidata and Panax ginseng, sources

f the key anticancer drug, paclitaxel and neuroprotective ginseno-ides, respectively, circumvented obstacles routinely associatedith the commercial growth of DDCs. Subsequent molecular

trategies uncovered a network of regulators that control thexpression levels of genes integral to the biosynthesis of these keyatural products. We envisage both natural and where appropriate,ngineered CMCs, will provide a cost-effective and environmen-ally friendly platform for the sustainable production of a wideariety of important plant-derived pharmaceuticals.


2-2

olecular marker-assisted selection and pyramidingffect of major QTLs for cotton fiber strength

oulu Yuan ∗ , Tiankang Wang, Yuzhen Shi, Haihong Shang, Aiy-ng Liu, Junwen Li, Juwu Gong, Tao Wang, Wan-kui Gong, Tingtinghen, Botao Li

Institute of Cotton Research of Chinese Academy of Agricultural Sciences/Stateey Laboratory of Cotton Biology/Key Laboratory of Biological and Geneticreeding of Cotton, The Ministry of Agriculture, China

Two elite fiber quality materials 0-153 and Xinluzao 24, andwo commercial cotton cultivars (lines) Lumianyan 28 and Jimian16 were used as parents to develop two double-cross com-inations (Jimian 516 × 0-153) × (Jimian 516 × Xinluzao24)Pop1,Lumianyan 28 × 0-153) × (Lumianyan 28 × Xinluzao 24)Pop2,
our SSR markers, which were linked to three QTLs of fiber strengthnd derived from 0-153 or Xinluzao 24, were used respectivelyo study the efficiency of molecular marker-assisted selection andyramiding effect of fiber strength. The result showed that assisted
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election of four markers had significant genetic effect. In the tworoups of ++/+ genotype, the average fiber strength of individualsere 31.21-32.62 cN/tex, and 30.77-32.50 cN/tex for the +/− geno-

ype, selection effects of single marker were 0.80-1.51cN/tex; Inhe groups for QTL-1 × QTL-3 and QTL-2 × QTL-3Combinations,he average fiber strength of individuals while pyramiding twoTLs were 33.40-34.08 cN/tex, the selection effects were 2.73-3.56

N/tex which compared to the plants without the two QTL, andhe selection effects of 1.12-3.02 cN/tex compared to the plantsith one of the two QTL. In addition, QTL-1 and QTL-2 might be

he same QTL, QTL-2 and QTL-3 had obvious epistasis effect. So,his shows it is feasibility to use the molecular marker for assistinghe pyramiding selection of fiber strength.


2-3

he CO2 microalgae biorefinery: high value products andiofuels using halophilic microalgae in the “D-Factory”

atricia Harvey1,∗ , David Bailey1, Ami Ben-Amotz2, Vitorerdelho3, Guy Harris4, David Rooke4, Herre Hoekstra5, Pauloacher6, Joao Crespi7, Guido Reinhardt8, Laura Martinelli 9,eclan Schroder10, Richard Pipe10, Nadine Igl-Schmid11, Antonisokossis12, Karin Perrson13

University of Greenwich, United KingdomNature Beta Technologies Ltd., (NBT), IsraelA4F AlgaFuel, PortugalDynamic Extractions, United KingdomEvodos, GermanyHafren Investments, United KingdomInstituto de Biologia Experimental e Tecnológica (IBET), PortugalIFEU – Institute for Energy and Environmental Research, United StatesIN SRL Italy, Italy

0 Marine Biological Association of the UK, United Kingdom1 NATECO2 GmbH & Co. KG, Germany2 National Technical University of Athens, Greece3 SP Technical Research Institute of Sweden AB, Sweden

Fuel-only algal systems are not economically feasible becauseields are too low and costs too high for producing microal-al biomass compared to using agricultural residues e.g. straw.iorefineries which integrate biomass conversion processes andquipment to produce fuels, power and chemicals from biomass,ffer a solution. The CO2 microalgae biorefinery (D-Factory) is a 10illion Euro FP7-funded project which will cultivate the microalgaunaliella in highly saline non-potable waters in photobioreactorsnd open raceways and apply biorefinery concepts and Europeannnovations in biomass processing technologies to develop a bas-et of compounds from Dunaliella biomass, including the highalue nutraceutical, �-carotene, and glycerol. Glycerol now findsarkets both as a green chemical intermediate and as a biofuel inHP applications as a result of novel combustion technology. Driv-

ng down costs by recovering the entire biomass of Dunaliella cellsrom saline cultivation water poses one of the many challenges
or the D-Factory because Dunaliella cells are both motile, and doot possess an external cell wall, making them highly susceptible
o cell rupture. Controlling expression of desired metabolic path-ays to deliver the desired portfolio of compounds flexibly and

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nvestment in algae biorefineries and in large-scale production oficroalgae.


2-4

xploiting nature’s chemists: high value bioactive com-ounds from algae

hristine Edwards ∗ , Linda Lawton

IDEAS/Robert Gordon University, United Kingdom

Algae are an essential component of global ecosystems usingight and carbon dioxide to produce organic carbon and oxygen.onsequently they are found in diverse habitats including ter-

estrial and aquatic habitats, hot springs to Antarctic mats. Thecological diversity has resulted in chemical diversity and theyncreasingly investigated as sources of new chemical entities inrug discovery programs, in particular anti-cancer.

In order to exploit the chemical diversity in target algae,00–500 L algae per month, are grown in parallel batch cul-ures. Cells are harvested and bioactive compounds are extractedsing methanol, cleaned up by automated flash chromatogra-hy with final polish by preparative high performance liquidhromatography (HPLC). Throughout the process quality anduantity of compounds in extracts/fractions are tracked by ultraerformance liquid chromatography–photodiode array-mass spec-rometry (UPLC-PDA-MS) to ensure high purity is achieved. Dueo the highly toxic nature of the compounds, extreme precautions

ust be exercised throughout processing, with frequent review.n addition, shipping is a challenge as they are categorised asxtremely dangerous according to transport regulations so theysual couriers such as FedEx will not take them. In addition, as theyre considered as potential weapons of mass destruction, exporticence and control is essential.

Despite all the challenges 14 products are globally available viafine biochemical company, generating in excess of £120 K per

nnum.


2-5

se of synthetic biology in creating high-value metalanoparticles from phytoremediated waste

atthew Edmundson ∗ , Michael Capeness, Louise Horsfall

Edinburgh University, United Kingdom

Metal and metalloid contamination of land is a major prob-
em on former industrial sites in the UK and in the wider world.hytoremediation has been used as a method to clean up this con-amination in the past but disposal of the contaminated plantaste is still a problem. The Cleaning Land for Wealth (CL4 W)
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SYMPOSIUM 2: PLANTS FOR THE PRODUCTIONOF HIGH VALUE CHEMICALS

onsortium is looking into financially incentivising land decon-amination by producing high-value products from the waste, withur initial case studies focusing on arsenic, platinum and palla-ium. CL4 W is following a number of research tracks, including

nvestigating the optimal plant species for phytoremediating aumber of different metals and developing cost-effective meth-ds of processing the phytoremediation waste. We are also usingynthetic biology tools to employ micro-organisms in harvest-ng heavy metals from the waste, engineering bacteria to converthese metals into high-value nanoparticles for use in medicine andndustry, and looking into extracting energy from the left-overlant material.

The data presented here will detail the progress made so far onhe CL4 W project, highlighting our work in evaluating contami-ated industrial sites and our current model of assessing the costsnd benefits of the project. As part of the nanoparticle productionesearch track we are working with bacteria able to reduce plat-num and palladium, and have identified a number of the genesnvolved in these pathways. We have also engineered a strain of. coli able to tolerate high levels of arsenic, and which at theame time produces nano-scale structures when in the presencef arsenic.


2-6

ene isolation and its identification of salinity stress on. hirsutum L.

uwei Ye

Institute of Cotton Research, CAAS, China

Soil salinization has become a serious global problem affect-ng the agricultural development and the ecological environment.alinityas one of the most important abiotic stresses in the world,everely limits the production of crop. Saline-alkali land in ourountry is widely distributed with the character of multi typesnd serious salt-deposition. In order to carry out the utilizationf saline-alkali land efficiently, it is necessary to develop the agri-ulture on the saline-alkali land. Cotton, the major cash crop inhina, is playing a crucial role in national economic development.hina, with less cultivated lands and more people, faces the con-

radiction between food and cotton, which seriously affects theultivation and production of cotton. Therefore, it is an effectiveay to farm saline land and to enhance the sustainable agriculturalevelopment by develop the salinity-tolerant varieties of cotton.dentification of salinity-tolerance also plays a vital role on cot-on breeding. 11 salt-tolerance related genes, H+-pyrophosphataseene and S-adenosylmethionine synthetase gene and others, wereloned from the salt-tolerance material on Gossypium hirsutum,hich were named GhVP and GhSAMS, respectively. The bioin-

ormatics analysis and their transformed accessions were tested



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YMPOSIUM 3: GLYCOBIOTECHNOLOGY

ymposium 3: Glycobiotechnology

3-1

evelopment of new synthetic and analytical tools inlycobiotechnology

abine L. Flitsch

The University of Manchester, United Kingdom

Carbohydrates provide the largest biomass on Earth and areentral to many aspects of biotechnology with applications iniofuels, biomaterials, food and medicine. Carbohydrates are com-lex biomolecules and there is an urgent need to develop robustynthetic and analytical methodologies to fully exploit oppor-unities presented by glycobiotechnology. We have developed aoolbox for the synthesis and analysis of complex carbohydratesnd their function with a focus on (i) chemoenzymatic synthesis oflycoconjugates such as glycopeptides, (ii) glycoarrays to study gly-oenzyme activity and discover carbohydrate-protein interactionsnd (iii) ion mobility mass spectrometry for high resolution struc-ural analysis of carbohydrates and (iv) mass spectrometry for theabel free identification of carbohydrate-binding proteins [1–5].

eferences

].Both, et al. Nat Chem 2014;6(1):65.].Noble, et al. J Am Chem Soc 2012;134(31):13010–7.].Sardzík, et al. J Am Chem Soc 2012;134(10):4521–4.].Castangia, et al. Angew Chem 2012;51(52):13016–8.].Rannes, et al. J Am Chem Soc 2011;133(22):8436–9.


3-2

ichia pastoris GlycoDelete: the way out when N-glycansre a burden

ram Laukens1,2,∗ , Katrien Claes1,2, Charlot De Wachter1,2, Nicoallewaert1,2

Unit for Medical Biotechnology, Inflammation Research Centre (IRC), VIB-Gent, Technologiepark 927, B-9052 Ghent-Zwijnaarde, BelgiumDepartment of Biochemistry and Microbiology, Laboratory for Protein Bio-hemistry and Biomolecular Engineering, UGent, K.L.-Ledeganckstraat 35,-9000 Ghent, Belgium

In order to simplify bio-production of glycoproteins pro-uced in Pichia pastoris, new approaches need to be developed toeal with the heterogeneity that arises from the fungal-type N-lycosylation. Here, we present a method that enables the in-vivoemoval of N-glycans during production of recombinant glyco-roteins in Fungi.

The methylotrophic yeast P. pastoris is an excellent host forecombinant protein production thanks to the strong, tightlyontrolled methanol-inducible promoter system and the well-stablished upscaling processes. Moreover, Pichia does not secrete

lot of endogenous proteins which facilitates downstreamrocessing. However, the sometimes very extensive oligomannose-ype N-glycosylation pattern can be of concern during downstreamrocessing and characterization of the recombinant product.



lthough some tools are available to produce proteins with tail-red N-glycans in P. pastoris (GlycoSwitch®), such engineeringrocess is costly and time-consuming.

We developed a method that enables the in vivo removal of-glycans by inducible co-expression of a fungal endoglycosi-ase, removing N-glycosylation associated heterogeneity without

mpairing strain viability and growth. This approach, called PichialycoDelete, drastically reduces sample heterogeneity, facilitatesrotein purification and alleviates many of the problems that aressociated with yeast-type N-glycosylation.


3-3

nzymatic remodelling of chitin for agrochemical appli-ations

émi Chambon1,∗ , Guillaume Despras2, Dominique Urban2, Borisauzeilles3, Jean-Marie Beau3, Sébastien Fort4, Sylvie Armand4,ylvain Cottaz4

CERMAV-CNRS, GrenobleUniversité Paris Sud and CNRS – Institut de Chimie Moléculaire et des Matéri-ux d’Orsay, FranceUniversité Paris Sud and CNRS – Institut de Chimie Moléculaire et des Matéri-ux d’Orsay – Centre de Recherche de Gif, Institut de Chimie des Substancesaturelles, FranceCentre de Recherches sur les Macromolécules Végétales – CNRS, France

Lipo-chitinoligosaccharides (LCOs) are produced by bacteriagenus Rhizobium) and arbuscular mycorrhizal fungi, and arenvolved in the establishment of symbiosis with plants. They pro-

ote the assimilation of atmospheric nitrogen in legumes (Nodactors [1]) and intake of water and nutrients in 80% of plantpecies (Myc factors [2]). These Nod and Myc factors consist of aackbone of four or five N-acetylglucosamine residues modified byfatty acid on the non-reducing unit and have various decorationsn some hydroxyl groups.

The LCOs, which are active at subnanomolar concentra-ion, are produced naturally in extremely small quantities. Theetailed analysis of their recognition mechanisms as well as theirgrochemicals applications requires the development of efficientynthetic methods.

In this context we developed a chemo-enzymatic chitinemodeling approach to access LCOs via combined use of thehitin-N-deacetylase NodB (Sinorhizobium meliloti) and chitinligomers chemically functionalized on the reducing unit [3].

eferences

].Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Prome JC, et al.Nature 1990;344(6268):781–4.

].Maillet F, Poinsot V, Andre O, Puech-Pages V, Haouy A, Gueunier M,et al. Nature 2011;469(7328):58–63.

].This project is supported by the ANR (ANR-10-CD2I-0008, BioCOS

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3-4

olysaccharides production by autotrophic cultures oficroalgae

iuseppe Olivieri 1 , Renato S. Coellho2, Telma T. Franco2, Antoninoollio3, Antonio Marzocchella1,∗

DICMaPI – Università degli Studi di Napoli Federico II, ItalyFaculdade de Engenharia Química – University of Campinas, BrazilBiological Science Department – Università degli Studi di Napoli “Federico II”,

taly

Light energy and CO2 may be considered as the most abun-ant and cheap feedstock for bioprocesses aimed to produce energyectors as well as green chemical building blocks. Microalgae cane cultivated on waste streams of gas (CO2 polluted) and liquidsindustrial effluents and salt supplements) for producing biofuelsnd carbohydrates, a building block for bioplastics production.

The aim of this contribute is to report about recent jointrogress of a research activity carried out in Campinas (BR) and inapoli (IT). The study regards autotrophic cultures of microalgae

elected to produce a significant amount of carbohydrates coupled,f possible, with lipids as biofuel resource.

Selected strains were autochthon in Brazil and fromhe collection available at the University of Napoli ACUFhttp://www.biologiavegetale.unina.it/acuf.html). Microalgaeere grown in flasks and then inoculated in the photobioreactors.oth the broth and the cells have been characterized for culturesarried out under a wide interval of operating conditions. Maineasured data were cell concentration, total organic carbon (TOC)

n the liquid phase, polysaccharide concentration and proteinoncentration. The attention was paid on both the extracellularnd intracellular polysaccharides.


3-5

ynthesis of potential prebiotics using Pseudomonasyringae DC3000 levansucrase Lsc3

riinu Visnapuu1,∗ , Anneli Aasamets1, Karin Mardo1, Eerik Jõgi1,eiki Vija2, Tiina Alamäe1

University of Tartu, EstoniaNational Institute of Chemical Physics and Biophysics, Estonia

Plant-derived inulin-type oligofructans are considered to beffective prebiotics that function as specific growth substratesor beneficial gut bacteria. The effects of levan-type FOS (fruc-ooligosaccharides) containing �-2,6 linkages are poorly studieds they are not commercially available. Some studies prove theirnhanced prebiotic effect [1]. Polymeric levan has potential appli-ations as anti-cancer, anti-inflammatory and immune stimulatinggent.

Levansucrases (EC 2.4.1.10) are bacterial enzymes belonging
o GH68 family of glycoside hydrolases. Pseudomonas syringaeC3000 encodes three levansucrases (Lsc1, Lsc2, Lsc3). Recom-inant Lsc3 splits sucrose and synthesizes �-2,6-linked FOS,olymeric levan and also heterooligofructans when transfructosy-
vtag

SYMPOSIUM 3: GLYCOBIOTECHNOLOGY

ating alternative acceptors [2]. Lsc3 has very high catalytic activitykcat ∼500 1/s), stability and therefore a high biotechnologicalotential.

In this study, we optimized levan and FOS production by Lsc3rotein. High-performance liquid chromatography system cou-led with ELS detector was used to detect and quantify saccharides,

evan was quantified spectrophotometrically.We showed that Lsc3 produced up to 15.4 g of FOS per mg of

rotein under optimized conditions. Product yield and spectrumepended on reaction conditions. Also, permeabilized bacterialells expressing levansucrase were shown to serve as effective cat-lyst for FOS production.

We developed a method for enzymatic production of levan-typeOS from sucrose and a simple yeast-based method for the removalf monosaccharides that form as by-products of the reaction.

Acknowledgements: This work was supported by ERF grant.2.0701.12-0041 (SLOMR12215T) managed by Archimedes Foun-ation and an ETF grant GLOMR9072.

eferences

].Marx SP, et al. FEMS Microbiol Lett 2000;182:163–9.].Visnapuu T, et al. J Biotechnol 2011;155:338–49.


3-6

-Hydroxydehydronuciferine induces human melanoma375.S2 autophagy and apoptosis and inhibits metasta-

is in vitro and in vivo

ui MinWang

Kaohsiung Medical University, Taiwan

Melanoma is the deadliest cancer. We identified 7-ydroxydehydronuciferine (7-HDNF) isolated from the

eaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be aioactive agent against human melanoma A375.S2 cells. 7-ydroxydehydronuciferine (7-HDNF) was known to induceutophagy and apoptosis response mechanisms, and anti-igratory activity of melanoma in vitro and in vivo. Cell

roliferation assay was used to test cell viability. Acridine orangeAO) staining and flow analysis were applied to observe cell

orphology. The apoptotic cell death ratio was measured viawo-dimensional flow cytometry by annexin V-fluorescein iso-hiocyanate (FITC)/propidium iodide (PI) double stained. Westernlot was applied to examine protein expressions whereas woundealing assay was to examine cell activity. Strong anticancer effectsf 7-HDNF exhibited in a dose-dependent manner, and displayedinor cytotoxicities on normal human skin cells.7-HDNF induced

he formation of intracellular vacuoles and the augmentation ofcidic vesicular organelles (AVO). 7-HDNF increased the cellularrrest in cell cycle at G2/M phase. Cellular membrane asymme-ry lost was confirmed. Protein expressions were discovered to
erify autophagy and apoptosis response mechanisms sharinghe associated pathways. 7-HDNFpresented the high-qualitynti-migratory activity. 7-HDNF inhibited melanoma tumorrowth in mice xenograft model, accompanied with a decrease

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YMPOSIUM 3: GLYCOBIOTECHNOLOGY

f phosphorylation of AKT. We demonstrated the mechanism
f this compound starting with the formation and accumula-ion of AVO leading to autophagy. 7-HDNF caused the cellular
embrane asymmetry lost, triggering the G2/M cell cycle arrestn caspase-dependent apoptosis. 7-HDNF presented high-quality

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ymposium 4: Robust biocatalysts for the pro-uction of novel bio-based products

4-1

olecular design of transglucosidases for polysaccharidend oligosaccharide synthesis

. Monsana,b,c,d,e,f

Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077oulouse, FranceCNRS, UMR 5504, F-31400 Toulouse, FranceINRA, UMR 792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400oulouse, FranceToulouse White Biotechnology, 3 rue des Satellites, F-31400 Toulouse, FranceINRA, UMS 1337TWB, F31400 Toulouse, FranceCNRS 3582, F-31400 Toulouse, France

Among the diversity of transglucosidase enzymes, a specificnterest is given to the glucansucrases of the GH70 family, whichre produced extracellularly by different lactic acid bacteria: Leu-onostoc sp., Lactococcus sp., Streptococcus sp., Weissella sp. Thesenzymes are able to use the energy of the osidic linkage of theucrose molecule (27.6 kJ/mol) to catalyse the efficient transfer ofts �-D-glucopyranosyl unit. This results in the synthesis of a wideariety of products, polysaccharides, oligosaccharides and gluco-onjugates, which contain a diversity of linkages: �-1,2; �-1,3;-1,4; �-1,6.

It is possible to combine the biochemical and structural char-cterization of these transglucosidases with gene sequence datand alignement analysis, to develop structure-function relation-hip studies and molecular engineering based on both rational inilico and combinatorial in vitro approaches. The resulting variantsresent new and improved catalytic properties, resulting in theynthesis of new products.

In addition, original transglucosidases have been obtained fromequencing the genome of several lactic acid bacteria and isola-ion of the corresponding genes. They are able to decorate dextranolysaccharides (�-1,6 main backbone linkage) with �-1,2 and �-,3 osidic branching and generate a new series of polysaccharidesnd oligosaccharides.


4-2

tructural studies on transaminase enzymes and appli-ations in biocatalysis

enny Littlechild1,∗ , C. Sayer1, M. Isupov1, J. Ward2, J. Littlechild1

University of Exeter, United KingdomUniversity College London, United Kingdom

The transaminases (TAs) catalyse the transfer of an amino grouprom an amino acid to a keto acid using the cofactor pyridoxal 5’-hosphate (PLP) and are important for the production of optically
ure amines and amino alcohols used in the synthesis of many
mportant drugs.

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4: ROBUST BIOCATALYSTS FOR THE PRODUCTIONOF NOVEL BIO-BASED PRODUCTS

We have solved the structures of TAs of the Pfam classes, III,V and V in order to further understand their mechanism andubstrate specificities.

The class III �-amino acid TAs catalyse transamination of �-mino acids such as �-alanine or �-aminobutyric acid where theransferred amino group is not adjacent to the carboxyl group.he crystal structures and inhibitor complexes of two �-TAs fromseudomonas aeruginosa and Chromobacterium violaceumhave beenetermined to understand differences in their substrate specificity.

The class V TA enzymes include the serine:pyruvate transami-ases having broad substrate specificity including a reaction with�-hydroxyl substrate. The structure of the thermophilic archaealulfolobus solfataricus serine TA has been determined to 1.8 A res-lution and in complex with the inhibitor, gabaculine. Thesetructures have shown the conformational changes in the enzymective site during the course of the catalytic reaction.

The structure of the class IV Nectria haematococca transam-nase enzyme has been determined in the holo and inhibitoround form which offers a detailed insight into the structuralasis for substrate specificity and enantioselectivity of (R)-selectivemine:pyruvate transaminases [1–4].

eferences

].Sayer S, et al. Acta Cryst 2007;F63:117–9.].Sayer S, et al. Acta Cryst 2012;D68:763–72.].Sayer S, et al. Acta Cryst 2013;D69:564–76.].Sayer S, et al. FEBS J 2014 [in press].


4-3

urrent developments on the engineering of Escherichiaoli biofilms for enzymatic biosynthesis of halotrypto-hans

saac Vizcaino-Caston1,∗ , James Thomas Leech1, Tania Triscari

arberi 2, Rebeca J.M. Goss2, Mark J.H. Simmons1, TimW. Overton1

The University of Birmingham, United KingdomUniversity of St. Andrews, United Kingdom

Halogenated molecules are being utilized on a daily basis byhe fine chemistry and pharmaceutical industries in diverse reac-ions for the production of enantiomerically pure compounds.he chemical synthesis of such molecules requires harsh solventsnd conditions which result in expensive and environmen-ally unfriendly processes. We propose an alternative to such

ethods utilizing recombinant bacterial biofilms to synthesise-halotryptophans. The plasmid pSTB7 expressing recombinantryptophan synthase TrpBA was transformed into diverse strainsf E. coli and used to generate engineered biofilms [1,2]. Theseiofilms were used as biocatalysts for the bioconversion of 5-aloindoles into 5-halotryptophans. Biofilm cultures showedigher yield than planktonic bacteria, bacterial lysates or immo-
ilised TrpBA enzyme [3]. Biofilm topology and viability weressessed using confocal microscopy before and after reactions. Inrder to enable scale-up, a novel method to adhere bacteria toupports was devised. We will discuss the ability of different cell

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mmobilization methods to develop biofilm communities as wells their ability to perform biotransformations.

eferences

].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH,Goss RJM. Engineering biofilms for biocatalysis. ChemBioChem2011;12:1391–5.

].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, SimmonsMJH. Characterisation of spin coated engineered Escherichia colibiofilms using atomic force microscopy. Colloids and Surfaces B: Bioin-terfaces 2012;89:152–60.

].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisationof engineered Escherichia coli biofilms for enzymatic biosynthesis ofL-halotryptophans. AMB Express 2013;3:66.


4-4

tructural and biochemical characterization of twoovel enzymes with promiscuous ene-reductase activity

ea Pavkov-Keller1,∗ , Alexandra Binter2, Steinkellner Georg1,hristian C. Gruber1, Kerstin Steiner2, Christoph Winkler3, Helmutchwab4, Kurt Faber3, Peter Macheroux5, Karl Gruber6

ACIB GmbH, C/o ZMB, AustriaACIB GmbH, AustriaDepartment of Chemistry, University of Graz, AustriaInstitute of Molecular Biotechnology, Graz University of Technology, AustriaInstitute of Biochemistry, Graz University of Technology, AustriaInstitute of Molecular Biosciences, University of Graz, Austria

An approach using three-dimensional motifs reflecting specificctive site arrangements (catalophore) was developed in our group.t does not depend on overall protein similarity and thereforenables the search across enzyme families and the detection ofotential catalytic promiscuity. This catalophore approach led tohe discovery of two novel enzymes with ene-reductase activity.nzymes of this family have recently been shown to possess a greatotential for (industrial) biotransformations. Neither the aminocid sequence of these two enzymes nor their overall structures related to those of the well-known old yellow enzymes (OYE).hese two flavoproteins contain FMN as a cofactor and exist asomodimers. We cloned, expressed and purified both enzymesnd subjected them to crystallization trials. Obtained crystalsere soaked with putative substrates/inhibitors. The enzymatic

haracterization was pursued by stopped-flow and difference titra-ion experiments. Additionally, several typical OYE substrates (i.e.lkenes bearing an electron-withdrawing activating group) wereested to assess the biocatalytic performance. The analysis showedome salient features of typical OYEs as well as some strikingifferences, i.e. a stereocomplementary behaviour. In conclusion,he two novel enzymes can be described as NADPH-dependent
uinone reductases with significant OYE-like side activities.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1657 Ca(iLT


DUCTS New Biotechnology · Volume 31S · July 2014

4-5

essons on directed evolution of hydrolases and glucosexidase

lrich Schwaneberg

RWTH – Aachen University, Chair of Biotechnology, Germany

Protein engineering by directed evolution and semi-rationalesign has become a standard method to tailor enzyme propertieso industrial demands. Improving thermal stability and activ-ty simultaneously is often challenging since high activity oftenequires flexibility whereas thermal resistance relies and ‘strong’nteractions within a protein. On the example of proteases (BgAP1,2], S41 [3]) and a phytase [4,5], lessons learned from improvingoth properties individually and simultaneously will be presented.ubsequently, lessons on improving detergent and salt (ionic liq-id) resistance of a protease (subtilisin E [6,7]) and a cellulaseCelA2 [8,9]) will conclude the hydrolase reengineering examples.

As a highlight, the generation of oxygen independent andighly active glucose oxidase variants (GOx from A. niger) [10,11]ill conclude the presentation.

eferences

]. Martinez R, et al. Biotechnol Bioeng 2013;110:711–20.]. Jakob F, et al. Appl Microbiol Biotechnol 2013;52:2359–63.]. Martinez R, et al. Protein Eng Des Sel 2011;24:533–44.]. Shivange A, et al. J Biotechnol 2014;170:68–72.]. Shivange AV, et al. Appl Microbiol Biotechnol 2012;95:405–18.]. Li Z, et al. ChemBioChem 2012;13:691–9.]. Li Z, et al. J Biotechnol 2014;169:87–94.]. Lehmann C, et al. Green Chem 2012;14:2719–3272.]. Pottkämper J, et al. Green Chem 2009;11:691–7.0].Arango Gutierreza E, et al. Biosens Bioelectron 2013;50:84–90.1].Prodanovic R, et al. Anal Bional Chem 2012;404:1439–47.


4-6

mmobilization of carbonic anhydrase for biomimeticO2 capture in slurry absorber

ara Peirce1,∗ , Maria Elena Russo2, Viviana De Luca3, Clementeapasso3, Mosè Rossi 3, Giuseppe Olivieri 4, Piero Salatino4, Anto-io Marzocchella4

Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industri-le – Università degli Studi di Napoli Federico II, ItalyConsiglio Nazionale delle Ricerche – Istituto di Ricerche sulla Combustione,

talyConsiglio Nazionale delle Ricerche – Istituto di Bioscienze e Biorisorse, ItalyDipartimento di Ingegneria Chimica, dei Materiali e della Produzione Indus-

riale – Università degli Studi di Napoli Federico II, Italy

Novel post-combustion treatments include biomimetic Carbonapture and Storage (CCS) processes based on CO2 absorption intoqueous solution assisted by enzyme catalysis. Carbonic anhydrase
EC 4.2.1.1) catalyzes CO2 hydration and it has been proposed asndustrial biocatalyst for biomimetic CCS processes [Lacroix andarachi, 2008, Recent Patent Chem Eng; Russo et al., 2013, Sep Purechnol].

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ew Biotechnology · Volume 31S · July 2014 SYMPO

The present contribution reports results of immobilization ofarbonic anhydrase (CA) on fine solids. The aim of the study iswofold: the improvement of the biocatalyst stability at the oper-ting conditions typical of CCS processes; the confinement of theiocatalyst in the CO2 absorption reaction volume during contin-ous operations. Studies reported in the literature suggest the usef slurry reactors as an effective technology for the maximizationf the absorption rate enhancement in the presence of heteroge-eous catalysis [Alperet al., 1980, Chem Eng Sci; Penders-van Elkt al., 2012–2013, Int J Greenhouse Gas Control; Russo et al., 2013]. Ingreement with observation reported in the literature, a covalentA immobilization technique has been investigated. The carriersave been fine paramagnetic, non-porous and polyglutaraldehyde

unctionalized particles. Bovine CA was used as model enzyme toptimize immobilization procedure and solid biocatalyst activityssay.

Results have pointed out that the activity of the immobilizednzymes is satisfactory. The immobilized enzymes may be effec-ively used in biomimetic CCS processes.

Further investigation will be focused on the immobilizationf thermostable recombinant carbonic anhydrase [Capasso et al.,012, Chem Eng Trans].


4-7

ngineering of pyranose 2-oxidase for modified oxygeneactivity

agmar Brugger1,∗ , Dietmar Haltrich2, Clemens Karl Peterbauer2

Food Biotechnology, BOKU Vienna, AustriaBOKU Vienna, Austria

The flavoprotein pyranose 2-oxidase (POx) [1] attracts inter-st as potential component in electrochemical devices [2]. POxatalyses the oxidation of aldopyranoses, preferentially D-glucose,
sing molecular oxygen, benzoquinone, radicals or chelated metal
ons [3] as e− acceptors. Reduced oxygen reactivity is a desir-ble property for applications in biosensors/biofuel cells becausef reduced H2O2 production and consequently reduced dam-

4: ROBUST BIOCATALYSTS FOR THE PRODUCTIONOF NOVEL BIO-BASED PRODUCTS

ges to the enzyme itself, the surrounding environment and aore effective electron transfer to the redox mediator or elec-

rode surface. In this study we aimed to identify amino acidesidues involved in oxygen reactivity. We applied a semi-rationalrotein engineering approach. Eleven amino acids around thective site were chosen as target positions for site-saturation muta-enesis. Using a screening assay in 96-well plates with ABTSnd 2,6-Dichlorophenolindophenol (DCPIP) [4] five variants withecreased oxidase activity (0.1 to 39%) and maintained dehydroge-ase activity were identified. Our results show that the exchange ofne amino acid can reduce the unfavourable production of H2O2

f POx without losing activity with alternative e− acceptors.

eferences

].Leitner C, Haltrich D, Nidetzky B, et al. Appl Biochem Biotechnol1998;70–72:237–48.

].Spadiut O, Brugger D, Vasile C, et al. Electroanalysis 2010;22:813–20.].Leitner C, Volc J, Haltrich D. Appl Environ Microbiol 2001;67:3636–44.].Brugger D, Krondorfer I, Zahma K, et al. Biotech J 2013,


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ymposium 5: Synthetic biology

5-1

esign and production of new-to-nature antimicrobialsy synthetic biology

scar P. Kuipers ∗ , Auke van Heel, Dongdong Mu, Liang Zhou,ndrius Buyvidas, Manuel Montalbán-López

Department of Molecular Genetics, University of Groningen, Germany

Antibiotic resistance in human pathogens is on the rise andhis rise is not met with new approved antibiotics to combathese resistant bacteria. To solve this growing problem of resis-ance, alternative sources of antibiotics should be explored. Onef these sources could be the class of ribosomally synthesized, post-ranslationally modified peptides called lantibiotics. We’ll describeour different approaches to develop novel antimicrobials:1) Developing a synthetic biology approach to efficiently use the

large amount of sequenced genomes (>4000) as a resource fornovel lantibiotics. Here we present the results of the first 25candidates.

2) Use of heterologously expressed post-translational modifi-cation enzymes to hypermodify lantibiotics. Various docu-mented posttranslational modifications have been introducedinto lantibiotic peptides broadening the antimicrobial spec-trum

3) Design and production of novel lantibiotics by ring module-and hinge-variation. Specific lantibiotic modules have beenrandomly fitted in a defined architecture, and the nisininduction and modification machinery are exploited for therequired modifications. The screening of more than 10,000chimeric molecules for biological activity has already ren-dered several more active antimicrobials.

4) Biomodules for introducing various types of circular andheterocyclic modifications in lantibiotics. Purpose is todesign three unique biomodules that introduce heterocyclic,cyclic and circular (head-to-tail) modifications in lantibiotics(already containing lanthionine rings


5-2

ynthetic transcription factors allow regulon wideontrol and shifting the nitrogen/carbon balance inacteria

org Schumacher1,∗ , Baojun Wang2, Ana Claudia Bonatto3, Martinuck1

Imperial College London, UKUniversity of Edinburgh, UKUniversidade Federal do Paraná, Curitiba, PR, Brazil

Synthetic approaches to enhance the production of a desired
roduct commonly involve altering the DNA control sequencesegulating expression of the enzymes required. However, manyetabolic pathways involve many core and functionally associ-
hafb



ted genes, so that optimising and testing each control elementtep by step to achieve desired outcomes is laborious. We havengineered chimaeric activators of the �54 RNA polymerase toontrol transcription of the bacterial ntr regulon, comprising over00 genes involved in the regulation, metabolism, scavenging andransport of nitrogen compounds.

We functionally combined the signal input and functional out-ut domains from various �54 RNA polymerase activators with theimerisation/promoter recognition domains of the master Ntr reg-lator NtrC and established orthogonal signal input/transcriptionutput transfer functions. Such transcription activators allow toctivate transcription ‘against the grain’ of the cell physiology.y force driving transcription of dozens of genes and measuringystems changes (transcriptomic, metabolomic and proteomic) weould observe which aspects of the cell system resisted or gave wayo the synthetic perturbation and at which level along the centralogma of molecular biology. This revealed inter-network connec-ions and control hierarchies between these levels and how theyontribute to cellular robustness. We found that in the syntheticell system, the intracellular carbon/nitrogen levels can be shiftedowards higher nitrogen assimilation compared to the wild type. coli.


5-3

ofactor uptake in 1,2-propanediol metabolising micro-ompartments

atthias Mayer1,∗ , Stefanie Frank2, Evelyne Deery2, Andrewawrence2, Mark Smales2, MartinWarren2

University of Kent/Warren Group, United KingdomUniversity of Kent, United Kingdom

Propanediol utilising microcompartments are proteinaceoustructures consisting of a shell that incorporates enzymes necessaryor 1,2-propanediol degradation. The shell is formed by 7 differentypes of proteins that form hexamers (assemble to form the faces ofhe shell) or pentamers (vertices of the shell) with central pores ands thought to protect the cell from the toxic propionaldehyde inter-

ediate as well as to transport substrate and cofactors across therotein shell. The dehydration of 1,2-propanediol to propionalde-yde, which is catalysed by the protein complex PduCDE, is B12

adenosylcobalamin) dependent. Previously it was shown that thedu microcompartment houses enzymes to regenerate B12. Uptakef B12 into enteric cells and reactivation are well described, but ittill remains unclear if B12 is transported or accumulated insidedu microcompartments.

We investigated if Pdu microcompartments take up orccumulate B12. Recombinantly expressed and purified emptyicrocompartments (shell proteins) and full Pdu microcompart-ents (fully expressed pdu operon) showed a higher content of B12

han the crude cell lysate, suggesting that Pdu microcompartments
ave an inherent ability to bind and gather B12. Different cobal-mins were found to be co-purified with both the empty and theully expressed microcompartments. This finding was confirmedy in vitro co-localisation studies of purified microcompartments

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ith fluorescently labelled B12. The uptake mechanism remainsnclear and is part of our current research.

An understanding of transport along the protein shell and itselectivity is an important landmark in our efforts to generate engi-eered compartments capable of sequestering complex syntheticetabolic pathways.


5-4

se of transporter plug-ins in building effective micro-ial cell factories for chemical and fuel production

hristopher Grant ∗ , PhattarapornMorris, Frank Baganz

University College London, United Kingdom

Synthetic biology hopes to predictably build cellular factoriesor the production of chemicals, pharmaceuticals and fuels fromheap renewable feedstocks. A critical, yet understudied, elementf this is control of compound transport both into and out ofhe cell as a means of avoiding issues such as substrate accessimitations, substrate and product inhibition and aiding productecovery.

We report here a successful strategy for the contextual evalua-ion of this topic using a library of transport modifying plug-insombined with multifactorial characterisation. Auxiliary plas-ids (pUMP) were used to express a library of transporters as

lug-ins alongside two biosynthesis plasmids pGEC41, which oxi-ises hydrocarbons into fatty alcohols and pADAR7942 whichynthesizes bioalkanes from metabolic fatty acid precursors. Weemonstrate here benefits of the transporter plug-in approach for:(i) Facilitated delivery hydrophobic substrates to improve

whole-cell biocatalysis rates by up to 70 fold(ii) Industrially relevant product yields of over 40 g/Lorganic phase

(8 g/Ltotal)iii) Reducing byproduct formation in whole-cell bioconversion

of alkanes to alkanolsiv) Improving bioalkane synthesis yields from glycerol by >5 fold(v) Reducing alkanol and aldehyde intermediate formation in

biosynthesis of bioalkanes by > 10 foldvi) The integration with in situ product removal strategies to

improve bioalkane yields by 10 fold compared to the startingprocess.

This library and plug-in approach is of broad appeal for bio-ogical production of hydrophobic compounds and could be a keynabling technology for biological routes for producing a widerange of hydrophobic compounds such as biofuels, fine and spe-
ialty chemicals and pharmaceutical intermediates.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1664apsirtGgt

SYMPOSIUM 5: SYNTHETIC BIOLOGY

5-5

ovel tuneable gene expression systems based on ortho-onal riboswitches

eil Dixon

MIB – Manchester, United Kingdom

Strategies that permit precisely controlled, differential, andimultaneous expression of multiple genes would be extremelyseful for a broad range of metabolic engineering, protein expres-ion and synthetic biology applications.

A new paradigm in genetic regulation emerged with the dis-overy of novel genetic regulatory elements within the 5′ UTRf bacterial mRNA. Upon binding to a specific metabolite, theseo-called ‘riboswitches’ change conformation, permitting differ-ntial gene regulation to occur. As these switches operate via amall molecule-dependent, protein-free mechanism, they presenthemselves as attractive targets for use as novel genetic controllements.

Previously, we showed that it is possible to re-engineeriboswitches so that they no longer bind to their original cognateigands, but are instead activated by synthetic ligands, and furtherhat these ‘orthogonal’ riboswitches can be used to control het-rologous gene expression in vivo [Dixon et al., PNAS 2010]. Weave further developed these into multi-component systems thatermit fine-tuning over co-expression output stoichiometry, withide ranging potential applications in functional and structuralnalysis [Dixon et al., Angew Chem, 2012].

Finally, I will discuss the development of these cellular systemsnd molecular devices from simply proof-of-principle studies, intohe tuneable modular expression system RiboTite, and demonstratehe application of this expression technology for the productionf proteins of biotechnological interest.


5-6

ignal transduction engineering: a powerful platformechnology for enhancing secondary metabolite produc-ion

ian-Jiang Zhong ∗ , Yi-Ning Xu, Gao-Yi Tan, Linquan Bai

Shanghai Jiao Tong University, China

Streptomycetes and higher fungi produce many bioactiveecondary metabolites. Ganoderic acids (GAs) produced by Gan-derma lucidum, a higher fungus, have significant anti-tumor andnti-metastasis activities. Validamycin, an anti-fungal antibioticroduced by Streptomyces hygroscopicus 5008, is an efficient riceheath blight controller, and can be used as the source for chem-cal synthesis of voglibos, an antidiabetic drug. Engineering ofegulatory mechanism was done to enhance the production ofarget secondary metabolites. The effects of metal ions on the
As biosynthesis in liquid cultures of G. lucidum were investi-ated, and the increased enzyme activities and up-regulation ofranscriptional levels of genes in the triterpene biosynthesis were

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bserved. The regulation mechanism of Mn2+ on the GA biosyn-hesis was found to be via calcineurin signaling transduction. Forhe validamycin biosynthesis, our previous study indicated thenvolvement of A-factor-like cascade. A recent genome-wide anal-sis reveals three pairs of afsA-arpA in S. hygroscopicus 5008. Thisork aims to decipher the regulatory role of the multiple afsA-arpAomologs in the A-factor-like cascade, and then to improve thealidamycin production by engineering the regulatory cascade. Byouble deletion of shbR1/R3, the transcriptions of adpA-H and thealidamycin biosynthetic genes were up-regulated, and the vali-amycin production and productivity were enhanced significantlyor both the wide-type and a high-producing industrial strain. Theranscriptomic analysis revealed that the engineering of A-factor-ike signaling cascade caused a shift from primary to secondary

etabolism. The signal transduction engineering proposed here isery useful for efficient production of those secondary metabolitesn cultivation processes.


5-7

evelopment of two continuous genome engineeringtrategies for efficient microbial evolution

hen Cai ∗ , Guodong Luan, Linjiang Zhu, Yin Li

Institute of Microbiology, Chinese Academy of Sciences, China

Engineering complex phenotypes of microbes, for instancetress tolerance, remains to be a big challenge in this field. Currentvolutionary engineering methods including physical and chem-cal mutagenesis, global transcription machinery engineering,nd artificial transcription factors engineering, use “Mutagenesisollowed-by Selection” as the core principle. That is, mutations orenetic perturbations are firstly introduced by exogenous muta-ens or genetic manipulations, followed by selection of desired
henotypes. Thus iterative rounds of mutagenesis-selection andrequent manual interventions are often required, resulting in dis-ontinuous and inefficient strain improvements. To address theiscontinuity of the existing evolutionary engineering approaches


o as to improve the engineering efficiency, we devised twoovel methods termed as “Genome Replication Engineeringssisted Continuous Evolution (GREACE)” and “Stress-Induced-utagenesis Based Adaptive Evolution (SIMBAE)”. Both methods

mplant controllable in vivo mutagenesis machineries into the cell.tarting the mutagenesis machineries under stressful conditionsill trigger continuous mutation generation andsynchronous

election process, and then result a continuous and efficient evolu-ion process which enabled “Mutagenesis coupled-with Selection”.pecifically, GREACE uses a library of activity-compromised proof-eading elements of the main DNA polymerase during genomeeplication as the mutagenesis machinery. In SIMBAE, the muta-enic state is generated by constructing a SIM module whiche-produces complex cellular stress-responses implemented by up-egulation and down-regulation of various genes. Either methods capable of increasing genomic mutation rate up to 3000-fold.ignificantly improved chemical tolerance (n-butanol, acetate,anamycin), themotolerance, and osmotic tolerance of E. coli were

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ymposium 6: Assimilation of CO2, CO andH4 into biobased products

6-1

icrobial fixation of CO2 in water bodies and in drylandso combat climate change, soil loss and desertification

oberto De Philippis

Department of Agrifood Production and Environmental Sciences, University oflorence, Florence, Italy

The growing concern for the increase of the global warm-ng effects raises the challenge of finding novel technologicalpproaches to stabilize CO2 emissions in the atmosphere.iological-CO2 mitigation, triggered through biological fixation,

s considered a promising and eco-sustainable method. Microor-anisms such as cyanobacteria, green algae and some autotrophicacteria could potentially fix CO2 more efficiently than higherlants, due to their faster growth. Some examples of the poten-ial of Biological-CO2 mitigation will be reported and discussed inhis lecture.

In arid and semiarid environments, soil carbon sequestra-ion (CO2 fixation) by cyanobacteria and Biological Soil Crustss considered an eco-friendly and natural process to increase soil

content and a viable pathway to soil restoration after oneisturbance event. Another way for Biological-CO2 mitigation

ntensively studied in the last few years is related to the possibilityo perform carbon dioxide sequestration using microalgae, obtain-ng at the same time bioproducts of industrial interest. Anotherossibility under study is the exploitation of specific chemotrophicacteria for CO2 fixation coupled with the production chemicalsuch as polyhydroxyalkanoates. In spite of the potential of theserocesses, multiple factors still have to be optimized in order tochieve a cost-effective CO2 sequestration.


6-2

icroalgal biofuels from native biological resource ofearl River Delta

aurycyDaroch ∗ , Zongchao Jia, Cong Shao, Hui Guo, Ying Liu, Jay. Cheng

Peking University School of Environment and Energy, China

Algal biofuels are seen as promising solutions of global energyrisis and climate change for the years to come. Major advantagesf algae are potentially high yield and no competition with foodrops for arable land and fresh water. Although the coastal areasf Pearl River Delta have long been known for huge diversity ofquatic life little work has been done to assess the possibility ofsing local microalgae resources for biofuel production. Our studylls this niche and tests the feasibility of producing renewable fuels
thanol and biodiesel from local algal strains.
A total of 89 unique algal strains from Peking University Algaeollection were isolated and screened as feedstocks for biofuel

SYMPOSIUM 6: ASSIMILATIONOF CO2, CO AND CH4 INTO BIOBASED PRODUCTS

roduction. Microalgal strains M. afer PKUAC 9 and S. abun-ans PKUAC 12 isolated from coastal waters of Pearl River Deltaegion were used for saccharification and subsequent fermenta-ive bioethanol production where as Hindakia sp. PKUAC 169 andhlorella sp. PKU AC 102 were optimal for biodiesel production.indakia sp. PKUAC 169 isolate was successfully cultivated in twoerivatives of BG11 medium: nitrogen starved and salt induced.oth methods yielded improved lipid accumulation, but only salt

nduction resulted in increased overall lipid productivity. Derivati-ation of algal lipids to FAMEs showed different lipid profiles underelected growth conditions and suggest that salt induced mediums better suited for biodiesel production than nitrogen starved

edium. Optimisation of heterotrophic cultivation of algal strainhlorella sp. PKUAC 102 resulted in tenfold higher lipid produc-

ivities than autotrophically grown strains.


6-3

xploring the potential of microalgae for bioenergy pro-uction

rank Baganz ∗ , Yanan Xu, Paul Hellier, Nicos Ladommatos, Saulurton

University College London, United Kingdom

Intensive research is being applied to biofuel production fromlgal biomass owing to their fast growth rates and high lipid con-ent. This work explores key steps in algal bioprocessing focusingn algal biomass harvesting through flocculation with chitosan;nd the use of algal biomass for engine combustion.

Initially flocculation with chitosan to harvest algal biomass ofhe green alga Chlorella sorokiniana was explored. Chitosan provedo be highly efficient in the induction of flocculation with the clar-fication efficiency reaching >99% below pH 7 at optimal dosage.nfluencing factors of flocculation efficiency and its effect on theubsequent dewatering process were also evaluated. It was showno reduce the volume to be processed by 20–50 folds, and signif-cantly reduce energy input and material costs of centrifugationr filtration operations. In order to reduce the complicated andostly downstream processes of algal biofuel production, this workxplores an alternative way of utilizing energy from algal cells bylending algal slurry into fossil diesel using specific combinationsf surfactants. We will show that this approach provides benefits byeducing the consumption of fossil fuel and also reducing exhaust


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6-4

evelopment of luminescent photobioreactors formproved microalgae cultivation

eyedeh FatemehMohsenpour ∗ , NikWilloughby

Heriot-Watt University, United Kingdom

Effect of light conditions on the growth, photosynthetic pig-ents and lipid production of green algae Chlorella vulgaris

nd cyanobacteria Gloeothece membranacea was investigated. Theicroalgae were cultivated in luminescent acrylic bubble column

hotobioreactors (PBR) under varying conditions. Luminescentcrylic PBR in blue, green, yellow, orange, and red ranges, capa-le of spectral conversion of simulated full-spectrum daylightere used. The results showed significant variations in culturerowth and photosynthetic pigmentation dependent on cultureonditions and illumination. The highest biomass productivity of84 mg L−1 day−1 was obtained in red luminescent PBR. Increas-ng the initial culture density reduced the rate of productivitylthough lipid accumulation was significantly induced. Blue andellow PBR doubled lipid accumulation up to 11% in high den-ity cultures. However, blue PBR provided the least efficient lightondition for bio-pigment production. Red PBR was the most effec-ive portion of PAR ranges to enhance chlorophyll productionp to 0.75% in G. membranacea whilst green PBR induced pig-entation in C. vulgaris. Phycobiliproteins were the dominant

igments in G. membranacea and red and green light favouredynthesis of these pigments. The average production of phyco-rythrin and phycocyanin was slightly improved in high densityultures.

The proposed illumination strategy offers improved microalgaerowth without resorting to artificial light sources, reducing energyse and costs of cultivation.


6-5

dentification of new auxiliary enzymes for the hydrol-sis of lignocellulose

riana Salazar ∗ , Gonzalo Carvajal, Javier Devia, Tania Quiroz

University of Chile, Chile

Despite important technological advances, the cost of lignocel-ulosic bioethanol as an energy source is still high compared toossil fuels. Cellulose hydrolysis is carried out by the action of cel-ulases and, given the recalcitrance of the biomass high loadingf enzymes is required, impacting significantly the total cost ofhe process. Efficient hydrolysis of lignocellulose depends on theoncerted action of cellulases and auxiliary proteins, which canelp to reduce cellulase loading in the conversion to monomerugars.

In order to identify auxiliary proteins from rot fungi and
mprove the action of cellulases on pretreated wheat straw, extra-ellular proteins from Gloeophyllum trabeum, Fusarium oxysporumnd Trametes versicolor cultured in wheat straw were fractioned by
fAd



xclusion chromatography. Individual fractions were analyzed forhe capability to increase the activity of a commercial cellulase

ix. Fractions showing the highest cellulolytic activity were ana-yzed by LC GC/MS. Two new polysaccharide monooxygenasesPMO, former GH61) and one xylanase were identified, amongstther hydrolytic and oxidative enzymes. Corresponding genesere cloned by reverse transcription and PCR, and sequenced. Pro-

eins GtLPMO9A, FoLPMO9A and GtXyl1 were produced in theeterologous host Pichia pastoris and purified. Different combina-

ions of these enzymes with cellulases, in reactions with wheattraw as substrate, were tested for production of glucose and xylose.esults indicate that GtLPMO9A, FoLPMO9A and GtXyl1 increasehe production of monomer sugars, suggesting that they could beseful tools for improvement of lignocellulose degradation.

cknowledgement

This work was financed by Fondecyt, Project 1121088.


6-6

ngineering biofuel producing microbes for efficientemicellulose utilisation using synthetic biology

avin Thomas1,∗ , Rosanna C. Hennessy1, Henrique I. Neves1,reben Krabben2, Elizabeth Jenkinson2

University of York, United KingdomGreen Biologics Limited, United Kingdom

Rapid depletion of fossil fuels and oil reserves is driving theevelopment of biofuels to replace reliance on petroleum usage.emicellulose is the second most abundant renewable polymern Earth, and thus an attractive resource for the productionf bulk chemicals and fuels (e.g. biobutanol). Development ofiomass-derived biofuels is however challenged by the recalci-rance of lignocellulosic materials to degradation. Harsh andostly pretreatment methods are required to break down struc-urally robust plant biomass to fermentable sugars, which canelease inhibitors and retard subsequent fermentations. An alter-ative method is microbial-based enzymatic degradation. Whileiverse hemicellulolytic bacteria have been identified, many areoorly characterised with limited genetic manipulation tools and

mportantly lack industrially relevant pathways. A synthetic biol-gy approach is therefore being used to engineer hemicellolyticbility into butanol producing bacteria. Synthetic gene clustersomprising a minimal enzyme system required to catalyse theommon hemicellulose xylan will enable such bacteria to trans-ort and metabolise hemicellulose-derived sugars more efficientlyo butanol. Synthetic gene clusters will include endoxylanases andeta-xylosidases to hydrolyse xylan into xylose monomers but alsonzymes that hydrolyse side chains or modifications. As the rate ofubstrate uptake is a key factor in determining the rate of product
ormation, transporters will be an important design consideration.t the bioprocess level, milder pretreatment procedures will enableeployment of a cost-competitive technology with reduced envi-

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onmental impact for the production of biofuels from renewableesources.


6-7

conomic assessment of microbial lipids for biodieselroduction: Competitiveness with microalgae and agri-ultural plant oils

o Nam Chang ∗ , GwonWoo Park

KAIST, Republic of Korea

For economic assessment of microbial lipids volatile fatty acids
VFAs) derived from waste organics was used as low cost carbonource and multi-stage continuous high cell density culture (MSC-CDC) process for high bioreactor productivity and titer. The
xperimental lipid yield was 0.27 g/g-VFA, and the experimental

SYMPOSIUM 6: ASSIMILATIONOF CO2, CO AND CH4 INTO BIOBASED PRODUCTS

ell mass yield of glucose and wood hydrolyzates were 0.5 g/g.ipid contents from 10 to 90% of cell mass, bioreactor productivityf 0.5–64 g/L/h and plant capacity of 20,000–1,000,000 tons/yearere assumed. A $0.902/kg-lipid was predicted with $0.2/kg woodydrolyzates and $0.1/kg VFAs with productivity, 12 g/L/h andapacity, 100,000 tonnes/year and 75% lipids content. The rawaterials accounted for 55.7% of total operating costs followed

y direct fixed cost dependent 17.4%. A 50% Chemical oxygenemand (COD) recycle of waste empty cell mass to VFAs furthereduced the cost to $0.886.A biodiesel from microbial lipid has a


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YMPOSIUM 7: APPLICATIONS OFMETABOLICMODELLING

ymposium 7: Applications of metabolic mod-lling

7-1

aking use of metabolic models – in silico driven designnd engineering of industrial microorganisms

hristophWittmann

Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken,ermany

Superior microorganisms that produce chemicals, materialsnd fuels from renewables are major drivers of the developingio-based economy. Firstly, they hold the key to the optimizedroduction of traditional bio-based products regarding key perfor-ance indicators such as titer, yield and productivity [1]. Secondly,

hey enable future production of important petrochemicals fromreen resources rather than from fossil fuels [2]. Without doubt,ystems metabolic engineering is changing the way to design andptimize such microbial cell factories for industrial production:he integration of systems biology and systems biotechnology withew concepts from synthetic biology enables the global analysisnd engineering of microorganisms–and bioprocesses at efficiencynd versatility otherwise not accessible. In this regard, the lec-ure will highlight the integration of model-based prediction andesign into industrial strain engineering: driven by massively

ncreasing genomic information, stoichiometric models providealuable insights into the properties of metabolic networks andive access to theoretical maximum yields, optimal pathways andreferred production hosts [3]. Most importantly, metabolic mod-ls can even guide smart and straightforward engineering of cellsnd processes [4]. The use of models for decision-making andtrategic developments appears particularly useful for industrialpplication. Model-based approaches are fast, feasible at limitedevel of knowledge in early project stages and allow the investiga-ion of different scenarios, all relevant for short development timend high performance required. Different examples from indus-rial biotechnology will demonstrate the power of using metabolic

odels: bio-based production of chemicals [5,6], polymers [7] andecombinant proteins [8]. Regarding industrial lysine productionodel-based design has provided synthetic strains, which reach

he high performance of classical producers derived over the pastfty years [9].

eferences

].Becker J, Wittmann C. Curr Opin Biotechnol 2012;23:718.].Becker J, Wittmann C. Curr Opin Biotechnol 2012;23:631.].Krömer JO, Wittmann C, Schröder H, Heinzle E. Metab Eng2006;8:353.

].Melzer G, Eslahpazir M, Franco-Lara E, Wittmann C. BMC Syst Biol2009;3:12.

].Buschke N, Becker J, Schäfer R, Kiefer P, Biedendieck R, Wittmann C.Biotechnol J 2013;8:557.

].Kind S, Neubauer S, Becker J, Yamamoto M, Völkert M, Weong WK,et al. Metab Eng 2014 [in press].

].Poblete-Castro I, Binger D, Rodrigues AL, Becker J, Dos Santos V,Wittmann C. Metab Eng 2013;15:113.

evii



].Driouch H, Melzer G, Wittmann C. Metab Eng 2012;14:47.].Becker J, Zelder O, Häfner S, Schröder H, Wittmann C. Metab Eng2011;13:159.


7-2

esign of optimally constructed metabolic networks ofinimal functionality

avid Ruckerbauer ∗ , Christian Jungreuthmayer, Jürgen Zanghellini

ACIB GmbH, Austria

Background: Metabolic engineering aims to design microor-anisms that will generate a product of interest at high yield. Thus,variety of in silico modeling strategies has been applied success-

ully, including the concepts of elementary flux modes (EFMs) andonstrained minimal cut sets (cMCSs).

The EFMs (minimal, steady state pathways through the system)an be calculated given a metabolic model. cMCSs are sets of reac-ion deletions in such a network that will allow desired pathwayso survive and disable undesired ones (e.g., those with low productecretion or low growth rates). Grouping the modes into desirednd undesired categories had to be done manually until now.

Results: Although the optimal solution for a given set ofathways will always be found with the currently available tools,anual selection may lead to a sub-optimal solution with respect

o a metabolic engineering target. A small change in the selectionf modes can reduce the number of necessary deletions while onlylightly reducing production. Based on our recently introduced for-ulation of cut set calculations using binary linear programming,e suggest an algorithm that does not require manual selection of

he desired pathways.Conclusions: We demonstrated the principle of our algorithm

ith the help of a small toy network and applied it to a model of. coli using different design objectives. Furthermore we validatedur method by reproducing previously obtained results withoutequiring manual grouping of modes.


7-3

hat is the relationship between intracellular andxtracellular metabolites? The theory of “metabolicverflow” put into test

ilas Villas-Boas1,∗ , Ting-Li Han1, Tim Liu1, Sang Kim1, Soniaarneiro2, Eugenio Ferreira2, Isabel Rocha2

The University of Auckland, New ZealandUniversity of Minho, Portugal

Compared to our knowledge on metabolic pathways and thestablishment of tools to manipulate these pathways, we know
ery little about the mechanisms behind the secretion of metabolicntermediates. Microorganisms secrete a wide range of metabolicntermediates, and many of them are of great industrial interest.

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espite the cellular process of metabolite efflux being ubiquitouso all microbial cells, we still do not know clearly how this processorks and how it is regulated. It is believed that small metabolites,ainly those end products of fermentation are excreted through

he plasma membrane passively, or they are secreted throughpecialised mechanisms such as vectorial reaction in response toypo-osmotic stress, or uniport and synport transport systems.owever, all of these mechanisms are based on the concept of

metabolic overflow”, which under specific metabolic conditionst is observed a massive excretion of some metabolic intermedi-tes due to their intracellular accumulation. Although this concepteems appropriate to explain the secretion of some intracellu-ar metabolites, it does not apply to many cases studied duringontinuous culture. Our metabolomics data obtained during dif-erent time-series studies of microbial growth under continuousnd batch cultures confirm that the concept of “metabolic over-ow” cannot be applied to explain the efflux of several intracellularetabolites found in the extracellular medium. Most of our studies

ndicate that microbial cells very often get rid of some intra-ellular metabolites in response to an environmental stimulus,ven if these metabolites are key intermediates of central carbonetabolism.


7-4

owards genome-scale metabolic pathway analysis:etabolome integration allows efficient enumeration of

lementary flux modes in metabolic networks

ürgen Zanghellini ∗ , Matthias P. Gerstl, David Ruckerbauer, Chris-ian Jungreuthmayer

Austrian Centre of Industrial Biotechnology, Austria

Background: Elementary flux mode (EFM) analysis is used todentify all non-decomposable steady state pathways in metabolicetworks. EFMs represent minimal functional building blocks.hey can be used to characterize metabolic phenotypes and cellu-

ar robustness or identify targets in metabolic engineering. Despitets potential EFM analysis is currently restricted to medium scale

odels as the number of EFMs in a network explodes with theetwork’s size.

Method: We present a novel computational method that inte-rates the cellular metabolome into an EFM analysis. As theetabolome directly maps to the Gibbs free energy surface of

he reaction network, we are able to identify and remove ther-odynamically infeasible EFMs during the runtime of an analysisithout impacting any biologically relevant EFMs. Thermody-amic infeasibility is determined in the framework of networkmbedded thermodynamics. In this way we curb the explosionf the number of EFMs in large networks.

Results: We present details of our implementation and demon-trate that our new approach successfully tackles the major
ottleneck strongly reduce the memory consumption. Thus annbiased characterization of large-scale metabolic models is madeossible. In fact, we present the first thermodynamically consis-ent EFM analysis of iJE660a, a genome-scale metabolic model of i
SYMPOSIUM 7: APPLICATIONS OFMETABOLICMODELLING

. coli. For instance, our analysis confirms the inactivity of gluta-ate dehydrogenase in E. coli grown on glucose.Conclusion: Thermodynamic EFM analysis successfully inte-

rates the metabolome into an EFM analysis. It is solely based onrst principles, and allows for an unbiased characterization of theetabolic capabilities in genome-scale metabolic networks.


7-5

enome scale metabolic modeling of recombinant pro-ein producing yeasts: prediction of process parametersnd metabolic engineering targets for efficient produc-ion

iethardMattanovich

University of Natural Resources and Life Sciences Vienna, Austria

Genome scale metabolic models have been successfullypplied to predict genetic interventions to redirect metabolicuxes towards desired products of the primary and secondaryetabolism. Complex polymeric products like heterologous pro-

eins equally demand redistributions of primary metabolic fluxes,heir rational design however is far less obvious. Therefore cellngineering for protein overproduction has concentrated mainlyo transcription, codon usage, protein folding and secretion.

We have developed the first genome scale metabolic model forhe yeast Pichia pastoris (PipMBEL1254), and integrated the synthe-is of heterologous protein. The model could successfully predicthe increase of protein production in oxygen limited conditions, asell as changes of central metabolic fluxes in production strains,s measured by 13C flux analysis. Metabolic engineering targetsor enhanced protein productivity were predicted with FSEOF forene overexpression and MOMA for gene deletion. After deletingr overexpressing the respective genes as predicted more than 50%f the interventions led to an enhanced production of cytosolicuman superoxide dismutase (hSOD), and similarly of other pro-

eins. Beneficial mutations were mainly related to reduction of theADP/H pool and the deletion of fermentative pathways.

We demonstrate that genome scale metabolic modeling is suit-ble to describe metabolic changes in recombinant strains andan be successfully applied to design genetic interventions to therimary metabolism to increase recombinant protein production.


7-6

se of a novel combinatorial genetics platform to rapidlylone, express and select target biocatalytic activities forultigenic metabolic pathway optimization

an Fotherigham

Ingenza, Ltd., United Kingdom

Replacement of petroleum-based products and manufactur-ng processes with competitive bio-based alternatives is attracting


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YMPOSIUM 7: APPLICATIONS OFMETABOLICMODELLING

ncreased attention due to environmental concerns surroundingetroleum sustainability and supply. Replacement of conventionalrocesses for manufacturing valuable industrial products and theelection of optimal biosynthetic routes requires the construc-ion, and in most cases subsequent context-dependent evaluation,nd optimization of multicomponent biosynthetic pathways toenerate intermediates and end products. This talk will presenthe use of Ingenza’s proprietary combinatorial genetics platforminABLE®) to rapidly clone, express, select and optimize targetctivities for many separate enzymatic reactions, from thousandsf independent genes derived from metagenomic and phyloge-etic discovery approaches. Multiple gene variants comprisingf up to ten individual genetic elements are combined in sin-le reactions, generating expression libraries with hundreds orhousands of members in diverse heterologous configurations forTP interrogation. Obvious synergy exists between this approach

nd versatile, solid phase screening and selection methods usingrowth-based, crossfeeding or colorimetric methods to identifyolonies of interest. This is illustrated through the rapid identifica-ion of critical pathway enzymes, optimal gene coding sequencesnd enzyme variants from inABLE®-derived high quality variantibraries for bio-based polymer production. The technology aimso bring increasing predictability and overcome limitations asso-iated with iterative and empirical processes for microbial strainmprovement. The successful realization of optimal target reac-ions enables rapid pathway definition and progression to processptimization and scale-up.


7-7

ethanol – a potential carbon source for Corynebac-erium glutamicum

abrinaWitthoff ∗ , JanMarienhagen, Michael Bott

Forschungszentrum Jülich, Germany

Methanol represents an interesting carbon source for microbialroduction processes, but cannot be assimilated by Corynebac-
erium glutamicum, an industrial producer of >4 million tons ofmino acids and a model organism in white biotechnology. Weherefore attempted to introduce the ribulose monophosphateRuMP) pathway for methanol assimilation in this species.


For implementation of the RuMP pathway, three heterol-gous genes encoding methanol dehydrogenase, 3-hexulose-6-hosphate synthase, and 6-phosphate-3-hexuloisomerase werexpressed in C. glutamicum wild type. All other enzymes requiredor conversion of fructose-6-phosphate and regeneration ofibulose-5-phosphate as C1-acceptor can be recruited from endoge-ous metabolism. The recombinant strain metabolized methanolt a rate of 500 �mol h−1 (g CDW)−1 during growth on glu-ose/methanol mixtures.

Further studies revealed, however, that C. glutamicum wild types able to oxidize methanol via formaldehyde and formate to CO2

ith a rate of 200 �mol h−1 (g CDW)−1. The alcohol dehydroge-ase AdhA is mainly responsible for the oxidation of methanol to

ormaldehyde, whereas the oxidation of formaldehyde is predomi-antly catalyzed by two enzymes, the acetaldehyde dehydrogenaseld and the mycothiol-dependent formaldehyde dehydrogenasedhE. The resulting formate is oxidized via formate dehydroge-ase FdhF to CO2 [1,2]. A C. glutamicum mutant lacking Ald anddhE activity is severely impaired in its ability to oxidize formalde-yde to CO2 and thus, represents a promising strain to enforceethanol assimilation and prevent dissimilation.

eferences

].Witthoff S, Eggeling L, Bott M, Polen T. Microbiology2012;158:2428–39.

].Witthoff S, Mühlroth A, Marienhagen J, Bott M. Appl Environ Micro-

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ew Biotechnology · Volume 31S · July 2014 SYMPOSIU

ymposium 8: Industrial biotechnology ofatural and synthetic polymers

8-1

reen polymer processing with enzymes

nrique Herrero Aceroa , Doris Ribitscha, Veronika Perza, Alessan-ro Pellisb, Antonino Biundob, Katrin J. Greimela,b, Gibson S.

yanhongob, GeorgM. Guebitza,b,∗

Austrian Centre of Industrial Biotechnology, Konrad Lorenz Strasse 20, 3430ulln, AustriaUniversity of Natural Resources and Applied Life Sciences, Vienna, Konradorenz Strasse 20, 3430 Tulln, Austria

Environmentally friendly reaction conditions and the highlypecific mode of action are among the advantages of enzyme basedrocessing of (bio)synthetic polymers when compared to chemi-al processes. In coatings, enzymes can replace toxic metal basedatalysts or improve dispersing properties of lignins. Their surfacepecific action is exploited in functionalization of synthetic mate-ials to impart e.g. antimicrobial properties. On the other hand,nzymes have a potential for specific recovery of polymer build-ng blocks from composite materials or mixed plastics waste. Tomprove the efficiency of enzymes on non-natural polymeric sub-trates, engineering enzyme surface properties and attachment ofinding modules are useful strategies together with engineering ofhe active site architecture [1–4].

eferences

].Herrero Acero E, et al. Biotechnol Bioeng 2013;110(10):2581–90.].Ribitsch D, et al. Biomacromolecules 2013;14(6):1769–76.].Greimel KJ, et al. Green Chem 2013;15(2):381–438.].Nugroho Prasetyo E, et al. Bioresour Technol 2010;101(14):5054–62.


8-2

pen mixed cultures for bioethanol production from lig-ocellulosic materials

avide Dionisi ∗ , TomAdams, Craig Dempster

University of Aberdeen, United Kingdom

This study investigates the feasibility of a new process forhe production of bioethanol from lignocellulosic wastes. In thenvestigated process, conversion of lignocellulosic materials tothanol is obtained by using microorganisms for all the requiredrocess stages, i.e. lignin and cellulose hydrolysis and carbohy-rates conversion to ethanol. The aim is therefore to avoid these of expensive chemical-physical pretreatments for lignin andellulose hydrolysis, replacing them with cheaper and more envi-onmentally sustainable microbial processes. A key aspect of thenvestigated process is the use of open mixed microbial cultures,
nstead of pure cultures of selected microorganisms.
This presentation will discuss the main challenges to be over-ome for the development of the investigated process and willresent experimental data on glucose and cellulose fermentation

INDUSTRIAL BIOTECHNOLOGYOF NATURAL AND SYNTHETIC POLYMERS

y open mixed microbial cultures. Lab-scale bioreactors, inocu-ated with soil as a source of microorganisms, were operated undernaerobic conditions and fed with either glucose or cellulose (twoifferent particle sizes) as only carbon sources. Ethanol yield up topprox. 20% of the theoretical value was obtained with glucose.n the cellulose-fed reactors, cellulose hydrolysis occurred and itas found to occur faster with the smaller particle size.


8-3

trategies for enzymatic functionalization of syntheticolymers

nrique Herrero Acero1,∗ , Caroline Gamerith1, Andreas Ortner1,oris Ribitsch1, Georg Steinkellner1, Karl Gruber2, Helmutchwab3, GeorgM. Guebitz4

ACIB GmbH, AustriaUniversity of Graz, AustriaGraz University of Technology, AustriaUniversity of Natural Resources and Applied Life Sciences, Vienna, Austria

Polymers have a wide range of uses because of their outstandingulk properties, but their surfaces are lacking of desired reactiv-ty or properties. Current activation processes rely in the use ofoncentrated acids/alkali solution or plasma approaches. Whilearsh chemicals typically decrease the polymer properties, plasma

reatment are very energy demanding and unable to function-lize complex geometries like tubes. Enzymes have revealed asowerful catalyst able to overcome all these limitations. Enzymesre able to partially hydrolase the upper most layers of polymersn a controlled manner, introducing anchor groups for subse-uent grafting. Following this approach it was possible to graftuman Serum Albumin (HSA) to polylactic acid (PLA) membranes

n which the polymer was activated in a first step with a cuti-ase [1]. The functionalized material had a higher hydrophilicity,ntioxidant capacity, which improved its biocompatibility. Usingmore elaborated double enzymatic reaction it was possible to

ovalently graft ferulic acid on polyamide (PA) fibers [2]. In ordero improve the enzymatic process we have improved the polymer-nzyme interaction via rationale design of the enzyme surface [3]nd by fusion of binding modules leading to increased adsorptions measured via Quartz Crystal Microbalance [4].

eferences

].Nyanhongo GS, et al. React Funct Polym 2013;73(10):1399–404.].Herrero Acero EJ. Mol Catal B: Enzym 2012;79:54–60.].Herrero Acero E. Biotechnol Bioeng 2013;110(10):2581–90.


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YMPOSIUM 8: INDUSTRIAL BIOTECHNOLOGYOF NATURAL AND SYNTHET

8-4

nfluence of nutritional and physicochemical variablesn PHB production from raw glycerol by a wild Bacillus

egaterium strain

arolinaGuzmánLuna1,∗ , PaaloAndreaMorenoYanez1, Camilo Joseanez Diaz1, Nilo Sérgio Medeiros Cardozo2, Humberto Escalante1,arianny Y. Combariza1

Universidad Industrial de Santander, BrazilFederal University of Rio Grande do Sul, Brazil

Reducing production costs and increasing strain productivityn biosynthetic processes are decisive factors linked to biodegrad-ble polymers worldwide production and usage. Agro-industrialyproducts as alternative carbon sources and optimization of cul-ure media composition are some of the strategies used to addresshese issues.

Amongst many available biopolymers polyhydroxybutyratePHB), intracellularly produced and accumulated by abundantacteria species, could become a substitute for synthetic plasticsue to its thermoplastic and mechanical properties. In this con-ribution we report on the influence of five nutritional and twohysicochemical variables in PHB production by Bacillus mega-erium B2, a recently isolated and characterized bacterium withHB accumulating ability using raw glycerol, from the biodieselndustry, as carbon source [1].

According to Plackett Burman and central composite designsemperature, glycerol and Na2HPO4 concentrations are the mostignificant variables on PHB production by B2, with optimal valuesf 34 ◦C, 7.6 g/L and 3 g/L, respectively. After 14 hours of fermenta-ion, in shake flasks with optimized medium, B2 produced 0.37 g/Lf PHB with an 18% w/w accumulation. This corresponds to an5% increase in PHB production when compared to initial cultureonditions. These results suggest the potential of B. megaterium B2s PHB producer using raw glycerol an inexpensive, abundant andeadily available carbon source.

eference

].Tarazona N, Moreno P, Yanez C, Combariza MY, Guzmán C. Micro-bial production of polyhydroxybutyrate by native Bacillus strains using abiodiesel by-product as carbon source. Lisbon, Portugal: Presented at theEuropean Symposium on Biopolymers; October 7–9, 2013. PS 2.1.


8-5

alue-added carotenoid production in the pennateiatom Phaeodactylum tricornutum with light emittingiode based photobioreactors

eiqi Fu1,∗ , Sigurður Brynjólfsson1, Bernhard Palsson2

University of Iceland, IcelandUniversity of California, San Diego, United States

Diatoms are one of the most promising feedstocks for pro-ucing feed supplements, bioactive pharmaceuticals and biofuels

n a bio-based economy. The marine pennate diatom Phaeodacty-


OLYMERS New Biotechnology · Volume 31S · July 2014

um tricornutum is able to accumulate large amounts of specialtyarotenoids. Among the valuable carotenoids present in diatoms,ucoxanthin has health promoting effects in humans, includingnti-cancer, anti-obesity, and anti-diabetic effects as well as anti-alarial activity. Fucoxanthin has also been observed to be more

otent than �-carotene and astaxanthin in terms of anti-obesitynd anti-proliferative effects on adult T-cell leukemia cells. Theemand for fucoxanthin in the global market has been increasingramatically. In this study, we characterized the biomass produc-ivity and biomass yield of P. tricornutum with light emitting diodeLED)-based photobioreactors with different light intensities andavelengths. Under red LED illumination, increasing the photonux from 85 to 255 �E/m2/s caused photoinhibition of Phaeodacty-

um cells when there was either 3.0 mM or 0.3 mM of metasilicaten the medium. In contrast, combined red and blue (50:50) LEDllumination produced a higher biomass yield and growth rate.urther, the presence of blue light enhanced the accumulation ofucoxanthin in comparison to illumination with red light only.hese results demonstrated the feasibility of fucoxanthin produc-ion in P. tricornutum, and a method combining LED technologynd synthetic biology approaches was proposed to facilitate itsevelopment.


8-6

ntibacterial and antifungal activity of charcoal mate-ials and microwave radiation

ee Jin Yang1,∗ , Yun Jeong Cha2, Hern Kim2, Shin Sik Choi2,3

Myoungji University, Republic of KoreaDepartment of Energy and Biotechnology, Myongji University, Republic oforeaDepartment of Food and Nutrition, Myongji University, Republic of Korea

Suppression of microbial contamination or growth usingntibacterial and antifungal materials has been required in vari-us commercial products including foods and cosmetics. In thistudy, we have investigated the inhibitory effect of charcoal poly-ers and microwave radiation on bacterial and fungal cell growth

r survival rate. When bacterial and fungal cells were cultured withharcoal polymers, the growth of some microbes was significantlynhibited by nano and micro sized charcoal particles protrudedrom the surface of polymers. Four species of fungi were also dra-

atically killed by microwave radiation for less than 5 min. Theseesults suggest that the use of charcoal plastics and microwave hasgreat potential for reducing microbial contamination in multiplereas.

Acknowledgements: This work was supported by NGV ofyundai Motors Company and BK21 Plus of Ministry of Education

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8-7

olyhydroxyalkanoates production by aerobic mixedicrobial cultures using crude glycerol

aulo Costa Lemos ∗ , Rita Moita, André Freches, Rita Pontes

REQUIMTE/CQFB, Portugal

Due to the prospective partial replacement of fossil fuelsy biodiesel, its production has continuously grown in theast decade. In order to biodiesel production to be carried outnder sustainable conditions, new application to their wastes/by-roducts need to be investigated, especially concerning their majory-product – crude glycerol. In this study the feasibility of produc-ng polyhydroxyalkanoates (PHA) by mixed microbial communitysing crude glycerol as feedstock was investigated.

The microbial population selected under aerobic dynamiceeding conditions had the ability to consume both major car-
on fractions present in the crude, glycerol and methanol. Twoiopolymers were stored, poly-3-hydroxybutyrate (PHB) and glu-ose biopolymer (GP), apparently using glycerol as the only carbonource for their production. The microbial enrichment obtained
INDUSTRIAL BIOTECHNOLOGYOF NATURAL AND SYNTHETIC POLYMERS

as able to accumulate up to 47% PHB of cell dry weight with aroductivity of 0.24 g HA/L d. The overall PHA yield on total sub-trate consumed (0.32 g COD HB/g COD crude glycerol) was inhe middle range of those reported in literature (0.08–0.58 g CODHA/g COD real waste). Molecular biology methods (FISH, DGGE)ere used to characterize the microbial population.

As opposed to many other reports with real wastes, crudelycerol can be directly used to produce PHA. Its use avoids andditional pre-fermentation step, making the overall productionrocess economically more competitive, reflected in a reductionf the polymer final cost. This was the first study that demon-trates the valorisation of the glycerol fraction present in the crude


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IOECONOMY

ioeconomy

P3-1

sian bioeconomy and biobusiness: current scenario anduture prospects

atyahari Dey1,2,∗

Indian Institute of Technology Kharagpur, IndiaDeputy Secretary General, Asian Federation of Biotechnology, India

The 20th century witnessed exciting inventions in biologyhat has resulted in innovations today. The recombinant DNAechnology, animal cell culture, bioprocess engineering of recom-inant cells and the deciphering of human genome are notable.plethora of ‘omics’ related platform technologies have enabled

recise and rapid probing of cellular processes facilitating hugepplications in 5 Fs (food, fuel, f(ph)armaceuticals, fabric and fod-er).

The revenue from bio-industries in some countries, including aew in Asian regions, was as high as 2.5% of GDP. The developingegions in Asia could not experience such a growth uniformly. Theluggish global market over the last 5 years was also a bottlenecko this aspiration. The world trade trends now to recover fromlowdown. It is important to set a realistic goal for biobusiness insia for 2025.

Asia in one hand is blessed with the highest number of biotech-ologists of the world in the younger age group, and the largestopulation of underprivileged people (having lower human devel-pment indices) on the other. The bountiful bioresources in Asia ifxploited in bioentrepreneurship could eliminate the disparities.nspiring young biotechnologists towards this goal is essential. Atrong academia-industry partnership could accelerate the success.he European Federation of Biotechnology and Asian Federa-ion of Biotechnology together can mobilize their members, andnterface with respective Governments facilitating favourable reg-latory and policy frameworks.


P3-2

iotech products on the market as a main driver fornowledge based bio-economy

leksandraMałyska ∗ , Tomasz Twardowski

Institute of Bioorganic Chemistry Polish Academy of Sciences, Poland

As biotechnology applications are present in broad variety ofectors it has been the key technology for realising the KBBE pro-iding a more cost-effective option and better quality products. Athe same time some of its products arouse great public controversy,n particular, the products of genetic engineering in the agri-foodector. Poland is no exception and international studies revealedhat attitudes of Poles towards biotech products reflect the views
f other Europeans. It is understandable because people are notware in how many everyday products biotechnology is alreadypplied.


The supply chain of biotech products involves many stakehol-ers, organizations, activities and resources, some of which havedirect and often significant influence on consumers purchase

ehaviors. One of them is the retailers, who are a key players inhe supply chain by providing link between the end-consumersnd the manufacturers as well as suppliers.

Our study took place in 2012 and 2013 and involved morehan 250 visits in various retail outlets in Poland and conductingtandardized interviews with merchants concerning their viewsnd knowledge of products referring to innovative biotechnology.ithin this study we were able to identify goods (manufactured in

ifferent EU countries) that were either direct applications of inno-ative biotechnology or unduly usurped to be such products Thetudy revealed a positive correlation between the level of knowl-dge and the opinion on biotech products. The results helpedo gain an understanding of reasons and motivations underlying

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ew Biotechnology · Volume 31S · July 2014 TUESDAY 15 JULY PLENA

uesday 15 July

lenary lecture: Prosthetic gene networks foriomedical applications

L2-1

rosthetic networks – synthetic biology-inspired treat-ent strategies for metabolic disorders

artin Fussenegger

ETH Zurich, Department of Biosystems Science and Engineering, Basel,witzerland

Since Paracelsus’ (1493–1541) definition that the dosing makeshe drug the basic treatment strategies have largely remainednchanged. We continue to use a precise prescribed dose of amall-molecule drug, a protein therapeutic or a therapeutic trans-ene to constitutively modulate or complement the activity of aisease-relevant target. However, this treatment concept does nei-
her consider the metabolic dynamics nor the interdependence ofhe most important pathophysiology’s of the 21st century such asbesity, diabetes and cardiovascular disorders. Synthetic biology-nspired prosthetic networks may act as metabolic prostheses that
CTURE: PROSTHETIC GENE NETWORKS FOR BIOMEDICAL APPLICATIONS

rovide the dynamic interventions, the immediate pre-diseasection and the multi-target capacity required to meet with thereatment challenges of the future. Prosthetic networks consistf synthetic sensor-effector gene circuits that (i) seamlessly oper-te in implanted designer cells, (ii) constantly sense, monitor andcore metabolic disturbances in peripheral circulation, (iii) pro-ess OFF-level concentrations of pathologic metabolites, and (iv)oordinate an adjusted therapeutic response in an (v) automaticnd self-sufficient manner. We will present our latest generationf synthetic mammalian gene circuits and provide a few examplesf prosthetic networks operating in animal models of prominentuman diseases to highlight the challenges and impact of syn-


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YMPOSIUM 9: BIOMARKERS ANDDIAGNOSTIC TOOLS

ymposium 9: Biomarkers and diagnosticools

9-1

esigning nanomaterials for ultrasensitive biosensing

olly Stevens

Imperial College London, UK

Bio-responsive nanomaterials are of growing importance withotential applications including drug delivery, diagnostics and tis-ue engineering. This talk will provide an overview of our recentevelopments in the design of materials for ultrasensitive biosens-

ng. Our recent simple conceptually novel approaches to real-timeonitoring of protease, lipase and kinase enzyme action usingodular peptide functionalized gold nanoparticles and quantum

ots will be presented. Furthermore we have recently developednew approach to ultrasensitive biosensing through plasmonicanosensors with inverse sensitivity by means of enzyme-guidedrystal growth as well as a “Plasmonic ELISA” for the ultrasensitiveetection of disease biomarkers with the naked eye. We are apply-

ng these biosensing approaches both in high throughput drugcreening and to diagnose diseases ranging from cancer to globalealth applications.


9-2

ntibodies by design

eter Tessier

Rensselaer Polytechnic Institute, United States

The ability of antibodies to recognize target molecules (anti-ens) with high affinity and specificity is central to theiridespread use in diagnostic and therapeutic applications. Theinding activity of antibodies is encoded in up to six of theirolvent-exposed peptide loops that directly contact antigens. Anti-odies are generated by randomly varying the sequences of theirntigen-binding loops and selecting rare variants that are com-lementary to target antigens. Due to the daunting number ofossible antibody sequences with variation only within theirntigen-binding loops (>1030 variants), it seems unlikely thathe needles (antibodies with desired binding activity) in theaystack (all possible antibody variants) can be predicted instead ofeing selected. We have challenged this conventional wisdom byeducing the seemingly intractable problem of designing multiplentibody loops to cooperatively bind antigens to a tractable one inhich we design individual antibody loops with binding activity.sing this simplified design strategy that is inspired by natural bio-

ogical interactions, we find that antibody fragments can be readilyngineered to recognize diverse aggregated proteins linked to neu-odegenerative disorders (e.g., Alzheimer’s disease) by targeting
nique structural features within such proteins. Our innovativepproach generates single- and multidomain antibodies that rec-gnize misfolded proteins not only based on their sequence, butlso based on their conformation. We also find that our antibodies b


ecognize sequence epitopes that are difficult to target using con-entional antibodies, and that these novel antibodies are potentnhibitors of protein aggregation.


9-3

ngineering cofactor specificity of methyltransferases

artin Tengg1,∗ , Yu Zheng2, Mandana Gruber-Khadjawi3, Elmareinhold1

Institute of Organic Chemistry, RWTH Aachen University, GermanyNew England Biolabs Inc., Ipswich, MA, USAACIB GmbH, Austrian Centre for Industrial Biotechnology, Graz, Austria

Biological methylations of various substrates occur in everyiving cell and are essential for cell survival. These transforma-ions are catalyzed by methyltransferases (MTases) which generallyransfer the activated methyl group from S-adenosyl-l-methionineAdoMet) to DNA, RNA, proteins and small biomolecules. Recenttudies demonstrated that many MTases can accept AdoMet ana-ogues for transfer of extended carbon chains to their substrates1,2]. The ability to accept a broad range of cofactor analogues isf great interest both in terms of DNA diagnostics and biocatalyticynthesis.

In order to increase transfer rates of extended groups fromdoMet analogues protein engineering approaches are performed.igh-throughput directed evolution of the DNA MTase M.SssI iserformed by in vitro compartmentalization. This method linksenotype and phenotype for an effective selection of active vari-nts. The bacterial enzyme M.SssI is of particular interest becauset performs the same reaction as mammalian DNA MTases by tar-eting 5′-CG-3′ (CpG) DNA sequences. CpG modifications are keypigenetic signatures in transcriptional regulation and genomemprinting. The transfer of functional groups will provide newools for DNA labelling as well as DNA modification detection.

.SssI evolution will also guide rational protein engineering of theomologous small molecule MTase NovO, for the synthesis of newne chemicals as well as bioactive intermediates and products.

eferences

].Dalhoff C, Lukinavicius G, Klimasauskas S, Weinhold E. Nat ChemBiol 2006;2:31–2.

].Stecher H, Tengg M, Ueberbacher BJ, Remler P, Schwab H, Griengl H,Gruber-Khadjawi M. Angew Chem Int Ed 2009;48:9546–8.


9-4

ynthetic bioreporters for detection of environmentalollutants

an Roelof van der Meer ∗ , DavideMerulla, Siham Beggah

University of Lausanne, Switzerland

One of the main immediate application areas for syntheticiology are bioreporters, living cells with simple designed genetic

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ircuits that permit detection of a specific chemical or group ofhemicals, under the concomitant production of an easily butccurately quantifiable reporter signal. Bioreporters have attractedonsiderable interest because they offer cheap alternatives forhemical analysis in remote areas where high-end instruments arenavailable. Such demands on bioreporters, however, require anlmost fail-proof and robust technology that goes much beyondhat traditional research “proof-of-principles” have been able toemonstrate.

We will show on the example of a bioreporter for arsenic, howmportant improvements can be made in the circuit design andn the optimization of the assay technology. Arsenic is a recurringoxious contaminant of drinking water in large areas of our planet,nd bioreporter technology can provide the means to rapidlyuantify its presence in the concentration range of 1–10 �g/L. First,e demonstrate how designing a feedback or an uncoupled circuitffects the signal output and detection sensitivity for arsenic. Sec-ndly, we show a new system to better control residual backgroundxpression in the circuit while maintaining optimal induction,nd we also provide experimental evidence to improve the circuitehaviour by avoiding cross-interference from the host. Finally, weresent the use of micro-engineered structures that would permitontinuous remote operation of a bioreporter sensor.


9-5

homogeneous quenching resonance energy trans-er assay for H-Ras activation cycle monitoring andnhibitor screening

ari Kopra1,∗ , Arjan van Aldrichem2, Markku Syrjänpää1,tefan Veltel 3, Pekka Hänninen1, Daniel Abankwa4, Urpoamminmäki5, Harri Härmä1

Laboratory of Biophysics, University of Turku, FinlandInstitute for Molecular Medicine Finland, University of Helsinki, FinlandUniversity Hospital Hamburg-Eppendorf, FinlandTurku Centre for Biotechnology, University of Turku and Åbo Akademi Univer-ity, FinlandDepartment of Biotechnology, University of Turku, Finland

Background: Recently, we have developed a homogeneousingle-label signaling technique, the Quenching Resonance Energyransfer (QRET). In this study, the homogeneous QRET technol-gy was applied to monitor GTPase activation cycle and activationycle inhibition.

Methods: The QRET system is based on the protection of tar-et protein bound Eu3+-GTP from a soluble quencher molecule. (1)he small GTPase cycle (nucleotide exchange and hydrolysis) cane monitored in the competitive assay by tracking GTP hydrolysisn the presence of nanomolar concentrations of H-RasWt, SOScat,nd p120GAP. (2) The nucleotide exchange can be monitoredsing a second assay with H-RasWt and SOScat. The increased time-esolved luminescence (TRL) signal is monitored when either GTP
ydrolysis (1) or Eu3+-GTP association (2) occurs.
Results: We have proven the suitability of the QRET systemo monitor GTP hydrolysis (1) and nucleotide exchange (2). The

SYMPOSIUM 9: BIOMARKERS ANDDIAGNOSTIC TOOLS

TP hydrolysis assay was used to screen 1280 compound smallolecule library whereof twelve inhibitors were found (average Z-

actor 0.78). The GTP hydrolysis assay can simultaneously detectnhibitors affecting either nucleotide exchange or GTP hydrolysis.herefore, same screening was performed by monitoring Eu3+-GTPssociation to H-RasWt (average Z-factor 0.78). From the screeningssays, seven same inhibitor hits were found. Additionally, fivenhibitors were found only in GTP hydrolysis assay.

Conclusions: These novel QRET assays for GTPase researchan be performed using nanomolar protein concentrations. Theresented QRET assays enable the study of whole small GTPaseycle by monitoring the guanine-nucleotide exchange factor (GEF)nduced nucleotide exchange and/or GTPase-activating proteinsGAP) catalyzed GTP hydrolysis.


9-6

omparative large scale microRNA expression profilesf cynomolgus monkeys, rat and human reveal miR-182ssociated with Type 2 diabetes

ongli Du ∗ , Jinghui Zhou, YuhuanMeng, XiaoningWang

South China University of Technology, China

Type 2 diabetes (T2D) is a prevalent disease that is presenthroughout the world, and is usually associated with insulin resis-ance. MicroRNAs (miRNAs) play important role in the suppressionf gene expression and have been shown to be implicated inuman diseases. We used a novel animal model, cynomolgus mon-ey fed with normal and high fatty diet (HFD) respectively, tonalyze the miRNA expression profile in whole blood by deep-equencing. Finally in total 24 miRNAs with differential expressionere filtered. Among them, mir-182 and mir-183, related to insulin

esistance by modulating FOXO1 and PI3 K/AKT cascade, had thereatest copy number in the whole blood. Decrease of mir-182n T2D cynomolgus individuals is completely consistent with therevious studies in human and rat. Integrating mir-182 tissuexpression profile, target genes and copy number in blood revealedhat mir-182 plays a key role in FOXO1 modulation that leadso potential hyperglycemia and modulates the insulin secretion.n addition, the possible miRNA regulation system of T2D underhe influence of different diet conditions was also interpreted inhe present study. The cholesterol content influences mir-182 andotentially other miRNAs expression level that causes the insulinesistance and the various miRNA regulation systems between nor-


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9-7

ssociation of apolipoprotein E gene polymorphismith serum lipid levels

ehrish Fatima ∗ , Syed Shahid, Obaid Khan, Abid Azhar

University of Karachi, Pakistan

Dyslipidemia has been marked to play an integral role in theevelopment of cardiovascular diseases. Various environmentalnd genetic factors may influence dyslipidemia. Polymorphism ofhe apolipoprotein E (apo E) gene may influence lipid metabolismy modulating lipid levels. This study was designed to investi-ate the role of genetic variants of apo E in dyslipidemic patients.
he study was carried out in equal number (n = 110) of normalubjects and dyslipidemic patients. Genotyping was performed byolymerase Chain Reaction and Restriction Fragment Length Poly-orphism (PCR- RFLP) at Single Nucleotide Polymorphisms (SNPs)
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s429358 (ApoE4) and rs7412 (ApoE2). The genotype frequenciesere: E3/E3 (75%), E2/E3 (4.1%), E3/E4 (20%), E4/E4 (0%), E2/E4

0%), and E2/E2 (0.45%). The allele frequencies were 0.025, 0.875nd 0.1 for �2, �3 and �4, respectively. In control group allele fre-uencies for �2, �3 and �4 were 0.045, 0.83 and 0.127, respectively,hereas in dyslipidemic patients group allele frequencies for �2, �3nd �4 were 0.045, 0.682 and 0.273 respectively. Total cholesterolTC) and low density lipoprotein cholesterol (LDL-C) levels wereignificantly high in �4 allele carriers (p < 0.001) and high densityipoprotein cholesterol (HDL-C) level was high in �2 allele carriersp < 0.05) indicating protective effect of �2 allele. Apo E polymor-hism has significant influences on lipid profile in dyslipidemicatients group as frequency of �4 allele was found to be prevalent

n patients group which indicates a relationship of �4 allele in apo
gene with dyslipidemia.

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ew Biotechnology · Volume 31S · July 2014 SYMP

ymposium 10: Biowaste biorefinery for aore sustainable bioeconomy

10-1

iowaste biorefinery for a more sustainable andiobased food industry

abio Fava

DICAM, School of Engineering, Alma Mater Studiorum-University of Bologna,ologna, Italy

Modern biobased industry is giving its close attention onrganic waste as a new bioresource. Specific biowaste valorizationathways are focusing on food processing waste, being food sectorhe first manufacture in Europe. Anyway they need to become inte-rated, combining biomass pretreatments and recovery of biogenichemicals with bioconversion processes in order to obtain a largelass of chemicals. This will help to (a) use the whole biowaste,y avoiding producing residues and providing to the approachhe required environmental sustainability, and (b) producing dif-erent biobased products that enter different markets, to get theossible economical sustainability of the whole biorefinery. How-ver, the costs of the developed integrated processes might be high,ostly for the fact that the industry dealing with such issues is

till underdeveloped and therefore dominated by high processingosts. Such costs can be significantly reduced by intensifyingesearch & innovation on process integration and downstreamrocessing. The low or no cost of starting material along with thenvironmental benefits coming from the concomitant biowasteisposal would offset the high capital costs for initiating such aiorefinery.


10-2

evelopment of an advanced biorefinery concept basedn valorization of pulp and paper industry wastetreams

postolis Koutinas , Mary Alexandri ∗, Chrysanthi Pateraki, Anestislysides, Harris Papapostolou

Agricultural University of Athens, Greece

The biorefinery concept is based on the efficient fractionationf a renewable resource followed by the conversion of individualractions into marketable products in a way that exploits the fullotential of the resource. Research at the Agricultural Universityf Athens focuses on restructuring conventional industrial plantsnto integrated biorefineries through the valorization of waste ory-product streams.

This study evaluates the potential restructuring of a pulp andaper process employing the sulfite process through fraction-tion of spent sulfite liquor (SSL) for the production of phenolic
ompounds as antioxidants, lignosulfonates and succinic acid.SL is a complex waste stream generated via wood chip diges-ion with calcium or magnesium sulfite where delignification
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M10: BIOWASTE BIOREFINERY FOR AMORE SUSTAINABLE BIOECONOMY

ccurs under high pressure and low pH conditions. SSL diges-ion leads to hemicellulose degradation to C6 and C5 sugarsnd lignin depolymerisation by sulfonation and hydrolysis. SSLas processed via ultrafiltration to produce a sugar-rich perme-te that is suitable as carbon source for fermentative productionf succinic acid. The retentate contained a high concentration ofignosulfonates. An antioxidant-rich fraction was extracted fromhe permeate stream via solvent extraction with ethyl acetate.he total phenolic content, the antioxidant activity and specifichenolic compounds were analysed in this stream. Productionf succinic acid was evaluated with the bacterial strains Acti-obacillus succinogenes and Basfia succiniciproducens in batch anded-batch bioreactor cultures using either free or immobilizedells. SSL valorisation could lead to a sustainable biorefineryoncept.

Acknowledgements: The authors gratefully acknowledgehe financial support by the FP7 research project BRIGIT (KBBE-012-6-311935, www.brigit-project.eu).


10-3

iotechnological conversion of spent coffee grounds intoolyhydroxyalkanoates

tanislav Obruca ∗ , Pavla Benesova, Sinisa Petrik, Dan Kucera,vanaMarova

Faculty of Chemistry, Brno University of Technology, Czech Republic

Coffee is one of the world’s most popular beverages and haseen growing steadily in commercial importance. Nowadays, cof-ee is, after petroleum, the second largest traded commodity in theorld. Hence, coffee industry is responsible for the generation of

arge amounts of waste, above all, spent coffee grounds (SCG).The aim of this work was to study conversion of SCG into

aluable product–polyhydroxyalkanoates (PHAs). These polyestersre accumulated by various bacteria as carbon and energy storingaterials. Due to their mechanical properties, they are considered

eing an alternative to petrochemical plastics.At first, oil extracted from SCG (approx. 15 wt% oil in SCG) was

fficiently (YP/S = 0.82) converted into PHA employing Cupriavidusecator H16. Further, the solid residues after oil extraction wereydrolysed (combination of chemical and enzymatic hydrolysis)ielding fermentable sugars, which were further used as a sub-trate for production of PHAs employing Bacillus megaterium andurkholderia cepacia. B. cepacia revealed significantly higher PHAsields and; moreover, it was capable of direct accumulation ofopolymer of 3-hydroxybutyrate and 3-hydroxyvalerate withoutddition of precursors.

Finally, solids after SCG hydrolysis possess high calorific valuend can be used as a fuel to at least partially cover energeticemands of the process. Hence, entire biomass of SCG can besed for sustainable production of PHAs employing bio-refinery
pproach.
Acknowledgement: This work was supported byrojects “Centre for Materials Research at FCH BUT” No.


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YMPOSIUM 10: BIOWASTE BIOREFINERY FOR AMORE SUSTAINABLE BIOE

Z.1.05/2.1.00/01.0012 from ERFD and “Excellent youngesearcher at BUT” No. CZ.1.07./2.3.00/30.0039.


10-4

xtraction of the protein fraction of dry distillers grainsith solubles, implementing biocatalytic and chemicalethods

aria Villegas Torres ∗ , Gary Lye, JohnWard

UCL, United Kingdom

Distillers dried grain and solubles (DDGS) is a by-product fromistilleries and breweries, currently used as animal feed. It con-ains mainly protein, fats and residual carbohydrates, making itn ideal feedstock for biocatalytic processes. In addition, due tohe current expansion of the biofuel industry in the UK, a dra-

atic increase in DDGS production is expected. Therefore, it is ofndustrial interest to develop manufacturing processes of indus-rially relevant compounds implementing DDGS as feedstock. Inhis work, we focused on the extraction of the protein fraction (upo 40%), which is expected to have a high frequency of specificmino acids, as wheat grain is mostly gluten -a highly elastic pro-ein mainly composed of glutamine and proline. Glutamine haseen extensively used as injury treatment, and muscle growth, andecause of its physical characteristics can be an ideal source for bio-aterials manufacture. We optimized a protein extraction process

nvolving both chemical steps combined with enzyme hydrolysis.he optimized extraction method is a novel approach that can bepplied at industrial scale.


10-5

uccinic acid production from raw materials by Acti-obacillus succinogenes

hristophe Roca1 , Margarida Carvalho2,∗, Maria A.M. Reis2

REQUIMTE, PortugalREQUIMTE/Universidade Nova de Lisboa, Portugal

Succinic acid (SA) is currently considered a key platform chemi-al as it is used in the production of a wide range of products, fromharmaceuticals to green solvents, fibers and bioplastics [1]. These of renewable feedstocks as carbon source for the production ofA via fermentation has been suggested as a solution to reduce pro-uction costs and develop a sustainable SA production process [2].n this work, Actinobacillus succinogenes was used as biocatalyst forhe conversion of two raw materials into SA: i) glycerol, byproductf biodiesel industry, is usually poorly metabolized to SA becausef a redox imbalance but here, it could be efficiently converted
o SA, reaching concentration as high as 49 g/L SA using DMSOs electron acceptor; ii) extracts from carob pods, a by-product ofarob locust bean gum industry were used as carbon source in smallcale batch cultivations. A simple aqueous extraction was used to

OMY New Biotechnology · Volume 31S · July 2014

btain extracts rich in sugars, containing around 12.5 g/L sucrose,g/L glucose and 2.5 g/L fructose. All sugars were consumed andA yield of 0.4–0.5 g SA/g sugar could be obtained, depending onxtract concentration. These results show that carob pulp waterxtracts or glycerol are promising substrates for SA production,emonstrating the high versatility of A. succinogenes.

eferences

].McKinlay, et al. Appl Microbiol Technol 2007;76:727–40.].Carvalho, et al. Nat Biotechnol 2014;31:133–9.


10-6

nhanced welan gum production using cane molasses asubstrate by Alcaligenes sp. ATCC31555

ufangWang ∗ , Hongxia Ai, Min Liu

South China University of Technology, China

Welan gum is a new high molecular microbial exopolysaccha-ide with a wide range of potential industrial applications in food,oncrete, petroleum, ink and for enhancing oil recovery. Welanum is more effective than any other polysaccharides in enhancingil recovery and expected to become a novel oil recovery agent forts excellent stability at high temperature. For new polymers to be aommodity product in the near future, it is crucial to enhance theroductivity and lower their production costs. In this work, caneolasses was selected for welan gum production by Alcaligenes sp.

TCC31555 in 5L fermentor. The pre-treatment of cane molasses,gitation and the additives for improving dissolved oxygen werenvestigated for process optimization. Sulfuric acid hydrolysis washe optimal molasses pretreatment for welan gum production withmaximum welan gum concentration (33.5 g/L), yield (0.62 g/g),roductivity (0.28 g/L/h), and broth viscosity (3.375 Pa s), whichre 50%, 138%, 47%, and 67% higher, respectively, compare tohose without treatment. The process was subsequently optimizedy agitation and adding additives for improving the dissolved oxy-en at special time point during the fermentation. Optimal welanum production (41.0 ± 1.41 g/L) was found at 600 rpm under0 ◦C with addition of 10% n-dodecane at 36 h in 5L fermentor,hich is the highest welan gum concentration so far. Moreover,elan gum from molasses displayed similar rheological propertiesnd thermal stability than that from glucose. It indicated that caneolasses may be an economical industrial substrate for welan gum

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10-7

nvestigating the biomass modifying and degradingnzymatic toolbox of Aspergillus japonicus var aculeatusEC 156 with quantitative proteomics and New Genera-ion Sequencing tools

eorge Anasontzis1,∗ , Thuy Nguyen Thanh2, Thanh Vu Nguyen2,isbeth Olsson1

Chalmers University of Technology, SwedenFood Industries Research Institute, Sweden

In the bio-based economy concept, the current hydrocarbonuels and non-biodegradable plastics will be replaced by new prod-cts which will derive from natural and renewable resources. Theynthesis of such biofuels and biochemicals is still challenged byhe difficulties to cost efficiently degrade lignocellulosic materials
o fermentable sugars or to isolate the intact polymers. Biomassegrading and modifying enzymes play an integral role both in theeparation of the polymers from the wood network, as well as inubsequent modifications, prior to further product development.
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M10: BIOWASTE BIOREFINERY FOR AMORE SUSTAINABLE BIOECONOMY

The type of application usually defines the conditions wherehe reactions should take place. Thus, novel enzymes with variableombined properties, such as different thermotolerance, pH rangef activity, substrate specificity and solvent tolerance, still needo be discovered and developed to achieve the highest possiblefficiency in each case.

We isolated an Aspergillus japonicus var aculeatus strain andvaluated its cellulase and hemicellulases activities. It was thenultivated in bioreactors with different carbon sources, such asheat bran, spruce and Avicel and its biomass degrading capac-

ty was determined and semiquantified with TMT (Tandem Massags), through cross species protein identification of its secretome.nformation on the genes involved in the different stages of theermentation and carbon sources have been acquired with nexteneration sequencing of its total transcriptome.

Interesting transcripts are being heterologously cloned andxpressed in order to identify their role and potential use in the
iorefinery concept.


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YMPOSIUM 11: NANOTECHNOLOGY: NEW BIOLOGICAL APPLICATIONS

ymposium 11: Nanotechnology: new biologi-al applications

11-1

esign, structure and assembly of superparamagneticore–shell nanoparticles

rik Reimhult

Institute for Biologically inspired materials, Department of Nanobiotechnology,niversity of Natural Resources and Life Sciences Vienna, Vienna, Austria

Nanoparticles with carefully controlled core–shell structuresan be used in biomedical applications, for example, for separa-ion, as contrast agents, for hyperthermia and in drug delivery1,2]. Exquisite control allows further applications through assem-ly into biomimetic membrane and vesicular structures for whichermeability externally can be controlled by applied magneticelds.

I will briefly describe a new synthetic toolkit based on nitro-atechol dispersants, monodisperse synthesis of Fe3O4 cores usingapping agents and new approaches to ligand grafting into bio-ompatible shells on such nanoparticles; these developmentsllow the synthesis of a range of new magnetic nanoparticlesor defined biotechnological applications and for fundamentalesearch. The emphasis is on the purification and characterizationf such nanoparticles and further on the assembly of nanopar-icles into membrane superstructures that can be magneticallyontrolled. The relationship between nanoparticle structure, thessembled membrane structure and the release of a model drugrom nanoparticle actuated vesicles will be highlighted.

eferences

].Amstad E, Reimhult E. Nanomedicine 2012;7:145–64.].Amstad E, Textor M, et al. Nanoscale 2011;3:2819–43.


11-2

roduction and characterization of HIV-1 virus-like par-icles using transient gene expression in mammalianells

onia Gutiérrez-Granados ∗ , Laura Cervera, Segura Maria de lasercedes, Francesc Gòdia

Universitat Autònoma de Barcelona, Spain

New vaccine strategies based on virus-like particles (VLPs) areapidly evolving. Upon expression in a number of heterologousost systems, Gag polyprotein of HIV-1 spontaneously assembles

n the vicinity of the plasma membrane and it is released by bud-ing producing enveloped particles that resemble immature HIV-1irions. These particulate immunogens have proven to be potenttimulators of both cellular and humoral immune responses in
nimal models. Besides, Gag-based VLPs do not contain the viralenome, minimizing biosafety concerns. Thus, VLPs offer greatromise as HIV-1 vaccines. Mammalian cells are the preferred sys-em as a cell factory for this kind of vaccines due to the complexity


f VLP assembly and release from cells. Transient gene expressionTGE) offers the flexibility to rapidly screen a number of productariants with sufficient quantity and quality for preclinical prooff concept studies. In this work, mammalian cells are used for theroduction of a fluorescent variant Gag-based VLP using a TGEpproach. A method based on GFP fluorescence has been devel-ped to quantify both the transfection efficiency and the Gag-GFPLP production. Additionally, the properties and purity of thebtained VLPs are further characterized by other techniques suchs transmission electron microscopy, nanoparticle tracking analy-is and size-exclusion chromatography. Discussion will be focusedn the characterization of the process as well as the obtained prod-ct.


11-3

acterial microcompartments moving into the world ofiotechnology

tefanie Frank1,∗ , Andrew Lawrence1, Allan Pang2

University of Kent, United KingdomUniversity of California, United States

Bacterial microcompartments (BMCs) are cytosolic proteintructures that are composed of a shell housing sequentially act-ng enzymes associated with particular metabolic pathways.BMCsermit the enhancement of metabolic processes, which is whyhey are expected to have great potential for biotechnologicalpplications. One of the most complex BMCs is associated with,2-propanediol utilisation (pdu) and the focus of our effortsor microcompartment engineering. The shell of the native Pdu

icrocompartment is composed of seven proteins (PduA, PduB,duJ, PduK, PduN, PduT and PduU); only five are required forhe self-assembly of empty heterologous microcompartments in. coli. PduA is one of the most abundant shell proteins and hashe ability to self-assemble into higher-order structures which weound can be manipulated by strategic intervention at the molec-lar level. Targeting of enzymes to the shell is mediated by shorteptide sequences. We have solved the structure for the N-terminalargeting peptide (P18) of PduP, an enzyme associated with thedu microcompartment and identified a main interaction part-er PduK, a protein of the outer shell. We have also fused twoeterologous enzymes required for ethanol production (pyruvateecarboxylase and alcohol dehydrogenase) with targeting peptidesnd found it has been possible to direct these enzymes to emptyMCs in vivo and to generate a functional ethanol bioreactor.

Ongoing research is striving to further understand protein-rotein interactions and assembly of microcompartments andelated higher order protein structures in molecular detail in ordero control and manipulate these structures for the development of

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11-4

rotein supramolecular engineering – applications iniotechnology

atrick Shahgaldian1,∗ , Maria Rita Correro2, Alessandro Cumbo3,hilippe Corvini 2

University of Applied Sciences and Arts Northwestern Switzerland,witzerlandUniversity of Applied Sciences Northwestern Switzerland, SwitzerlandINOFEA AG, Switzerland

A large number of living organisms possess the capabilityo produce intricately patterned and hierarchically structurediogenic silica. Imitating natural systems’ ability to produce hier-rchical silica structures may provide the possibility to designunctional (nano)materials with the same degree of complexity. Inhis lecture, two novel design strategies of nanomaterials capablef either molecular recognition or biocatalysis will be discussed;oth approaches are based on the self-assembly of organo-silicarecursors using protein templates [1].

The first part of this presentation will be dedicated to the devel-pment of a novel class of nanomaterials possessing selectiveolecular recognition properties of viruses. The synthetic strat-

gy to produce those nanoparticles is based on the formation ofchemical imprint of the template virion at the surface of silicaanoparticles. It is demonstrated that the so-produced particlesossess enhanced molecular recognition properties for their tar-et, even in complex media (e.g. human serum). The second partf this lecture will be dedicated to the development of a chemicaltrategy to produce nanobiocatalysts with enhanced biochemical,hysical and chemical stabilities. It is based on the formation of arotective shell at the surface of enzyme proteins that provides aomfortable medium that allows for enzyme stabilization. It wille demonstrated that those systems can find a number of biotechpplications.

eference

].Cumbo A, Lorber B, Corvini PF-X, Meier W, Shahgaldian P. Nat Com-mun 2013;4:1503.


11-5

iological production of stable copper nanoparticles

ikolaos Pantidos ∗ , Louise Horsfall

University of Edinburgh, United Kingdom

Many nonferrous industries such as mining and surface treat-ent plants produce co-products that are high in heavy metals

nd therefore toxic to the environment. A less obvious producer ofeavy metal containing co-products is the whisky industry. Cur-
ent methods of copper removal from such co-products includelectrolysis and membrane filtration which are reported to bempractical and costly. Alternatively, when copper is found as aalt, current methods of removal include settlement, filtration and
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SYMPOSIUM 11: NANOTECHNOLOGY: NEW BIOLOGICAL APPLICATIONS

recipitation. Biological copper ion removal from effluents haseen shown to be quite effective.

There are two biological methods to remove copper fromffluent which involve biosorption and reduction. Biosorptionnvolves bacteria binding to copper via the cysteine-rich transportroteins that are associated with the cell membrane to precip-

tate it. Some bacteria are also able to reduce higher valencynsoluble copper ions into zero valency insoluble forms of the

etal.Here we present a metal-reducing bacterium Morganella psy-

hrotolerans, which is able to reduce Cu2+ to insoluble Cu0

anoparticles. The copper nanoparticles are produced as part ofhe bacterium’s defence mechanism against the toxic effects of

etal ions. M. psychrotolerans grows at low a temperature whichakes it ideal for industrial applications as energy for heating is

ot required; hence it can be potentially cheaper than currentethods of copper removal. The copper nanoparticles produced

uring the distillery co-product retreatment can be isolated andubsequently used for purposes such as optics, catalysts, antimi-robials or recycled in order to make new copper stills for whiskyistilleries.


11-6

nsight into the physiological role of a compartmen-alised ferritin like protein in Rhodospirillum rubrum

onMarles-Wright ∗ , Didi He, Atanas Georgiev, David Clarke


All domains of life have evolved systems for the com-artmentalisation of enzymes and metabolic pathways. These

nclude the lipid-bounded organelles in eukaryotes and extendo protein-shelled microcompartments, such as carboxysomes, inrokaryotes. Rhodospirillum rubrum, which is a model organismor the study of bacterial nitrogen fixation, possesses two dis-inct metabolic compartments. We have characterised the simplestf these compartments, which is encoded in a two-gene locusnd comprises a linocin-family shell protein and a ferritin-likerotein. Using a combination of structural biology techniques,

ncluding X-ray crystallography, native mass-spectrometry, andlectron microscopy, in combination within vivostudies we haveetermined the structure and function of this intriguing metabolicompartment.

The compartment shell is build from 60 subunits of the linocinrotein and forms a 25 nm icosahedron, much like the capsid of airus. The ferritin is encapsulated co-translationally through thenteraction of a short signal sequence at its terminus with theinocin. It forms a donut shaped structure and presents a uniquenverted ferroxidase site, which is formed through the interactionf two protein monomers. The activity of this ferritin is mediatedy encapsulation within the linocin shell and is distinct to the fer-
itins, bacterioferritins and DPS proteins that make up the rest ofhe family of ferritin-like proteins.
This metabolic compartment is widely distributed across bac-erial groups and is highly conserved, indicating a common


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YMPOSIUM 11: NANOTECHNOLOGY: NEW BIOLOGICAL APPLICATIONS

hysiological purpose. The promiscuous metal ion binding sitesn the ferritin and localisation mechanism give hints into theotential for engineering these compartments.


11-7

atalytic properties improvements of Alcaligenes faecalisitrilase by self-assembly induced aggregation

huang Li ∗ , Xiaofeng Yang

School of Bioscience and Bioengineering, South China University of Technol-gy, China

A large number of studies have been devoted to improveatalytic properties of enzymes by protein engineering. How-
ver, these conventional methods, i.e. site directed, random andaturated mutagenesis, always rely on the correlation betweentructure and function or high-throughput screening approaches,hich make it hard to obtain positive mutants in a short time. Here


e fused the nitrilase from Alcaligenes faecalis with an amphipathiceptides. Results showed that approximately 90% of nitrilase wereroduced as active inclusion bodies (aggregrates) in Escherichia coliells. The thermal stability of the self-assembly induced nitrilaseas increased by 6.8 and 4.3 folds at 45◦C and 50◦C, respectively.hen the nitrilase aggregrates are purified by differential cen-

rifugation with sucrose density gradient and negatively stained,highly ordered amyloid fibrils structure is observed under TEM.fter two steps of washing with buffers, the nitrilase aggregatesere purified and further immobilized with sodium alginate as

he carrier, then designated as iSEA. Thermal stability was fur-her increased by 1.5-fold compared to the aggregrates. Also theSEA showed good progress in the tolerance of mandelonitril from0 mM to 200 mM. High expression yields, simple recovery stepsf aggregates from the host cells and the favorable catalytic char-

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ymposium 12: Stress responses in microbialioprocessing

12-1

echanisms of protein folding and quality control inacteria

ernd Bukau

Center for Molecular Biology of the University of Heidelberg, German Can-er Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, D-69120eidelberg, Germany

Protein homeostasis is established by a complex cellularachinery which assists and regulates regular folding pathways

nd counteracts protein misfolding and aggregation. A particularlyritical process in the life of a protein is the native folding of newlyynthesized proteins, which therefore is tightly controlled. Alreadyuring ongoing synthesis by the ribosome nascent polypeptidesre subject to enzymatic processing, chaperone-assisted folding tohe native state or targeting to translocation pores at membranes.he ribosome itself plays a key role in these different tasks by serv-

ng as platform for the regulated association of enzymes, targetingactors and chaperones that act upon the nascent polypeptidesmerging from the exit tunnel. The molecular mechanisms inte-rating the different co-translational processes leading to theaturation and native folding of nascent chains will be described.


12-2

ngineering customised cell signalling circuits and theiriotechnological applications

aojunWang


Cells live in an ever-changing environment and continuouslyense, process and react to environmental signals using their inher-nt signalling and gene regulatory networks. Here I will presenthe construction of synthetic gene circuits to customise cellularnformation processing and responses by harnessing the inher-nt modularity of signalling networks [1–3]. In particular, a set ofodular and orthogonal genetic logic gates, e.g. AND and NAND,

nd analogue circuits such as a gain-tunable genetic amplifier werengineered to modulate multiplein vivo transcriptional signals inither digital-like or bespoke analogue manner. I will then showhat how these gene circuits can be used to enhance the specificitynd sensitivity of synthetic cell-based biosensors for detectingeavy metal ions and bacterial signalling molecules in an aqueousnvironment, and to realise robust gene expression control andensing in single cells over a range of abiotic conditions. Further-ore, we are engineering modular genetic controllers that can act

s dynamic stress sensor-regulators to achieve adaptive control of
ellular pathway gene expression flows for optimised biomoleculeroduction.
SYMPOSIUM 12: STRESS RESPONSES INMICROBIAL BIOPROCESSING

eferences

].Wang B, Kitney R, Joly N, Buck M. Engineering modular and ortho-gonal genetic logic gates for robust digital-like synthetic biology. NatCommun 2011;2:508.

].Wang B, Buck M. Customizing cell signalling using engineeredgenetic logic circuits. Trends Microbiol 2012;20(8):376–84.

].Wang B, Barahona M, Buck M. A modular cell-based biosensor usingengineered genetic logic circuits to detect and integrate multipleenvironmental signals. Biosens Bioelectron 2013;40:368–76.


12-3

tochastic activation of the GlpR-controlled glp geneluster in Pseudomonas putida KT2440 results in aistable growth pattern on glycerol

ablo Ivan Nikel ∗ , Victor de Lorenzo

Centro Nacional de Biotecnologia (CNB-CSIC), Spain

Phenotypic variation is a widespread trait among prokary-tes, and the molecular mechanisms underlying the phenomenonnclude genetic changes (e.g., genomic inversions and strand-lippage processes), epigenetic variations (e.g., distinct patterns ofNA methylation), as well as feed-back-based multi-stability. All

hese mechanisms are known to ultimately lead to the appearancef at least two distinct phenotypes within an otherwise isogenicopulation. However, this complex trait has been scarcely explored

n microorganisms that have environmental and industrial inter-st. The soil bacterium Pseudomonas putida exhibits promisingiotechnological potential, together with its generally regarded-s-safe certificates, resistance to endogenous and exogenous stress,menability to genetic manipulation, and suitability as a hostor heterologous gene expression. Growth of P. putida KT2440n glycerol is characterized by an unexpectedly long lag phaseNikel PI, Kim J, de Lorenzo V. Metabolic and regulatory rear-angements underlying glycerol metabolism in Pseudomonas putidaT2440. Environ Microbiol 2014;16:239-54). In the present con-

ribution, the growth of individual P. putida KT2440 cells wasssessed on glycerol to further explore this phenotypic behavior.t was found that the physiological properties of cells growingn this carbon source resulted in the appearance of two distinctell sub-populations which significantly differed in their metabolicctivity. These macroscopic properties are wired to the dual logic ofhe GlpR regulator, repressing the transcription of the cognate glpenes, encoding the enzymes needed for glycerol catabolism. Ana-yzed in perspective, these results suggest that P. putida is subjectedo a carbon-source-dependent bet-hedging strategy that might be


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12-4

he adaptation of the intestinal sulphate reducing bac-erium, Desulfovibrio desulfuricans, to nitrosative stressnduced by nitric oxide

atthew Faulkner ∗ , Jeff Cole

The University of Birmingham, United Kingdom

Desulfovibrio desulfuricansis an environmentally importantrganism that causes widespread bacterial corrosion to metal struc-ures. It is useful for the bioremediation of heavy metals in thenvironment. The presence of D. desulfuricans populations in theuman gut has been correlated with gastro-intestinal disease: itsetabolism is thought to form inflammatory products. Unlikeesulfovibrio vulgaris, D. desulfuricans reduces both sulphate anditrate as terminal electron acceptors. Nitrate is reduced via nitrite

o ammonia, a process that generates nitric oxide as a side prod-ct. A strong adaptive nitrosative stress response is essential forrganisms living in nitrate-rich, micro-oxic environments. Nitricxide is generated during nitrate reduction in this, and other, orga-isms. Nitrosative stress, caused by NO production, is inducedy macrophages to kill pathogens during human infection. It isherefore important to understand how D. desulfuricans respondso nitrosative stress.

The tolerance of D. desulfuricans ATCC 27774 to nitric oxide wasefined for bacteria with various histories of exposure. Culturesrown with nitrate as terminal electron acceptor showed a 10-foldncrease in tolerance to NO compared to sulphate grown cultures.itrate grown cells also reduced nitric oxide at a 5-fold higher rate

han sulphate grown cells. These phenotypic changes were inves-igated in the transcriptome of cells from stressed and unstressedonditions by qPCR. The data reveal that exposure to nitrosativetress induced a global response, that includes the induction ofenes encoding proteins of previously unknown function.


12-5

rigins of Escherichia coli growth rate and cell shapehanges at high external osmolality

euta Pilizota1,∗ , Joshua Shaevitz2

University of Edinburgh, United KingdomPrinceton University, United States

In line with the challenge of designing robust synthetic sys-ems, there has been an increased interest in understandingacterial growth rates. Growth can be modulated in different ways,ncluding temperature, antibiotics, toxins, nutrients and osmolar-ty. While the effects of nutrient availability have been extensivelyharacterized, an almost equally common modulation of bacte-ial growth occurs in response to changes in external osmolarities.n Escherichia coli, a sudden increase in external concentration
auses a pressure drop across the cell envelope, followed by anctive recovery. Post recovery E. coli cells have been shown torow slower, smaller and at a reduced turgor pressure. Despite the


act that the active recovery is a key stress response, the naturef these changes is not understood. The biotechnology indus-ry is heavily involved in engineering bacteria for production ofseful compounds. High-level excretion of substrate feed duringiotechnological applications constantly increases medium con-entration. As a result, limited cell growth and low volumetricroductivity has been reported. Here, we use fluorescence imagingf single cells during hyperosmotic shocks, combined with customade microfluidic devices, to show that cells fully recover their

olume to the initial value and continue to grow slower immedi-tely after the recovery. We also show that turgor pressure recoverso the initial value along with the cell volume and, unlike previ-usly thought, does not cause the reduction in growth rates. Weresent results that point to changes in cellular energetics as theain cause of growth slow down at high external osmolarities.


12-6

ngineering global regulator cAMP receptor proteinCRP) of E. coli for improved biobutanol tolerance

ongrong Jiang ∗ , Hefang Geng, Huiqing Chong

Nanyang Technological University, Singapore

The toxicity of biofuel, including isobutanol and 1-butanol,o microbial host is a major drawback during fermentative bio-uel production. Previously, UV/chemical mutagens and metabolicngineering have been used for strain engineering. However, thesepproaches are either labor intensive or time consuming, andequire comprehensive metabolic information. In this work, weimed to improve E. coli tolerance towards isobutanol and 1-utanol by rewiring its global transcription factor cAMP receptorrotein (CRP). Error-prone PCR was performed to mutate crp, andhe random mutagenesis libraries were subjected to isobutanol or-butanol stress for screening. After 3–5 subcultures with increasedtressor concentration, the mutants with improved tolerance werenriched in cell culture. Mutant IB2 (S179P, H199R) and MT5G71D, T127N, D138V, T208N) exhibited enhanced isobutanol/1-utanol tolerance respectively. In the presence of 1% (v/v, 9.6 g/L)sobutanol, IB2 had the growth rate of 0.18 hour−1, 3.6-timesaster than the control (0.05 hour−1). Similarly, when challengedith 1.2% (v/v, 9.6 g/L) 1-butanol the growth rate of MT5 was.18 hour−1, double that of the control (0.09 hour−1). Genomeide DNA microarray revealed that IB2 had 366 differentially

xpressed genes (>2 fold, p-value < 0.05) under isobutanol stress,ncluding acid resistance genes (gadABCE, hdeABD) and trans-orters (proVWX, manXYZ). Quantitative real-time PCR showedhat 1-butanol stress response genes (rpoH, ompF, sodA, manX,

arA) in MT5 demonstrated differential expression compared tohe control. Therefore, we believe that engineering CRP can serve

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12-7

eat shock at higher cell densities improves the translo-ation of measles virus hemagglutinin into the yeastndoplasmic reticulum

imantas Slibinskas ∗ , Edita Bakunaite, Ruta Zinkeviciute,aimundas Razanskas, Evaldas Ciplys

Vilnius University Institute of Biotechnology, Lithuania

The yeast Saccharomyces cerevisiae is a widely used cell factoryor the production of heterologous proteins. However, secretory
xpression of the heterologous proteins in yeast is often subjecto several bottlenecks that limit yield. Translocation of newly syn-hesized proteins into endoplasmic reticulum (ER) is the first stagef the secretion pathway. It was earlier shown that synthesis of
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SYMPOSIUM 12: STRESS RESPONSES INMICROBIAL BIOPROCESSING

easles virus hemagglutinin (MeH) is inefficient mostly due toottleneck in the translocation of precursors of viral protein intohe ER of the yeast cells. The aim of this study was to improveranslocation of MeH in S. cerevisiae by manipulating cell cultureonditions. Induction of MeH expression under various conditionsas tested to establish factors that influence the efficiency of MeH

ranslocation. We found that heat shock with subsequent induc-ion of MeH expression at 37◦C improved translocation of MeHrecursors at the higher cell densities. The amount of MeH gly-oprotein increased about three-fold after the heat shock in theate-log phases of both glucose and ethanol growth. Our resultsuggest that the heat shock may be employed for the improve-ent of expression of the heterologous proteins in the S. cerevisiae

ecretory pathway.



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UTURE COLLABORATIONS OF AFOB-EFB ANDMOU SIGNING CEREMONY

uture collaborations of AFOB-EFB and MOUigning ceremony

P6-1

sian Federation of Biotechnology (AFOB) as a drivingorce for collaborative growth of biotechnology in Asiand beyond

o Nam Chang , Jian-Jiang Zhong ∗, Tai Hyun Park, Hyun Jung Kim

Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12aetbeol-ro, Yeonsu-gu, Incheon, Republic of Korea

Asian Federation of Biotechnology (AFOB, homepage:ww.afob.org) is a non-profitable organization established in008. Since then, 13 Asian regions have joined AFOB as memberegions and currently 3000 individual members from Asia arectively involved in AFOB activities. Of course, the number ofoth member regions and individual members is still increasing.

Following the mission to promote co-operation and to expandetwork between academia and industry of biotechnology, AFOBolds various events including congresses and symposia for scien-

ists and engineers in all areas of biotechnology. Especially, AFOBas established a tradition in Asian Congress on Biotechnology

ACB) since 1990 when APBioChEC was its predecessor, Youngsian Biochemical Engineers’ Community (YABEC) since 1995,FOB Regional Symposium (ARS) and AFOB International Sym-osium.

AFOB also organizes Annual Meetings and Summits for eachivision to promote their activities and collaboration with relatedivisions in partner organizations.

In addition, Biotechnology Journal publishes AFOB special issueswice a year as an official partnership between AFOB and the Wileyublisher, enhancing the international status of Asian regions iniotechnology and exhibiting top biotechnology achievementsrom member institutions and organizations to the world beyondhe Asia. All the members of AFOB are working hard for innovativend practical biotechnology for the benefits of our human beingsor better life, clean environment and sustainable development ofhe society.


P6-2

cademic Division of AFOB and Possible Collaborationith EFB

en-Chien Lee ∗ , Hyung Joon Cha, Jian-Jiang Zhong, Ho Nam

hang

Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12aetbeol-ro, Yeonsu-gu, Incheon, Korea

Asian Federation of Biotechnology (AFOB) has set up ancademic committee to coordinate and support its academic Divi-
ions. Totally twelve academic Divisions are established to coverll areas of applied biotechnology. These are Divisions of (1)gricultural and Food Biotechnology, (2) Applied Microbiology,


3) Biopharmaceutical and Medical Biotechnology, (4) Biocatal-sis and Protein Engineering, (5) Bioprocess and Bioseparationngineering, (6) Bioenergy and Biorefinery, (7) Environmentaliotechnology, (8) Marine Biotechnology, (9) Nanobiotechnology,iosensors and Biochips, (10) Systems and Synthetic Biotech-ology, (11) Tissue Engineering and Biomaterials, and (12) Asiaioeconomy and Biobusiness. Currently, some AFOB Divisionsave started to work. For example, the Bioenergy and Biorefin-ry Division will have the annul division meeting and summit inoming August, in which the leader of EFB Environmental Biotech-ology Section has been invited to join as an invited speaker.he division meeting of Bioprocess and Bioseparation Engineer-

ng Division is to be held in this September. AFOB and its Divisionsave strong interest in (co-)hosting various seminars or symposiaith EFB and other societies. In addition to collaborations on

ongresses of AFOB and EFB, the collaborations between similarivisions from two parties will also be beneficial. For collaboration

n Division/Section level, we are expecting academic Divisions ofFOB and their counterpart Sections of EFB to jointly organizecademic activities and establish close collaborations.


P6-3

dvances in Biotechnology in Asia and Future Collabo-ation with EFB

ai Hyun Park ∗ , Teruyuki Nagamune, Jung-Keug Park, Jian-Jianghong, Ho Nam Chang

Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12aetbeol-ro, Yeonsu-gu, Incheon, Korea

The fast advancement of biotechnology around the worldrovides us with new products, ideas, methods, and tools.iotechnology in Asia is also growing very fast, and abundant bio-iversities and biomass resources are located in Asian region. AFOBnd EFB are able to develop a partnership to provide their membersith the opportunities for the collaborative research. In this sense,cademic societies can play an important role. As an example, dur-ng last three years the American Institute of Chemical EngineersAIChE) has been organizing an International Forum: Biotechnol-gy in Asian Countries. The topics of the International Forum atIChE annual meetings were as follows: Biotechnology in China

2011), Biotechnology in Taiwan and Southeastern Asia (2012),iotechnology in South Korea and Japan (2013). In this presenta-ion, we are going to introduce some activities of AFOB and theiotech researches in Asian countries. We would also like to sug-est some possible strategies for strengthening the collaborationetween AFOB and EFB, and any feedback from EFB colleagues is

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dvances in omic technologies

P4-1

ntegrating omics to study human biology and disease

athias Uhlen

Science for Life Laboratory, Sweden

The human proteins constitute the major building blocks forhe function of the various processes necessary for human life.he mapping of the human genome has allowed us to predict thatach human has approximately 20 000 genes encoding for pro-eins. In the field of proteomics, these proteins are studied usingarious tools such as mass spectrometry, antibody-based profiling,hromatography, bioimaging, crystallography and spectroscopy.n addition, the new tools in genomics based on next generationequencing have open up the possibility to study human variationnd expression levels in a quantitative manner not possible onlyfew years ago. We have classified all the protein coding genes inumans using a combination of genomics, transcriptomics, pro-

eomics and antibody-based profiling. We have used this data totudy the global protein expression patterns in human cells, tissuesnd organs as well as a discovery tool to find potential biomarkersor disease, such as cancer [1–9].

eferences

].Mardinoglu, et al. Nat Commun 2014;5:3083.].Agren, et al. Mol Syst Biol 2014;10:721.].Stadler, et al. Nat Meth 2013;10(4):315–23.].Danielsson, et al. Proc Natl Acad Sci U S A 2013;110(17):6853–8.].Mardinoglu, et al. Mol Syst Biol 2013;9:649.].Paik, et al. Nat Biotechnol 2012;30(3):221–3.].Uhlen, et al. Nat Biotechnol 2010;28(12):1248–50.].Bourbeillon, et al. Nat Biotechnol 2010;28(7):650–3.].Vashisht, et al. Science 2009;326:718–21.


P4-2

he use of Genome-scale metabolic models for drug tar-et and biomarker identification

dil Mardinoglu1,∗ , Mathias Uhlen2, Jens Nielsen1

Chalmers University of TechnologyRoyal Institute of Technology

The elucidation of diverse disease mechanisms for identifica-ion of novel drug targets and discovery of biomarkers has beenmajor focus in medicine, and further research efforts are stilleeded for developing efficient diagnostic and treatment strate-ies. Genome-scale metabolic models (GEMs) can aid in this byroviding a scaffold for integration of omics data and revealinghe molecular mechanisms involved in the occurrence of theisease (J Intern Med 271, 142;Biotechnol J 8,985). My presen-
ation will cover the reconstruction and the use of functionalEMs for adipocytes (iAdipocytes1809) (Mol Syst Biol 9, 649),epatocytes (iHepatocytes 2322) (Nat Commun 5,3083) and per-onalized GEMs for Hepatocellular carcinoma (HCC) patients (Mol
ADVANCES IN OMIC TECHNOLOGIES

yst Biol 10,721). iAdipocytes1809 was employed for gaining fur-her insights from transcriptomics data obtained from Swedishbese Subjects (SOS) Sib Pair study whereas iHepatocytes2322 was

mployed for the analysis of transcriptomics and metabolomicsata obtained from NAFLD patients, and thereby we identifiedoth putative biomarkers as well as therapeutic targets. Person-lized GEMs for HCC patients were employed for identifyingnticancer drugs that are specific to individual patients using theoncept of antimetabolites i.e. drugs that are structural analogs toetabolites. The toxicity of each antimetabolite was predicted by

ssessing the in silico functionality of 83 healthy cell type specificEMs. Finally, I will present the integration of data from RNA-

eq and antibody-based immunohistochemistry across all majoruman tissues and organs to explore the human tissue proteomeith enriched expression (Mol Cell Proteomics 13, 397;Faseb

, doi:10.1096/fj.14-250555) and its use for the validation and


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PONSORED SYMPOSIUM

ponsored Symposium

S-1

ecent developments in scaling down and using singlese probes for measuring the live cell concentration byielectric spectroscopy

ohn Carvell , Matt Lee, Pardip Sandhar ∗

Aber Instruments Ltd, United Kingdom

Real-time bioprocess monitoring is fundamental for maxi-izing yield, improving efficiency and process reproducibility,inimizing costs, optimizing product quality, and full under-

tanding of how a system works. Bioreactors that are monitoredontinuously and in real-time offer the advantage of meeting cur-ent and future supply demands with biological product of thetmost quality and safety, achieved at the lowest overall cost andith least risk. This paper will focus on the latest developments

n dielectric spectroscopy for live cell concentration measure-ent and how the technology has been scaled down allowing

ioreactors with less than 100 ml working volume to be mon-tored in real time. The presentation will also focus on howielectric spectroscopy can also be applied to single use biore-ctors in a cGMP environment and on samples down to 100 �lolume.


S-2

cale-down study of oscillations in oxygen and substrateupply for Corynebacterium glutamicum

arco Oldiges1,∗ , Friedrich Käß1, Ioanna Hariskos1, Andreaichel1, Robert Spann2, Peter Neubauer2, Stefan Junne2, Wolf-angWiechert1

Forschungszentrum Jülich/Institute of Bio- and Geosciences – IBG-1: Biotech-ology, GermanyChair of Bioprocess Engineering/TU Berlin, Germany

The platform organism Corynebacterium glutamicum has a suc-essful history of biotechnological application and prevailingignificance in industrial biotechnology with applications at biore-ctor scales of several hundred cubic meters. Studies in metabolicnd bioprocess engineering, however, are mostly limited to cul-ivation at laboratory scale. An often neglected upscaling effects that physical transport and metabolic uptake processes of, forxample oxygen and substrate lead to stronger concentration gra-ients with increasing bioreactor working volume. This causesscillating microenvironments for individual cells. The metabolicmpact of such oscillations can be monitored in specialized scale-own bioreactor setups, where large scale effects are simulated in

aboratory-scale experiments.In this work, two-compartment reactor systems (stirred-tank-

eactor connected to plug-flow-reactor) have been used to generateefined oscillations in oxygen and substrate supply during cul-ivation of C. glutamicum ATCC13032 and lysine producing

ip



train C. glutamicum DM1933. Within oxygen-limited plug-flow-ompartment coupled to aerobic stirred-tank-reactor, primary (i.e.eliberately induced) and secondary (resulting from primary)scillations of substrate and dissolved oxygen concentration asell as pH and metabolic side product concentrations are studied.ith anaerobic residence times from 40 s up to several minutes,

obustness and performance of microbial metabolism is challengedt continuously oscillating microenvironments.

Carbon flux, respiratory activity and side product concentra-ions show fundamental differences under oscillating conditions.

ost strikingly, Omics-data from the metabolome, proteome andranscriptome show only marginal effects with respect to oscil-ation. This indicates that the regulon of C. glutamicum is veryobust against oscillatory conditions, which constitutes an impor-ant mechanism for robustness in industrial processes.


S-3

utomated development of recombinantioprocesses–from vision to mission

lorian Glauche1,∗ , Andreas Knepper1, Michael Heiser1, Lorenzheuer1, Fabian Wollny1, Susan Bigesse1, Antje Neubauer2, Sarinarain3, Gernot Thomas John3, Joachim Aschoff4, Birgit Stehlik4,ngrid Schmidt4, Rudibert King5, Norman Violet5, Detlef Goelling6,ndreas Raab6, Gregor Kiesewetter6, Rick Nolte6, Peter Neubauer1

Chair of Bioprocess Engineering, TU BerlinBioSilta Europe GmbHPreSens Precision Sensing GmbHInfoteam Software AGChair of Measurement and Control, TU BerlinOrganobalance GmbH

The development of recombinant protein production processess a time and labor-intensive task. Implementing Quality by DesignQbD) and Design of Experiments (DoE) principles, already at thecreening stage can significantly reduce development risk, timend costs. With the recent advances in laboratory automation,ost steps of up- and downstream bioprocess development can be

utomated with implementation of DoE and QbD. At TU Berlin, inlose collaboration with industrial partners, a reference laboratory,hich focuses on the automation of recombinant protein pro-uction process development, was established. Key features of theystem are (i) development of software solutions in order to applyoE for screening and optimization procedures, (ii) automated at-

ine and on-line analyses by the use of analytical procedures andensors, (iii) software-supported model creation and developmentf process models for advanced process control at the bioreac-or scale. Microbial cultivations are performed in 96 and 24 welllates as well as in a 10 mL 48-bioreactor system. The applicationf EnBase® technology to maintain controlled growth is one ofhe key technologies in this concept. Growing cells in fed-batchultures over the whole developmental line ensures consistency
n operation procedures from microliter and milliliter scales toilot plant scale. The overall strength of the automation concept

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s demonstrated on a number of difficult to produce proteins inscherichia coli and Saccharomyces cerevisiae.


S-4

cale up of chito-oligomer production via bacterial fer-entation

endrik Waegeman1,∗ , Ko Wisse2, Saskia Vander Meeren2, Anermeulen2, Brecht Vanlerberghe2

Bio Base Europe Pilot Plant vzw, BelgiumBio Base Europe Pilot Plant, Belgium

Chito-oligomers constitute an interesting class of specialty car-ohydrates, among other applications used in plant protectionnd wound healing products. Today’s commercially available chi-osans are produced chemically from chitin isolated from shrimphell wastes. They can be well defined concerning their degree ofolymerisation and degree of acetylation, but they are invariablyharacterised by a random pattern of acetylation (PA), despite thisnfluences the activity greatly.

Together with other partners within the ERA-IB ChitoBioEngi-eering and FP7 Nano3Bio project, Bio Base Europe Pilot Plantims at establishing, through genetic, metabolic and enzymengineering, biotechnological ways of producing fully defined,artially acetylated chitosans, of which no production methodsre available to date. Our specific goal, is to employ the strainsested at laboratory scale, to develop an industrially viable fer-

entation and downstream purification process for every specifichito-oligomer. Processes, meeting defined requirements in termsf titer and production rate are scaled up in our facilities. This pre-entation will show the results of the successful scale up of the first
roduct developed by BBEPP and its partners.

SPONSORED SYMPOSIUM

S-5

assive protein superfamily data integration applied tomart library design

enk-Jan Joosten

Bio-Prodict, Nijmegen, Netherlands

Nature offers a wide variety of enzymes that can be utilizedn many different processes, but often these enzymes need to beptimized by introducing mutations to meet the requirements ofhese (biotechnological) processes. In many cases a combinationf multiple mutations are needed to reach these goals, but findinghe right combination of mutations still is problematic. More and

ore protein engineers use a strategy referred to as “smart libraryesign”. Smart mutant libraries contain only a small number ofutants and are designed such that they contain a high number of

ctive clones with mutations at positions (hotspots) that are likelyo show the desired effect. The quality of a smart library dependsn: 1. The selection of hotspots and 2. The prediction of the bestmino acid changes at these hotspots. Recently it was shown thathe vast amounts of data nowadays available for protein super-amilies can be used for the prediction of both these steps andherefore for the design of high quality smart mutant libraries.ere we present how 3DM, a protein superfamily analysis plat-

orm that integrates many different data types for complete proteinuperfamilies, can be used to design smart libraries. Using this strat-gy different enzymes features, such as enantioselectivity, activitynd thermostability have been optimized. Comparisons with ran-om designed libraries show that, by using 3DM when designing amart library, libraries are of high quality, which reduces not onlyhe number of clones that need to be screened but it also increases


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YMPOSIUM 13: BIOMEDICAL RESEARCH

ymposium 13: Biomedical research

13-1

acteria fabricate 3D scaffolds for organ regeneration

aul Gatenholm ∗ , Hector Martinez, Johan Sundberg, Saraohannesson, Daniel Hägg

BBV Laboratory and WWSC, Department of Chemical and Biological Engineer-ng, Biopolymer Technology, Chalmers University of Technology, Göteborg,weden

In recent years, stem cells and biomaterials have emerged asools to regenerate damaged or diseased tissue. Typically, a bio-ompatible material that can be engineered to match the shape ofhe defect and can function as a vehicle to deliver cells and/or bio-ogically active factors that promotes tissue regeneration is used.acterial nanocellulose is emerging biomaterial which combineshe nanofibril network with hydrogel like behavior which makes itdeal for Tissue Engineering applications. We have previously usedacteria to fabricate artificial blood vessels and menisci substitutes,nd excellent biocompatibility has been shown in rat, hamster,ig and sheep. In the present study we describe a novel biofabrica-ion method, which uses the Gluconoacetobacter Xylinus machineryo engineer 3D scaffolds with features at different length scales –anging from the nano, subcellular to the macroscale. Examplesf 3D Bacterial nanocellulose scaffolds prepared in our laboratorynclude multichannel scaffolds for preparation of microvasculartructures, highly porous 3D structures for growth of cartilage tis-ue and combination of 3D multichannel scaffold with highlyorous architecture for growth of vascularized tissue such asone and adipose tissue. 3D Bacterial nanocellulose scaffolds havehown to support neural network formation and enable stem cellifferentiation. Human tissues grown on 3D Bacterial nanocel-

ulose scaffolds show great potential of this new biomaterial-cellonstructs for applications in reconstructive surgery and as in vitroodel of diseases such as Alzheimer and Osteoarthritis.


13-2

nzyme immobilized polymeric biomaterials

ukesh Doble ∗ , Prabhawathi Veluchamy

IIT Madras, India

Polymers are widely used in the biomedical technology forhe fabrication of medical implants and devices. Implant basednfection is highly prevalent in biomaterial which leads to itsarly rejection. Several physical, chemical and biological meth-ds are practiced to overcome the bacterial attachment and theubsequent formation of biofim. Antibiotics are used as mainorm of therapy, but emergence of antibiotic resistance bacterias forcing the researches to search for other methods. Biomateri-
ls are hydrophobic, with no labile chemical groups that mighte used with conventional immobilization strategies. Papain, anntimicrobial and biocompatible enzyme is immobilized on two
Mi



olymers namely, poly urethane and polycaprolactam, which arepproved by FDA for medical applications. The enzyme is immo-ilised using glutaraldehye as the fixing agent. Presence of thenzyme is confirmed using FTIR. The conditions namely, temper-ture, pH and time of reaction for immobilization are optimizedo achieve maximum enzyme activity. The storage stability of theurfaces is tested successfully. The formation of Staphylococcusureus and Escherichia coli biofilm on these surfaces are tested.hese organisms are widely found in implant associated infection.oth the surfaces reduced the number of live colonies (by about 30imes), amounts of biomass, protein and carbohydrate content (bytimes) in the bacterial biofilm. The composition of the biofilmas probed using FT-Raman spectroscopy. This study indicates that

table antimicrobial surfaces could be prepared using this enzyme.uch surfaces could find applications in the design of implantabler topical biomaterials which require prevention of biofilm.


13-3

steoblast cell proliferation on magnesium-substitutedydroxyapatite (Mg-HA) coatings

atma Nese Kok1,∗ , Sakip Onder2, Ayse Ceren Calikoglu3, Kursatazmanli 4, Mustafa Urgen4, Gamze Torun Kose3

Istanbul Technical University, TurkeyIstanbul Technical University, Molecular Biology Genetics and Biotechnologyrogramme, TurkeyYeditepe University, Genetics and Bioengineering Department, TurkeyIstanbul Technical University, Department of Metallurgical and Materials Engi-eering, Turkey

Hydroxyapatite (HA; Ca10(PO4)6(OH)2) is a well-known bio-ompatible material commonly preferred in surface modificationf hard tissue implants to ensure better osteointegration with theurrounding bone tissue. Properties of HA may also be improvedia doping different ions into HA structure for better osteoin-egration and cell proliferation properties. In our study, cellroliferation studies on magnesium substituted HA coatings (Mg-A) that were deposited on different Mg2+ containing (Ti,Mg)N

hin film coatings (0, 4.24 and 10.42% at) were studied to deter-ine the effect of Mg2+ on cell proliferation. Mg-HA coatings and

haracterization studies were conducted as our previous study [1].hen, osteoblast cells were seeded on HA and Mg-HA coatingsnd MTS studies were conducted for 1, 4 and 7 days. Resultshowed that cell proliferation was better on Mg-HA coatings thatere deposited on low Mg2+ (4.24 at%) containing surfaces com-ared with the HA coatings that were deposited on Mg2+ freeurfaces. Cell proliferation slowed down on Mg-HA coatings thatere deposited on high Mg2+ (10.42 at%) containing surfaces. Highg content also accelerated the corrosion so this was taught to be

he major reason for the decrease in cell survival. Studies on the

g presence in the coatings. The grant from TUBITAK (112M339)s gratefully acknowledged.

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eference

].Onder S, et al. Mater Sci Eng C 2013;33(7):37–42.


13-4

one regeneration through facile binding of bone graftubstitute particles using mussel adhesive protein

yung Joon Cha1,∗ , Bong-Hyuk Choi1, Hogyun Cheong1, Yun Keeo1, Jin-Soo Ahn2, Sang-ho Jun3

Pohang University of Science and Technology, Republic of KoreaSeoul National University, Republic of KoreaKorea University Anam Hospital, Republic of Korea

To date, hydroxyapatite and related calcium phosphates haveeen intensively investigated as the bone substitutes for boneissue engineering. Among these, inorganic bovine bone is the

ost commonly used bone graft material. However, lack of cellecognition motifs and/or biochemical factors has been consid-red a limitation. Mussel adhesive proteins (MAPs) are one ofost remarkable and powerful adhesive materials in nature. Pre-

iously, recombinant MAPs were successfully demonstrated to beunctional cell adhesion materials on various surfaces due to theireculiar adhesive properties. Herein, MAPs were applied as surfaceoating and functionalization biomaterials to xenograft materials.e successfully coated MAPs onto xenograft surfaces by sim-

ly mixing xenografts with the MAP solution. Through in vitrotudy using mouse osteoblast cell line MC3T3-E1, significantnhancement of cellular activities such as attachment, prolifer-tion, spreading, and osteogenic differentiation was observed onAP-coated xenografts. In addition, we found that in vivo implan-

ation of MAP-coated xenografts enhanced bone regeneration inrat calvarial defect model. These results collectively demonstrate

hat facile coating of xenografts using biofunctional MAP would bepromising strategy for successful bone tissue engineering appli-

ations.


13-5

he stabilisation of red blood cells in a powdered form

rishnaaMahbubani ∗ , Nigel K.H. Slater

Department of Chemical Engineering and Biotechnology, University ofambridge, United Kingdom

In recent years apprehension surrounding blood transfusionsave significantly reduced. However, fears around the supply oflood have become a concern. In metropolitan areas, managinghe blood inventory is critical to ensure emergency and elec-ive surgeries can take place without shortages while military
lood banking faces a slightly different challenge, in obtaininghe required amount and type of blood products at specific timesnd often in remote areas.
SYMPOSIUM 13: BIOMEDICAL RESEARCH

Current practice is to refrigerate blood (from donor centres),ith an anticoagulant, where it is retained for a mere 42 days.o ensure a readily available safe blood supply, preservation tech-iques and methods for long-term storage need to be investigated.

Cryopreservation in glycerol has shown some success inxtending the shelf life of blood. However, prior to use, the glycerolust be removed. This is a demanding, labour intensive and costly

rocess resulting in a limited 24-hour shelf life of deglycerolisedample making it an unfeasible procedure.

We present the use of freeze-drying with intracellular sugars as aethod for stabilisation. A two part process; freeze-drying involvesfreezing step followed by a sublimation process that takes placeith the sample held in a vacuum. The ideal product would bedried powder capable of room temperature storage. With the

ppropriate additives, the hydrated blood would be suitable forirect transfusion. While the process is still in its infancy; freeze-rying has the potential to change the future of blood banking.


13-6

table aqueous solutions of keratin polypeptides: theirroperties in solution and at interfaces

ang Pan ∗ , Jian Lu

The University of Manchester, United Kingdom

Keratins are important structural fibrous proteins and are theain constituting components in skin, hair, wool, feathers, nails,

orns and connecting tissues. Their physical and biological studiesave direct relevance to health and disease and also bear impor-

ant implications to hair and skin care. In addition to personalare, there are many other reasons that require us to understandow keratins behave physically and how they interact with otherolecules. Wool is predominantly composed of keratin proteins

hat provide desirable properties. The purpose of this work iso develop water-soluble keratin polypeptides from sheep wool,hich can be used to form smooth molecular layers and opti-

ally flat thin films. These model biointerfaces can facilitate varioushysical and biological measurements that would otherwise be tooifficult to contemplate. Successful preparation of keratin samplesas demonstrated by identification of molecular weights of theey components from gel electrophoresis and measurements ofheir surface tension and basic solution properties. Zeta poten-ial measurements from keratin samples prepared demonstratedlmost identical pH dependent surface charge distributions withsoelectric points around pH 3.5, showing that during purificationy dialysis has completed removal of SDS used during wool fibre


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YMPOSIUM 13: BIOMEDICAL RESEARCH

13-7

rehalolipid biosurfactants from Rhodococcus ruberith anti-adhesive and immunomodulatory activities

aria Kuyukina1,∗ , Irena Ivshina1, Tatiana Baeva1, Olesiaochina1, Sergey Gein2

Institute of Ecology and Genetics of Microorganisms, RussiaPerm State University, Russia

In recent years, glycolipid biosurfactants traditionally con-idered as emulsifying and solubilizing agents are attractingn increasing attention as possible biomedical agents withxpressed biological activities [1]. Trehalolipids (TL) produced byembers of closely related actinobacterial genera Rhodococcus,ocardia, Corynebacterium, Gordonia, Mycobacterium, Tsukamurella,nd Arthrobacter include �,�′-D-trehalose, a nonreducing disac-haride, which is linked by an ester bond to long-chain fatty
cids [2]. Well-known TL of pathogenic Mycobacterium tuberculo-is, Corynebacterium diphtheriae play a key role in the infectionsaused by these actinobacteria, and they are characterized byigh immunomodulatory activity [3]. However the pathogeni-
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ity of producers and high cytotoxicity of produced TL limitheir biomedical applications. Therefore, the search for TL pro-ucers among nonpathogenic actinobacteria is essential. In in vitroxperiments, TL biosurfactants from Rhodococcus ruber IEGM 231timulated both proinflammatory (interleukin (IL)-1b, IL-6, tumorecrosis factor-a (TNF-�) and anti-inflammatory (IL-12, IL-18)ytokine production of human monocytes, depending on cellulture composition and induction. Also, diverse anti-adhesiveffects of TL towards human monocytes and bacterial species wereevealed. Since TL from R. ruber displayed no cytotoxicity againstuman lymphocytes or bacterial cells, they could be proposeds potential immunomodulatory, antitumor and anti-adhesivegents.

Research was funded by the RAS MCB and RF President Leadingcience School programs.

eferences

].Kitamoto, et al. J Biosci Bioeng 2002;94:187–201.].Kuyukina, Ivshina. Microbiol Monographs 2010;16:292–313.
].Ryll, et al. Microbiol Immunol 2001;45:801–11.

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ymposium 14: Plant genetic engineering

14-1

enetic engineering of secondary metabolism for plantrotection

.A. Pickett

Rothamsted Research, Harpenden AL5 2JQ, United Kingdom

Only recently has secondary plant metabolism become a targetor genetic modification, initially to investigate the role of metabo-ites and now in crop plants for pest management. By generation inhe plant, chemically unstable and even highly volatile metabo-ites can be exploited beyond the toxicants traditionally used toontrol pests. Expression of the aphid alarm pheromone, (E)-�-arnesene, in wheat will be described leading on to the need fornduced or primed expression. Examples of other semiochemicals,n addition to pheromones, such as stress related homoterpenes,hat are already exploited in crop protection, demonstrate alsonduction or priming as a consequence of stress related plantignalling involving secondary metabolism. Such signals them-elves then provide alternatives to constitutive expression for usen GM crops and already provide evidence of likely success asell as offering new types of sentinel plants and potentially solu-

ions for problems of perennialisation of arable crops. Secondaryetabolites, exploited in these ways, also show promise for not

nly disease and weed management, but in dealing with otherequirements of more sustainable agriculture relating to non-CO2

reenhouse gas production.


14-2

ranscriptome and small RNA sequencing analysis of aew dwarf mutant in Gossypium hirsutum L

iongming Du1,∗ , Wen-yan An2, Jun-ling Sun1, Wen-fang Gong1,hou-pu He1, Zhao-e Pan1

Institute of Cotton Research of Chinese Academy of Agricultural Sciences (ICR,AAS), ChinaInstitute of Cotton Research of Chinese Academy of Agricultural Sciences

ICR, CAAS)/College of Life Science and Technology, Huazhong Agriculturalniversity, China

Plant height is an important trait in upland cotton. Ari1327as a dwarfed mutant derived from an American upland cot-

on varieties-Ari971 by 60Co �-ray irradiation. Ari1327 exhibitedwarf trait during germination and cotyledon periods. Using theext generation high-throughput sequencing technology, threeDNA and small RNA libraries of the dwarfed mutant Ari1327,igher plant mutant Ari3697 and their wild type Ari971 wereonstructed and sequenced with Illumina HiSeqTM2000 system.hrough comprehensive analysis with transcriptome and miRNA
egulation level for the plant high mutant, it will be beneficial toeveal the molecular mechanism dwarf mechanism and clone theelated genes for dwarf plant breeding in cotton.
SYMPOSIUM 14: PLANT GENETIC ENGINEERING

After comparing the transcriptome data of Ari1327 with Ari971nd Ari3697, 13919 differentially expressed unigenes (DEGs) weredentified, of which5406 up-regulated while 8513 down-regulatedn Ari1327. The 16 genes in plant hormone signal transductionathway was validated and analysed using quantitative real timeCR (qRT-PCR). After analysis of the differential expression ofiRNA families in each sample, 16 miRNA families were found

o up-regulate in Ari1327 and down-regulated in Ari3697. In threeiRNAs libraries, we found 25 miRNAs only expressed in Ari1327

nd 13 miRNAs only expressed in Ari3697. These 16 miRNA fami-ies and 38 specific expressed miRNAs may play significant role inegulating plant height of Ari1327. Some miRNAs were selected toalidate the reality of small RNA sequencing by using stem-loopT-PCR. The target genes of these differentially expression miRNAsere also annotated in A and D genomes in cotton.


14-3

an plant still be a major and cost-effective source forhe supply of artemisinin, the most potent anti-malariarug

exuan Tang ∗ , Qian Shen, Fangyuan Zhang


Artemisinin isolated from Artemisia annua L., is most potentn treating malaria. Since the discovery of artemisinin in 1970s,. annua has been the only source for artemisinin supply. Recentreakthrough in transgenic yeast has broken the traditional sup-ly way of artemisinin. According to the report, transgenic yeastystem can supply 50–60 tons of artemisinin annually at the salerice of $350–400/kg, which is lower than the current artemisininroduction cost by using traditional plant varieties. Data showed

n this year A. annua plantation has been reduced by over 60%.t brings concerns if farmers are not willing to grow A. annuanymore, is the yeast system ready for providing all the amountf artemisinin the world needs and when? Will plant system toroduce artemisinin be out of the stage?

Here, we report the development of three strategies, includ-ng spraying fertilizer technology, developing high artemisininontent varieties, and transgenic plant technology to increasertemisinin content in A. annua, which dramatically reduces pro-uction cost, and could supply artemisinin in large quantity athe sale price of $250 in 3 years. Transgenic lines have beenpproved for environmental release, the first GM medicinal plantso be approved in the world. Environmental test and animal testemonstrate transgenic lines are safe and artemisinin extractedrom transgenic lines has same function with that extracted fromon-transgenic varieties. We believe these strategies, together with

ransgenic yeast system, will secure artemisinin supply enough at


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14-4

ngineering barley for increased drought resistance

vo Frébort ∗ , Hana Pospísilová, Petr Galuszka

Palacky University in Olomouc, Czech Republic

Barley is an agriculturally important crop and the production oflants with enhanced stress tolerance is one of the important goals

n barley breeding. New techniques of molecular cloning and plantransformation accelerate classical breeding techniques helping toroduce barley plants with required enhanced traits. Morphologynd development of the barley plants have been altered by geneticanipulation with genes coding for cytokinin dehydrogenase (EC

.5.99.12; CKX), a principal enzyme controlling cytokinin levelsn plants.

Three unique transgenic barley lines (Hordeum vulgare cv.olden Promise) transformed with an expression cassette consist-

ng of �-glucosidase root specific promoter, modified CKX generom Arabidopsis thaliana with engineered protein targeting toytoplasm, vacuoles or apoplast, and NOS terminator were pre-ared and T2 generations of homozygous plants were analyzed.elected transgenic lines, distinctive with altered morphology ofhe root system, showed higher resistance to drought conditionshan wild type plants, both when grown in soil or in hydro-onic culture. This method of conveying drought resistance maye further exploited in order to create a usable trait that can beransferred to commercial cultivars of barley.


14-5

ecombinant protein expression in the chloroplast ofhe green microalga Chlamydomonas reinhardtii: A casetudy using a novel green fluorescent protein as aeporter

tephanie Braun Galleani ∗ , Frank Baganz, Saul Purton

UCL, United Kinglom

Unicellular algae such as Chlamydomonas reinhardtii are attract-ng increasing interest as low cost, GRAS platforms for theynthesis of high-value heterologous proteins. Foreign genesntroduced into its chloroplast genome can be targeted to a preciseocus and expressed without suffering gene silencing issues.

Fluorescent proteins such as green fluorescent protein (GFP)rovide a simple tool for monitoring protein synthesis in vivo.owever, the level of fluorescence from GFP expressed in the C.

einhardtii chloroplast has proved disappointingly low. A newly dis-overed protein called Verde Fluorescent Protein (VFP) has showeduperior fluorescence in other organisms, so we are currently inves-igating its utility as reporter.

A codon-optimised gene encoding VFP was synthesised anduccessfully introduced into the chloroplast genome under the
ontrol of the atpA promoter/5′UTR. Protein expression was con-rmed by western blotting, and fluorescence was demonstrated byonfocal microscopy and flow cytometry. We explored whether


rotein expression and fluorescence levels could be improved by:i) expressing VFP under the control of the psaA promoter/5′UTR;ii) fusing the VFP coding sequence to that of an endogenous generbcL); (iii) co-expressing a bacterial chaperone in the chloroplast,btaining increased protein expression for (i) and (iii).

These constructs have been grown using different media andemperatures, in order to evaluate growth rate together with levelf protein expression and fluorescence.

This approach merges both cell engineering and bioprocessptimisation of a strain of algae widely used in research and has theotential to be applied to other recombinant proteins expressed in. reinhardtii.


14-6

omparative evaluation of bacterial diversity from gmnd Non-GM maize rhizosphere

aseer Ahmad1,∗ , Naseer Ahmad2, Zabta Khan Shinwari 3

Department of Environmental Sciences, COMSATS Institute of Informationechnology (CIIT), PakistanCOMSATS Institute of Information Technology (CIIT), PakistanQuaid-i-Azam University, Islamabad, Pakistan

The rhizosphere is a critical interface supporting the exchangef resources between plants and their associated soil environment.hizosphere microbial diversity is influenced by the physical andhemical properties of the rhizosphere, some of which are deter-ined by the genetics of the host plant. However, within a plant

pecies, the impact of genetic variation in the composition of theacterial biota of GM and Non-GM maize rhizosphere is poorlynderstood. Here, we studied the bacterial diversity and popula-ion dynamics in the rhizosphere of one GM and two Non-GM

aize cultivars (IG and IW) grown under field conditions, by tradi-ional cultivation techniques and 16S rRNA gene-based molecularnalysis of DNA directly extracted from pre cultivated soil andhizosphere samples. Rhizosphere and pre cultivated soil samplesere taken at three different plant growth stages. The isolatedacterial strains were further screened for different functional char-cterization. From this study, we find that the transgenic crop has

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14-7

nderstanding the interactions between plant bioticnd abiotic stress through characterization of microRNAffectors in jute (Corchorus spp.) – Macrophomina phase-lina interaction system

alit Kharbikar ∗ , Pran Gobinda Karmakar

Central Research Institute for Jute and Allied Fibres (Indian Council of Agricul-ural Research), India

Jute (Corchorus spp.) is an important fibre crop in India. Thismallholder’s crop is vulnerable to most devastating pathogen,acrophomina phaseolina. It causes damping off, root rot and col-

ar rot collectively known as stem rot, which results in up to0% yield loss and low fibre quality. The pathogen first causesoot rot upon drought stress and then colonizes physiologically
ltered stem causing stem rot at maturity, under favorable weather.owever, the molecular mechanisms underlying are unknown.lant–pathogen–weather interface is regulated by a number ofenes. Existing, probe-dependent gene regulation analysis meth-
ib

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SYMPOSIUM 14: PLANT GENETIC ENGINEERING

ds require plant and pathogen cells to be physically separated.owever, probe-independent RNA sequencing method allowslant and pathogen transcripts to be analyzed simultaneously.icroRNA effectors serve the conserved gene regulatory mech-

nisms which have been reported to control plant–pathogennteractions. Most works on miRNAs have focused only on ini-ial pathogen infection processes under controlled environment.owever, miRNA-mediated regulation of disease development

hroughout life cycle of plants under the influence of abiotictresses, such as drought has not been studied so far. Since, stemot becomes severe at maturity under hot and humid weather,espite the colonization of roots by M. phaseolina at early growthtages of jute, under high soil temperature and low soil moisturedrought) conditions, the jute–M. phaseolina interaction systemould provide a good model to study whether physiologicallterations induced by pathogen colonization or drought stressacilitate the development of disease. Sequencing of miRNAs dur-ng this could elucidate molecular interactions between plant
iotic and abiotic stress.


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YMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION

ymposium 15: Recombinant protein produc-ion

15-1

etting a grip on complexes: tools and technologies forultiprotein complex research

mre Berger

EMBL, Grenoble

Most eukaryotic proteins exist as large multicomponent assem-lies with many subunits, which act in concert to catalyze specificellular activities. Many of these are only present in low amountsn their native hosts, impeding purification from source material.nraveling their mechanisms of action will therefore often dependn heterologous overproduction.

Complex proteins, involved in disease causing processes, arelso entering center-stage as key drug targets of the future. Inddition to their essential role as tools for drug discovery, suchomplex biologics themselves are increasingly being employed asedicines, for example in the form of multicomponent vaccines.

hese are predicted to dominate the next generation of drugs.My laboratory develops advanced methods for producing mul-

icomponent protein biologics for studying their structure andunction at molecular resolution. We have created MultiBac, aaculovirus/insect cell system designed for high-quality multi-rotein complex production, and have installed MultiBac as alatform technology at the Eukaryotic Expression Facility (EEF) inur laboratory. More recently, in the context of the EC FP7 projectomplexINC, we have extended our technology concept to mul-

iprotein production in other host systems including mammalianells. Our technologies and successful applications in academicnd industrial R&D will be presented [1–3].

eferences

].Bieniossek, et al. The architecture of human general tran-scription factor TFIID core complex. Nature 2013;493(January(7434)):699–702.

].Barford D, Takagi Y, Schultz P, Berger I. Baculovirus expres-sion: tackling the complexity challenge. Curr Opin Struct Biol2013;23(3):357–64.

].Bieniossek C, Imasaki T, Takagi Y, Berger I. MultiBac: expandingthe research toolbox for multiprotein complexes. Trends Biochem Sci2012;37(February (2)):49–57.


15-2

aking life better for Escherichia coli cells that produceoxic membrane proteins

imitra Gialama1,∗ , Fragiskos Kolisis 2, Georgios Skretas1

National Hellenic Research Foundation, GreeceNational Technical University of Athens, Greece

Membrane proteins (MPs) constitute an exciting world thatncludes protein families such as G protein-coupled receptorsGPCRs) and transporters. Currently, there is increasing scientific

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nterest in the field and as MPs are extremely important drug tar-ets they attract the attention of the pharmaceutical industry. Forharacterization of MPs and acquisition of information for theational design of drugs large amounts of protein are required. Asheir natural abundance is usually very low, MPs are produced byverexpression in heterologous hosts. However, they are notori-usly difficult to express as the yields per cell are typically lownd MP production is highly toxic to cells. The aim of the presentork is to engineer strains of Escherichia coli to overcome MP-verexpression toxicity. The ultimate goal is to use these strains forarge-scale production of MPs. Also, our goal is to investigate the

echanism behind the observed cytotoxicity. For this purpose weave generated libraries of mutant bacteria carrying different typesf genetic modifications and used appropriate genetic screens tosolate the desired clones. The human GPCR bradykinin recep-or 2 that shows very high overexpression toxicity in E. coli wassed as our model MP. We have identified a number of strains thatvercome the toxicity upon BR2 overexpression while more over-xpressed protein is produced. The strains have also been showno have a more general application for several GPCRs in E. coli. Thelones are being characterized using various methods. A potentialhysiological role of the identified mutations is proposed.


15-3

opulation heterogeneity in Pseudomonas putida ana-yzed on the single cell level using proteomics and digitalCR

ichael Jahn1,∗ , Carsten Vorpahl1, Dominique Türkowsky1, Martinindmeyer2, Bruno Bühler2, Hauke Harms1, SusannMüller1

Helmholtz-Centre for Environmental Research - UFZ Leipzig, GermanyTU Dortmund University, Germany

Recombinant protein production using microbial cells is a keylayer for the chemical and pharmaceutical industry of the future.he efficiency of such biotechnological processes is usually bench-arked using bulk parameters. However, the catalytic unit drivingprocess is the single cell. Even clonal populations show high cell-

o-cell variability, e.g. caused by genetic mutations, cell cycling oregulatory decisions.

As a model system for population heterogeneity, we use Pseu-omonas putida with plasmid-based production of an EGFPused recombinant protein (StyA). With EGFP as a marker, flowytometry shows large proportions of cells (30–60%) not able toroduce functional protein even under high induction regimes.e used cutting edge cell sorting of fluorescent (EGFP+) and

on-fluorescent cells (EGFP−) to identify the cause for impairedrotein production. Sorted sub-populations were analyzed by pro-ein mass spectrometry to reconstruct pathways, and by digitalCR to determine the plasmid copy number (PCN) with extraor-inary precision.

In summary, only minor changes in the protein inventory wereound, most of them related to stress as a result of protein aggre-ation. In contrast to that, we identified unequal distribution oflasmids as a major cause for heterogeneity: The non-fluorescent

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15-4

ynthesis of antibacterial bacteriophage proteins inicroalgae

aura Stoffels1,∗ , Bambos Charalambous2, Saul Purton3

University College London, UKResearch Department of Infection, University College London Medical School,KInstitute of Structural and Molecular Biology, University College London, UK

Widespread antibiotic resistance among pathogenic bacteriand the low specificity of these drugs cause a pressing need for theevelopment of novel antibacterial agents. Endolysins are antibac-erial proteins that are produced by bacteriophages to digest theacterial cell wall for phage progeny release at the end of the lyticycle. These efficient enzymes are highly specific for the cell wallf the target bacteria without affecting other species. Developmentf resistance against endolysins is very rare, because they evolvedo target molecules in the cell wall that are essential for bacte-ial viability. Taken together, this makes them promising novelntibacterial agents. The eukaryotic microalga Chlamydomonaseinhardtii offers already established techniques for the expressionf foreign genes in the chloroplast and is an attractive expressionlatform for therapeutic proteins, due to the lack of endotoxinsnd potentially infectious agents. Furthermore it can be inexpen-ively cultivated in full containment and under sterile conditionsn simple photobioreactors. Two bacteriophage endolysins tar-eting two major human pathogens were successfully expressedn the chloroplast of C. reinhardtii, purified and their specificitynd efficiency in killing the target bacteria was assayed in vitro.urthermore the cyanobacterium Synechocystis sp. PCC6803 wasnvestigated as an alternative expression platform for endolysins.


15-5

ntegrative ‘-omic’ approach to explore molecular mech-nism of miRNA engineered Chinese hamster ovary cell

aibhav Jadhav1,∗ , Matthias Hackl1, Deniz Baycin-Hizal 2, Geraldlanert3, Michael Betenbaugh2, Johannes Grillari 1, Nicole Borth3

University of Natural Resources and Life Sciences, Vienna, AustriaJohns Hopkins University, Baltimore, USAAustrian Center for Industrial Biotechnology (ACIB), Austria

MicroRNAs (miRNA) are noncoding regulators of translation,ontrolling a broad range of physiological functions. They haveeen shown to regulate large number of mRNA targets simulta-
eously, thus acting as global regulators to alter cellular functions.he application of miRNAs to engineer Chinese hamster ovaryCHO) cells is an emerging strategy to improve the bioindustriallyelevant characteristics of CHO cells, which are one of the most
bcd

SYMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION

mportant biopharmaceutical cell factories. In this regard, recentlye have developed stable engineered miR-17 overexpressing CHO

ells, which exhibit both enhanced growth performance andncrease in specific productivity. This lead to overall 3-fold increasen recombinant protein (EpoFc) titers, this parallel enhancementf both cell-specific growth rate and productivity is very unique.he underlying molecular mechanism that controls such pheno-ype is generated by co-regulations various biological networksnd understanding such molecular regulation is hindered by theack of a miRNA-mRNA interaction database for CHO cells. Forhis reason, we applied transcriptomics (mRNA and miRNA) using

icroarray platform and quantitative proteomics using iTRAQechnology. These data sets are ultimate readout of miRNA activitynd developed a bioinformatics pipeline to handle such datasetshere the rules of interaction are not a priori known. This novel

nter-omics approach is described as well as an interpretation of theffects caused by miR-17 overexpression that led to the interestinghenotype described above.


15-6

nvestigating the physiological effect of increasedeterologous gene dosage in Pichia pastoris using tran-criptomics

lena Camara1,∗ , Landes Nils 2, Lluís Revilla Sancho3, Joan Albiol 4,iethardMattanovich2, Pau Ferrer4

UAB, United StatesDepartment of Biotechnology, University of Natural Resources and Life Sci-nces Vienna/Austrian Centre of Industrial Biotechnology (ACIB GmbH), AustriaSchool of Bioengineering, University of Applied Sciences FH Campus Vienna,ustriaDepartment of Chemical Engineering, Autonomous University of Barcelona,pain

The alcohol oxidase (AOX1) promoter of Pichia pastoris is onef the strongest promoters for heterologous gene expression inethylotrophic yeast, allowing the methanol-regulated expres-

ion of the gene of interest. Previous studies have shown thatncreased heterologous gene dosage often leads to the overloadf the secretory pathway and metabolic burden.

The expression of Rhizopus oryzae lipase (Rol) in P. pastoris haseen previously shown to trigger the unfolded protein responseUPR) [1]. To assess that physiological response in P. pastoris iselated to the increased ROL gene dosage, a subset of strains with 1,, 4, 8 and 15 copies was constructed and tested in carbon-limitedhemostat cultures, using a mixed glycerol:methanol substrateeed. Once the system was at steady state a transcriptomic analysisas performed using DNA microarrays.

The macroscopic physiological parameters revealed that growthield and carbon uptake rate are gene dosage dependent, and theighest productivity was found at 2 copies of ROL. These resultsere supported by the transcriptomic data, showing a correlation
etween the regulation of central carbon metabolism and the geneopy number. The number of regulated genes increased with geneosage. In addition to carbon metabolism, transcriptional changes

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YMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION

n other cellular processes such as peroxisome biogenesis and stressesponses involving the UPR were observed.

eference

].Resina D, Bollók M, Khatri NK, Valero F, Neubauer P, Ferrer P. Trans-criptional response of Pichia pastoris in fed-batch cultivations toRhizopus oryzae lipase production reveals UPR induction. Microb CellFact 2007;6:21.


15-7

nhanced membrane protein expression by engineeringncreased intracellular membrane production

atrien Claes1,2,∗ , Mouna Guerfal 1,2, Nico Callewaert1,2

Unit for Medical Biotechnology, Inflammation Research Center (IRC), VIB,elgiumLaboratory for Protein Biochemistry and Biomolecular Engineering, Depart-ent of Biochemistry and Microbiology, Ghent University, Belgium

Membrane protein research is frequently hampered by low nat-ral abundance of these proteins in cells and typically requiresecombinant expression. Different expression systems have been
sed to date, but only little research is directed towards the specificustomization of the host cell itself.
We hypothesized that increasing the intracellular membraneontent would increase the membrane area available to accom-

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odate recombinant membrane proteins. Hereto, we inactivatedhe phosphatidic acid phosphatase gene, PAH1, which codes forkey enzymatic and regulatory factor in lipid biosynthesis. The

esult is a shift of lipid metabolism away from triacylglycerol- andterylester-storage towards membrane phospholipid synthesis. Wehose Yarrowia lipolytica as a test organism, as this yeast prefer-ntially grows on fatty acids and a compatible strong induciblerotein expression system is available.

Electron microscopic imaging of the knock-out revealed strongembrane proliferation upon growth on oleic acid, without any

igns of ER-phagy. Guided by this, we analyzed membrane proteinxpression in the PAH1 knock out strain of Yarrowia lipolytica.nalysis of eight representatives of different integral membranerotein families showed enhanced protein accumulation levels forll of them and in some cases also reduced proteolysis. Compli-entary to this improvement, UPR co-induction further enhances

he quality of the membrane proteins in terms of proper foldingnd biological activity.

These results allow to conclude that re-routing of lipidetabolism is a valid strategy to increase membrane protein pro-

uction and quality. As this pathway is conserved in eukaryotes,imilar strategies can be explored in other frequently used expres-


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ymposium 16: Bioprocessing

16-1

acterial enzymes for lignin degradation: production ofromatic chemicals from lignocellulose

imothy D.H. Bugg

University of Warwick, Department of Chemistry, Coventry CV4 7AL, UK

The lignin content of lignocellulose and lignin-containingastes represents a possible resource for production of aromatic

hemicals, if efficient biocatalytic routes for lignin degradationan be found. The enzymology of fungal lignin degradation isell studied, but the enzymology of bacterial lignin degradation

s much less well known. In Rhodococcus jostii RHA1 we have iden-ified a dyp-type peroxidase DypB as a lignin peroxidase that isctivated by Mn2+, and shows activity with a �-aryl ether ligninodel compound, with Kraft lignin, and with lignocellulose.sing a colorimetric assay as a screen, we have also identifiednumber of novel lignin-degrading bacteria from soil samples,hich show higher activity for lignin degradation, including three

trains of Microbacterium and a thermotolerant strain of Sphingob-cterium, in which strains we are currently investigating furthernzymes for lignin degradation. Deletion of the gene encodinganillin dehydrogenase in Rhodococcus jostii RHA gives a mutanttrain which, when grown on minimal media containing wheattraw lignocellulose, accumulates up to 96 mg/L of vanillin, a highalue chemical, highlighting the potential for pathway engineer-ng to be used for conversion of lignin into renewable aromatichemicals [1–4].

eferences

].Ahmad M, Taylor CR, Pink D, Burton K, Eastwood D, Bending GD,et al. Mol Biosyst 2010;6:815–21.

].Ahmad M, Roberts JN, Hardiman EM, Singh R, Eltis LD, Bugg TDH.Biochemistry 2011;50:5096–107.

].Taylor CR, Hardiman EM, Ahmad M, Sainsbury P, Norris PR, BuggTDH. J Appl Microbiol 2012;113:521–30.

].Sainsbury PD, Hardiman EM, Ahmad M, Otani H, Seghezzi N, EltisLD, et al. ACS Chem Biol 2013;8:2151–6.


16-2

uantitative single cell analysis of isolated microbes inontrolled microenvironments

hristian Dusny ∗ , Frederik Fritzsch, Katrin Rosenthal, Andreaschmid ∗

TU Dortmund University, Laboratory of Chemical Biotechnology, 44227 Dort-und, Germany

Single cell analysis (SCA) has been recognized as the key tech-ology for an unbiased disclosure of cellular functionality. In
ontrast to conventional bulk analysis strategies, SCA grants accesso mechanistic data of individual cells–the minimal functionalnit of cell based bioprocesses. These data are usually maskedehind an average value of a clonal, but heterogeneous popula-
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SYMPOSIUM 16: BIOPROCESSING

ion. It was shown that the extent of heterogeneity is stronglyinked to frequency and amplitude of physicochemical changesn the extracellular environment. Hence not only the identifica-ion of individual physiological phenotypes, but also the abilityo analyze single cells in a controlled and steady environment is aundamental requirement for the accurate description of cellulareatures. We tackle the challenge of creating controlled physico-hemical microenvironments for a precise analysis of individualellular physiology with the Envirostat single cell analysis sys-em [1,2]. In contrast to most other microfluidic systems for SCA,hich mostly rely on the mechanical trapping of cells, the Envi-

ostat uses contactless retention via negative dielectrophoresis,hich excludes cell-surface interaction and resultant changes in

he cellular phenotype. Furthermore, the immediate removal ofotentially inhibiting metabolites and unlimited availability ofutrients and dissolved oxygen is guaranteed by a continuousedium flow. CFD simulations indicate that the microenviron-ent of the target cell has indeed a virtually static composition

1]. We experimentally validated the Envirostat principle with sin-le cell growth studies [3]. We observed significant differencesetween specific growth rates of single cells and populations inulk cultivations. Two yeast species and one bacterial strain con-istently exhibited increased specific growth rates of up to 120%hen cultivated with the Envirostat system [3]. Our results imply

hat the extracellular environment can dictate the specific growthate of unicellular microbial eukaryotes and prokaryotes and aonstant extracellular environment diminishes the influence ofhysiological cell-to-cell differences. Moreover, our experimentsemonstrate the Envirostat as a platform for systems biology thatay be used for disclosing the impact of controlled perturbations

n cellular physiology unbiased by population activity.

eferences

].Kortmann H, et al. Lab Chip 2009;9:576.].Fritzsch F, et al. Lab Chip 2013;13:397.].Dusny C, et al. Appl Environ Microbiol 2012;78:7132.


16-3

ontinuous precipitation of recombinant antibodiesrom CHO cell culture supernatant by calcium-hosphate flocculation and cold ethanol precipitation

ikolaus Hammerschmidt1,∗ , Anne Tscheließnig2, Bernhard Helk3,lois Jungbauer2

Centre of Industrial Biotechnology (ACIB), AustriaUniversity of Natural Resources and Life Sciences Vienna, AustriaNovartis Pharma, Austria

We have developed two precipitation steps that could replacehe costly protein A affinity chromatography as capture step in theurification of recombinant antibodies, relying solely on cheapineral salts (CaCl2) and organic solvents (ethanol). Both steps

an be performed in continuous mode using tubular reactors with-ut changes in performance compared to batch mode. The startupime until steady state conditions were reached was very short andoth reactors were operated for several hours at steady state with-


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ut manual intervention, delivering antibody at constant yieldnd purity. An overall yield of >90%, a host cell protein reduc-ion to 9000 ppm and a DNA reduction to 7 ppm (for a titer of.7 g/L) could be achieved for the antibody investigated. The coldthanol precipitation step can be used for concentrating the anti-ody to >45 g/L, thereby making a subsequent concentration stepnnecessary. Furthermore, the antibody can be readily dissolved

n a variety of buffers of high and low ionic strength optimized forhe next purification step. Cell culture supernatants with high anti-ody titer can be processed with constant tubular reactor size andithout changing any parameters or increasing precipitant con-

umption. Continuous precipitation allows the use of disposableeactors, allowing flexible operation.


16-4

odelling of mixing and microbial growth in bubble col-mn bioreactors using computational fluid dynamics

ale McClure ∗ , John Kavanagh, David Fletcher, Geoffrey Barton

The University of Sydney, Australia

Bubble columns are widely used in the bio-processing indus-ry for large-scale aerobic fermentations. In order to maximisehe yield of many bioprocesses, it is desirable to achieve a homo-eneous distribution of substrate; a task of some complexity athe industrial scale. Computational Fluid Dynamics (CFD) offers aromising approach as a tool to model such processes, as it enablesow patterns inside the reactor to be readily visualised, somethinghich is very difficult to achieve experimentally. A further advan-

age of a CFD model is that it allows different designs and operatingonditions to be evaluated in silico, minimising the need for costlynd time-consuming experimentation. By including growth kinet-cs into the CFD model, it becomes possible to quantify the effectf poor mixing on the overall yield of the process. As part of a long-erm industrial collaboration we have developed and validatedCFD model of the complex and time-varying hydrodynamics

including mixing) inside bubble columns operating at industri-lly relevant superficial velocities. Here, we extend this model byncluding the growth kinetics for two processes of industrial inter-st; namely the production of baker’s yeast and the production ofrecombinant protein using Escherichia coli. The impact of sub-

trate gradients on the yield of each process is examined, as wells the potential for employing CFD as a tool to model industrial
io-processes.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1754cddphmccct



16-5

ioprocess strategies for production of xylanase on agro-esidual products with Aureobasidium pullulans

irma Yegin1,∗ , Sayit Sargin2, Yekta Goksungur1

Ege University, Food Engineering Department, TurkeyEge University, Bioengineering Department, Turkey

Xylanases are mainly used in pulp and paper industry andecently found widespread applications in food and feed indus-ries. They are also used in waste clarification processes andioethanol production.

The aim of this study was to enhance production of xylanaseith A. pullulans by evaluating the effects of different fermen-

ation parameters. Among the A. pullulans strains tested, NRRL2311-1 provided the highest activity on both xylose and xylanrown cultures. Maximum activity was observed after 96 h of cul-ivation at 28 ◦C. The optimized initial medium pH and shakingpeed for cultures grown on xylan were 3.0 and 200 rpm, respec-ively. After elucidating the effects of fermentation parameters onylanase production in synthetic medium, the potential of sev-ral agro-residual products were examined for the production ofylanase. The highest activity was obtained when wheat bran wastilized. Further optimization studies were performed by responseurface methodology for cultures grown on wheat bran. By fit-ing the experimental data to second-order polynomial equation,he optimum levels of initial medium pH (4.2), temperature (30.3)nd incubation time (126.7 hours) were determined. The maxi-um xylanase activity at optimum process conditions reached to

51.95 ± 28.70 U/ml. The model obtained satisfactorily explainedhe effect of process variables on enzyme production.

Acknowledgements: This study was supported by TUBITAK-OVAG 112O521 and COST TD1203.


16-6

ioconversion of lignocellulosic hydrolysates: strategieso overcome the inhibitory effects at high gravity pro-esses

harilaos Xiros ∗ , Lisbeth Olsson

Chalmers University of Technology, Sweeden

High-gravity (HG) technology aims at generating final ethanoloncentrations above 50 kg m−3 in order to reduce the cost of theistillation step. The generation of higher amounts of inhibitorsuring the pretreatment step is one of the challenges that accom-any the increase in initial dry matter. Detoxification of spruceydrolysate, adaptation of the cells before fermentation, supple-entation with nutrients, and washing of solids were the strategies

ompared in this study. They represent different approaches toope with the inhibitory effects, and we compared their efficien-
ies using a thermotolerant strain of Saccharomyces cerevisiae atemperatures from 30 ◦C up to 40 ◦C.

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ater-insoluble solids) when no improvement method waspplied. In HG simultaneous saccharification and fermentationt 30 ◦C combined with a 24 h pre-hydrolysis step, the detoxifi-
ation of pretreated spruce with reducing agent (Na2S2O4) gavehe best result with an ethanol yield of 57% (on total sugars)f the maximum theoretical and a volumetric productivity of.58 g dm−3 h−1. In HG separate hydrolysis and fermentation,
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SYMPOSIUM 16: BIOPROCESSING

utrients supplementation gave better final ethanol yields thanetoxification of the material, reachingan ethanol yield of about0% of the theoretical (on total sugars). The results obtained,howed an increase in severity of inhibitory effects with tem-erature increase. Improved cell viability was observed whenetoxified material was used and also when yeast extract addition
as coupled with adaptation of the cells to the hydrolysate.


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YMPOSIUM 21: BIODEGRADATION AND BIOMEDIATION

ymposium 21: Biodegradation and bioreme-iation

21-1

ioremediation for resource recovery

ouise Horsfall


In order to move towards a sustainable and circular economye need to start viewing waste differently. Through biotechnol-gy research we have the potential to use waste as a feedstockather than it being an end product. A limiting factor in usingaste in this way is the presence of minor amounts of metal

ontaminants, which inhibit bioprocesses and kill bioremediat-ng microorganisms. There are, however, bacteria that are toleranto high concentrations of metal ions due to a resistance mecha-ism that involves metal reduction and nanoparticle formation.e are investigating this pathway to enable the bioremediation ofaste, water and land; employing the techniques, tools and prin-

iples provided by synthetic biology to increase the value of theetal recovered. To achieve this we are taking a modular approach

o identify and optimise genetic elements that have transferableetal ion use, with an aim to control production, size, shape

nd homogeneity, tailoring the nanoparticles to their ascribedse. Our particular case studies under investigation are platinumroup metals, nickel, copper and arsenic. However, since we arettempting to develop a flexible process, there is huge potential toransfer the knowledge gained in our biomineralisation studies tother metal waste streams.


21-2

eduction of hexavalent chromium using combinationf nanoscale zero-valent iron and biological treatmentn situ

omás Cajthaml1,∗ , Jan Nemecek2, Petr Pokorny2, Lenka Lacinová3

iroslav Cerník3, ZuzanaMasopustová3, Ondrej Lhotsky 4

Institute of Microbiology v.v.i., ASCR, Czech RepublicENACON s.r.o, Czech RepublicTechnical University of Liberec, Czech RepublicDEKONTA a.s., Czech Republic

Hexavalent chromium is considered as a priority pollutant dueo its high toxicity and mobility. The aim of this study was to set uppilot-scale in situ remediation experiment in the saturated zonef a historically Cr(VI)-contaminated site. Two geofixation meth-ds were used – chemical reduction of Cr(VI) with commerciallyvailable nanoscale zero-valent iron (nZVI) and consequent bio-ogical reduction of Cr(VI). Combination of the methods resultedn a rapid decrease of Cr(VI) concentrations in the groundwa-
er. The process was monitored using standard chemical analyses,
easurement of redox potential, standard ecotoxicity tests andhospholipid fatty acid analysis (PLFA) supported with 454 pyrose-

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uencing. The result of PLFA revealed that the application of nZVItimulated significantly growth of autochthonous microorganismsndicating general reduction of toxicity of the site in the first phase.he consequent injection of an organic substrate enabled furtherransformation of Cr(VI) below the respective quantification limitven after 9 months of the single organic substrate injection. Theesults of the pilot test indicated a synergic effect of both of thebiotic and biotic phases. nZVI that had been oxidized during thebiotic phase was afterwards partially recovered during the biotichase due to the substantial decrease of the redox potential. The

mmobilized Fe (III) was probably microbially reduced to Fe(II) thatcted further as the reducing agent for Cr(VI) even when microbialensity was already low due to depletion of the organic substrate.


21-3

solation of PAH dwelling Penicillium for application inioremediation processes

lisabet Aranda1,∗ , Patricia Godoy1, Rocio Reina1, Marina Badia-abregat2, Mónica Rosell 3, Regina Michela Wittich1, Ernest Marco-rrea2, Inmaculada García-Romera1

Spanish National Research Council (CSIC), SpainUniversitat Autònoma de Barcelona, SpainUniversitat de Barcelona, Spain

Fungi represent the living dominant biomass in soils and arebundant in aqueous systems. In addition, they possess a highotential for degrading environmental organic chemicals. The aimf this study is to find polycyclic aromatic hydrocarbon (PAH)egrading fungi, which are adapted to polluted environments,sing culturing-based techniques. In this study, a total of 12 fun-al cultivable species have been isolated from a PAH contaminatedond. The isolated fungi were genetically identified by amplifying,loning and further sequencing fragments corresponding to theTS1-5.8S-ITS2 (internal transcribed spacer ITS) region of each cul-ivable fungal strain. We tested their ability to convert anthracene,n time courses of 42 days. Among the 12 screened fungal species,enicillium oxalicum showed remarkable conversion ability, degrad-ng 100 �M in 5 days in a rich carbon source medium. The use ofdefined mineral medium with 13C-labelled anthracene showed

hat P. oxalicum can cometabolize anthracene, leading to the for-ation of anthraquinone, anthrone and hydroxyderivatives, as

evealed by nuclear magnetic resonance (NMR) analysis. The lastetabolites could indicate the further ring cleavage of anthracene

hat could be mediated by hydrolase or dioxygenase enzymes. Theonversion level of anthracene was reduced to 50% in presence ofn inhibitor of cytochrome P450 monooxigenase, suggesting itsarticipation in the first oxidation step. Our results show the highffectiveness in PAHs conversion by the isolated fungi P. oxalicumy cometabolic strategies, indicating its potential application in


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21-4

otential of ectomycorrhizal fungus Pisolithus tincto-ius to tolerate and to degrade trifluoroacetate intouoroform

aula Castro1,∗ , Albina Franco2, Miguel Ramos2, Sara Cravo3, Car-os Afonso3

CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado,scola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto,ortugalCBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado,scola Superior de Biotecnologia, Universidade Católica Portuguesa, Portugal2CEQUIMED-UP, Laboratório de Química Orgânica e Farmacêutica, Centro

nterdisciplinar de Investigacão Marinha e Ambiental (CIIMAR/CIMAR), Depar-amento Ciências Químicas, Faculdade de Farmácia, Universidade do Porto,ortugal

Trifluoroacetate (TFA) is a persistent fluorinated organic com-ound originated from the degradation of fluorinated compounds,uch as HCFC and isoflurane, or as a side product from the ther-olysis of fluoropolymers, like Teflon. TFA can reach soil through

recipitation, where it persists in water and soil, and may con-ribute to forest decline. In this study, we assessed the capacityf P. tinctorius, an ectomycorrhizal fungus (ECMF), to toleratend/or degrade TFA. In vitro studies in glucose-supplemented solidedium showed that the fungus tolerated up to 8.77 mM TFA. P.

inctorius also degraded 88.3%, 89.9%, and 42.1% of 0.88, 2.39,nd 4.39 mM TFA, respectively, in liquid cultures. No TFA accu-ulation was detected on the fungus mycelium, suggesting that

FA depletion was due to fungal degradation. Defluorination wasot detected. A volatile compound with a structure and behav-

or compatible with fluoroform (CHF3), a potent greenhouse gas,as detected using GC-MS/MS, only in the gas phase of sealed P.

inctorius cultures supplied with TFA. Further confirmation of thisompound is needed. Nevertheless, the study shows that P. tincto-ius was capable to degrade TFA possibly through a similar pathwayo that found on marine sediments. The results evidence the rolef ECMF may play in the degradation of fluorinated organic com-ounds, enhancing their potential contribution on establishingree growth in soils exposed to organic contamination.

Acknowledgements: A. Franco thanks FCT the grantFRH/BD/47722/2008. This work was supported by FCT Project
PTDC-AGR-CFL-111583-2009 and PEst-OE/EQB/LA0016/2011.

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SYMPOSIUM 21: BIODEGRADATION AND BIOMEDIATION

21-5

valuation of the biological sulfamethoxazole degrada-ion mechanism for biotechnological applications

enjamin Ricken1,∗ , Markus Lenz1, Danuta Cichocka1, Hans-Peterohler2, Boris Kolvenbach1, Philippe Francois-Xavier Corvini 1

Institute for Ecopreneurship/University of Applied Sciences and Art North-estern Switzerland, SwitzerlandEawag, Swiss Federal Institute of Aquatic Science and Technology, Depart-ent of Environmental Microbiology, Switzerland

Sulfonamide antibiotics are of rising concern as their releasento the environment has been suspected to enhance the for-

ation of resistant pathogenic bacterial strains [1]. Amongulfonamides, antibiotics such as sulfamethoxazole (SMX) arerousing public interest since they are photo- and thermostable asell as recalcitrant to traditional, biological waste water treatmentrocesses [2].

Our studies focused on SMX as it is the most used sulfonamidentibiotic in human medicine. Even though extensive research haseen done on SMX degradation by bacterial consortia or isolates,he complete pathway is not yet understood, let alone the proteinsnvolved.

We were able to identify the first crucial degradation step ofeveral sulfonamide antibiotics [3] by means of isolating Microbac-erium sp. strain BR1, proven to partially mineralize SMX forhe first time [4]. Concerning the pathway, molecular oxygen isncorporated into the sulfonamide antibiotic at its aniline moi-ty through ipso-substitution, which is an uncommon process foriodegradation. This leads to the release of the verified metabolites:-amino-5-methylisoxazole, p-aminophenol and sulfite.

Further investigations are currently under way in order to iden-ify the involved protein and its cofactor dependencies.

To conclude, it is postulated that there is one common sul-onamide degradation mechanism among various bacteria [3]. Itslucidation and the determination of involved proteins are essen-ial for upcoming biological sulfonamide removal approaches.

eferences

].Hruska, Franek. Vet Med 2012;2012:1–35.].Gros, et al. Environ Int 2010;36:15–26.].Ricken, et al. Appl Environ Microbiol 2013;79:5550–8.
].Bouju, et al. Appl Environ Microbiol 2012;78:277–9.


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EDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEWVACCINES A

ednesday 16 July

ymposium 17: Development of new vaccinesnd antimicrobials

17-1

nsect cell technology as a vaccine producing platform

aula Marques Alves

IBET/ITQB-UNL, Apartado 12, Oeiras, Portugal

The insect cell-baculovirus system is today a well-accepted uni-ersal manufacturing platform as demonstrated by the number ofpproved veterinary and human vaccines. The key advantage ofhis platform is that a universal “plug and play” process may besed for producing a broad range of biologicals while offering theotential for low manufacturing costs. Its major downside residesn the quality and quantity of the generated product, two criticalariables that seldom pose problems to the translation of the pro-uction process from lab to industrial scale. Aiming at closing thisap, our group has been focusing on (1) the application of systemsiology tools and (2) the development of stable insect cell lines toetter understand and optimize this producing platform.

Taking advantage of recent advances in systems biology toolsor insect cells we have conducted a metabolomics study with thewo most used cell lines, Sf9 and Hi5 cells, with the objective ofne-tuning the insect cell-baculovirus system for production ofnveloped virus-like particles (VLPs) and retrovirus like particlesunpublished data). Results demonstrate that although Sf9 cellsave improved growth and metabolic efficiency, Hi5 cells wereetter recombinant protein producers for all tested targets withroductivities 3- to 4-fold higher. In addition, data points outowards the potential of media manipulation strategies as a way toeinforce specific cellular pathways associated with higher produc-ivity and thus optimize the production of complex biologicals.

In parallel, we have been developing stable insect cell linessing targeted approaches based on the flp recombinase-mediatedassette exchange (RMCE) system. Sf9 and Hi5 cells populationsfter flp-mediated cassette exchange were compared for complexroteins expression, including influenza VLPs. Specific proteinroductivities in Hi5 cells were markedly higher than in Sf9 cellsor all proteins tested. Noteworthy, the metabolic efficiency off9 cells allowed to extend the production phase and to deliverimilar protein titers at the end of the process. Overall, a compar-son of Sf9 and Hi5 cells expression capabilities with our flexible
lug-and-play Flp-RMCE platform will be discussed.

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ANTIMICROBIALS New Biotechnology · Volume 31S · July 2014

17-2

ngineering of factor H binding protein, a key vaccinentigen for the prevention of meningococcal disease

ayley Lavender ∗ , Susan Lea, Christoph Tang

University of Oxford, United Kingdom

Neisseria meningitidis is a leading cause of bacterial sepsis andeningitis in children. There are effective vaccines available to

revent meningococcal disease caused by strains expressing cer-ain polysaccharide capsules. However, capsule-based approachesannot be used against serogroup B N. meningitidis because its cap-ule is identical to a molecule found in the developing humanrain. Therefore there have been intense efforts to identify proteinntigens that provide protective immunity against this importantuman pathogen.

Meningococcal factor H binding protein (fHbp) is a surfaceipoprotein that elicits serum bactericidal responses in mice anduman volunteers. This antigen is a key component of Bexsero, a

icensed vaccine for the prevention of meningococcal disease.There is considerable sequence variation of fHbp among strains

f N. meningitidis, affecting predicted coverage of fHbp-based vac-ines. Also fHbp binds the human complement regulator factor HfH); there is evidence that fH binding impairs immunogenicity ofHbp. Finally, certain fHbps display inherent instability, makinghem unsuitable as vaccine antigens.

Based on the structure of the fHbp:fH complex and muta-enesis studies, we have identified non-functional fHbps for allhree variant groups, and generated stable versions of the proteins.dditionally, we found that a homologue of fHbp in Neisseria gon-rrhoeae fails to bind fH. Given the specificity of fHbp for humanH, the vaccine candidacy of these antigens has been evaluated intransgenic mouse model.

In conclusion, we have employed structure-based design toodify and characterise novel fHbps as antigens in next gener-

tion vaccines against meningococcal disease.


17-3

ntimicrobial properties of sophorolipids produced byandida Bombicola ATCC 22214 against gram positivend Gram-negative bacteria

ayri Alejandra Diaz De Rienzo1,∗ , Ben Dolman1, Fernandouzman1, Candice Kaisermann1, James Winterburn1, Ibrahim M.

anat2, Peter Martin1

University of Manchester, United KingdomUniversity of Ulster, United Kingdom

Biosurfactants are amphipathic, surface-active molecules oficrobial origin which accumulate at interfaces, decreasing sur-

ace and interfacial tensions and forming aggregated micellular
tructures in solution. Some are reported to have antimicro-ial properties and anti-adhesive/disruption abilities on biofilmormation. Sophorose lipids are glycolipids Biosurfactant con-

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ew Biotechnology · Volume 31S · July 2014 WEDNESDAY 16 JU

isting of a dimer sugar, sophorose and a long-chain fatty acidhat are produced by yeasts belonging to the Candida genus.ntimicrobial properties and the ability to disrupt biofilms usingophorolipids produced by Candida bombicola ATCC 22214 duringfed-batch fermentation, using glucose 100 g/L as carbon source

nd glucose/rapeseed oil as feed at 47 and 120 h and sodiumodecyl sulfate (SDS) at different concentrations were investi-ated. Growth of Ralstonia eutropha ATCC 17699 was inhibitedy sophorolipids and SDS at concentrations > 1% (v/v) and therowth of B. subtilis BBK006 was inhibited by sophorolipids andDS at concentrations > 0.5%, v/v. Sophorolipids were able to dis-upt biofilm formation (at concentrations higher than 1%, v/v)nd the effect was evidenced through fluorescence microscopy. Its concluded that sophorolipids are promising compounds for thenhibition/disruption of biofilms formation by Gram-positive andram-negative microorganisms which could be enhanced by theresence of a combination of biosurfactants.


17-4

ailoring Streptomyces: producing novel minor grooveinder antibiotics

milio Cortes Sanchez1,∗ , Sara De Ornellas2, John May2, Glennurley2, Paul Hoskisson3

University of Strathclyde, United KingdomUniversity of Strathclyde, Chemistry, United KingdomUniversity of Strathclyde, SIPBS, United Kingdom

The rise of antibiotic resistant strains and the decline of antibi-tic discovery have resulted in an urgent need to develop andiscover novel antimicrobials. Minor groove binders (MGBs) areompounds that shown antibiotic, anticancer and antiviral activ-ty by binding to the minor groove of the DNA helix.

MGBs are naturally produced by Streptomyces, and recentlyome of the clusters encoding for those antibiotics have been char-cterised. In order to produce a new drug, we are using syntheticiology and combinatorial biosynthesis to modify a pathway of atreptomyces strain, forcing the assembly of a new antibiotic.

For this we transformed several Streptomyces strains with anGB producing biosynthetic cluster containing four independent

on-ribosomal peptide synthetases. These strains were fermentednd sampled at different time points and the organic extractionsere analysed looking for the production of novel compounds.he HPLC, LCMS and HRMS data suggest the assembly of a novelompound, which exhibits antibiotic activity according to ourioassays. Further characterisation of the molecule will be done byMR .We have also cloned one of the non-ribosomal peptide syn-

hetases in order to engineer this enzyme to broaden its substrate
pecificity.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1765p

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YMPOSIUM 17: DEVELOPMENT OF NEWVACCINES AND ANTIMICROBIALS

17-5

rogesterone biosynthesis by combined action of adrenalteroidogenic and mycobacterial enzymes in fast grow-ng mycobacteria

icolai Strizhov1,∗ , Victoria Fokina1, Galina Sukhodolskaya1,mitry Dovbnya1, Mikhail Karpov1, Andrey Shutov1, Ludmilaovikova2, Marina Donova1

Institute of Biochemistry & Physiology of Microorganisms, Russian Academyf Sciences, RussiaBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow Stateniversity, Russia

We have for the first time demonstrated the use of mycobac-eria as a harbor for synthesis of C21-steroids by heterologousxpression of mammalian steroidogenesis key proteins. Proges-erone biosynthesis was successfully reconstructed in recombinant

ycobacterial cells. The first stage of mammalian steroidogen-sis, cholesterol conversion to pregnenolone is carried out byeterologously expressed cytochrome P450scc cleaving the sub-trate side chain. cDNA copies of the CYP11A, Adx and AdR genesncoding mature forms of cytochrome P450 cholesterol hydrox-lase/20, 22-lyase (P450scc), adrenodoxin (Adx) and adrenodoxineductase (AdR) from bovine adrenal cortex were transferred withNS11 construct to Mycobacterium smegmatis mc2155. Acetamidehemoinduction resulted in high expression level of all threeroteins. Pregnenolone oxidation to progesterone in the subse-uent step is mediated by innate mycobacterial 3�-hydroxysteroidehydrogenase (3�-HSD). However, no progesterone synthesisas detected in mycobacteria with pNS10 construct expressing450scc alone without Adx and AdR redox partners. We concludedhat bacterial ferredoxins and ferredoxin reductases ubiquitouslyresent in mycobacteria fail to deliver electrons to P450scc. Pro-esterone was also produced by recombinant pNS11 mycobacterialells with sitosterol as a substrate for bioconversion though withower yields.

The highest progesterone production level achieved is 25 mg/lt 7.5 mM cholesterol load. This yield is 60 fold higher thanhe maximum pregnenolone concentration we obtained earlierith recombinant Escherichia coli (pBar Triple). The level differenceetween two hosts clearly reveals prospects of the mycobacterialrogesterone biosynthesis for biotechnological purposes. Our find-

ngs pave the way for future exploration of these lipophilic bacteriaor production of valuable active pharmaceutical ingredients.


17-6

NA vaccine expressing ubiquitin-conjugated multi-ragments antigens protects BALB/c mice against Toxo-lasma gondii infection

ua Cong ∗ , Quan Yuan

Shandong University, China

Toxoplasma gondii, an intracellular parasite with a complex lifeycle, is highly prevalent in humans and animals and causes


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EDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEWVACCINES A

oonotic toxoplasmosis. In order to prevent from I infection,n ideal vaccine that can elicit protective cell-mediated immuneesponses is greatly needed. In our study, we selected seven frag-ents derived from seven T. gondii surface antigens (SAG3, ROP18,IC6, GRA7, MAG1, BAG1, SPA), which was linked to ubiquitin

rotein. DNA vaccines encoding 7 fragments and ubiquitin pro-ein were constructed. BALB/c mice were immunized with p-Ub-Tgag, pVAXI, or PBS and challenged with highly virulent T. gondiiH strain or the cyst of T. gondii genotype II strain of PRU. Vaccina-ion with p-Ub and p-Tgag both showed high ability to generate atrong Th1 cell response with significant production of IFN-�, IL-2nd low levels of IL-10, and protected mice against high parasiteurden when challenged with T. gondii compared with PBS andVAX1. Furthermore, the result of vaccination with p-Ub demon-trated that it was more effective than p-Tgag in protection of T.ondii infection. We conclude that the DNA vaccine we constructedn this study is efficacious and in particular, ubiquitin-conjugatedaccines showed a stronger Th1-type immunity and significantesistance to parasite challenge.


17-7

roteome-wide analysis of the functional roles ofacilysin biosynthesis in Bacillus subtilis

ulay Ozcengiz1,∗ , Asli Aras Taskin2, Mustafa Demir1, Ayten Yaz-an Karatas3

Department of Biol. Sci., METU, TurkeyUniversity of Freiburg, GermanyMol. Biol. Gen. Department, Istanbul Technical University, Turkey

Bacilysin, being produced and excreted by certain strains ofacillus subtilis, is a dipeptide antibiotic composed of L-alaninend L-anticapsin. We previously showed that the biosynthesis ofacilysin is positively regulated by quorum sensing regulatory cir-


ANTIMICROBIALS New Biotechnology · Volume 31S · July 2014

uit and sporulation regulator Spo0A while negatively regulated byransition regulators CodY, ScoC and AbrB. In this study, a compar-tive proteomic analysis of the bacilysin producer B. subtilis PY79nd its bacilysin non-producer derivative OGU1 (bacA::lacZ::erm)as performed in order to gain a deeper insight into the func-

ional roles of bacilysin biosynthesis in its producer. 2-DE gellectrophoresis coupled to MALDI-TOF/MS within different pHanges separated more than 1900 protein spots. Of these, 159rotein spots were identified which corresponded to 123 distinctroteins. 60 out of 123 distinct proteins were down-regulated inGU1 strain as compared to the wild-type. Thirty-five of 123 pro-

ein spots were expressed more abundantly while 19 protein spotsere found to be absent and one protein spot was newly induced in

he mutant strain. To avoid any experimental limitations, we alsoerformed 1-DE gel electrophoresis coupled to LC–MS/MS analy-is which led to the identification of 1282 proteins from the totaloluble proteome of PY79 and OGU1. 76 of those proteins wereound to be differentially expressed as deduced from their relativebundance.

The analysis of the results obtained from 2-DE gel electrophore-is coupled to MALDI-TOF/MS and GeLC–MS/MS revealed thempact of the absence of bacilysin biosynthesis on the expres-ion of sporulation, two component-regulatory system, peptide

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ew Biotechnology · Volume 31S · July 2014 SYMPOSIUM 18: EXPL

ymposium 18: Exploitation of metagenomicsor environmental and biocatalytic applica-ions

18-1

etagenomics: mining for novel catalysts

lizaveta Bonch-Osmolovskaya

Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow,ussia

About three decades back the analysis of environmental 16SRNA clone libraries revealed that roughly 95% of microorgan-sms in natural microbial communities had never been obtainedn laboratory cultures, and so their catalytic capacities were notnown. The metagenomic approach, actively developing over theast 10 years, provides access to genes of microorganisms that

ight never be isolated. This became possible as a result fastrogress of the sequencing technologies, leading to their signifi-ant cost reduction. The contemporary state of technology makeseasible efficient sequencing of environmental DNA with multipleoverage, allowing consequent alignment and assemblage of largeenome fragments. Metagenomic libraries became an object forene mining, both for predicting novel metabolic pathways, notnown in cultured prokaryotes, and for searching new catalystsor biotechnology. For the latter purpose, microbial communitiesf extreme environments have been investigated, including hotprings, acidic mines, soda lakes, and polar soils; and new enzymeshighly stable hydrolases, esterases, DNA polymerases with new

apacities have been cloned and expressed. One example of suchork is a current FP7 project “HotZyme – Systematic screening ofovel hydrolases from hot environments”, in which an interna-

ional team covers ground from environmental samples to purifiednd crystallized new proteins with target activities.


18-2

etagenomics unveils bacterial and fungal communi-ies response to mycoremediation of polychlorinatediphenyl-contaminated soil

atiana Stella1,∗ , Stefano Covino1, Monika Cvancarová1, Maurizioetruccioli 2, Alessandro D’Annibale2, Tomas Cajthaml1

Institute of Microbiology AS CR, v.v.i., Czech RepublicTuscia University, Italy

The remediation of sites contaminated by polychlorinatediphenyls (PCBs) is a major priority due to the teratogenic, car-inogenic and endocrine-disrupting features of these xenobiotics.ithin this frame, the present work was aimed at assessing the

echnical feasibility of both bioaugmentation (white-rot fungirpex lacteus and Pleurotus ostreatus) and biostimulation treatments
f three different samples of a historically PCB-contaminated soilbulk soil, topsoil and rhizosphere soil). The highest PCB depletionields (41 and 51%) were observed in P. ostreatus-augmented micro-
nuf

ION OFMETAGENOMICS FOR ENVIRONMENTAL AND BIOCATALYTIC APPLICATIONS

osms with topsoil and rhizosphere soil, respectively. Several PCBegradation intermediates were detected (i.e. chlorobenzoates,ydroxylated and methoxylated PCBs) and the initial acute tox-

city was reduced mainly in the rhizosphere soil. Furthermore,o gain new insights into the biota composition throughout theemediation processes, the diversity and dynamics of both bacte-ial and fungal communities were estimated via high-throughput54-pyrosequencing method. Metagenomic analysis showed thatirmicutes relative abundance increased when the bulk and rhizo-phere soils were treated with P. ostreatus. Conversely, in all soilamples augmented with I. lacteus an initial rise in the relativebundance of the Proteobacteria phylum was observed, whereasacteroidetes predominate in the topsoil and rhizosphere soil athe end of incubation. P. ostreatus was able to compete with theutochthonous fungi, its relative abundance being higher than0% along the whole incubation period. By contrast, the abun-ance of I. lacteus sequences tended to decline during the lastweeks of incubation. In biostimulated microcosms, the largeajority of detected sequences belonged to the phyla Ascomycota

nd Zygomycota.


18-3

ining alginate lyases in sediment metagenomes fromour geographically distant cold coastal environments

ebe Dionisi 1,∗ , Marina Matos1, Luciano Anselmino1, Marianaozada1, Walter Mac Cormack2, Jolynn Carroll 3, Leif Lundgren4,ara Sjöling5, Krystle Chavarría6, Bernard Henrissat7, Janetansson6

Patagonian National Research Center (CENPAT-CONICET), ArgentinaArgentinean Antarctic Institute and National University of Buenos Aires,rgentinaUniversity of Tromsø, Norway and Akvaplan-niva AS, FRAM – High Northesearch Centre for Climate and the Environment, Tromsø, NorwayStockholm University, SwedenSödertörn University, SwedenLawrence Berkeley National Laboratories, USACentre National de la Recherche Scientifique, Marseille, France

Brown macroalgae are considered an attractive option as sus-ainable feedstock for the production of biofuels and commodityhemicals due to their high carbohydrate content. Microbial com-unities from cold coastal environments represent promising

ources of novel enzymes depolymerizing brown algal polysac-harides such as alginates, as these organisms constitute a largerimary biomass in these environments. We used a nested samp-

ing strategy to obtain sediment samples from four high-latitudeoastal environments (Svalbard Archipelago, Norway; Baltic Sea,weden; Ushuaia Bay, Argentina and Potter Cove, Antarctica).wenty-three samples were sequenced using Illumina HiSeqTM

500, assembled and annotated using the IMG/M pipeline. Theomplete assembled metagenome dataset contains 5.6 Gb and.4 × 107 protein coding genes. With the goal of identifying algi-
ate lyase homologs in the metagenomes, we mined this datasetsing both blastp searches and product names assigned in theunctional annotation. We retrieved 2,705 sequences between

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YMPOSIUM 18: EXPLOITATIONOFMETAGENOMICS FOR ENVIRONMENTAL AND BIO

00 and 1,166 amino acids in length, mostly belonging to theAZy polysaccharide lyase families PL17 (30.4%), PL7 (28.2%)r PL6 (22.1%). When normalized with the protein coding geneumbers of the assembled metagenomes, Antarctic samples con-

ained the highest abundance of identified sequences, and Balticea samples contained a larger proportion of novel sequencesKruskal–Wallis test, p < 0.05). Different levels of gene order con-ervation were found among scaffolds containing these genes, andith genomes of isolated alginate-degrading bacteria. This study

evealed a large diversity of alginate lyase homologs from yet-to-beultured marine microorganisms, which could aid in the engineer-ng of microbial platforms for biorefineries.


18-4

esigned sensor cells for direct detection of microbialolonies with target enzyme activities on solid plates

aseong Kim ∗ , Eugene Rha, Bong-Hyun Sung, Seung-Goo Lee

KRIBB, Republic of Korea

Screening of new enzymes from vast genetic resources is indis-ensable for environment-friendly, cost-effective, and sustainablehemical industry. Here is a new method to explore the variety

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YTIC APPLICATIONS New Biotechnology · Volume 31S · July 2014

f microbial colonies on solid plates for the existence of targetnzyme activity using designed sensor cells. When an effector,uch asp-nitrophenol, is released from a microbial colony, it williffuse out to the vicinity surrounding the colony and the sensorells in the vicinity turn on the expression of a fluorescence andn auxotrophic genes. The expression of these reporters enableshe sensor cells to grow rapidly and to display strong fluores-ence signals near the original colonies. So, by simply co-culturinghese sensor cells with any microbes from nature, the sensorells will directly mark the locations of microbial colonies witharget enzyme activity, which is responsible for the buildup of p-itrophenol. We tested this proof-of-concept using Escherichia coliensor cells with a phenol-sensitive promoter, dmpR, to detect Cit-obacter freundii cells expressing tyrosine phenol-lyase. Also, theensor cell was applied to examine diverse microbial cells fromoils, to find a cellulase activity using p-nitrophenyl-cellobiosides a substrate. We isolated seven cellulase producing bacteria anddentified one of them identified as a novel species of Pseudomonas,ased on 16s rRNA/NGS analysis along with morphological, bio-hemical, and physiological properties. Lastly, the sensor cell wassed to isolate new cellulases from metagenome libraries, whichere confirmed to have the hydrolyzing activity and specificity on

ellulose by conventional methods.


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ew Biotechnology · Volume 31S · July 2014 SYM

ymposium 19: Evolutionary strategies forell factory development

19-1

volutionary and reverse metabolic engineering of Sac-haromyces cerevisiae

ack T. Pronk

Department of Biotechnology, Delft University of Technology, Delft, Theetherlands

Laboratory evolution is a powerful, versatile approach formproving and expanding the capabilities of industrial micro-rganisms which, in contrast to targeted genetic modification,oes not require a detailed a priori understanding of the molec-lar basis for the trait of interest. Cultivation in bioreactors offersany options to design and implement culture conditions thataximize the selective advantage of spontaneous mutations that

onfer a specific, industrially relevant trait. I will briefly discusspplications of this ‘evolutionary engineering’ approach to theeast Saccharomyces cerevisiae for optimizing its sugar fermentationinetics and for increasing its robustness to industrially relevanttresses.

Until recently, expression profiling with DNA micro-arraysas the only cost-affordable genome-wide analytical technique

or analysing the molecular basis for improved performance ofeast strains derived from evolutionary engineering experiments.owever, such transcriptome analyses tended to generate largeumbers of targets, often without a clear lead to the responsibleutation(s). Based on recent studies from the Delft yeast group,will illustrate how the advent of whole-genome sequencing has

ransformed the molecular analysis of evolved genotypes. Analysisf multiple, independent evolution experiments and integrationf classical genetics approaches were shown to greatly amplify theower of whole-genome resequencing of evolved strains.


19-2

daptive evolution of saccharomyces cerevisiae to earlytage of an alcoholic fermentation

naMangado ∗ , Pilar Morales, Jordi Tronchoni, Ramon Gonzalez

ICVV/CSIC, Spain

Experimental evolution was used to identify genes involved inhe adaptation to the early stages of wine fermentation. Evolutionxperiments were performed in continuous culture for 150-250enerations, in conditions emulating the initial stages of alcoholicermentation. We performed three independent experimental evo-ution experiments of a haploid laboratory strain BY4741 andne evolution with a haploid strain (not adapted to winemakingrowth conditions) obtained by meiotic segregation of the wine
east EC1118. By the end of the experiments, colonies were pheno-ypically characterized, and strains that showed improved initialrowth rates were selected. We used next generation sequenc-
ase

IUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT

ng techniques in order to find the genetic changes. Four strainsere selected from the evolution of BY4741. The mutations found,ointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the pre-erred evolutionary target under these experimental conditions.sp5p is a multifunctional enzyme able to ubiquitinate target pro-eins participating in different cellular processes, while Bul1p isn Rsp5p substrate adaptor involved in the ubiquitin-dependentnternalization of Gap1p and other plasma membrane permeases.ur results might be related to increased halftime of plasma mem-rane amino acid permeases. Apparently the genetic backgroundf the laboratory strain is conditioning the result. However, weave shown the strength of this approach. The evolution of theaploid segregant of EC1118 was run for 250 generations, threedapted strains were selected. Preliminary bioinformatic analysisighlighted changes in chromosome numbers in mutant strains.e are currently testing these results by karyotyping, qPCR, flow

ytometry and other techniques.


19-3

enome dynamics of the human embryonic kidney 293HEK293) lineage in response to cell biology manipula-ions

organe Boone1,2,∗ , Yao-Cheng Lin3,4, Leander Meuris1,2, Irmaemmens5,6, Nadine Van Roy7, Arne Soete8, Joke Reumers9,atthieu Moisse9,10, Stephane Plaisance11, Radoje Drmanac12,ason Chen12, Frank Speleman7, Diether Lambrechts9,10, Yves Vane Peer3,4,13, Jan Tavernier5,6, Nico Callewaert1,2

Unit for Medical Biotechnology, Inflammation Research Center (IRC), VIB,elgiumLaboratory for Protein Biochemistry and Biomolecular Engineering, Depart-ent of Biochemistry and Microbiology, Ghent University, BelgiumDepartment of Plant Systems Biology, VIB, BelgiumDepartment of Plant Biotechnology and Bioinformatics, Ghent University,elgiumDepartment of Medical Protein Research, VIB, BelgiumDepartment of Biochemistry, Faculty of Medicine and Health Sciences, Ghentniversity, BelgiumCenter for Medical Genetics, Ghent University Hospital (MRB), BelgiumBioinformatics Core Facility, Inflammation Research Center (IRC), VIB, BelgiumLaboratory for Translational Genetics, Department of Oncology, KULeuven,elgium0 Vesalius Research Center, VIB, Belgium1 VIB Bioinformatics Training and Services (BITS), VIB, Belgium2 Complete Genomics, USA3 Genomics Research Institute, University of Pretoria, South Africa

The HEK293 human cell lineage is widely used in cell biol-gy and biotechnology. We used whole genome resequencingethods in six 293 cell lines to study the dynamics of this ane-

ploid genome in response to the cell biology manipulations thatere used to generate common derivatives of 293 cells, such as

ransformation and stable clone generation (293T); suspensionrowth adaptation (293S) and cytotoxic lectin selection to isolate
glycosylation-homogenous clone (293SG). While the chromo-
omal structure of single 293 cells within a culture appears to bextremely diverse, our analysis suggests that standard cell culture


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YMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELO

rocedures (passaging and cell banking) do not affect the ‘average’enome structure and sequence to a great extent. The extraordi-ary chromosomal plasticity of this genome, however, seems to be

he driving adaptive force when cells are put through a bottleneck.his feature underlies a novel application for which we provideroof of concept here: selection of 293 clones surviving stringentelective conditions (eg ricin toxin), followed by whole-genomenalysis of copy number alterations, can effectively pinpoint theenomic region(s) that contain the gene(s) required for adaptationo those selective conditions. Furthermore, up to the level of sen-itivity afforded here (single copy plasmid insertions were easilyetected), these cell lines have no inadvertent virus insertions. Inerms of tools, we optimized a workflow to detect human/vectorenome breakpoints, and enabled visualization of the 293 genomeata both through a user-friendly visualization web page, as wells through the Integrative Genome Browser (IGV) for rich dataining.


19-4

ersatile and stable vectors for efficient gene expressionn Ralstonia eutropha H16

teffen Gruber , Jeremias Hagen, Helmut Schwab, Petra Koefinger ∗

Graz University of Technology, Austria

The gram-negative �-proteobacterium Ralstonia eutropha H16 isrimarily known for polyhydroxybutyrate (PHB) production and

ts ability to grow chemolithoautotrophically by using CO2 and

2 as sole carbon and energy sources. Up to now some basic sys-ems for targeted genetic manipulation of this bacterium werelready established. However, the majority of metabolic engineer-ng and heterologous expression studies conducted so far rely on amall number of suitable expression systems. Particularly the plas-id based expression systems already developed for the use in R.

utropha H16 suffer from high segregational instability and plas-ids loss after a short time of fermentation. In order to develop

fficient and highly stable plasmid expression vectors for the usen R. eutropha H16 a new plasmid design was created includinghe RP4 partitioning system, as well as various promoters andrigins of replication. The application of minireplicons derivedrom broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa forhe construction of expression vectors and the use of numerous,ersatile promoters extend the range of feasible expression levelsonsiderably. Moreover, the implementation of the RP4 partitionequence in plasmid design increased plasmid stability signifi-antly and enables fermentations with marginal plasmid loss ofecombinant R. eutropha H16 for at least 96 hours. The utility ofhe new vector family is demonstrated by providing expression
ata with different model proteins.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1776btsb


NT New Biotechnology · Volume 31S · July 2014

19-5

ovel human kidney epithelial cell line in pharmaceuti-al biotechnology

ukas Fliedl 1,∗ , Matthias Wieser1, Gabriele Manhart1, Matthias. Gerstl 1, Florian Kast1, Abdulhameed Khan2, Renate Kunert2,ohannes Grillari 2, Regina Grillari-Voglauer2

ACIB, AustriaDepartment of Biotechnology, University of Natural Resources and Life Sci-nces Vienna, Austria

Mammalian cells are used as model systems, products them-elves and as producers of recombinant proteins and vaccines.n these different applications a variety of different cell linesre used and although all have proven valuable for their spe-ific application, there is still room for improvement in terms ofosttranslational modifications. Especially novel human cell linesre of ever increasing importance since they ideally represent then vivo situation and might produce high quality biopharmaceu-icals as similar to endogenous proteins as possible.

Therefore we established a novel human continuously grow-ng renal proximal tubular epithelial cell line (RPTEC) that has

aintained many differentiated characteristics of the normal non-ransduced counterpart and tested its performance in the differentelds of pharmaceutical biotechnology.

A complex model protein, erythropoietin, was stably producedn this cell line and the quality of the recombinant protein wasompared to CHO derived product by analysis of isoforms as wells specific non-human glycopatterns. Additionally, we used ourell line to produce influenza virus and proved high capabilities ofur cell line in this application.

Finally, we used our cell line to get insights into nephrotoxi-ity induced by cisplatin, which has important implications as ahemotherapeutic drug and hypothesize that especially epithelialarrier formation and polarity of RPTECs need to be considered inoxicity models to validly predict the in vivo situation.

Therefore, the here established kidney epithelial cells combinepplicability in various fields of pharmaceutical biotechnology, ashey are capable of production as well as of pre-clinical testing ofiopharmaceuticals.


19-6

e novo production of geranic acid with Pseudomonasutida

ens Schrader ∗ , Jia Mi, Daniela Becher, Patrice Lubuta, Markusuchhaupt, Dirk Holtmann

DECHEMA Research Institute, Germany

Production of plant terpenes by engineered microbes hasecome a prime example of applied synthetic biology with
remendous progress being made during the last decade. Whereasesquiterpene titers reported have already reached g/L values in theioreactor, efficient monoterpene production seems to be more

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ew Biotechnology · Volume 31S · July 2014 SYM

ifficult with conventional host strains due to product toxicity.ence, we set out to investigate to potential of solvent tolerantseudomonas putida for monoterpene production. P. putida DSM2264 shows a pronounced robustness in the presence of monoter-enes. Recent results revealed that the wildtype strain efficientlyonverts geraniol to geranic acid. The monoterpenoic acid showsnteresting properties as a fungicidal agrochemical and may besed as a natural preservative for foods and cosmetics. Comparinghe growth pattern with E. coli and S. cerevisiae, P. putida revealedseveral times higher tolerance towards geranic acid. Following

he cell factory idea, we functionally expressed plant geraniol syn-hase in P. putida, which led to the production of small amounts ofhe desired geranic acid from glycerol as the C-source indicatingxploitation of terpene precursors derived from the endogenousXP pathway. The cellular precursor supply was improved by

xpression of the mevalonate (MVA) pathway from Myxococcusanthus. With this P. putida strain, a product concentration of ca.30 mg/L was obtained in a fed-batch bioreactor. This is the firstxample of de novo monoterpenoic acid production with an engi-eered microbe. We intend to further improve the product titersy pathway engineering to eventually take advantage of the host’sonoterpene tolerance.


19-7

igh-throughput nL-reactor screening for antimicrobialeptides

teven Schmitt 1,∗ , Manuel Montalban Lopez2, Oscar Kuipers2,ven Panke1, Martin Held1

ETH Zürich, Department of Biosystems Science and Engineering, SwitzerlandUniversity of Groningen, Molecular Genetics Group, Netherlands

The number of multi drug resistant pathogens is constantlyrowing and novel antibiotic substances are desperately needed inrder to at least maintain the status quo. Ribosomally synthesizedntimicrobial peptides are not yet exploited for human applica-

IUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT

ions despite an indisputable potential. While peptide engineeringrotocols allow for the generation of thousands of novel, putativective antimicrobial peptides at ease, the assessment of their activ-ty is a rather laborious procedure limiting the assay throughputo 102 variants per day.

We will present a platform based on nL-sized reaction vesselsnL-reactors) that are used for peptide production and activity-creening in a single step and at rates of 105 variants per day.uring screening, library cells are grown to microcolonies withinL-reactors along with a sensor strain serving as a model for aathogen. Library cells secreting an active antimicrobial peptide,ill deactivate the sensor cells within the encircling nL-reactor.learance of an nL-reactor from the sensor thus indicates the pres-nce of a strain secreting a highly active peptide. We use largearticle flow cytometry and fluorescently labeled cells in order to

solate promising candidates.The power of the technology will be demonstrated by screening

f libraries containing ∼105 peptide variants, generated by site-aturation mutagenesis or synthetic biology approaches based onhe blueprint of natural antimicrobial peptides. We show thathe assay has not only a high throughput but also a high accu-acy, making this technique a valuable contribution for futurepproaches targeting the design of highly active antimicrobial


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YMPOSIUM 20: SYSTEMS BIOCATALYSIS

ymposium 20: Systems biocatalysis

20-1

ombination of the two ‘worlds’ chemo- and biocatalysisowards multi-step one-pot processes

arald Gröger

Faculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld,ermany

Multi-step one-pot processes represent an attractive syntheticoncept for the improvement of overall process efficiency byecreasing the required number of work up and purification steps.y avoiding such time-, capacity- and solvent-intensive processteps, multi-step one-pot syntheses contribute to a significantlymproved process economy as well as to more sustainable syn-hetic routes. A key criterion for multi-step one-pot processes ishe compatibility of the individual reaction steps with each other.ccordingly, most of today’ known multi-step one-pot processesre based on either chemocatalytic multi-step reactions or ‘pure’iotechnological processes such as, for example, fermentation. Inontrast, successful combinations of chemo- and biocatalytic reac-ions, in particular in aqueous reaction media, are much less widelynown.

In this contribution strategies for the combination of chemo-nd biocatalysts towards the development of multi-step one-ot processes in aqueous reaction media are presented.ince palladium-catalyzed cross-coupling reactions are of partic-lar importance in the field of metal catalysis, as enzymaticeductions are in the field of biocatalysis, we were interested inhe investigation of the compatibility of these types of reactionsith each other in water. As an example for such a one-pot process

he synthesis of chiral biaryl-containing alcohols via Suzuki-ross-coupling reaction and subsequent asymmetric enzymaticeduction is shown [1]. A further example for the combination ofalladium catalysis and a biotransformation is a one-pot processomprising a Wacker oxidation and subsequent enzymatic reduc-ion [2]. Very recently we could also demonstrate the compatibilityf a metal-catalyzed cross-metathesis reaction with a biotransfor-ation [3]. A further research focus is on the combination of

nzyme-compatible organocatalytic reactions with biotransforma-ions towards multi-step one-pot syntheses. It turned out that aeaction mixture resulting from an asymmetric organocatalyticldol reaction is compatible with a direct subsequent enzymaticeduction without the need for a work-up step of the aldol reac-ion [4,5]. In addition, an organocatalytic nitroalkene synthesisas been successfully combined with its subsequent ene reductase-atalyzed asymmetric reduction, leading to the correspondingitroalkane with high enantioselectivity [6].

eferences

].Burda E, Hummel W, Gröger H. Angew Chem 2008;120:9693–6;Angew Chem Int Ed 2008;47:9551–4.

].Schnapperelle I, Hummel W, Gröger H. Chem Eur J 2012;18:1073–6.].Tenbrink K, Seßler M, Schatz J, Gröger H. Adv Synth Catal2011;353:2363–7.

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].Baer K, Kraußer M, Burda E, Hummel W, Berkessel A, Gröger H. AngewChem 2009;121:9519–22;Angew Chem Int Ed 2009;48:9355–8.

].Rulli G, Duangdee N, Baer K, Hummel W, Berkessel A, Gröger H.Angew Chem 2011;123:8092–5;Angew Chem Int Ed 2011;50:7944–7.

].Burda E, Ress T, Winkler T, Giese C, Kostrov X, Huber T, et al. AngewChem 2013;125:9493–6;Angew Chem Int Ed 2013;52:9323–6.


20-2

ngineering artificial metabolisms in vitro

olf-Dieter Fessner

Technische Universität Darmstadt, Institut für Organische Chemie und Bio-hemie, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany

Systems Biocatalysis is a new concept [1] of organizing bestnzymes in vitro to construct novel artificial metabolisms for anfficient, sustainable synthesis of valuable chemical products [2].he strategy merges the synthetic focus of chemistry with theodular design of biological systems, which is similar to Synthetic

iology but can be realized at a far lower level of complexity fromtrue reductionist approach.

A particular advantage of the concept arises from the inherentotential to construct novel biocatalytic reaction systems for thefficient synthesis of non-natural products by using enzymes engi-eered for non-natural substrate promiscuity. Such operations are

ree from material erosion by competing metabolic pathways androm kinetic restrictions by regulating circuits, which are notoriousroblems in cellular production systems (cell factories).

Examples illustrating the technology will be discussed.

eferences

].http://www.cost.eu/domains actions/cmst/actions/CM1303.].Fessner W-D, Walter C. “Artificial metabolisms” for the asymmetricone-pot synthesis of branched-chain saccharides. Angew Chem Int EdEngl 1992;31:614–6.


20-3

xpanding the diversity of diketopiperazines biosynthe-ized by cyclodipeptide synthases

sabelle Jacques ∗ , Jérôme Seguin, Mireille Moutiez, Emmanuelavry, Muriel Gondry, Pascal Belin

CEA, iBiTec-S, Service d’Ingénierie Moléculaire des Protéines (SIMORPO), France

Cyclodipeptides and more complex diketopiperazines (DKPs)re secondary metabolites mostly synthesized by microorganisms.hey are well known for their wide range of noteworthy biological
ctivities including antibacterial, antifungal, antiviral and anti-ancer effects [1]. In the last decade, an important effort has beenade to elucidate the biosynthesis pathways of these compounds.ur group and others characterized five novel pathways [2–4] that

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re dependent on cyclodipeptide synthases (CDPSs) [5,6], enzymeshat catalyze the formation of the DKP scaffold by condensingwo aminoacyl-tRNAs. Herein, I will describe our medium-highhroughput method to characterize 46 putative CDPSs identifiedy bioinformatics. Our work confirms thereby 40 new CDPSs andeveals a large variety of produced cyclodipeptides. This studypens the ways to a future characterization of the CDPS-dependentathways, enabling combinatorial biosynthesis to produce unnat-ral natural DKPs.

eferences

].Borthwick AD. Chem Rev 2012;112:3641–716.].Belin P, et al. Nat Prod Rep 2012;29:961–79.].Giessen TW, et al. Chem Biol 2013;20:828–38.].Giessen TW, et al. Biochemistry 2013;52:4274–83.].Gondry M, et al. Nat Chem Biol 2009;5:414–20.].Seguin J, et al. Chem Biol 2011;18:1362–8.


20-4

mproving the performance of coupled race-ase/acylase systems: new structural insights and

ovel tools for high-throughput screening

uiomar Sanchez Carron1,∗ , Dominic Campopiano1, Toni Fleming2

University of Edinburgh, United KingdomDr. Reddys Laboratories, India

A coupled enzymatic dynamic kinetic resolution (DKR) sys-em to allow the preparation of synthetically useful amino acidsas been developed and improved. We combined a previouslyngineered N-acetyl amino acid racemase (NAAAR G291D/F323Y)ith an L- or D-specific acylase and used them in a preparative

cale DKR to generate enantiomerically pure amino acids. In thisork we describe the latest efforts to expand the synthetic utilityf this process. We present two novel spectrophotometric assayso measure NAAAR activity towards different substrates, replac-ng the existing HPLC and In vivo selection assays. One of theminks the NAAAR/acylase couple to an L-amino acid oxidase anderoxidase. The other uses a D-amino acid dehydrogenase withroad substrate specificity. These assays allow us to identify novelAAAR substrates and facilitate the high-throughput screening ofAAAR saturation mutagenesis libraries. X-ray structural analysisf NAAAR mutants in complex with N-acetyl-D-naphthylalanineeveals active site residues involved in the accommodation ofulkier substrates. These assays, combined with structural insights,uide the engineering of new NAAARs with catalytic potentialcross a larger range of amino acids.

Acknowledgements: This work has been funded by the
BSRC and Dr Reddys Laboratories.

4

mctum

SYMPOSIUM 20: SYSTEMS BIOCATALYSIS

20-5

rtificial enzyme cascade to the polymer building block-amino caproic acid

olfgang Kroutil 1,∗ , Johann Sattler2, Michael Fuchs1, Verenaesch1, Joerg Schrittwieser1

University of Graz, AustriaACIB GmbH, Austria

The monomer units for polyamides may be �-amino-carboxyliccids, lactams or diamines and dicarboxylic acids. Caprolactam, forxample, is produced chemically at 4.2 million ton/year mainlyrom cyclohexanone via its oxime and Beckmann rearrangement.

Biocatalytic cascades involving redox steps [1] allow to performxidation and reduction reaction in the linear sequence simulta-eously. [2,3] Oxidation and reduction reactions might be coupled

n that way that the electrons gained in the oxidation step areonsumed in the reduction step turning the overall process redoxeutral and cost efficient.

Here we report a biocatalytic redox cascade starting fromyclohexanol to yield �-amino caproic acid, thus the hydrolysedquivalent to caprolactam. The designed cascade involves fouredox steps and additional hydrolytic steps, whereby the cas-ade was designed in that way, that no external redox reagentsere required, thus the cascade was redox neutral or redox self-

ufficient. Since an intermediate in the reaction sequence causednhibition for a later enzyme in the cascade an in situ functionalroup protection strategy was established to avoid the formationf the inhibiting compound.

eferences

].Schrittwieser H, Sattler J, Resch V, Mutti FG, Kroutil W. Curr OpinChem Biol 2011;15:249–56.

].Staudt S, Burda E, Giese C, Müller CA, Marienhagen J, SchwanebergU, et al. Angew Chem Int Ed 2013;52:2359–63.

].Sattler JH, Fuchs M, Tauber K, Mutti FG, Faber K, Pfeffer J, et al. AngewChem Int Ed 2012;51:9156–9.


20-6

ncovering the broader roles of redox partner proteinsor cytochrome P450 enzymes

hengying Li1,∗ , Wei Zhang1, Yi Liu1, Yojiro Anzai2, Fumio Kato2,arissa Podust3, David Sherman4

Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academyf Sciences, ChinaToho University, JapanUniversity of California, San Francisco, USAUniversity of Michigan, Ann Arbor, USA

The superfamily of cytochrome P450 enzymes is one of theost versatile biocatalytic systems in nature. P450 enzymes are

apable of catalysing many distinct types of industrially impor-ant reactions such as regio- and stereoselective oxidation ofnactivated C−H bonds, dealkylation, decarboxylation, and aro-atic coupling. For most P450 enzymes, redox partner proteins


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YMPOSIUM 20: SYSTEMS BIOCATALYSIS

re required for the electron transfer during catalysis. Conven-ionally, it has been thought that these auxiliary proteins onlynfluence catalytic efficiency and/or product distribution, but nothe type and selectivity of reactions catalysed by P450s. However,ur recent study (J. Am. Chem. Soc. 2014, 136, 3640) on MycG,hich is the multifunctional P450 monooxygenase involved in the
iosynthetic pathway of 16-membered ring macrolide antibioticsycinamicins in the rare actinomycete Micromonospora griseoru-
ida, has provided solid evidence to challenge this generallyccepted “postulate”. Moreover, we have found that some redox

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artner proteins are able to support the activity of a biofuel related450 peroxygenase from Jeotgalicoccus sp. ATCC 8456 that nor-ally uses hydrogen peroxide as cofactor (Biotechnol. Biofuels

014, 7, 28). These results highlight broader roles of redox part-er proteins for modulating the catalytic activity of P450 enzymesia alternative protein-protein interactions that have not been well
nderstood so far.

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ew Biotechnology · Volume 31S · July 2014 BIO-BASED PRODUCTIONOF C

io-based production of chemicals, fuelsnd materials by metabolically engineeredicroorganisms

L3-1

io-based production of chemicals, fuels and materialsy metabolically engineered microorganisms

ang Yup Lee

Department of Chemical and Biomolecular Engineering (BK21+ Program),ioProcess Engineering Research Center, Center for Systems and Syntheticiotechnology, Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu,aejeon, Republic of Korea

Many chemicals, fuels and materials we use every day are
erived from fossil resources. Relying on the current produc-ion system is not sustainable and has been raising concernsue to the climate change and other environmental problems.o address this issue, there has recently been much interest in
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CALS, FUELS ANDMATERIALS BYMETABOLICALLY ENGINEEREDMICROORGANISMS

eveloping bio-based processes for the production of chemicals,uels and materials from renewable non-food biomass. In ordero maximize the efficiencies of bioconversion process, metabolicngineering has become an essential practice. Metabolic engineer-ng has recently become more powerful through the integrationith systems biology, synthetic biology and evolutionary engi-eering, which led to the birth of systems metabolic engineering.

n this lecture, systems metabolic engineering approaches taken toevelop strains capable of efficiently producing various chemicals,uels and materials will be described together with several exam-le cases. Through systems metabolic engineering, it is possibleo achieve cost-effective production of desired bioproducts, whichre either natural or nonnatural products.

Acknowledgements: This work was supported by the Tech-ology Development Program to Solve Climate Changes onystems Metabolic Engineering for Biorefineries of Ministry of Sci-
nce, ICT & Future Planning.


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iocatalysis

A-01

nhancing of enzymatic palmitoylation of racemic 9-2,3-dihydroxypropyl)adenine in co-solvent mixture ashe reaction media

ana Brabcova1,∗ , Jiri Blazek1, Marcela Krecmerova1, Mariearevucka1, Jose M. Palomo2

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of thezech Republic, PragueDepartamento de Biocatálisis. Instituto de Catálisis (CSIC).Campus UAM Can-

oblanco, Madrid, Spain

A comparative study of racemic 9-(2,3-dihydroxypropyl)denine (DHPA) palmitoylation, a potential prodrug, catalysed byeveral lipases in DMF/co-solvent mixtures and in pure organicolvent was performed. The optimal conditions were investigateds follows: optimal co-solvent mixture, initial aw (water activity),inyl palmitate/DHPA molar ratio, temperature, enzyme dosage,nd chemical modification of biocatalysts. It was shown that annhancement in the substrate conversion could be achieved withMF/hexane (4:1) co-solvents as the reaction medium instead ofure organic solvent with immobilized Candida antarctica B (CALB)

ipase, although in very low yield. The chemical modification ofhe lipase had strong positive effect on its activity. The reactionield was successfully increased (50% after 24 hours) when CALBlycosylated with dextran polymer was used under optimal con-itions. The results described further highlight the versatility of

ipases and the potential of rational substrate, solvent and enzymengineering for modulating enzymatic reactions.

Acknowledgments: The authors are grateful for the financialupport of the Academy of Sciences of the Czech Republic (project
o. M200551203).


A-02

imultaneous improvement of specific catalytic activitynd thermal stability of MBP fusion heaprinase II byodifying amino acid residues in 758 site

an Su ∗ , JingjunWu, Chong Zhang, Xin-Hui Xing

Key Laboratory for Industrial Biocatalysis, Ministry of Education of China,epartment of Chemical Engineering, Tsinghua University, Beijing, China

Heparinase II (HepB) is an important polysaceharide lyase,hich plays an vital role in the development of anticancer drugs,roduction of low molecular weight heparin (LMWH) and qual-

ty control of heparins. At present, the main technical bottleneckhich has restrained its industrial application is the high produc-

ion cost, the poor level of heterologous recombinant expressionnd lack of the thermal stability. In a systematic design for con-truction of fusion expression system with the MBP tag in E.coli,e found that heparinase II could be expressed efficiently withigh activity in the soluble form, and firstly observed that themino acid residues of the 758 site in the HepB sequence greatlyffected the catalytic activity and thermal stability of this enzyme,hich were closely related with the protein dimmer formationf HepB. Furthermore, we systematically studied the fusion pro-ein design by changing the linker, indicating that linker propertyould significantly affect both the fusion enzyme activity duringhe expression and the thermo stability. As a result, the total MBP-epB activity reached 3674.82IU/L, and half-life of the enzyme in0 ◦C reached more than 250 h which increased 25 times compared


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A-03

ffective encapsulation of biocatalysts in sol–gel silicaanosheet prepared by peptide catalysts

atsuya Kato ∗ , Hitomi Nakamura, Fukue Nagata

National Institute of Advanced Industrial Science and Technology (AIST), 2266-8 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan

Encapsulation restricts the mobility of the biomolecules con-ained within a confined space and can prevent denaturationven in harsh environmental conditions related to temperature,H, and solvents. For this reason, many biomolecules have beenrapped within sol–gel silica, and this research has led to some ofhe most interesting and important applications of these materi-ls, paving the way for their use in the design and fabrication ofiocatalysis and biosensing devices [1]. Very recently, we reported2] the mesoporous silica (MPS) sheet (thickness < 100 nm) was syn-hesized using a novel approach for the immobilization of enzyme.nzyme immobilized on the MPS sheet exhibited a remarkablyigher catalytic oxidation activity than native enzyme. From theseesults, the sheet morphology significantly influences the activityf immobilised enzyme. In this report, we present the details of ournvestigation on the immobilization of the enzyme, glucose oxi-ase (GOX), in a sol–gel silica matrix using poly-L-lysine assistedirect condensation reactions of silicon oxide, trimethoxysilane.he resulting GOX-silica composites with hexagonal sheet mor-hologies were thoroughly characterized in terms of morphology,ize and surface structure. The activity and stability of the immo-ilized GOX were also investigated in details.

eferences

].Kato K, et al. J Asian Ceram Soc 2014. DOI:10.1016/j.jascer.2013.12.004.

].Nakanishi K, et al. RSC Adv 2014;4:4732.


A-04

ovel mesoporous silica sheet as a protein carrier fornhancement of catalytic activity

azuma Nakanishi 1,2,∗ , Masahiro Tomita1, Katsuya Kato2

Department of Chemistry for Materials, Mie University, 1577 Kurimamachiya-ho Tsu city, Mie 514-8507, JapanNational Institute of Advanced Industrial Science and Technology (AIST),266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan

Porous materials have much attention because they can interactith atoms, ions, molecules, and nanoparticles, at their surfacess well as throughout the bulk of the material. Mesoporous silicaMPS) (pore size 2–50 nm) with great mechanical stability has beenpplied in many fields including enzyme immobilization, catalysisnd chromatography. Recently, we reported the enhanced activ-
ty and stability of enzymes and proteins on MPS as an effectivearrier. [1] The presence of pore in nanostructured materials foriological applications must be promoted. Then we focused onhe morphologies of materials in order to increase the mass trans-
BIOCATALYSIS

er of substrate, which have important role in immobilized enzymectivity. Only few MPS sheet have been reported because they areuite difficult to prepare. On these bases, we introduce a new syn-hetic approach to MPS sheets (thickness under 100 nm) by usinghe carboxylate surfactant (N-palmitoyl-L-alanine) and Pluronic123 as dual-templating agents. Cytochrom c (cyt c, molecular sizef 2.5 × 2.5 × 3.7 nm) immobilised on the MPS sheet exhibited aemarkably higher catalytic oxidation activity than native cyt c.

eference

].Nakanishi K, et al. Mater Chem B 2013;1:6321.


A-05

hysiological responses of Pinus sylvestris var. mongolicaeedlings to the interaction between Suillus luteus andrichoderma virens

uiqing Song1,∗ , Dachuan Yin1, Xun Deng2

Northeast Forestry UniversityForestry Protection Institute, Heilongjiang Academy of Forestry

The effects of the interaction between Suillus luteus (L.) Rousselnd Trichoderma virens (J.H. Mill., Giddens & A.A. Foster) Arx oninus sylvestris var. mongolica Litv. were studied using plant phys-ology, mycorrhizal science, forest pathology and biochemistry.eedling growth and physiological parameters were determined,ncluding the colonization rate of mycorrhizal fungi, biomass, rootctivity, photosynthetic pigment content, soluble protein content,ntioxidant enzyme activities, rhizosphere soil enzyme activitiesnd protective enzyme activities. In addition, an optimal resis-ance system involving T. virens, mycorrhizal fungus (Suillus luteus)nd P. sylvestris var. mongolica seedlings was constructed. Syner-ies between S. luteus and T. virens were observed and most of thearameters of Pinus sylvestris var. mongolica seedlings inoculatedith S. luteus 30 days + T. virens were higher than other treatments.fter three months, when compared the control, the S. luteus0 days + T. virens treatment gave increases in: height (42.3%);ollar diameter (66.7%); fresh weight (54%); dry weight (50%);oluble protein content (69.86%); root activity (150%); chloro-hyll a (77.6%); chlorophyll b (70.5%); carotenoids (144%); CATctivity (876.9%); POD activity (268.3%); SOD activity (66.18%);-1,3-glucanase activity (125.8%); chitinase activity (40%); rhi-osphere soil catalase activity (97.8%); and phosphatase activity266.7%). These results indicate that there may be a stimulatingactor between S. luteus and T. virens when they are inoculatedogether (S. luteus 30 days + T. virens)

Keywords: Suillus luteus; Trichoderma virens; interaction;


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A-06

urification and characterisation of a �-galactosidaserom the thermoacidophilic bacterium Alicyclobacillusulcanalis DSM 16176

aryWalsh ∗ , JayneMurphy

University of Limerick

Thermoacidophiles are microorganisms capable of optimumrowth under a combination of high temperature and low pH.hese microorganisms are a rich source of thermo- and acid-ctive/stable glycosyl hydrolases. Such enzymes could find uses novel biocatalysts in some industrial processes, as operationt elevated temperature can increase substrate solubility, decreaseiscosity and reduce the risk of microbial contamination [1,2].e report the purification and characterisation of an intracel-

ular �-galactosidase from the thermoacidophile Alicyclobacillusulcanalis DSM 16176. The enzyme was purified 110-fold, with5% yield. Denatured (84 kDa) and native (179 kDa) molecularasses were determined by SDS-PAGE and gel filtration, respec-

ively and suggest the enzyme functions as a homodimer. Highestctivity was measured at 70 ◦C and pH 6.0. The Km on the sub-trates ONPG and lactose were, respectively, 3.8 and 425.3 mM.his enzyme is thermostable, retaining 76, 50 and 42% rela-ive activity after 30, 60 and 120 min, respectively, at 70 ◦C. Thisroperty could lend its use to high-temperature industrial pro-esses requiring a thermo-active �-galactosidase, including theroduction of lactulose and in the manufacture of lactose-freeilk and dairy products. To date, there have been no reports

n the literature on the characterisation of a glycosyl hydro-ase from A. vulcanalis DSM 16176. Funded by IRCSET EMBARKnitiative.

Key-words., �-galactosidase, thermoacidophile, proteinurification.

eferences

].Niehaus F, Bertaldo C, Kahler M, Antranikian G. Applied Microbiologyand Biotechnology 1999;51(6):711–29.

].Turner P, Mamo G, Karlsson EN. Microbial Cell Factories 2007;6:9.


A-07

egioselective hydroxylation of aromatic carboxyliccids by cytochrome P450 CYP199A2 and its mutants

oshiki Furuya ∗ , Kuniki Kino

Waseda University

Hydroxy-aromatic carboxylic acids are practically or poten-ially important industrial chemicals since the functional groupslay key roles in biological activities and physical propertiesnd also can be utilized for further modification of the chemi-
als. Cytochrome P450 monooxygenases are promising catalystsor use in the hydroxylation of chemicals. We found thatYP199A2 from Rhodopseudomonas palustris has catalytic activity
or the hydroxylation of 2-naphthoic acid to 7- and 8-hydroxy-2-

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aphthoic acids. [1]. Furthermore, CYP199A2 was able to catalyzehe regioselective hydroxylation of hydroxy-2-naphthoic acids,ndolecarboxylic acids, and a quinolinecarboxylic acid [2,3]. Theseesults indicate that CYP199A2 should be an efficient biocata-yst for the hydroxylation of various aromatic carboxylic acids4].

The crystal structure of CYP199A2 determined by Bell et al. (J.ol. Biol., 383, 561 (2008)) shows that Phe at the 185 position is

ituated directly above the heme iron. When this Phe185 residueas replaced with hydrophobic or hydroxylated amino acids,

everal mutants predominantly produced 5-hydroxy-2-naphthoiccid from 2-naphthoic acid. Interestingly, these Phe185 mutantslso exhibited novel substrate specificities. In particular, thehe185Leu mutant exhibited high hydroxylation activity for cin-amic and p-coumaric acids [5]. The Phe185Leu whole-cell catalystchieved gram-per-liter-scale production of caffeic acid from p-oumaric acid.

eferences

].Furuya T, Kino K. ChemSusChem 2009;2:645–9.].Furuya T, Kino K. Biosci Biotechnol Biochem 2009;73:2796–9.].Furuya T, Kino K. Appl Microbiol Biotechnol 2010;85:1861–8.].Furuya T, Kino K. Appl Microbiol Biotechnol 2010;86:991–1002.Review.

].Furuya T, et al. Appl Environ Microbiol 2012;78:6087–94.


A-08

ew biotransformation process for production of �-actones from hydroxy fatty acids by permeabilized

altomyces lipofer cells induced with oleic acid

ung ung An ∗ , Deok kun Oh

Konkuk university

The production of the flavor lactone was developed by usingermeabilized Waltomyces lipofer, which was selected as an efficient-lactones producing yeast among 10 oleaginous yeast strains.he cells were permeabilized using 50% ethanol and 0.5% Tri-on X-100, sequentially. Among several fatty acids tested, oleiccid was selected as the most efficient inducer for the produc-ion of �-lactones. The cells were induced by incubation for 12 hn a medium containing 10 g l−1 yeast extract, 10 g l−1 peptone,g l−1 oleic acid, 1 g l−1 glucose, and 0.05% (w/v) Tween 80. Theptimal reaction conditions for �-lactones production by wholealtomyces lipofer were pH 6.5, 35 ◦C, 200 rpm, 0.71 M Tris, 60 g

−1 hydroxy fatty acid, and 20 g l−1 cells. Under these conditions,on-treated cells produced 30 g l−1 �-dodecalactone from 60 g l−1

0-hydroxystearic acid after 30 h, with a conversion yield of 63%w/w) and a productivity of 1.3 g l−1 h−1, whereas treated cells pro-uced 51 g l−1 �-dodecalactone from 60 g l−1 10-hydroxysteariccid after 30 h, with a conversion yield of 85% (w/w) and a pro-
uctivity of 1.7 g l−1 h−1. The conversion yield and productivityf treated cells were 22% and 1.3-fold higher, respectively, thanhose of non-treated cells. The treated cells also produced 28 g l−1
-decalactone and 12 g l−1 �-butyrolactone. These are the highsest

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A-09

ioconversion of linoleic acid to 5,8-dihydroxy-,12(Z,Z)-octadecadienoic acid by 5,8-diol synthaserom Aspergillus nidulans

eoMin-Ju ∗ , Shin Kyung-Chul, OhDeok-Kun

Konkuk university

The fungus diol synthase from Aspergillus nidulans was clonednd expressed in Escherichia coli. Recombinant E. coli cells con-erted linoleic acid to 5,8-dihydroxy-9,12(Z,Z)-octadecadienoiccid, which was identified by LC-MS/MS. The recombinant cellsnd the purified enzyme showed activity for linoleic acid. The reac-ion using purified diol synthase stoppedafter 15 min, whereas theeaction using recombinant cells expressing diol synthase from. nidulans continued over 60 min, indicating that recombinantells expressing diol synthase were more stable than the puri-ed enzyme. The optimal reaction conditions for the productionf linoleic acid to 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acidsing whole recombinant E. coli cells were pH 7.5, 35 ◦C, 250 rpm,3 g l−1 cells, 5 g l−1 linoleic acid, and 20% (v/v) dimethyl sulfoxiden a 250 ml-baffled flask. Under these optimized conditions, wholeecombinant cells produced 4.98 g l−1 5,8-dihydroxy-9,12(Z,Z)-ctadecadienoic acid for 150 min, with a conversion yield of 99%w/w) and a productivity of 2.5 g l−1 h−1. This is the biotechnolo-ical production of dihydroxy fatty acid using whole recombinantells expressing diol synthase.


A-10

roduction of compond K from major protopanaxadiolinsenosides by combined enzymes, including �-L-rabinofuranosidase and �–galactosidase from Caldicel-ulosiruptor saccharolyticus and �–glucosidase fromulfolobus acidocaldarius

yung-Chul Shin ∗ , Hye-Jin Oh, Baek-Joong Kim, Deok-Kun Oh

Konkuk university

The ginsenoside compound K has diverse pharmaceutical activ-ties such as anti-tumor, anti-inflammatory, anti-allergic, andepatoprotective effects. To increase the production of com-ound K from major protopanaxadiol ginsenosides in ginseng rootxtract, �-l-arabinofuranosidase (CS-abf) and �–galactosidase (CS-gal) from Caldicellulosiruptor saccharolyticus were mixed with–glucosidase (SA-bglu) from Sulfolobus acidocaldarius. The opti-
um conditions for the production of ginsenoside compound K
rom major protopanaxadiol ginsenosides in ginseng root extractere determined to be pH 6.0 and 75 ◦C with 10% (w/v) ginseng

oot extract and 10.5 U ml−1 CS-abf and 10.5 U ml−1 CS-bgal sup-

ctc

BIOCATALYSIS

lemented with 4.5 U ml−1 SA-bglu. Under optimum conditions,ajor protopanaxadiol ginsenosides in ginseng root extract were

ompletely converted to compound K after 20 h with the respec-ive productivities of 144 mg l−1 h−1.


A-11

tructural conformation of enzyme in ionic liquids:olecular dynamic simulation study

oon-Mo Koo

Inha University

Enzymatic reactions in ionic liquids have been gaining increas-ng interests during the last decade due to their unique properties.t is well documented that, in some cases, enzymes showednhanced activity, selectivity and stability in ionic liquids. How-ver, there were very few studies investigating the structure ofnzymes in ionic liquids. In this study, the conformational struc-ure changes of Candida antarctica lipase B (CALB) in differentmidazolium-based ionic liquids observed by molecular dynamicimulation are discussed. The results showed that two isoleucines,LE-189 and ILE-285, in CALB played critical role in the open-losed conformations of the catalytic cavity. The ILE-285 situatedn �-10 helix region (residues 268-287) where its conformationan be significantly changed in different solvents. For example,n 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) conforma-ion of �-10 helix changed to a turn as a result of direct interactionsith chlorine anions gave rise to a closed conformation of the

atalytic cavity, corresponding to lower enzyme activity.


A-12

onjugation of chitooligosaccharides and glucosamineo bovine trypsin. Effects on stability and functionality

örður Filippusson ∗ , Jóhann G.K. Gizurarson

University of Iceland

The improvement of protein stability is important in relation tohe use of enzymes as practical biocatalysts and in the use of pro-eins as pharmaceuticals. All proteins are unstable, especially whenn solution. Among the processes that can affect protein stabilityre proteolytic degradation in vitro or in vivo, thermal denaturation,nd antigenicity after injection. Attempts to improve proteintability include protein engineering, immobilization, chemicalodification and the use of cosolutes.In this study bovine trypsin was modified by coupling

he enzyme to either D-glucosamine or a partially acetylatedhitoologosaccharide via binary carbodiimide/succinimide ester
onjugation. D-glucosamine was found to conjugate, on average,o 12 residues on trypsin. The oligosaccharide coupling producedross-linked polydisperse complexes with hydrodynamical radii

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[2[3].Francois AA, et al. Drug Metabol Disposit 2009;37(1):178–86.


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IOCATALYSIS

rom, on average, from 218 to 330 nm for 1:5 and 1:10 cross-linkedrypsin, repectively.

The physical properties of the enzyme species were studiedy electrophoresis, gel filtration, circular dichroism, nanoprticleracking analysis and MALDI-TOF mass spectrometry. The stabil-ty of these enzyme species was studied by activity assays, ureaenaturation, diffferential scanning calorimetry and autolysis.

The modified trypsin showed increased resistance against ther-al inactivation and autolysis, better storage stability but stability

gainst urea inactivation was unchanged. The proteolytic activ-ty against azocasein improved for the cross-linked trypsins butas slightly reduced for D-glucosamine conjugated trypsin. The

pecies were found to become basophilic upon conjugation andross-linking. D-glucosamine conjugated trypsin was found to belightly structurally altered which and as consequence displayed.2 times higher catalytic efficiency (kcat/Km) than native trypsingainst the substrate L-BAPNA.


A-13

,8-cineole-hydroxylating cytochrome P450s from Sphin-obium yanoikuyae

irgitUnterweger1,∗ , David J.Midgley2, PaulGreenfield3, DieterM.ulach4, Dena Lyras5, Priscilla Johanesen5, Geoffrey J. Dumsday6

Department of Microbiology, Monash University, Clayton, VIC 3800, Australiand CSIRO Future Manufacturing Flagship, Clayton, VIC 3168, AustraliaCSIRO Animal, Food and Health Sciences, North Ryde, NSW 1670, AustraliaCSIRO Computational Informatics, North Ryde, NSW 1670, AustraliaVictorian Bioinformatics Consortium, Monash University, Clayton, VIC 3800,ustraliaDepartment of Microbiology, Monash University, Clayton, VIC 3800, AustraliaCSIRO Materials Science and Engineering, Clayton, VIC 3168, Australia

In Australia, extensive Eucalyptus plantations have been estab-ished to prevent dry land salinity and novel applications forhis renewable resource are being sought. One approach is todd value to the leaf oil from these plantations via the oxyfunc-ionalisation of its chemically unreactive component 1,8-cineole.ydroxyl-groups create starting points for further modification

nd/or incorporation into more complex molecules such as poly-ers. Biooxidation of 1,8-cineole promises a more sustainable

oute to hydroxylated intermediates with greater stereospecificityompared to conventional chemistry. The availability of biocat-lysts known to catalyse the oxidation of 1,8-cineole, however,s limited. To expand the range of biocatalysts, several microbesapable of hydroxylating 1,8-cineole were isolated including aphingobium yanoikuyae strain. Genome sequencing revealed theresence of multiple genes encoding P450s and potential elec-ron transport partners. To confirm the identity of genes encoding,8-cineole-hydroxylating P450s, proteins exhibiting the char-cteristic spectroscopic shift upon addition of 1,8-cineole wereurified from S. yanoikuyae cultivated in the presence of 1,8-
ineole. In addition to this the genes were cloned and the generoducts heterologously expressed in Escherichia coli and puri-ed from clarified cell lysates. Spectroscopic characterisation andhole-cell biotransformation have demonstrated that the proteins


an hydroxylate 1,8-cineole. Compared to the well characterised450cin from Citrobacter braakii, the S. yanoikuyae enzymes haveimited amino acid sequence identity, appear to have differingtereo- and regioselectivity and based on testing completed soar the preferred substrate is 1,8-cineole. Current investigationsnvolve mining the S. yanoikuyae genome for the natural electronransport partners to optimise the hydroxylation process.


A-14

uman flavin monooxygenase 2: Heterologous expres-ion in E. coli and API modification

argitWinkler1,∗,1 , ThorstenBachler2,2, MartinaGeier2,2, StevenP.anlon3,3, Matthias Kittelmann4, Anton Glieder2,2, Stephan Lütz4,eat Wirz3,3

ACIB GmbH c/o Institute of Molecular Biotechnology, Graz University of Tech-ologyACIB GmbHF. Hoffmann-La Roche LtdNovartis Pharma AG

Abstract The flavin monooxygenase (FMO) isoenzyme 2 isnown as the “pulmonary” FMO because it is expressed predomi-antly in the human lung. FMO2*1 is the active FMO2 allele found

n Africans and Hispanics [1]. The enzyme is membrane associ-ted and oxidizes xenobiotics with soft nucleophiles such as sulfurnd nitrogen at the expense of NADPH and oxygen. The aim ofhe current study was to generate simple and efficient whole celliocatalysts for the preparation of FMO2 drug metabolites on theulti-milligram scale. Recently, we demonstrated the possibility

o express human FMOs in E. coli, however, the expression levelf the FMO2*1 isoform was very low in comparison to FMO3 andMO5 [2]. This prompted us to investigate truncated variants ofMO2*1 and to compare their performance in whole cell biotrans-ormations of different active pharmaceutical ingredients such as.g. ethionamide–an antitubercular drug [3]

Key-words: Biocatalysis, flavin monooxygenase, drug metabo-ites, oxidation, membrane protein.

eferences

].(a) Dolphin C, et al. J Biol Chem 1998;273(46):30599–607;(b) Krüger SK, et al. Drug Metabol Disposit 2004;32(12):1337–40.

].Hanlon SP, et al. Chem Commun 2012;48(48):6001.

1 ACIB GmbH, Petersgasse 14/III, 8010 Graz, Austria2 F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland3 Novartis Pharma AG, 4070 Basel, Switzerland

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A-15

o-factor regeneration at the cell surface–upgrading theiotechnological potential of whole cell biocatalysts

an Schüürmann ∗ , Joachim Jose

Westfälische Wilhelms Universität Münster

Whole cell biocatalysts offer significant advantages comparedo purified enzymes, specifically cheap and efficient productionombined with obsolete purification. However, there are limi-ations to the applicability of whole cells. Cross reactions withndogenous enzymes and membrane impermeability of substratesnd products might complicate their use.

Autodisplay presents enzymes on the surface of E. coli cells elim-nating mass transfer problems and possible side reactions. Theechnique replaces the passenger domain of a native autotrans-orter protein by a peptide or protein of choice. However, a majorhallenge for some enzymes to be used in biotechnological appli-ations is the regeneration of co-factors, which are too expensiveo be added stoichiometrically. Here, we report the developmentf a cell surface based NADPH regeneration system. To circum-ent laborious protein purification and keep the advantages ofurface displayed catalysts we used the Autodisplay technologyo present various dehydrogenases on the cell surface of E. coli.urface display was confirmed by protease accessibility tests andACS analysis. NADPH production was measured photometricallyt 340 nm. We first concentrated our efforts on an engineered for-ate dehydrogenase. The enzyme showed similar specific activity

ompared to the soluble enzyme, but the efficiency of the systemas limited by the number of enzymes on the bacterial surface andigh amounts of whole-cell catalysts were needed for effective pro-uction of NADPH. Thus, we are currently exploring alternativenzymes with higher specific activities for surface display and cellssociated co-factor regeneration.


A-16

urface display of enzymes on Zymomonas mobilis andymobacter palmae using the autotransporter secretionathway

asson E.P. Tozakidis1,∗ , Annika Meyers1, Tatjana Brossette2,oachim Jose1

Institute of Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms-niversität MünsterAutodisplay Biotech GmbH

Displaying enzymes on a microbial cell surface has the potentialo significantly reduce the costs of large scale biocatalytic conver-ion processes. In contrast to intracellularly expressed enzymes,urface displayed enzymes do not have to be purified prior to theirse. Instead, the host cells can directly be employed in a reac-
ion, and neither substrates nor products need to cross a membranearrier. Additionally, whole cells can easily be harvested and usedn multiple process cycles, whereas the recovery of free enzymes
BIOCATALYSIS

rom a reaction mixture is often difficult. The gram-negative bacte-ia Zymomonas mobilis and Zymobacter palmae are promising hostrganisms for industrial applications due to their high ethanol pro-uctivity and resistance towards rough reaction conditions. Untilow, no functioning surface display system for these organismsas been reported. For this reason, we tested if the autotransporterecretion pathway can be utilized in Z. mobilis and Z. palmae.ere we present the surface display of two industrially relevant

nzymes, namely Burkholderia gladioli esterase EstA and Bacillusubtilis endoglucanase Cel5A, and demonstrate that the enzymesetain their activity on the cell surface of both organisms. Thisork represents the first step towards using Z. mobilis and Z. palmaes expression platforms for surface display applications.


A-17

ombined Surface Display of Cytochrome P450 1A2 andts Reductase on Escherichia coli

aul Quehl ∗ , Joachim Jose

University of Münster

Human cytochrome P450 monooxygenases (CYPs) play arominent role in drug metabolism as these enzymes are involved

n the breakdown of literally every drug. They catalyze a variety ofxidations of a broad range of substrates and have a major impactn bioavailability and drug-drug interactions. CYPs obtain thelectrons from the NADPH-dependent cytochrome P450 reduc-ase (CPR). However, recombinant expression of both enzymes inacteria is often not feasible and they exhibit poor stability afterurification. Moreover, the membrane associated proteins CPR andYPs require membrane surroundings for activity. CYPs alone cane displayed functionally active on the surface of Escherichia coliith externally added CPR. Here, we tested the co-expression ofYP1A2 and its NADPH dependent Cytochrome P450 reductase

CPR) on the E.coli outer membrane. Surface display is facilitatedy usage of the autotransporter secretion pathway for which thenzyme of interest is combined with an N-terminal signal peptide,C-terminal linker and a beta-barrel domain. Surface presenta-

ion was confirmed by FACS analysis and protease accessibilityests. Both CYP1A2 and CPR bind their cofactors and the sur-ace displayed NADPH-dependent cytochrome P450 reductase isble to react with Cytochrome C. The functional interaction ofYP1A2 with the CPR is currently under investigation by testing

he enzymatic activity towards the substrates 7-ethoxyresorufin


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A-18

iochemical and Structural Characterization of ahermophilc L-Arabinose Isomerase from Geobacillusaustophilus

ong-Woo Lee1,∗ , Yong-Jik Lee1, Jin Myung Choi2, Sun-Mi Shin1,ang-Jae Lee3, Han-Seung Lee3, Sang Jun Lee4, Sung Haeng Lee2

Kyungpook National UniversityChosun UniversitySilla UniversityKRIBB

Thermophilic L-Arabinose isomerase (AI), that catalyzes thenterconversion of L-arabinose to L-ribulose, can also isomerize-galactose to D-tagatose as a natural sugar substitute, which

s of commercial interest in the food and healthcare indus-ries.Recently, biochemical and mutational studies revealed thatnlike mesophilic AIs, thermophilic AIs showed the distinctetal dependence for their catalytic activity and thermostabi-

ity at elevated temperatures. However, it still remains unclearow mesophilic and thermophilic AIs showed different substratereferences and metal requirements at molecular levels. Hereine characterized a thermophilic AI from Geobacillus kaustophilus

GKAI) and presented the first crystal structures of the apo and holoorms of GKAI by X-ray crystallography to 2.40 and 2.30 A, respec-ively. We also determined the crystal structure of holo enzymeound to L-arabitol as a substrate analog at 2.25 A resolution.Inombination with biochemical and site-directed mutagenesis stud-es, the structures identified the structural elements of metalinding and substrate recognition.In comparison with the crystaltructures of Escherichia coli AI (ECAI) as a mesophilic counterpart,he GKAI structures revealed quite conserved structural featuresor substrate and metal binding, except forsubtle interactions of aew polar residues with water molecules near the substrate bind-ng region.Our comparative analysis proposes a metal-mediatedubstrate binding model for the isomerization reaction at ele-ated temperatures, providing a versatile strategy to engineer theromiscuity of substrate specificity for sugar isomerases as wells thermostability for mechanistic studies and industrial applica-
ions.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1804P

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A-19

unctional Characterization of Putative UDP-Glucose-Epimerase (TM0509) from the Hyperthermophilicubacterium Thermotoga maritima

un-Mi Shin1,∗ , Jin Myung Choi2, Yong-Jik Lee1, Sang-Jae Lee3,ang Jun Lee4, Sung Haeng Lee2, Dong-Woo Lee5

Kyungpook National UniversityChosun UniversitySilla UniversityKorea Research Institute of Bioscience and BiotechnologyKyungpook National Univ

UDP-glucose 4-epimerase (GalE; EC 5.1.3.2) catalyzes thenterconversion of UDP-glucose (UDP-Glc) and UDP-galactoseUDP-Gal), which is a pivotal step in the Leloir pathway for galac-ose metabolism. Although GalEs are widely distributed in Bacteriand Eukaryotes, there is little information on hyperthermophilicalE. Herein we cloned and overexpressed the TM0509 gene

ncoding a putative GalE from Thermotoga maritima (TMGalE) asfusion protein containing an N-terminal hexa-histidine sequence

n Escherichia coli. This gene encodes a 309-amino acid proteinith a calculated molecular weight of 34899 and a theoreticalI of 5.72.The recombinant protein was purified to homogene-

ty by heat precipitation, Ni2+ affinity chromatography followedy size-exclusion chromatography. The native enzyme was esti-ated to be a homodimer with a molecular mass of 70 kDa. The

ecombinant TMGalE could reversibly catalyze the epimerizationf UDP-Gal and UDP-Glc in the presence of NAD+ at elevatedemperatures. The apparent optimal temperature and pH for epi-

erization activity were 85 ◦C and pH 7.0, respectively. In ordero further characterize TMGalE at molecular levels, we determinedot only the crystal structure of TMGalE at 1.9 A resolution, butlso the co-crystal structure of TMGalE bound to UDP-glucose at.0 A resolution. These biochemical and structural data showedhat TM0509 is an UDP-galactose 4-epimerase involved in galac-ose metabolism, which is the first detailed characterization of ahermostable GalE from hyperthermophilic bacterium.


A-20

icroscale tools for evaluating the biological and pro-ess options of alkane biooxidations

ohannes Kolmar1,∗ , Frank Baganz1, Philip Engel2

University College LondonEvonik Industries AG

The direct �-oxyfunctionalisation of aliphatic alkanes in aegio- and chemoselective manner remains difficult to performy industrial organic chemistry. Monooxygenases such as thelkB enzyme complex from Pseudomonas putida efficiently catal-
se these readily available substrates to primary fatty alcohols andcids under mild conditions. These are of considerable interests potential intermediates in the chemical, pharmaceutical andosmetics industry.

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The ability to rapidly screen biotransformation reactions forharacterisation and optimisation is of major importance in pro-ess development and biocatalyst selection. Studies at lab scalere time consuming and labour intensive with low experimentalhroughput. The feasibility of a parallel microwell platform forwo-liquid phase whole-cell bioconversions has previously beenhown for longer chain alkane substrates [1]. However, excessivevaporation has limited the use of the microscale approach forolatile substrates.

This study developed a microwell system for use with highlyolatile n-alkane substrates. Particular attention was paid to mate-ial compatibility, evaporation and oxygen transfer. It was shownhat the use of 24 deep square microwell plates machined fromfluoropolymer alleviates problems of organic phase permeation

nd absorption. In combination with a new sealing approacho avoid substrate evaporation, the reproducibility of results wasncreased. However, a comparison with unsealed plates revealedhat, despite similar cell growth the bioconversion productivityas lower in sealed plates. The effect of sealing the plates on oxy-en transfer will be discussed.

eference

].Grant C, Pinto A, Lui H, Woodley J, Baganz F. Biotechnol Bioeng2012;109(9):2179–89.


A-21

hemical modification of lipase B from Candidantarctica for improving biochemical properties ofctivity, stability and selectivity

odrigo Torres Sáez1,∗ , Claudia Ortiz López2, Oveimar Barbosa3,oberto Fernández-Lafuente4

School of Chemistry. Universidad Industrial de SantanderSchool of Bacteriology and Clinical Laboratory. Universidad Industrial deantander, ColombiaSchool of Chemistry. Universidad Industrial de Santander, ColombiaDepartment of Biocatalysis. ICP-CSIC, Spain

Chemical modification of enzymes can be used to modulatenzyme properties by means of modification of protein surface orey residues from the enzyme structure [1,2]. In this work, it wasarried out chemical modifications of Candida antarctica lipase BCALB) preparations immobilized on octyl-agarose, BrCN-agarosend Eupergit-C supports using different chemical compounds,.g. ethylenediamine (EDA), succinic anhydride (SA) and 2,4,6-rinitrobenzensulfonic acid (TNBS). These modifications of thenzyme surface caused changes in physical properties such asharge (isoelectric point) or hydrophobicity (solubility), androved to be practical methods to enhance the biocatalyst per-ormance (stability, activity and enantio-selectivity) when thenzyme preparations were submitted to different ranges of pH (4-9)nd temperature (25-70 ◦C) and organic co-solvents such as ace-
onitrile or tetrahydrofuran at 50% (v/v). These immobilized andhemically modified CALB preparations displayed high enantiose-ectivity during the kinetic resolution of (R/S)-methyl mandelaten aqueous solution and esterification of beta-blocker drugs such as
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BIOCATALYSIS

tenolol and propranolol. These alterations in enzyme propertiesy chemical modification should be due to changes in the struc-ure of the active form of CALB. Therefore, solid phase chemical

odification of immobilized lipases may become a powerful tooln the design of lipase libraries with very different properties.

eferences

].Rodrigues RC, Ortiz C, Berenguer-Murcia A, Torres R, Fernández-Lafuente R. Modifying enzyme activity and selectivity by immobi-lization. RSC 2013;42:6290–307.

].Davis BG. Chemical modification of biocatalysts. Curr Opin Bio-technol 2003;14:379–86.


A-22

ewly engineered diol dehydrogenase from Clostridiumutyricum as promising agent in cell-free biosystems foriomanufacturing

arta Jankowska ∗ , Włodzimierz Grajek, Agnieszka Olejnik-

chmidt, Marcin Schmidt

Poznan Uniwersity of Life Sciences

Economical and environmentally friendly production of bio-hemicals and biotechnology-based polymers is a critical goal ofodern biotechnology. The use of enzymes as catalysts for chem-

cal transformations has emerged as more ecological synthesis.enerally, naturally occurring enzymes do not have appropriate

eatures necessary for their use in chemical industry. The devel-pment in protein engineering allows optimization of particularnzyme traits which make them more suitable to chemical process.

1,3-Propanediol is a key chemical bulk for the synthesisf polytrimethylene terephthalate with desired properties forarge volume markets. 1,3-Propanediol dehydrogenase (PDOR,C 1.1.1.202) encoded in Clostridium butyricum by dhaT gene isnvolved in the conversion of glycerol into 1,3-propanediol.

The study aimed to improve enzyme activity by directed evo-ution and rational design methods to generate a more efficient,3-propanediol production.

Error-prone PCR, employed to re-engineer PDOR resulted innzyme variants with higher enzymatic activity. Variants withighest activity showed twice higher oxidative activity and twelve

imes higher reductive activity, respectively. Subsequently three-imensional structure of PDOR was predicted by homologyodeling with Lactaldehyde reductase from Escherichia coli as tem-

late. Location of the mutations, important for activity and theirotential impact on the structure and function of the enzyme werepecified.

Results of study indicate mutations that might influence theffectiveness of cofactor binding to protein surface as well as onhe enzymatic activity of PDOR.

This work was part of POIG 01.01.02-00-074/09 project co-
unded by The European Union through The European Regionalevelopment Fund within the framework of the Innovative Econ-my Operational Programme 2007-2013.


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A-23

equence and function relationship of Escherichia coliavin mononucleotide binding fluorescent protein

yung-Kwan Cho ∗ , Hyeonseok Shin

Korea Advanced Institute of Science and Technology

Flavin mononucleotide (FMN)-binding fluorescent proteinsan provide in vivo reporter system, without oxygen. Here,e present the functional landscape of individual amino acid

equence of Escherichia coli FMN-binding fluorescent protein (EcF-FP). We used random mutagenesis to generate the mutantibraries, which were screened by function loss or retained. Theunction and sequence relationship of the mutants were analyzedn single high-throughput sequencing, resulting in 329 tolerant

utations and 259 sensitive mutations that show retained flu-rescence or loss of fluorescence respectively. In addition, thenrichment of tolerant or sensitive mutations in each amino acidesidues were weighed to find functionally important residues ncFbFP. The mutation enrichment analysis show that the positionsritical to the function of EcFbFP lies among the FMN bindingocket, turns and loops of the protein where dynamic confor-ational changes occur and the Glu56-Lys97 salt bridge which

s critical to structural stability of EcFbFP. Collectively, the muta-ional scanning results provide a functional landscape of eachmino acid of EcFbFP.

This work was supported by Intelligent Synthetic Biology Cen-er of Global Frontier Project (2011-0031957, 2011-0031962).


A-24

nalysis and Optimisation of the Physiology of Engi-eered Biofilms for Biotransformations

ames Thomas Leech1,∗ , Isaac Vizcaino-Caston1, Tania Barberi 2,ebecca Goss2, Mark Simmons1, TimOverton1

University of BirminghamUniversity of St Andrews

Engineered biofilms formed from reaction-competent recombi-ant Escherichia coli provide a useful platform for biotransforma-

ion reactions [1,2]. Biofilms form in response to chemical andhysical stresses such as organic solvents and shear, and thusre able to resist conditions encountered in flow reactor bio-ransformation reactions far better than planktonic cultures [3].mproving attachment and maturation of engineered biofilms isaramount to the optimisation of their use in biotransformations.hrough the use of fluorescent reporter genes, flow cytometry andonfocal laser scanning microscopy, the physiology and compo-ition of the biofilm has been studied, in addition to the spatialnd temporal expression of biofilm constituents such as curli,oly-N-acetlyglucosamine (PGA/PNAG) and colanic acid. Molec-
lar biology techniques have been used to bypass normal biofilmignals, creating increased biofilm formation. Furthermore, thehysiological effects of biotransformation reactions on the biofilm


re being studied with an aim to optimise biotransformation pro-esses, and to observe the effect of increased biofilm productionn biotransformations.

eferences

].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH,Goss RJM. Engineering Biofilms for Biocatalysis. ChemBioChem2011;12:1391–5.

].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, SimmonsMJH. Characterisation of spin coated engineered Escherichia colibiofilms using atomic force microscopy. Colloids and Surfaces B: Bioin-terfaces 2012;89:152–60.

].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisationof engineered Escherichia coli biofilms for enzymatic biosynthesis ofl-halotryptophans. AMB Express 2013;3:66.


A-25

ibrary sequencing strategies for comparative analysisf stress resistance mechanisms in Escherichia coli strains

ebecca Lennen ∗ , Ida Bonde, Anna Koza, Markus Herrgård

Novo Nordisk Foundation Center for Biosustainability, Technical University ofenmark

Transposon insertion sequencing (Tn-Seq) has recentlymerged as a powerful next-generation sequencing methodhat enables querying the contributions of all genes in a bacte-ial genome toward the fitness of a growing organism. In thisethod, transposon insertion mutant libraries are constructed

nd subjected to growth selections. Following selection, theocations of all insertions in the population are counted and cane compared between a control and a target condition, enablinghe identification of genes that are both conditionally essentialnd conditionally detrimental. We have exploited Tn-Seq torobe the basis for the large variations in osmotic and acetatetress tolerance of different laboratory strains of Escherichia coliK-12 MG1655, BL21(DE3), W, and Crooks). Little is currentlynown to explain the source of this variation and to enableational engineering to impart stress tolerance. Tn-Seq revealedany differences and similarities in resistance mechanisms at the

enetic level across strains, allowing correlations to be made withrowth phenotypes. Cross-strain comparisons of conditionallyssential genes and their relative essentiality also suggest a largeegree of variation in metabolic flux distributions and regulationf gene expression between strains. A number of direct targetsor metabolic engineering of stress resistance via loss-of-function

utations were also discovered, and we show that deletion of aelection of these genes results in improved growth under the

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A-26

icroorganisms respond in different ways to oscillationsn large-scale bioreactors: Conclusions from scale-downpproaches

eter Neubauer1,∗ , Anja Lemoine2, Sergej Trippel2, Eva Brand2,obert Spann2, Dennis Runge2, Ping Lu2, Basant El Kady2, Chris-ian Reitz2, Stefan Junne2

TU Berlin, Chair of Bioprocess EngineeringTU Berlin

Gradients of various growth related parameters are observedn large scale bioreactors by the limited mass transfer and arenhanced by the application of the substrate limited fed-batchechnology. Consequently, cells are exposed to oscillations, whichause a specific physiological adaptation.

Our studies with different scale-down approaches indicate thaticroorganisms adopt with different strategies to oscillations,hich is closely connected to their response to oxygen limitation.hile Escherichia coli, similar to Saccharomyces cerevisiae reacts with

ncreased glycolytic fluxes, Bacillus subtilisdecreases the maximumlucose uptake capacity. In contrast, Corynebacterium glutamicumhows a high robustness to oscillations.

In all cases, the oscillations influenced the pools of variousmino acids, which are closely connected to the central carbonetabolism. When we connected the two-compartment scale-

own bioreactor with the rapid sampling unit Bioscope to use3C-labelling for a study of the metabolic fluxes in E. coli, webserved that the preferred route towards the synthesis of branch-hain amino acids after a pulse depends on the history of growthonditions.

The presented methodology, which also includes other novelnalytical instruments, provides a bridge between systems biol-gy for the investigation of the cellular regulatory networks underndustrially relevant conditions.


A-27

xploring the new threonine aldolases with broad donorpecificity

ateryna Lypetska (Fesko) ∗ , Gernot Strohmeier, Rolf Breinbauer

Graz University of Technology

Threonine aldolases have great biotechnological potential, ashey catalyze the formation of unnatural amino acids with highnantioselectivity. The enzymes have been efficiently applied forhe aldol condensation of an aldehyde and glycine to produce L-nd D-�-hydroxy-�-amino acids.[1] Aldolases are tolerant towardscceptor aldehyde, but they are quite rigid for the amino acidonor. In our previous work we have isolated two natural thre-nine aldolases, which were able to accept alanine and serine as
onor.[2] Here we present the identification and characterizationf a range of L- and D-threonine aldolases with broad donor speci-city. The kinetic properties and the substrate specificity of new
BIOCATALYSIS

nzymes were investigated and the biocatalytic method for thetereoselective synthesis of a-quaternary a-amino acids was devel-ped.

Acknowledgements: The research leading to these resultsas received funding from the Innovative Medicines Initiative

oint Undertaking under grant agreement n◦115360, resources ofhich are composed of financial contribution from the Euro-ean Union’s Seventh Framework Programme (FP7/2007-2013)nd EFPIA companies’ in kind contribution.

eferences

].(a) Steinreiber J, Fesko K, Reisinger C, Schurmann M, van Assema F,Wolberg M, Mink D, Griengl H. Tetrahedron 2007;63:918–26;(b) Steinreiber J, Fesko K, Mayer C, Reisinger C, Schürmann M,Griengl H. Tetrahedron 2007;63:8088–93.

].Fesko K, Uhl M, Steinreiber J, Gruber K, Griengl H. Angew Chem2010;122:125–8. Angew. Chem. Int. Ed. 2010, 49, 121–124.


A-28

he effect of salt-preconditioning Torulaspora del-rueckii cells on fermentation performance

tilianos Logothetis1,∗ , Fotini Drosou1, Arhodoula Hatzilazarou1,anagiotis Tataridis1, Anastasios Kannelis2, Elias Nerantzis1,raemeWalker2

TEI of Athens Department of Enology and Spirit TechnologyAbertay University of Dundee

Abstract This paper concerns research into the influence ofalt on physiology of the yeast, Torulaspora delbrouekii. Specifically,he work focused on how NaCl affected the growth, viability andermentation performance of this yeast in laboratory-scale experi-

ents. One of the main findings of the research presented involvedhe influence of salt “preconditioning” of yeasts which repre-ents a method of pre-culturing cells in the presence of salt in anttempt to improve subsequent fermentation performance. Suchn approach resulted in preconditioned T. delbruekii yeasts havingn improved capability to ferment high-sugar containing mediaup to 30% w/v of glucose) with increased cell viability and withlevated levels of produced ethanol. Salt-preconditioning mostikely influenced the stress-tolerance of yeasts by inducing theynthesis of key metabolites such as trehalose and glycerol whichct to improve cells’ ability to withstand osmostress and ethanoloxicity. Overall, this research has demonstrated that a relativelyimple method designed to physiologically adapt yeast cells–byalt-preconditioning–can have distinct advantages for alcohol fer-


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IOCATALYSIS

A-29

rotein engineering of arylmalonate decarboxylase vari-nts with promiscuous racemising activity

obert Kourist ∗ , Sarah Gaßmeyer, Nadine Hülsemann, Robin Dorau

Ruhr-Universität Bochum

Enzymatic racemization allows the smooth interconversion oftereocenters under very mild reaction conditions. Racemases findrequent applications in deracemization and dynamic kinetic reso-utions [1]. Arylmalonate decarboxylase (AMDase) from Alcaligenesronchosepticus [2] has high structural similarity to cofactor-freemino acid racemases. The racemase-like catalytic machinery ofutant G74 C conveys it a unique activity in the racemisation

f pharmacologically relevant derivates of 2-phenylpropionic acidprofenes), which makes AMDase G74 C an interesting object forhe mechanistic investigation of cofactor-independent racemases.

While the racemase has high activity towards small ary-aliphatic acids, larger substrates such as ibuprofen are converted

uch slower. Objective of this study is the extension of the sub-trate range by rational design, either by creating space in theinding pocket or by stabilizing the substrate by the introductionf hydrophobic interactions.

eferences

].Felfer U, Goriup M, Koegl ME, et al. Adv Synth Catal 2005:951–61.].Kourist R, Miyauchi Y, Uemura D, Miyamoto K. Chem Eur J2011:557–63.


A-30

nzymatic Hydrolysis of PET: Structural Diversity andinetic Properties of Cutinases from Thermobifida

ltijana Hromic1,∗ , Doris Ribitsch2, Andrzej Lyskowski1, Georgteinkellner1, Helmut Schwab3, Georg Gübitz2, Karl Gruber1

ACIB GmbH c.o. IMB GrazACIB GmbHACIB GmbH c.o. Institute of Molecular Biotechnology, Graz University of Tech-ology

Poly(ethyleneterephthalate) (PET) is one of the most widelysed polymers worldwide. Its use ranges from different medi-al and thermoforming applications to fibers for textiles. It isighly hydrophobic which makes this polymer difficult to be func-

ionalized. Therefore, industrial applications often involve surfacectivation prior to final treatment.

Plasma or aggressive chemistry methods are energy consum-ng or environmentally harmful and in some cases they leado a decrease in polymer weight or strength. The exchange ofnpleasant chemical by enzymatic processes would avoid theseroblems. Cutinases from Thermobifida cellulosilytica DSM44535
Thc Cut1 and Thc Cut2) hydrolyze PET. Their ability to hydrolyzehe polymer was compared with other enzymes hydrolyzing natu-al polyesters including the PHA depolymerase from Pseudomonasuorescens and two other cutinases from Thermobifida fusca KW3.


he two isolated Thermobifida cutinases are very similar but hadifferent kinetic parameters on soluble substrates. We determinedhe structure of both enzymes. Structural analysis revealed surfaceegions of Thc Cut1 and Thc Cut2, which differ in electrostaticnd in hydrophobic properties. Modeling studies suggest thathese regions interact with PET during turnover which may explainhe differences in hydrolysis efficiencies which were observed forhe two enzymes.


A-31

haracterisation of a recombinant patchoulol syn-hase for the biocatalytic production of high valuableesquiterpenes

hore Frister1,∗ , Steffen Hartwig2, Katharina Schnatz2, Thomascheper2, Sascha Beutel2

Leibniz University Hannover, Institute of Technical ChemistryLeibniz University Hannover

Sesquiterpenes are a structurally diverse class of secondaryetabolites, which are mainly produced by various plants. Due

o their special odor sesquiterpenes are widely used as fragranceompounds in exclusive perfumes, scented household goods ands naturally flavor additives in food. These days the majority ofesquiterpene production is still based on isolation techniquesuch as solvent extraction and steam distillation. Recent biotech-ological approaches for the production of sesquiterpenes require

ime and cost-extensive pathway engineering.We hereby present a biocatalytic method for the production

f high valuable sesquiterpenes such as patchoulol and germa-rene A by converting synthetic farnesyl diphosphate (FPP) withrecombinant patchoulol synthase (PTS). [1] For the productionf the PTS we have established a bioprocess combined with a reli-ble purification strategy. The product spectrum, substrate scopend important parameters concerning the kinetic properties andnzyme stability have been studied. FPP is produced in a shortnd fast synthesis starting from chemically derived, cheap farnesolsing an optimized method according to Keller et al. [2] The bio-onversion of FPP was performed in different multi-phase modeleactor in order to increase sesquiterpene yield and maintain anasy product recovery.

eferences

].Deguerry F, Pastore L, Wu S, Clark A, Chappell J, Schalk M. Archivesof Biochemistry and Biophysics 2006;454:123–36.

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tr2ational Program funded by ERDF.



A-32

uclear Magnetic Resonance Spectroscopy: An Alter-ative Fast Tool for Quantitative Analysis of theolvent-free Ethanolysis of Coconut Oil Using Fungalesting Cells

amon Canela Garayoa1,∗ , Edinson Yara-Varón2, Mercè Balcells 2,ercè Torres2, Jordi Eras2

The University of Lleida-DBA CenterThe University of Lleida

Ethyl fatty esters (EFE) from coconut oil have broad applicationsn the flavouring and fragrance industries. A solvent-free synthesisf EFE using fungal resting cells was conducted. A reliable and fastnalytical method was needed to optimize the biocatalytic process.he method had to allow the quantification of the starting materialTAG), the various intermediates (monoacylglycerols–MAG- andiacylglycerols–DAG-) and the EFE.

The analysis of acylglycerols and EFE has been carried out bymploying analytical techniques such as high performance liquidhromatography (HPLC), and gas–liquid chromatography (GLC).owever, these methods are time-consuming. Recently, nuclearagnetic resonance spectroscopy (NMR) has been proposed in the

reparation of biodiesel and DAG as an alternative analytical tool1,2]. The usefulness of NMR has been increasingly recognized forts non-invasiveness and rapidity to detect a wide range of com-ounds that can be detected in a single measurement (spectrum),hereas little or no need for sample pre-treatment is required.

In the present study, a single NMR experiment was used as a fastnd effective method for studying the progress of the biocatalyticynthesis of EFE. A new algorithm was developed to determine theontents of TAG, DAG, MAG, EFE and free fatty acids in the crudeamples resulting from various experimental conditions. Resultsere in accordance with those resulting from GLC.

eferences

].Hatzakis E, Agiomyrgianaki A, Kostidis S, Dais P. J Am Oil Chem Soc2011;88:1695–708.

].Prakash P, Aulakh SS. J Basic Microbiol 2011;51:607–13.


A-33

nhancing hydrocortisone transformation to6a-hydroxy hydrocortisone by Streptomyces roseochro-ogenes

aola Diana ∗ , Odile Francesca Restaino, Mariacarmela Marseglia,aria Giovanna Borzacchiello, Chiara Schiraldi

Second University of Naples

In the last years the biotechnological production of steroids byicrobial transformations has became a common practice and a

eliable method to produce structural tailored-cut molecules alson large scales. Hydrocortisone is a pharmaceutical active moleculeenerally used as drug; its hydrolylated forms, like 16a-hydroxyydrocortisone, have higher and more efficient anti-inflammatory

BIOCATALYSIS

ctivity. Streptomyces roseochromogenes could be used as microbialhole cell catalyser for the biotechnological transformation ofydrocortisone to 16a-hydroxy hydrocortisone by using fermen-

ation technologies. Previous studies demonstrated the possibilityo obtain 0.508 ± 0.01 g·L−1 of 16�-OH-HC in 2-L pulsed batchermentations at 30 ◦C and pH 7, by using a malt extract- andeast extract-based medium. In this study the influence on theonversion ratio and the by-product formation of growing con-itions, like pH and temperature, was investigated in both shakeask experiments and 2-L batch fermentations. Once determinedhe optimal conditions for both bacterial growth and steroid con-ersion,different feeding strategies and substrate addition profilesere exploredin pulsed batch fermentations, and a 16�-OH-HCaximum production of 0.630 ± 0.02 g·L−1 was reached. Fed-

atch experiments, carried out scaling-up the process in a 15-Lermentor, allowed to reach a maximum of 0.804 ± 0.05 g·L−1

f 16�-OH-HC, while further investigations will be performed torive the process on industrial scales.


A-34

mmobilization of cells and enzymes to PVA gel

artin Rebros1,∗ , Michal Rosenberg1, Radek Stloukal2

Slovak University of TechnologyLentiKats

Compared to conventional free cell and enzyme processesmmobilized one offers several important advantages such as: theossibility of repetitive use of immobilized cells and enzymes, the

mprovement of enzyme stability, the reduction of non-productiveell growth phases, faster reaction rates at increased cell densitynd higher yields. A veryeffective and useful matrix for entrapmentmmobilization is polyvinyl alcohol (PVA). The gelation of PVAydrogel is based on partial drying at room temperature and there-

ore is very gentle to both types of biocatalysts. Due to cell growthithin the immobilizates the volumetric productivities may grad-ally increase and reached higher values compared to free cellrocesses. By immobilization enzymes improved their stability.hese phenomena were successfully applied to various whole-cellnd enzyme biocatalysts.

Acknowledgement: This work was done during implemen-ation of the project Development of Competence center foresearch and development in molecular medicine, ITMS code6240220071, supported by the Research and Development Oper-


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A-35

creening of Lipase Production Among Differenticroorganisms

uygu Elif Yılmaz1,∗ , H. Tansel Yalcın2, Nihat Alpagu Sayar1

Marmara UniversityEge University

The design and manifecture of single enantiomers of chiralntermediates have been attracting attention in the pharmaceu-ical industry during the last decades. Single enantiomers thatre potential biocatalysts due to their higher degree of enantio-electivity and regioselectivity are usually prepared by chemicalatalysis.

L-amino acid esters are promising intermediates for variousctive drug components and their catalytic biosythesis throughransesterification reactions could be a rational alternative way.esides, they can be effectively producted by using lipase fromarious microorganisms as a biocatalyst. The selection of the mostppropriate organism among different strains can be a valuabletarting point for this study. Various isolates from various potantialicroorganisms which differ significantly from each other, may

xhibit different catalytic effectiveness.The microbial isolates for this study are maintained by monthly

ransfers on MGYP agar slants and stored at + 5◦C. For the pro-uction stage, microorgansims are transferred to MGYP precultureedium. After overnight incubation, this culture is inoculated to

roduction medium including different types of oil. The culture isncubated in appropriate conditions. Samples are taken at differentime intervals to determine the growth and lipase activity.

The enzyme activity is shown by p-nitrophenyl palmitatepNPP) as substrate. P-nitrophenol is obtained from pNPP andbsorbance is measured spectrophotometrically against an enzymeree sample. One unit of lipase activity (U) is defined as the amountf enzyme which liberates 1 �mol p-nitrophenol/min under assayonditions. Protein is measured by the Coomassie Blue G-250 bind-ng method using bovine serum albümin as standard.


A-36

cutinase from Fusarium oxysporum with potential forET surface modification

vangelos Topakas ∗ , Efstratios Nikolaivits, Maria Kanelli, Paulhristakopoulos

National Technical University of Athens

Cutinases are small extracellular serine hydrolases whose natu-al function is the hydrolysis of the polyester cutin. Their ability toydrolyze a wide range of substrates (from low molecular weightsters to high molecular weight polymers) makes cutinases veryseful biocatalysts for various applications. In the present study,
cutinase (FoCut5a) from the ascomycete fungus Fusarium oxys-
orum was functionally overexpressed in Escherichia coli BL21,arboring pET22b(+)-cut16606. In order to improve enzyme sta-

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ility, the folding of the expressed protein took place also in theeriplasm in addition to cytosolic protein production by cloningut16606 gene downstream of the pelB signal peptide. The het-rologous expression was induced with IPTG in 16оC for 20 hf incubation. The recombinant cutinase was purified from theytosol or culture supernatant using immobilized-metal affinityhromatography (IMAC), resulting in a monomeric protein of ca.3 kDa that is optimally active at 40 ◦C.

FoCut5a cutinase was tested for its potential use in sur-ace modification of PET fabrics, with the intention to increaseheir hydrophilicity and to improve their properties. Towardshis direction, the enzymatic hydrolysis of two model sub-trates bis(benzoyloxyethyl)terephthalate (1) and commercialis(2-hydroxyethyl)terephthalate (2) was attempted, in order tovaluate the capability of the recombinant cutinase in PET modifi-ation. FoCut5a succeeded in hydrolyzing both models, releasingis(2-hydroxyethyl)terephthalate and benzoic acid from substrateand a bis(2-hydroxyethyl)terephthalate derivative in case of sub-

trate 2. Experiments are in progress for the surface modificationnd functionalization of PET fibers.


A-37

ynthesis of biological active compounds using carbohy-rate esterases as biocatalysts

aul Christakopoulos1,∗ , Io Antonopoulou1, Evangelos Topakas2

Luleå University of TechnologyNational Technical University of Athens

Various fungal and bacterial carbohydrate esterases representppealing biocatalysts that have the ability not only to deconstructlant biomass but also to modify compounds with a potential use

n food, cosmetic and pharmaceutical industries. Feruloyl esterasesFAEs, E.C. 3.1.1.73) have been proved promising candidates forhe enzymatic synthesis of antioxidants allowing more flexiblerocess configurations. Among the advantages they provide arese of lower temperatures (50-60 ◦C) comparing to the coun-erpart chemical process (150оC), one step production of oneroduct instead of mixtures and no need of by-product and cata-

yst residues removal in order to produce clean and high qualityubstances. Glucuronoyl esterase (GE) synthetic ability needs to bexplored towards the production of alkyl branched glucuronic aciderivatives which are non-ionic surfactants and have good surfaceroperties, including biodegradability. In addition, due to theirastelessness, non skin-irritation and non toxicity, these bioactiveompounds find diverse uses in the cosmetic and pharmaceuticalndustries.

Aim of this work is the development of competitive andco-friendly bioconversions based on transesterification reactionsatalyzed by FAEs and GEs, for the production of moleculesith antioxidant activity, such as phenolic fatty and sugar esters.
he synthesis of four biological active compounds (prenyl fer-late, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, andlyceryl ferulate) was evaluated using recombinant FAEs fromyceliopthora thermophila and Fusarium oxysporum, while the

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A-38

iodegradation of esfenvalerate by bacteria from Brazil-an biome mangrove

ndré Luiz Meleiro Porto1,∗ , Willian G. Birolli 2, Eloá B. Meira2,itschkeMarcia2, Luciene P.C. Romão3

Institute of Chemistry of São Carlos - University of São PauloInstituto de Química de São Carlos, Universidade de São PauloDepartamento de Química, Universidade Federal de Sergipe

Esfenvalerate is a widely used pyrethroid insecticide. Thisork aimed the biodegradation of the commercial formulationf esfenvalerate (EsfCom) by environmental bacteria isolate fromrazilian biome mangrove, especially from turfa soil. The turfaoil is a material of plant origin, partially decomposed, foundn layers, usually in swampy areas and mountains. The fouracterial strains (P5CBNB, P5MNB, P8CNB, R5RaNB) assessedn this work were able to grow in a solid and liquid culture

edium (nutrient agar) in presence of esfenvalerate (100 mg.L−1).he insecticide and its main metabolites [3-phenoxybenzoic acidPBAc), 3-phenoxybenzaldehyde (PBAld), and 2-(4-chlorophenyl)--methylbutyric acid (ClAc)] were quantified. It was observed thatll the evaluated strains promoted the esfenvalerate biodegra-ation after 14 days.The residual esfenvalerate ranged between03.0 mg.L−1 (similar to method recovery) and 41.6 mg.L−1, whilehe PBAc formation was 8.1-1.2 mg.L−1, ClAc was 10.6-0.0 mg.L−1.BAld was not detected in any of the biodegradation experiments.he EsfCom biodegradation promoted the accumulation of PBAcnd ClAc, which are considered toxic compounds. The formedBAld was completely transformated by strains and it was the mainetabolite in the abiotic degradation. It was observed that some

trains were efficient in esfenvalerate biodegradation and might besed in bioremediation. A biodegradation pathway for esfenvaler-te by bacteria isolated from turfa soil was proposed.

Acknowledgements: FAPESP, CNPq, CAPES, USP-SGA


A-39

ipase Catalyzed Esterification Reactions–A Kineticodel

eyda Kula ∗ , Nihat Alpagu Sayar

Marmara University Bioengineering Department

Biocatalysis has attracted important interest in chemistry
nd engineering fields. Substrate specificity, enantioselectivity,egiospecificity, chemoselectivity and soft reaction conditions aremportant advantages for enzyme-based synthesis in an indus-
iwi

BIOCATALYSIS

rial process. Compared to chemical catalysts, the limited ratend low throughputs are amongst the challenges which needo be addressed for broader application of biocatalysis. And alsoiocatalyst and process development require expensive and timeonsuming experimentation.

Mathematical modeling and simulation tools will be used asolution for process development duration problems. The use ofodel based techniques will facilitate the reduction of unneces-

ary experimentation accelerate optimisation and automation ofrocesses, resulting in a reduction in cost and time.

Enzyme-catalyzed esterification has gained increasing attentionn many applications, due to the significance of the derived prod-cts. More specifically, the lipase-catalyzed esterification reactionsave attracted research interest during recent years, due to an

ncreased use of organic esters in biotechnology and the chemicalndustry.

Selective substrate and desired product have also significantalue for scientific field. The final products may be new moleculesith different characteristics and properties.

We aim to focus on kinetic and mathematical modeling of aipase catalyzed esterification reaction as a model system. Esterifi-ation will be carried out with various substrates such as differentmino acids and carbohydrates.

The kinetic model will be developed using Matlab program-ing environment with extra possible use of Mathematica

oftware. Depending on the type of process designed for the ester-fication reaction a process model will be developed using theinetic model as a basis.


A-40

luconobacter oxydans used to production of natu-al aroma - 2-phenylacetic acid in immobilized systemLentiKats form)

onika Vidová1,∗ , Ivana Slezáková2, Martin Rebros 2, L’udmilristofíková2, Michal Rosenberg2

Institute of Biotechnology and Food Science, Slovak University of TechnologyInstitute of Biotechnology and Food Science, Faculty of Chemical and Foodechnology, Slovak University of Technology

System of generation of natural aromas using G. oxydans innteresting in many cases. Acetic acid bacteria G. oxydans specif-cally oxidates in unresting condition 2-phenylethanol PEA intohenylacetic acid PA on membrane surface, so product is eas-

ly remove to reaction mixture which significantly facilitates theeparation. Both PEA and PA have aromatic properties and iniotransformation step with whole cells biocatalysts change kindf flavor (from rose to honey like) and physical properties also.or highlighting is the fact that biomass in fermentation step isbtaied by using renewalbe source, cheap waste product - glyc-rol. Immobilization of G. oxydans into biocompatible polyvinyllcohol gel in form of LentiKats may increase the benefits of prepar-
ng natural flavorings repeated using of biocatalysts cells. Theork presents comparison of repeated bioconversion with free and
mmobilized cells, benefits of immobilized system and measured


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IOCATALYSIS

echnologically important factors - the effect of pH, oxygen, differ-nt concentrations of the substrate and also scale-up experimentsn controlled fermentation unit.

Keywords: G. oxydans, 2-phenylethanol, 2-phenylacetic acid,mmobilization, LentiKats, glycerol

Acknowledgment: This study was supported by the grantf Agency for Research and Development of the Slovak RepublicTMS APVV-0302-10 and by the grant of VEGA - Scientific Grantgency of the Ministry of Education, Science, Research and Sportf the Slovak Republic VEGA 1/0229/12.


A-41

nzyme mixture production from Pycnoporus sanguin-us DMSZ 3024 using a lignocellulosic waste, Hazelnutusk: A case study for laccase and cellulase

rkun Pinar1,∗ , Basak Kochan2, Nihat Alpagu Sayar2, Kübra Karaos-anoglu2, Dilek Kazan2

Marmara University, Bioengineering DepartmentMarmara University

Introduction: Hazelnut husk is a promising important lig-
ocellulosic material for fermentable sugar production. According
o Fiskobirlik, 774,000 to 1,032,000 tons of hazelnut husk are leftn fields as waste or incinerated. In different methods, cellulosend hemicellulose are converted into fermentable sugars by the

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retreatment of lignocellulosic waste and hydrolysis. In this con-ext, hydrolysis must be successful, inexpensive, and have lownergy requirements with lower fermentation time and less by-roducts. In hydrolysis, it is very difficult to obtain higher yieldf fermentable sugars due to lignin. Sugars obtained with enzy-atic hydrolysis can be fermented easily because of the relative

bsence of by-products and avoidance of undesirable operatingonditions. Pycnoporus sanguineus is a white rot fungus with highignocellulolytic potential.

Aim: Production of enzyme mixture (laccase and cellulase)rom P. sanguineus DMSZ 3024 by the hydrolysis of hazelnut husks aimed in this study.

Methods: Both laccases that catalyze the oxidation of ligninelated compounds and cellulases that hydrolyse cellulose tobtain smaller �-glucose oligomers and �-D-glucose were producedrom P. sanguineus by the hydrolysis of hazelnut husk. Enzyme

ixture and hazelnut husk concentration for hydrolysis condi-ions were optimized at different ratios and reaction times. Allnzyme activities were measured. Sugar and total sugar contentere determined by the phenol-sulfuric acid and DNS method.

Results and Conclusion: An appropriate method for hydrol-sis of hazelnut husk with enzyme mixture using, P. sanguineus wasbtained to overcome hydrolysis problems.

Acknowledgement: This work was supported by the Turkish


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B-01

inimization of Bacterial Contamination with Higholid Loading during Ethanol Production from Lignocel-ulosic Materials

ofoluwake Ishola1,∗ , Tomas Brandberg2, Mohammadaherzadeh2

Swedish Centre for Resource Recovery, University of BoråsUniversity of Borås

Abstract Ethanol is the most important renewable fuel in theransportation sector. Its production from lignocellulosic mate-ials, commonly referred to as second generation ethanol, isonsidered more attractive than production from starch and sugarrops. Bacterial contamination by lactic acid-producing bacteria istill a major problem during ethanol production processes. Bacteriaompete with the yeast by consuming the sugars and the nutrientsequired by the yeast for efficient ethanol production. This oftenauses substantial economic losses at industrial fermentations.n this study, without any sterilization of the substrate, simul-aneous saccharification and fermentation (SSF) was performedsing cellulase Cellic® Ctec2 enzyme for hydrolysis and Baker’seast, Saccharomyces cerevisiae, was used as the fermenting organ-sm with different loads of suspended solids - 8%, 10% and 12%.

ith 8% and 10% SS, there was a significant contamination, whichaused consumption of both hexoses pentose sugars in the fer-entation medium, this resulted in lactic acid concentrations of

3 g/L and 36 g/L from 10% SS and 8% SS respectively. In con-rast, only 2.9 g/L lactic acid was observed with 12% SS. An ethanoloncentration of 47 g/L was produced from high solid loading of2% SS while just 26 g/L and 23 g/L were produced from 10% and% SS respectively. Our results show that SSF with 12% SS hasn increased concentration of inhibitors, particularly acetic acidhich selectively inhibited the bacterial growth without affecting

he metabolic activities of the yeast during the fermentationKeywords Bacterial contamination; Lignocellulosic ethanol;

accharomyces cerevisiae, lactic acid.


B-02

reeding low temperature resistant Camelina sativa foriofuel production

lorentina Matei1,∗ , Florentina Sauca2, Paul Dobre3, Stefanaurcoane2

University of Agronomical Sciences and Veterinary Medicine Bucharest, Fac-lty of BiotechnologiesCentre of Microbial Biotechnology Biotehgen, Bucharest, RomaniaUniversity of Agronomical Sciences and Veterinary Medicine Bucharest, Fac-lty of Agriculture, Bucharest, Romania

In the European effort to produce sustainable aviation fuel ouream is involved in a FP7 project (ITAKA) in breeding activitiesf Camelina sativa L. as main feedstock. One of our purposes it

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BIOFUELS, BIOCHEMICALS AND BIOENERGY

s to obtain a variety with high productivity and resistant to lowemperatures specific to continental winters as can occur in Roma-ia. This variety may be seeded as autumn culture under minimal

illing conditions.As starting material have been used a local camelina cultivar

esistant to low temperatures and an international variety GP202ith high productivity and oil content. It has been taken intoccount classical approach, the use of immature embryo rescueechnique. The first hybrid generation was obtained by castra-ion and pollination; the immature embryos were cultivated in

S medium w/o hormones; new plantlets were cultivated till fullaturity under greenhouses conditions. The obtained seeds have

een used in open field for other randomized hybridization.The new hybrid registered the following characteristics: the

lant lengths and the branching level are non-significantly dif-erent compared to the parental lines (ANOVA tests); in terms ofroductivity, GP202 variety kept its top position, while the newybrid has proven top position for oil content (2.13% higher in

otal fat). Regarding the winter resistance, the hybrid has registered0% more survived plants in open land. As a remark, during thexperiments GP202 showed the highest sensibility to the mildewttack. The further aim of the work is to fix the new characters inrder to homologate the new variety.


B-03

dvanced Biomass Value: Microalgae biomass as a newource of sustainable aviation biofuels and lubricantroduction

elix Bracharz ∗ , Jan Lorenzen, Farah Qoura

TUM, Industrial Biocatalysis

The aviation industry grows 5% p.a. and by 2020 has to complyith strict governmental emission standards that mandate a 20%O2 reduction compared to the emission levels measured at 2005.ogether with the eminent end of fossil resources these driversorce the development of sustainable aviation fuel alternativeshat are carbon neutral and in compliance fuel standard regula-ions such as Jet A. In this study a new, mass-and energy efficientlgae biorefinery concept for the integrated production of aviationuels, industrial lubricants and CO2 adsorbing building materialsas been developed. The process chain is based on production of

ast growing microalgae biomass containing up to 20%w/w lipids.ubsequently, algae lipids are separated from the biomass frac-ion and converted to high performance, high value lubricantsy targeted functionalisation using a cascade of optimized biocat-lytic processes. The biomass residue is enzymatically hydrolysednd used as a fermentation substrate for oleaginous yeast strains.ince these fast growing, oleaginous yeasts can accumulate up to0% w/w lipids, they are the ideal biomass base for the produc-ion of drop-in aviation fuels. Conversion of wet yeast biomass
s accomplished using a streamlined thermochemical process thateatures optimized heterogenous catalysts. In this process carbonich coke is a residue of the thermochemical biomass processing.his residue stream is used as a settling modifier in the production

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f CO2 adsorbing building materials. The integrated biorefineryrocess does not produce any waste streams and adds value tovery process intermediate.


B-04

nzymatic production of biodiesel that avoids glycerols byproduct, by using immobilized Rhizopus Oryzaeipase

arlos Luna1,∗ , Cristobal Verdugo2, Enrique D. Sancho3, Diegouna4, Juan Calero4, Alejandro Posadillo5, Felipa M. Bautista4,ntonio A. Romero4

University of CordobaCrystallographic Studies Laboratory, Andalusian Institute of Earth Sciences,SICDepartment of Microbiology, University of CordobaDepartment of Organic Chemistry, University of CordobaSeneca Green Catalyst S.L

Immobilized Rhizopus oryzae lipase (ROL) was used as bio-atalyst on different supports in the selective transesterificationeaction of sunflower oil with ethanol to generate a new secondeneration biodiesel. As ROL it was applied a low cost pow-ered enzyme preparation from Biocon®-Spain (BIOLIPASE-R),multipurpose additive used in food industry. In this respect,

t was carried out a study to optimize the support used andhe immobilization process, as well as the best pH conditionsn each case. It has been also evaluated the physical adsorp-ion of this enzyme on a demineralised sepiolite as well as theovalent immobilization of this lipase on amorphous AlPO4 sup-ort by using two different linkers (p-hydroxybenzaldehyde andenzylamine-terephthalic aldehyde, respectively).On the otherand, the resulting new biofuel, already patented as ECODIESEL®

s composed by a mixture of fatty acid ethyl esters and monoacyl-lycerols (FAEE/MG) blended in a 2/1 molar relation. This noveliofuel, which present the advantage of integrating glycerol asonoacylglycerols (MG) into biofuels composition, exhibits sim-

lar physicochemical properties than the conventional biodieselnd it is produced through a process which minimizes wasteeneration and maximizes efficiency. Finally, one of these immo-ilization processes of lipases not only was effective with vegetalils, it has been even successfully carried out the transesterifica-ion reaction with animal fat from butchery wastes, using thisiocatalyst covalently immobilized with p-hydroxybenzaldehyde,chieving a viable and functional biofuel from low quality raw
aterials as animal waste.

f3ioeflsTl



B-05

he development of biodiesel production using lipaserom mutant and selected yeast

ree Rittiboon ∗ , Wannisa Pansuk, Marisa Jatupornpiput

King Mongkot’ s Institute of Technology, Ladkrabang

The aim of our research was to select and characterise yeastsolates from soil and other materials produced in the oil palmndustry. Of the strains isolated from palm kernels after com-ression, strain SLP27 produced the highest lipase activity (0.28nit/ml). Strain SLP27 was mutated sequentially with ultraviolet

ight for 38 seconds. The resulting strain, UV79, was mutated withthyl methane sulfonate for 58 minutes to produce strain EM107,hich was then mutated with gamma rays at 2 kGy to yield strainAM47. The maximum lipase activities of these strains were 0.30,.36 and 0.70 unit/ml, respectively. Lipase from GAM47 could besed as a catalyst in the transesterification reaction for biodieselroduction. This selected strain was identified to species level bynalysis of the D1/D2 domain of 26S ribosomal RNA sequence asandida orthopsilosis.


B-06

roduction of 3-hydroxybutyrate from waste biomass byetabolically engineered Escherichia coli

ohan Jarmander1,∗ , Mónica Guevara1, Mariel Perez Zabaleta1,ustav Sjöberg1, Jaroslav Belotserkovsky1, Jorge Quillaguamán2

en Larsson1

Industrial biotechnology, School of biotechnology, KTHCenter of biotechnology, Faculty of science and technology, San Simón Uni-ersity

There is a vast interest in establishing biorefineries that relyn microbial conversion of biomass for production of fuels andalue-added compounds. Especially waste biomasses, such a lig-ocellulose waste from e.g. agriculture, and municipal food wastere, due to their low cost, attractive substrates for productionf biochemicals. Escherichia coli is one of the most commonlysed organisms for production of recombinant products since it

s well documented, easily manipulated and fast growing in sim-le media. In this work we aim to use E. coli for production of thehiral compound 3-hydroxybutyrate from waste biomass. This wasone by introducing part of the pathway to poly-hydroxybutyraterom the halophilic bacterium Halomonas boliviensis into E. coli.-hydroxybutyrate is an industrially important compound ast can be utilized as a drug, as well as in synthesis of vari-us co-polymers. By employing flux modelling and metabolicngineering, the objective is to identify how the internal
uxes and concentrations of metabolites and co-factors can behifted towards an increased productivity of 3-hydroxybutyrate.his knowledge is also applied in the process design, where
imiting one or several elements in the cultivation medium

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an have the desired effect of increased carbon flux towards-hydroxybutyrate.


B-07

chievement of a biofuel-like biodiesel by regioselectiveransesterification of sunflower oil with mucor mieheiipase

uan Calero1,∗ , Juan Calero2, Diego Luna2, Enrique D. Sancho3,arlos Luna2, Cristóbal Verdugo4, Alejandro Posadillo5, Felipa M.autista2, Antonio A. Romero2

University of CordobaDepartment of Organic Chemistry, University of CórdobaDepartment of Microbiology, University of CórdobaCrystallographic Studies Laboratory, Andalusian Institute of Earth Sciences,SICSeneca Green Catalyst S.L

In previous researches, we have developed a biofuel that avoidhe production of glycerol as by-product. This biofuel is similaro the conventional biodiesel, being in the same way applicableo diesel engines. Thus, glycerol is kept as monoglyceride (MG),ogether to two fatty acid ethyl esters (FAEE) molecules. In thisespect, this biofuel is obtained by a partial ethanolysis of sun-ower oil with Mucor Miehei lipase as biocatalyst.

Results obtained by using M Miehei lipase have shown thathis lipase is an efficient biocatalyst in the 1,3-selective enzy-

atic ethanolysis reaction of triglycerides. Thus, reactions wereerformed to determine the optimal conditions, such as amountf lipase, volume of NaOH 10 N aqueous solution, temperature andil/ethanol molar ratio. It was always used 12 mL of sunflower oil,n a 25 mL round bottom flask, with a conventional magnetic stir-er at 300 rpm, during 2 h. Finally, a study of reuses is also carriedut.

The optimized conditions obtained were 3.5 mL of absolutethanol (oil/ethanol molar ratio 1/6), 37.5 �l of NaOH 10 N solu-ion, temperature of 30 ◦C and 15 mg of M Miehei lipase. Operatingnder these experimental conditions, eighteen successive reac-ions were efficiently carried out after recovering the lipases byentrifugation.

cknowledgements

Grants from the Spanish Ministry of Economy and Com-etitiveness (Project ENE 2011-27017), Spanish Ministry ofducation and Science (Projects CTQ2010-18126 and CTQ2011-8954-C02-02), FEDER funds and Junta de Andalucía FQM 0191,
O8-RMN-03515 and P11-TEP-7723.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1834htdfMfrf


B-08

he usage of carrot pomace as a feedstock for bioethanolroduction

kinDemiray ∗ , Sevgi Ertugrul Karatay, GönülDönmez, SedatDönm

Ankara University

eywords: Bioethanol; Saccharomyces cerevisiae

In today’s world there is urgent need for alternative energyources due to rapid depletion of the fossil fuels. Renewable sourcesre good candidates instead of fossil fuels because of their environ-entally friendly and less toxic properties. It has been estimated

hat bioethanol will be the most widely used renewable source inhe near future. The usage of agricultural wastes for bioethanolroduction has some advantages such as lower production costs.

Therefore in this study we investigated the potential of carrotomaces as a feedstock for bioethanol production by using Saccha-omyces cerevisiae. For obtaining fermentable sugars carrot pomacesere hydrolysed in 1.5% H2SO4 (v/v). The yeast growth, initial and

onsumed sugar concentrations were monitored periodically. Theioethanol concentration was determinated with gas chromotog-aphy. The microbial growth media containing carrot pomace ascarbon source and different nitrogen sources were prepared to

ncrease the bioethanol production.It has been observed that yeast cells used 35.4 g/L sugar in

he medium containing 0.5 g/L KH2PO4 and1 g/L (NH4)2SO4. Asresult the media that were prepared with carrot pomace sug-

rs supported the growth of Saccharomyces cerevisiae. These resultshow that carrot pomaces are suitable feedstocks for bioethanolroduction.


B-09

actic acid production from lignocellulosic hydrolysatesnder non-sterilized conditions using Bacillus coagulansPE22

inhuaWan ∗ , Yuming Zhang, Xiangrong Chen, Benkun Qi, Yi Su

Institute of Process Engineering, Chinese Academy of Sciences

A thermophilic lactic acid (LA) producer was isolated anddentified as Bacillus coagulans strain IPE22. The strain showedemarkable capability to ferment pentose, hexose and cel-obiose, and was also resistant to inhibitors from lignocellulosicydrolysates. Based on the strain’s promising features, it was used

o produce lactic acid (LA) from mixed sugar and wheat strawydrolysates under non-sterilized conditions. In order to eliminate

he sequential utilization of mixed sugar and feedback inhibitionuring batch fermentation, membrane integrated repeated batchermentation (MIRB) was used to improve LA productivity. With

IRB, a high cell density was obtained and the simultaneous
ermentation of glucose, xylose and arabinose was successfullyealized. The separation of LA from broth by membrane in batchermentation also decreased feedback inhibition. MIRB was carried

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ut for 5 cycles repeated culture using wheat straw hydrolysates29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose) as car-on source, a 2.33-folds increase of LA productivity (2.35 g/L/h)as obtained as compared with conventional batch fermentation.


B-10

olid state fungal fermentation as a biological pre-reatment strategy to convert lignocellulosic raw mate-ial into fermentable sugars

henyu Du ∗ , Nattha Pensupa, Siobhan Knight, Jwan Abdullah

The University of Nottingham

Pre-treatment is one of the key challenges in the conver-ion of lignocellulosic raw materials into bioethanol. Variousre-treatment strategies have been developed, such as dilute acidre-treatment, alkali pre-treatment, steam explosion and ammo-ia explosion pre-treatment. These methods are energy intensives these are normally operated at high temperature, high pres-ure or high chemical loading rate. Inhibitor formation duringhe pre-treatment is another concern in these methods, whichignificantly impacts the efficiency of the following fermentationrocesses.

In the University of Nottingham, we developed a solid-stateungal fermentation-based pre-treatment strategy to convert ligno-ellulosic raw materials into a fermentable hydrolysate. Aspergillusiger was firstly cultured on biomass for production of cellulolyticnzymes and then the biomass was hydrolyzed by the enzymeolution into a fermentable hydrolysate. Various lignocellulosicaw materials have been tried, including wheat straw, willow, mis-anthus, palm oil tree branches, napier, sago extract and municipalolid waste. In solid-state fermentations of most of these rawaterials, around 4-10 U/g cellulase activity could be obtained

fter 5 days’ culture. The acid or alkali modification of biomasst mild condition improved the cellulase production to over 10/g. The addition of yeast extract (0.5% w/v) and minerals sig-ificantly improved the cellulase production, for examples, to4 U/g in wheat straw, 22 U/g in napier. The fungal culture fil-rate from the solid-stage fermentation showed higher cellulolyticydrolysis ability then the commercial cellulase Ctec2 at the samenzyme loading rate. Moreover, no inhibitor was detected in theydrolysate examined.


B-11

etabolic Engineering of E. coli for the production oflkanes

ong Jun Choi ∗ , Sang Yup Lee


Our increasing concerns on limited fossil fuels and global envi-onmental problems are urging us to develop sustainable biofuels

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rom renewable resources. Although microbial production of dieselas been reported, production of another much demanded trans-ort fuel, gasoline, has not yet been demonstrated. Here we reporthe development of platform Escherichia coli strains that are capa-le of producing short chain alkanes (gasoline). The b-oxidationathway was blocked by deleting the fadE gene to prevent theegradation of fatty acyl-CoAs generated in vivo and the activityf 3-oxoacyl-ACP synthase (FabH) was enhanced to promote thenitiation of fatty acid biosynthesis by deleting the fadR gene. A

odified thioesterase was employed to convert short chain fattycyl-ACPs to the corresponding FFAs, which were consequentlyonverted to short chain alkanes by the sequential reactions ofatty acyl-CoA synthetase, fatty acyl-CoA reductase and fatty alde-yde decarbonylase. The final engineered strain produced up to80.8 mg l−1 of SCAs consisting of nonane (327.8 mg l−1), dode-ane (136.5 mg l−1), tridecane (64.8 mg l−1), 2-methyl-dodecane42.8 mg l−1) and tetradecane (8.9 mg l−1) together with smallmounts of other hydrocarbons.

[This work was supported by the Advanced Biomass Researchnd Development Center of Korea (ABC-2010-0029799) throughhe Global Frontier Research Program of the Ministry of Sci-nce, ICT and Future Planning (MSIP) through the Nationalesearch Foundation (NRF). Systems metabolic engineering workas supported by the Technology Development Program to Solvelimate Changes on Systems Metabolic Engineering for Biorefiner-

es (NRF-2012-C1AAA001-2012M1A2A2026556) by MSIP throughRF].


B-12

tatistical optimization of critical parameters for alka-ine treatments of canola agricultural residue bydvanced regression model

eung Wook Kim1,∗ , Hah Young Yoo1, Da Un Jung1, Sung Bongim1, Ja Hyun Lee1, Chulhwan Park2

Korea UniversityKwangwoon University

In this study, canola agricultural residue from Honam areaf Korea was pretreated by alkaline reagents (ammonium andodium hydroxide) in order to enhance the enzyme accessibil-ty, and the treatment conditions were optimized by statistical

ethod. Generally, most researches only focused on the enhance-ent of enzymatic digestibility in the process but if not mentioned

bout solid recovery then the overall sugar yield can be reduced.herefore, a novel experimental response (biomass to glucose con-ersion; BtG) which modified from solid recovery and enzymaticigestibility is applied toregression analysis, in current study. As aesult, the optimal conditions were carried out by BtG model andhe sugar yield was improved compared with the previous work.n optimal conditions, the predicted solid recovery, enzymatic
igestibility and BtG were 75.8%, 78.3% and 25.7% at ammo-ium hydroxide treatment and 49.2%, 86.4% and 24.3% at sodiumydroxide treatment, respectively. The experimental results show

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umerically over 90% in general. Finally, under the overall massalance, glucose yield was about 3 fold increased by the treatments.


B-13

iogas production from 3 strains of Napier grass

ramote Sirirote1,2,∗ , Farida Promma2, Dusanee Thanaboripat2

King Monkut’Institute of Technology Ladkrabang, Biogas production from 3trains of Napier grass, (Pennisetum purpureum)Department of Biology, Faculty of Science, King Mongkut’s Institute of Tech-ology Ladkrabang, Bangkok, Thailand

eywords: Biogas; Napier grass; Batch anaerobic digestion

Abstract The aim of this research was to study the potentialsf biogas production from 3 strains of Napier grass (Pennisetumurpureum); i.e, King grass, Napier Pakchong1 and Alafal. Batchnaerobic digestion was performed on five ratios of different grassnd inoculum volume at 1:1, 1:2, 1:3, 2:1 and 3:1, and performedn working volume of 5 L digestors for 45 days at room temper-ture (29-34 ◦C). The results showed that the highest cumulativeiogas production was 22.45, 26.25 and 24.29 L at the ratios of 1:3,:2 and 1:2 for King grass, Napier Pakchong1 and Alafal, respec-ively. Initial pH was 6 - 6.5 and biogas yield were 0.37, 0.53 and.47 L biogas/g VS. The efficiencies for the COD removal were2.8, 76.9 and 85.0% at the best grass inoculum ratios, respec-ively.Therefore, Napier Pakchong1 and Alafalat grass to inoculumatio of 1:2 were selected for further experimentation.The diges-ion was performed on working volume of1 L and adjusted initialH to 7.1 for 36 day. The resulst showed that the cumulative bio-as production were 2.46 and 6.97 L for Napier Pakchong1 andlafal, respectively. Therefore, the results indicated that Alafal hasigher potential for biogas production than King grass and Napierakchong1.


B-14

ontrolled localization of functionally active enzymes tonclusion bodies using leucine zippers

eung-Goo Lee ∗ , Haseong Kim, Bong-Hyun Sung

Korea Research Institute of Bioscience and Biotechnology

Inclusion bodies (IBs) are typically non-functional particlesf aggregated proteins. However, some proteins in fusion withmyloid-like peptides, viral coat proteins, and cellulose bind-ng domains (CBDs) generate IB particles retaining the originalunctions in cells. Here, we attempted to generate CBD IBs display-ng functional leucine zipper proteins (LZs) as bait for localizingytosolic proteins in E. coli. When a red fluorescent protein was
ested as a target protein, microscopic observations showed thathe IBs red-fluoresced strongly. When different LZ pairs with KDsf 20–1,000 mM were tested as the bait and prey, the localization
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f the red fluorescence appeared to change following the affinitiesetween the LZs, as observed by fluorescence imaging and flowytometry. Currently, we are applying the proposed method toonstruct an in vivo matrix to entrap different enzymes in E. coliytosole while maintaining their catalytic activities. In addition,asy detection of localization to IBs provides a unique platformor the engineering and analyses of protein-protein interactionsn E. coli.


B-15

irect biocatalytic conversion of methane-to-methanolsing methane and ammonia-oxidizing bacteria

un Yeol Lee ∗ , In Yeub Hwang

Kyung Hee University

Production of methanol from methane is the first step forchieving methane-based production of a wide range of chemicals.ethanol is a precursor to various chemicals and a liquid fuel that

an be blended with gasoline. In this study, we employed methanend ammonia-oxidizing bacteria to partially oxidize methaneo methanol. To prevent methanol catabolism, thus allowing

ethanol accumulation in the medium, various inhibitors forethanol dehydrogenase were used. Further, sodium formateas supplied as reducing power regeneration because methaneoonoxygenase-catalyzed oxidation of methane to methanol was

imited by reducing equivalents supply.

cknowledgment

This work was supported by the New & Renewable Energy ofhe Korea Insitute of Energy Technology Evaluation and PlanningKETEP) grant funded by the Korea government Ministry of Knowl-dge Economy (No. G031595311).


B-16

ncapsulation of Candida rugosa lipase in chitosaneads as biocatalyst for biodiesel production via non-lcohol route

eri Hermansyah ∗ , Merisa Bestari Faiz, Intan Sipangkar, Ritarbianti

Department of Chemical Engineering, Universitas Indonesia, Depok 16424,ndonesia

Lipase-catalyzed biodiesel production offers many advantagesuch as high level of selectivity and it has good ability to catalyzerganic reactions in aqueous or non-aqueous media. Unfortu-
ately, lipase is expensive and it cannot be reused because it isissolved in reaction media. To overcome the problem, in thisesearch, we immobilized Candida rugosa lipase (CRL) in chitosaneads through encapsulation method. The optimum condition of

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nzyme to support mass ratio, immobilization time and cross-inking agent concentration were investigated in the range of:4 to 1:8, 50 to 150 minutes and 0.6% to 3%w/v, respectively.nzyme concentration was measured through spectrophotometryethod. The highest enzyme loading was 97.24% when the

perating conditions were 1:6 mass ratio of lipase to chitosan,20 minutes immobilization time and 0.6%w/v of cross-linkinggent concentration. The immobilized enzyme then was used asiocatalyst in biodiesel production. Non-alcohol route was useds a route for biodiesel production because it can prevent enzymeeactivation which is commonly found in conventional route.he operating condition for biodiesel production process was7◦ C, 4%w/w biocatalyst and 1:12 mole ratio of used cooking oilas triglyceride source) to methyl acetate (as acyl acceptor). Theiodiesel, fatty acid methyl ester (FAME), concentration was deter-ined using High Liquid Performance Chromatography (HPLC).

hrough 50 hours batch production process, 94.06% biodieselield was achieved. Whereas through continuous process in aacked bed reactor sized 11 mm ID and 150 mm length withml/hour flow rate, 93.67% biodiesel yield was achieved.

Keywords: Biodiesel, immobilization, biocatalyst


B-17

nhancing energy yield through saccharification oforghum bagasse and second generation bio-ethanol pro-uction

eodor Vintila ∗ , Adrian Trulea, Daniela Vintila, Georgeta Pop, Iosifergen, Kornel Kovacs

University of Agricultural Science Timisoara

Sweet sorghum is an important source of sugar that can betilized for ethanol production. Sorghum bagasse resulted afterweet juice extraction can be further processed to obtain morenergy as lignocellulosic ethanol. In our study, we used six com-ercial products consisting of biomass degrading enzymes to

ydrolyze cellulose from pretreated sorghum bagasse and obtainermentable sugars. Tests containing combinations of steam-lkaline, mechanical pretreated bagasse and different enzymesocktails were conducted. The results indicated the combinationf steam-alkaline pretreatment and NS22086 cellulase complexNovozymes) as the most efficient. Next, these conditions werepplied to hydrolyze three types of Sorghum bicolor bagasse (Sugarraze, Jumbo and Fundulea FT132). The hydrolysis rates obtained

n the three sorghum types are between 32% and 40%. Concentra-ion of total sugars and glucose released in hydrolysis buffer was

onitored. The conversion process continued with fermentationf hydrolyzates by Saccharomyces cerevisiae in 500 ml fermentersquipped with NIR sensors (BlueSens) to evaluate in real time thethanol and CO2 concentration. Ethanol concentrations between.65 g·ml−1 and 1.96 g·ml−1 were obtained in fermentation media
f the hydrolyzed bagasse from three sorghum varieties, represent-ng ethanol yields between 330 g·g−1 and 392 g·g−1 reported at DMagasse. Applying biotechnology developed in this study in combi-ation with current sugar extraction method applied in sorghum,
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he overall ethanol production efficiency and energy yield wouldncrease.


B-18

ffect of hydraulic retention time on the performance ofnovel tubular MFC fed with petroleum hydrocarbons

luwaseun Adelaja ∗ , Godfrey Kyazze, Taj Keshavarz

University of Westminster

Pollution of groundwater by petroleum hydrocarbons is aerious threat to human health as the hydrocarbons are toxic,utagenic and carcinogenic. Microbial fuel cells (MFCs) could be

mployed in the treatment of these recalcitrant pollutants withoncomitant bioelectricity generation. For practical applicationhe MFCs would have to be effective, efficient and robust.

This study investigated the performance of a novel tubularFC, operated in a continuous mode at different hydraulic reten-

ion times, HRT, in the range 2.5 to 10 days at room temperature.romate was employed as the catholyte and the inoculum wasn adapted anaerobic microbial consortium. A mixture of ben-ene and phenanthrene was used as the substrate. Total chemicalxygen demand removal efficiencies and peak power densitiesecreased from 74 to 57% and 3.4 to 1.1mW/m2 respectivelyhen HRT was decreased from 10 to 2.5 days.The removal

fficiencies were higher than 90% for both phenanthrene and ben-ene.Bromate removal efficiencies increased from 52.5-78.6% asRT was raised from 2.5-10d.

The outcome of this study suggests the application of MFCsn the simultaneous removal of petroleum hydrocarbons andromates (with concomitant power production) in anoxic envi-onments, especially deep groundwater reservoir. MFC technologyould possibly be a substitute for the more expensive conventionalechnologies such as permeable reactive barrier (PRB) and elec-roremediation which are currently employed in remediation ofydrocarbon pollutants in subsurface environments.


B-19

n integrated lignocellulose-based bioprocessing for theroduction of a generic microbial feedstock

hen-Wei Chang ∗ , ColinWebb

University of Manchester

Conversion of ligno-cellulosic materials to sugars through aurely biological treatment is a key to sustainable chemicals pro-uction. Sequential solid-state fermentation of sugarcane bagasseith soybean hull and enzyme hydrolysis using Trichoderma lon-

ibrachiatum was performed. Microorganism incubation througholid-state fermentation resulted in process, not only producingn abundant enzyme complex, but also degrading the recalcitranttructure of the ligno-cellulosic materials.

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The process includes three stages: (1) Simultaneous decon-truction of recalcitrant lignocellulosic materials and cellulolyticnzyme production through filamentous fugal bioconversionsingTrichoderma longibrachiatum; (2) generic fermentation feed-tock production through enzymatic hydrolysis of fermented solidssociated with fungi autolysis; (3) subsequent fermentation byative Saccharomyces cerevisiaefor ethanol production. This processrovides cost-competitive and environmental friendly alternativeo produce chemical and biofuel from lignocellulosic wastes.


B-20

synthetic biology approach towards improved cellu-olytic activity of Clostridium acetobutylicum ATCC 824

atalin Kovacs ∗ , BenjaminWillson, Nigel Minton

University of Nottingham

Several Clostridia species employ large, self-assembled multi-nzyme complexes, or cellulosomes, for efficient decomposition ofhe plant cell wall cellulosic polysaccharides cellulose and hemicel-ulose. Clostridium acetobutylicum, the best studied of the butanolroducing Clostridia, produces small amounts of a non-functionalellulosome and is therefore unable to grow on cellulose. In ourtudies, we employed synthetic biology approaches to create sta-le C. acetobutylicum strains with the potential to utilise variousellulosic substrates by genome integration of synthetic celluloso-al subunits derived from various cellulosic degrading bacterial

pecies (Clostridia and other species) as well as integrating non-ellulosomal cellulolytic enzymes (bacterial and other sources).e use standardised synthetic parts (optimized DNA sequences) in

ioBrick2 format to assemble a range of synthetic genes encodingellulosomal scaffoldin proteins, glycoside hydrolases (GHs) andynthetic cellulosomal operons. All synthetic genes and operonsre integrated into the C. acetobutylicum genome using the recentlyeveloped allele-coupled exchange (ACE) technology. Heterolo-ous protein expression levels of the synthetic genes and theelf-assembly of the mini-cellulosomes are assayed by Westernlot, native PAGE and enzyme activity. We have demonstratedhe successful expression, secretion, self-assembly and activity ofhe mini-cellulosomes produced by recombinant C. acetobutylicumtrains, providing a platform for the construction of novel strains
ith finely tuned cellulolytic properties

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B-21

tudies on the role of different culture conditions on therowth and biochemical composition of marine microal-ae Nannochloropsis sp

aria Savvidou ∗ , Fragiskos Kolisis

National Technical University of Athens

The climate change, the rising fossil-fuel prices and overallhe societal concerns have raised attention to the sustainableroduction of advanced biofuels, as well as high added valueioproducts such as fatty acids (�-linolenic, eicosapentaenoic,ocosahexaenoic acids, etc.) pigments (carotenoids, ficobilins),itamins and metabolites.

The rapid growth rates, the accumulation of large quan-ity of lipid and other compounds of interest and the abilityo be cultivated in seawater or brackish water on non-arableand make algae an exciting addition to the sustainable fuelortfolio. Microalgae are sunlight-driven cell factories that usehotosynthesis to efficiently and quickly convert CO2 and otherreenhouse gasses into potential biofuels, foods, animal feedsnd bioactive compounds, the cultivation conditions and theole of nutrient media characteristicsare still under investigation.mongst various microorganisms, Nannochloropsis sp., an oleagi-ous eukaryotic marine alga, is well appreciated in aquaculture due

o its nutritional value and the ability to produce valuable chem-cal compounds, such as pigments, proteins and polyunsaturatedatty acids.

The aim of the present study is to quantify some biochemi-al compounds (lipids, chlorophyll a, carotenoids, proteins, totalell biomass) in cultures of Nannochloropsis sp. grown in dif-erent culture conditions. The microorganism is cultivated inhotobioreactors containing sterilized seawater enriched with f/2edium nutrients under standard illumination conditions. The

ffect of parameters such light intensity, pH, sodium bicarbonateupplementation and sodium nitrate concentration on growth andiochemical composition of Nannochloropsis sp. are extensivelytudied.


B-22

ffect of different components of media prepared withugar beet hydrolysate on cell growth and ethanol pro-uction

eltem Yesilcimen Akbas ∗ , Sar Taner

Gebze Institute of Technology

Ethanol is an alcohol made from the fermentation of thearbohydrate or sugar fraction in biomass materials. Sugar beetomposition makes it a potential and attractive raw material forhe production of the second generation bioethanol. The aim
f this research was to assess the usefulness of dilute enzymaticydrolysis of sugar beet molasses and the different media compo-ents for enhancement of cell growth and ethanol production of

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thanologic E. coli strain (FBR5). Sugar beet molasses was hydrol-sed with a dilute sulfuric acid at room temperature overnighto convert sucrose to glucose and fructose, followed by auto-laving. The enhancement of growth and ethanol production oftrain FBR5 were investigated in a variety of growth media compo-ents including nutrients (peptone and yeast extract) or thiaminr/and trace elements. The growth properties of the strain werelso evaluated before and after overliming with Ca(OH)2 treat-ent. Overliming treatment did not affect the growth and the

thanol production. The hydrolysate (diluted to 20%; v/v) mediumncluding nutrients, thiamin and trace elements enhanced the celliomass and ethanol yield.


B-23

otential use of different hydrolyzing methods of potatond corn processing industry wastes for ethanol produc-ion

eltem Yesilcimen Akbas ∗ , Fatma Sumer

Gebze Institute of Technology

Bioethanol produced from renewable sources, such as starchr lignocellulosic materials are promising sources of alternativenergy resources. Potato and corn processing industry waste (PCW)s a zero value waste rich in starch and lignocellulosic material.Theurpose of present research was to investigate the potential usef different hydrolyzing methods of PCW. The cell growth andthanol production of ethanologic E. coli strain (FBR5) were com-ared. PCW was hydrolyzed with different methods by usingifferent concentrations of dilute acid and/or enzymes such asylanase, cellulase or both enzymes. The sugar contents (glucose,ylose and arabinose) of different hydrolysates were investi-ated by Thin Layer Chromatography. The dilute acid treatmentethodology yielded the highest levels of glucose, xylose and

rabinose; 7.6%, 2.7% and 1.8%, respectively. The growth prop-rties and ethanol production of the strain FBR5 were evaluatedy using hydrolysate medium including nutrients, thiamin andrace elements. The experiments resulted in higher cell numbersnd ethanol yield.


B-24

otential application of liquor from sisal pulp hydroly-is as alternative substrate for biosurfactant production

arcia Nitschke ∗ , Claudia Marin Abadia, Joice Kaschuk, Elisabeterollini

University of São Paulo

Microbial-derived surfactants are more environmentally-riendly, biocompatible and biodegradable in comparison withonventionally produced detergents from petroleum sourcesoreover, their production supports the concept of biorefinery.

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uch characteristics have increased the interest in these moleculesowever, their large scale production is currently more expen-ive comparatively to synthetics. The use of cellulosic materialan improve economics of biosurfactant production and alsoontribute to reducing greenhouse gas emissions. This work inves-igates the production of surfactin by Bacillus subtilis using theiquor from sisal (Agave sisalana) pulp hydrolysis as substrate.he liquor deriving from acid and enzymatic hydrolysis of sisalellulose was utilized as carbon source in culture media. Theartially-purified product obtained using acid hydrolysate showedsurface tension (ST) of 29.8 mN/m, interfacial tension (IT) againstexadecane of 5.7 mN/m and a critical micelle concentration

CMC) of 1394.0 mg/L whereas when enzymatic hydrolysate wastilized the product showed a ST of 28.7 mN/m, IT of 3.8 mN/mnd a CMC of 64.0 mg/L. The enzymatic derived liquor demon-trates to be more suitable generating the BS with the best surfacective properties. In conclusion, the liquor from sisal celluloseydrolysis can be explored as an alternative sustainable substrate

or biosurfactant production.


B-25

se of hardwood sulphite spent liquor for acclimatingpolyhydroxyalkanoate storage capacity of a mixedicrobial culture

iogo Queirós1 , Luísa Seuanes Serafim1,∗, Simona Rossetti 2

CICECO, Chemistry Department, University of AveiroWater Research Institute, CNR

Polyhydroxyalkanoates (PHAs) are biodegradable and bio-ompatible biopolymers that emerge as a possible solution asubstitutes of petroleum-based plastics. PHAs can be producedithin the Biorefinery concept, in which wastes and by-productsf numerous industries are used as carbon source. The effectivenessf PHAs production process includes a first stage of selection of aMC with a stable PHAs-producing capacity, which determines

he success of subsequent PHA accumulation step. This could beone resorting to hardwood spent sulphite liquor (HSSL) which

s a complex feedstock originated from the pulp industry andeets the concept of a lignocellulosic-based biorefinery, due to its

bundance and affordability, wide variety and good marketing ofhe bio-based products. To complement the selection of the PHA-toring populations, the evolution of microbial community muste evaluated in order to identify the best producers and determinehe individual relative abundance, allowing for the design of oper-ting conditions favoring the most important PHA-accumulatingicroorganisms.In this project, a MMC collected in a wastewater treat-

ent plant was submitted to Aerobic Dynamic Feeding (ADF)n a Sequencing Batch Reactor (SBR) in order to select PHA-ccumulating organisms using HSSL. The reactor has been
perating for near 500 days in a steady state condition, produc-ng a copolymer, poly(hydroxybutyrate-co-hydroxyvalerate). The
edium, rich in xylose, acetic acid and lignosulphonates, led toselection of a co-dominant culture between Alphaproteobac-

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eria and Betaproteobacteria, determined by fluorescent in situybridization. A clone library was constructed and genera likegrobacterium, Rhizobium, Brachimonas, Acidovarax, Flavobacterium,eadbetterella and Dyadobacter were identified.


B-26

ioethanol Production: Adaptation of Scheffersomycestipitis to Hardwood Spent Sulfite Liquor

na M R B Xavier1,∗ , Cláudio J R Frazão1, Susana R Pereira2, Violetaanches i Nogué3, Luísa S Serafim2, Marie F Gorwa-Grauslund3

CICECO, Chemical Department, University of Aveiro, PortugalChemical Department, University of Aveiro, PortugalDivision of Applied Microbiology, Department of Chemistry, Lund University

Biofuels, which consist of fuels produced from biomass, areuitable renewable alternatives to conventional motor fuels (e.g.asoline, diesel). Bioethanol and biodiesel are the most promisingiofuels. Biofuels can be generated from various raw materials, likeignocellulosic biomass and its derivatives.

Hardwood Spent Sulfite Liquor, HSSL, is the side-product of thecidic sulfite pulping process, and it is rich in sugars (40–45 g.L−1),ainly the pentose sugar xylose that can be converted to ethanol

y yeast Scheffersomyces stipitis. However, HSSL contains toxicompounds (e.g. acetic acid and phenolics) that inhibit yeastetabolism.The aim of this study was the use of evolutionary engineer-

ng to generate a S. stipitis strain with increased tolerance to HSSLnhibitors and maintained xylose conversion rate.

A continuous reactor with increasing HSSL concentrations (20-0% (V/V)) was operated. The final population, POP, obtained after82 generations in HSSL, was characterized and compared to thearental strain, PAR.

By using this approach, improved fermentation performanceas obtained. POP showed a higher xylose consumption rate

0.33 g.L−1.h−1) and maximum ethanol concentration (6.93 g.L−1)han PAR (0.10 g.L−1.h−1 and 1.76 g.L−1, respectively).


B-27

icrobial Consortium with High Cellulolytic ActivityMCHCA) for enhanced biogas production

ukasz Drewniak1,∗ , Krzysztof Poszytek2, Martyna Ciezkowska2

Faculty of Biology, University of WarsawLaboratory of Environmental Pollution Analysis, Faculty of Biology, Universityf Warsaw

In our previous work we have constructed Microbial Consor-ium with High Cellulolytic Activity (MCHCA). This consortium
nvolves twenty two bacteria strains, which were isolated on
inimal salt medium containing carboxymethylcellulose as theole carbon source. Isolates were derived from: sewage sludge,


ydrolyzer of agricultural biogas plant, cattle slurry and manure,nd represents the following genera: Bacillus, Providencia, Ochrobac-rum.

The main aim of this study was to verify whether the con-tructed consortium can degrade lignocellulosic material andnhance the production of biogas from maize silage and cattleanure. The enhancement of the methane production by supple-entation with the MCHCA consortium was monitored in batch

ioreactors with the use of maize silage as substrate and cattleanure as methanogens inoculum. In the first step of the pro-

ess, maize silage (1% d.m.) was pretreated by 5% (v/v) or 10%v/v) MCHCA consortium (with a density ∼108cells/ml) for 72 ht 300C, and then pretreated substrate was subjected to anaero-ic digestion with cattle manure (20% v/v) for 21 days at 370C.uring the experiment the following parameters were monitored:

ellulolytic activity, pH, level of volatile fatty acids and stability ofhe consortium structure by DGGE (denaturing gradient gel elec-rophoresis) analysis. Methane concentration and volume of gasn the culture was measured after 7, 14 and 21 days.

The experimental results showed that constructed MCHCAonsortium can increase the efficiency of biogas production upo 20-30%, when used 5% addition of the consortium.


B-28

roduction of 1,3-propanediol from glycerol by C.utyricum: Optimization of medium composition andinetic studies

ojciech Białas ∗ , Marta Pikuła, Katarzyna Mroczyk, Włodzimierzrajek

Poznan University of Life Sciences

A two-step approach was employed to optimize culture mediumomposition for enhanced production of 1,3-propanediol (1,3-D) by Clostridium butyricum. In the first step, a simple two-levelcreening experimental design was used to evaluate the influencef individual components of the medium on 1,3-PD production.n the second step, a central composite experimental design andesponse surface methodology were employed to derive a statisticalodel describing the impact of yeast extract, potassium phosphate

ibasic and iron (II) sulfate on the 1,3-PD production.The final titer of 1,3-PD produced by C. butyricum cultured in

he optimized medium, with the starting glycerol concentration of0 g/L, was of 38.8 ± 1.23 g/L, resulting in the process yield of 0.49.he results obtained under the optimal fermentation conditionsere used as a starting point for development of a kinetic modelescribing the glycerol utilization, 1,3-PD and biomass production


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B-29

ignin utilization by Bacillus sp. associated with therowth enhancement and the molecular weight distri-ution change of lignin

yeongtaek Gong1,∗ , Han Min Woo1, Youngsoon Um1, Tai Hyunark2

Korea Institute of Science and TechnologySchool of Chemical and Biological Engineering

Lignin is one of the major components in lignocellulosiciomass and is the most abundant natural aromatic polymer. Totilize lignocellulosic biomass efficiently, lignin-degradation is an

mportant issue; however, lignin is known to be recalcitrant foriodegradation. Previously, we isolated Bacillus sp. GWD275 fromud flat thorough the culture in kraft lignin agar plate and thezure B decolorization screening method for lignin degradation.

n this study, lignin utilization by Bacillus sp. GWD275 was investi-ated by observing growth enhancement in the presence of lignin.lso, the molecular weight distribution of lignin in culture mediaas analyzed by Gel Permeation Chromatography (GPC) during

he growth of Bacillus sp. GWD275 in the presence of lignin. Therowth of GWD275 was compared with Luria-Bertani (LB) and LBupplemented with lignin (1 g/L). The growth in LB supplementedith lignin was increased by 1.8-fold compared with LB mediumnly, implying lignin utilization by Bacillus sp. GWD275. Further-ore, when the defined medium with glucose or xylose without

omplex nitrogen source was used, the growth of GWD275 waslso enhanced in the presence of lignin (0.5 g/L), possibly bysing lignin as a co-substrate for growth. Interestingly, the molec-lar weight distribution of lignin in the cultures with the definededium was shifted to a lower molecular weight portion, while itas shifted to a higher molecular weight portion in cultures withB medium. The results presenting here would provide an insightor efficient use of limited lignocellulosic bio-resources by lignintilization using Bacillus sp. GWD275.


B-30

iosynthesis of nylon precursor dodecanedioic acid fromatty acid

iang-Jung Chien ∗ , Szu-Min Yu

Ming Chi University of Technology

Aliphatic a,w-dicarboxylic acids (DCA) of the type addressed byhis program are used in a wide variety of plastics and other chem-cal applications. The DCA12 produced in the largest quantity>40 MM lb/yr) as a pure chemical intermediate is dodecanedioiccid (C12); it is used in polyamides such as nylon 6,12, whichs noted for high moisture resistance. The dodecanedioic processs based on non-renewable petrochemical feedstocks. The multi-
tep conversion process produces unwanted byproducts such asyclooctadiene and vinyl cyclohexene, which result in yield losses.he nitric acid oxidation step yields NOx, which is either released


o the atmosphere or must be destroyed in a reduction furnace.iotechnology offers an innovative way to overcome the limita-ions and disadvantages of the chemical processes to make diacids.arrowia biocatalyst are able to convert long-chain fatty acidsirectly to long-chain diacids through overexpressing �-oxidationathway and blocking the �-oxidation pathway. This study waseveloped and demonstrated key biocatalyst to produce cost-ompetitive long-chain dodecanedioic acid, DCA12. The focus ofhis study was to increase the rate of conversion of glucose into theorresponding fatty acid feedstocks through overexpress the genef fatty acid synthesis, Acetyl-CoA carboxylase (AccD) and Fattycid synthase (FA-1, FA-2, FB-1), in the yeast biocatalyst (Yarrowiaipolytica). In order to enhance DCA12 content, the Yarrowia-odon acyl-carrier protein thioesterase gene, BTE from Umbellulariaalifoenica, FatB3 from Cocos nucifera were also expressed. Finally,he RNA inference technology was also used to enhance DCA12roduction in this research.


B-31

utanol production by metabolically engineeredlostridium acetobutylicum with in-situ butanol removal

ang-Hyun Lee1,∗ , Min-A Kwon1, Yong-An Shin1, Kyoung Heon

im2

GS Caltex CorporationKorea University

Clostridium acetobutylicum is industrially important strain forutanol production. However, economically feasible productionf butanol by wild type Clostridium acetobutylicum is still limitedy butanol toxicity and by-product formation, resulting in lowutanol yield and productivity. To overcome the cellular toxicity ofutanol, we developed fermentation strategy with in-situ butanolecovery (ISBR) by addition of butanol selective synthetic resino the bioreactor. Also, we constructed the recombinant strain,hich are deletion mutant of acid formation pathways to reducey-products, acetic acid and butyric acid. Fed batch ISBR fermen-ation profiles showed the increse in yield, butanol selectivity androductivity of butanol production. In continuous fermentationith ISBR for 140 h, the recombinant strain showed that the over-ll yield, butanol selectivity and volumetric producivity were 35%,8%, and 2.6 g/L/h, respectively, when feeding 200 g/L glucoseolution containing 3% corn steep liquor as a nutrient

Acknowledgement: This work was supported by the Centeror Organic Wastes to Energy Business under the Environmen-al Technology Development Program funded by the Ministry of

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B-32

roduction of gaseous biofuels and fine chemicals fromood industrial wastes

abor Rakhely1,∗ , Balazs Balint2, Rita Beres2, Agnes Kis3, Kornelovacs2, Krisztian Laczi 2, Andrea Nyilasi 4, Andras Fulop2, Zoltanagi2, Etelka Kovacs2, Gergely Maroti 5, Katalin Perei 6

Institute of Environmental Sciences and Department of Biotechnology, Uni-ersity of SzegedDepartment of Biotechnology, University of SzegedInstitute of Biophysics, Biological Research Center, Insitute of Environmentalciences, University of SzegedInstitute of Biophysics, Biological Research CenterInstitute of Biochemistry, Biological Research CenterDepartment of Biotechnology, Institute of Environmental Sciences, Universityf Szeged

Food industrial activity is accompanied by the emission ofarious kinds of organic wastes including protein, fatty and sugar-ased materials. These substrates have distinct values, some ofhem could be utilized as animal nutrients but this is often limitedy public sanitation.

Protein containing wastes could be used for both biohydrogennd biogas production. Keratin was a substrate of a two-stage pro-edure for biohydrogen production. Alternatively, various proteinubstrates could be converted into biogas via a microbial adapta-ion processes which were monitored by metagenomic approach.

Sugar-based biowastes could also be used for either biohydrogenr biogas production and the yield could be stimulated by additionf either nutrients or properly chosen microbes. Both processesere monitored by new generation sequencing based approaches.

Fatty acids could be degraded by several microbes. Unctuousastes were effectively utilized by Rhodococcal strains and the pro-

ess was monitored by whole cell transcriptome analysis. Duringermentation, surfactant molecules were produced which mighte utilized in e.g. cosmetics.

Food industry and dark fermentation processes producedumerous organic acids which can be utilized either for biohy-rogen production or for synthesizing bioplastics. The bioplasticetabolism - also related to CO2 capture - was shown to be in con-

ection with hydrogen metabolism in purple phototrophic cells.hus, the overall process has benefits in recovery of food industrialastes in valuable products and also in CO2 capture.

cknowledgments

“The Project is supported by the European Union ando-financed by the European Social Fund (grant agreementso. TÁMOP-4.1.1.C-12/1/KONV-2012-0012, TÁMOP-4.1.1.C-
2/1/KONV-2012-0014).”


B-33

he role of adhe in thermophile ethanol production

ianyong Zheng ∗ , Daniel Olson

Dartmouth College

Clostridium thermocellum is a thermophilic, gram-positive obli-ate anaerobe that is a candidate organism for converting cellulosiciomass into ethanol through consolidated bioprocessing. C. ther-ocellum has one of the highest rates of cellulose utilization

nown, but ethanol production in wild type C. thermocellum waseported to be < 30 g/L. This is relatively low comparing to an engi-eered strain of Thermoanaerobacterium saccharolyticum, which hascheived theoretical yields of ethanol. However, because T. sac-harolyticum cannot use cellulose as an energy source, we seek toeconstruct the T. saccharolyticum ethanol production pathway in. thermocellum. The bi-functional alcohol/acetaldehyde dehydro-enase AdhE was shown to be a key enzyme in ethanol productionn many organisms, but this enzyme has never been individu-lly studied in C. thermocellum or T. saccharolyticum. Therefore,his study characterized and compared the activity of AdhEs inifferent strains of C. thermocellum or T. saccharolyticum, throughxpression in E. coli and purification. The Km values for substratesnd product inhibition were measured, the enzyme specificity foro-factors (NADH/NADPH) were also determined. The end goal ofhis study is to find a better performing AdhE that can be expressed


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tem cells, gene therapy, biomarkers andiagnostic tools

C-01

ysophosphatidic acid enhanced angiogenic capabil-ty of chondrocytes by regulating Gi/NF-kB-dependentngiogenic factor expression

ung-Chih Hsu1,∗ , Pey-Jium Chang2, Chang-Zern Hong3

Chang Gung Memorial HospitalGraduate Institute of Clinical Medical Sciences, College of Medicine, Changung UniversityDepartment of Physical Therapy, Hung Kuang University, Taichung, Taiwan

Lysophosphatidic acid (LPA) regulates myeloid differentiation,steogenesis, cell proliferation and migration, and inhibits apopto-is in chondrocytes. We investigated the effect on the angiogenicapability of human chondrocytes and the underlying mecha-ism using human chondrocyte cell line, CHON-001, and humanascular endothelial cells (HUVECs). Angiogenic capability wasetermined by capillary tube formation, monolayer permeability,ell migration and cell proliferation. Angiogenesis protein arrayit used to evaluate the angiogenic factors secretion. Angiogenin,nsulin-like growth factor-binding protein 1 (IGFBP-1), interleukinIL)-8, chonocyte chemoattractant protein-1 (MCP-1), matrix met-lloproteinase (MMP)-9 and vascular endothelial growth factorVEGF) mRNA and protein expression were evaluated by EIAnd Q-RT-PCR, respectively. LPA receptor (LPAR) expression wasetermined by RT-PCR. Signalling pathways were clarified using

nhibitors, Western blot analysis and reporter assays. LPA pro-otes the angiogenic capability of CHON-001 cells, resulting in

nhanced HUVEC capillary tube formation, monolayer permeabil-ty, migration and cell growth. Angiogenin, IGFBP-1, IL-8, MCP-1,

MP-9 and VEGF mRNA and protein expression were signifi-antly enhanced in LPA-treated chondrocytes. In addition, LPA2,, 4 and 6 were expressed in CHON-001 cells. Pre-treatment withhe Gi/o type G protein inhibitor, pertussis toxin (PTX) and theF-kB inhibitor, PDTC, significantly inhibited LPA-induced angio-enin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF expression. PTXre-treatment also inhibited LPA-mediated NFkB activation, sug-esting the presence of active Gi/NF-kB signaling in CHON-001ells.

The effect of LPA on inducing chondrocyte angiogenesis may beue to increased angiogenesis factor expression via the Gi/NF-kB
ignaling pathway.
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C-02

enotyping of Campylobacter jejuni using a novelolymerase-Chain-Reaction-Microsphere approach

oss Barnard1,∗ , Fang Liang2, Lawrence Wong2, Anna Weis2, Jil-ian Templeton3, Patrick Blackall 4

The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandDepartment of Agriculture Fisheries and ForestryQueensland Alliance for Agriculture and Food Innovation, The University ofueensland

Campylobacter is a major cause of foodborne disease, withampylobacter jejuni contributing more than 90% of reported cases.or diagnosis and monitoring of transmission, several genotypingethods have been developed, including multi-locus sequence

yping (MLST) [1]. However, there is still a need for fast, costffective methods for routine analysis. We developed a techniqueombining allele-specific PCR with a microsphere-flow cytome-er system [2]. Seven loci with single-nucleotide polymorphismsSNPs) with the highest Simpson’s index of diversity (D) wereelected from an MLST database. With these loci as a target, multi-lex allele-specific PCR was conducted on microspheres in a singleeaction and fluorescence signal was detected in a flow cytome-er. The signal on the microspheres indicated which allele specificrimer was consumed in the PCR. By this means all seven lociould be determined. This approach has a turnaround time of 4 h.

Results To date the method has been tested with two strainsf Campylobacter jejuni possessing known SNP patterns and six ofeven loci have been correctly determined for these strains in aingle PCR reaction.

Conclusion A new allele specific PCR-microsphere-basedethod for genotyping Campylobacter jejuni is under development.

his approach will be useful in laboratories utilizing PCR and flowytometers.

eferences

].Cornelius AJ, et al. Applied and environmental microbiology2010;76:1533–44.

].Liang F, et al. Anal Biochem 2013;432:23–30.


C-03

mmunocapture and labeling of cd133- and cd54-positiveells by using magnetic microspheres coupled with anti-ody via oriented immobilization

en-Chien Lee1,∗ , Wei-Chih Kuan1, Daniel Horák2

National Chung Cheng UniversityInstitute of Macromolecular Chemistry, Academy of Sciences of the Czechepublic

Magnetic microspheres coupled with cancer biomarkers can
e very useful for clinical cancer diagnosis. Cancer stem cellsCSCs) have been identified as a subpopulation of cells withinumor which are responsible for tumor growth and metasta-is. CD133 is considered a marker of CSCs in many malignant

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umors. Furthermore, to detect the presence of circulating tumorells (CTSs) in cancer patients, ICAM-1 (intercellular adhesionolecule-1) also known as CD54 is a potential target since CD54

s expressed on endothelial cells in attracting CTCs in blood.D54 was also found to be highly expressed on cell surface inepatocellular carcinoma. In the present work, anti-CD133 andnti-CD54 antibodies were separately immobilized on the amino-ontaining magnetic poly(glycidyl methacrylate) (NH2-PGMA)icrospheres. Antibody molecules were oxidized on their carbo-

ydrate moieties and bound to the magnetic microspheres viasite-directed (oriented) procedure. Experimental results suggest

hat the orientation immobilization could preserve the antigenecognizable site of antibody for binding with its antigen. Also,esults indicate that antibody molecules were evenly fixed onhe surface of magnetic microspheres. A bound antibody den-ity up to 104.2 mg/g-magnetic microspheres was achieved. Thenti-CD133 antibody-immobilized microspheres were found effec-ively for capturing CD133 positive cells from human cancer cellines. Flow-cytometric analysis confirmed the immunolabeling ofD54-positive cell and the enrichment of CD133-expressing cellsy using these antibody-bound magnetic microspheres.


C-04

pplication of CO-amplification at Lower Denaturationemperature (COLD) PCR principle to DNA profiling byTR genotyping

ereza Tichá1,∗ , Jirí Drábek2, Filip Kokás 1, Karolína Burdová1, Jantránská1, Veronika Holinková2, Miroslava Rabcanová2

Palacky University, OlomoucInstitute of Molecular and Translational Medicine

Introduction and aims The sensitivity of DNA profilingethods based on microsatellite genotyping process has increased

ince the introduction of the first commercial fluorescent mul-iplex kit through improvements in PCR (polymerase chaineaction) or primer chemistry and low-template modificationsf protocol (i.e. increase of cycle number, post-PCR cleaning ofmplicons, and/or increase of injection time). These changes witharious successes tried to faithfully generate electrophoreogramepresenting signals from minute amounts of single or mixed bio-ogical sample. However, it is sometimes desirable to increase the

inor profile in the mixture (i.e. to boost a signal of felon over sig-al of victim). In this project, we tested whether CO-amplificationt Lower Denaturation temperature (COLD) PCR that is used inlinical genetics settings able to improve the detection of singleucleotide mutant tumour DNA in a surplus of wildtype DNA,nd if so,whether it is amenable to STR (short tandem repeats)yping.

Materials and methods DNA was extracted from cell linesnd/or EDTA (ethylenediaminetetraacetic acid) treated blood
sing QiaGen kits, amplified in mixture or unblended eithery homemade fluorescent singleplex (locus DXS101) or byommercial multiplex GenomeLab Human STR Primer Set (Beck-an Coulter, USA) to see if COLD PCR protocol bring any
STEM CELLS, GENE THERAPY, BIOMARKERS ANDDIAGNOSTIC TOOLS

mprovements over standard PCR in terms of minority profileignal.

Results and discussion Our preliminary data show thatOLD PCR principle is not fully transferable from SNP (singleucleotide polymorphism) typing to STR typing.

cknowledgements

This project was partially supported by grants CZ.1.07/2.3.00/0.0004, CZ.1.07/2.3.00/30.0041,and CZ.1.05/2.1.00/01.0030.


C-05

he detection of TNT by the periplasmic ribose bindingrotein RbsB of Escherichia coli is not yet solved

rtur Reimer ∗ , Shantanu Roy, Manupriyam Dubey, Vladimirentchilo, Jan Roelof van der Meer

UNIL

There exists a wide range of naturally occurring biologicalensing systems, which can be combined in biosensors with versa-ile application possibilities. In addition, it has been proposed thathe natural range of biological sensing proteins can be expandedhrough predictions from computational simulation algorithms.

A landmark paper in 2003 described the computational simu-ation and subsequent construction of mutant periplasmic riboseinding protein from Escherichia coli to create a biosensor capablef detecting TNT (trinitrotoluene). The results from this work sug-ested that periplasmic binding proteins can be used as universalcaffolds for new target specificities, which could then be pluggednto a unique single E. coli host cell that carries a hybrid chemo-axis/osmoregulation cascade by which de novo gene expressionan be induced.

Here we describe an independent reconstruction and exami-ation of the scaffold idea and of the quality of the producedNT biosensor. The reconstructed E. coli hybrid scaffold is an excel-

ent biosensor for ribose in the nM-range, using wild-type RbsB. Inontrast, both in vitro and in vivo results suggest that the mutantbsB protein is not able to bind TNT, nor able to induce reporterene expression in presence of TNT. In order to better understandhe importance of the various amino acid residues in protein fold-ng, stability and receptor binding, we substituted each residue ofbsB by alanine and measured gene expression through the hybridignaling pathway. This information will be implemented in thesed simulation algorithm to increase the predictive power of the


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C-06

igh Performance Computing Based Smart Scan for thedentification of Species Based Unique DNA Sequences

ivanc Bilecen1,∗ , Behnam Rahnama2, Ender Altiok2

(1) Okan University, (2) Duzen Laboratories GroupOkan University

Nucleic acid based tools and techniques such as PCR, RT-CR, DNA microarrays and lately DNA-hybridization-on-a-chipevices provide reliable detection and identification of microor-anisms. These platforms rely on the presence of target specificNA sequences to be known in advance. These sequences, how-

ver, can be hard to identify in close species or on the strain level.n this work we have developed a parallel algorithm to accuratelypecify and classify species and strain specific DNA sequences.

The parallel implementation of the intelligent search algo-ithm runs on the parallel GPGPU cores on an HPC server. Eachepler K20X computational card provides 2688 fine cores acces-ing to 6 GB of DDR5 shared memory. Such massive parallelizationllows us to compare variable window size of base pairs in annmatched performance in comparison with conventional meth-ds. The intelligent scan results bidirectional and circular streamearch as well as comparing not only the same instance but alsoimilarity scan up to a defined threshold. Well-established CUBLASibrary allows comparison of the determinant of sample matrices inortion of microseconds rather than sequential scanning of eachase pare.

Our algorithm successfully identified unique markers (70 to20 bp) to differentiate Bacillus cereus and B. subtilis. Locationsf these markers on the chromosome have also been taken intoccount. Our second set will include Salmonella group as thisroup is highly important for the food industry. Our results willompare marker selection and validation studies, and also theirdentification power in PCR and RT-PCR.


C-07

nfluence of analgesia on circulating tumor cells inatients with colorectal carcinoma

anus Slavík1,∗ , Emil Berta2, Josef Srovnal2, Andrea Prokopova2,enka Radova2, David Vrana3, Marian Hajduch2

Palacky University OlomoucInstitute of Molecular and Translational Medicine, Faculty of Medicine andentistry, Palacky University, Czech RepublicDepartment of Oncology, Faculty of Medicine and Dentistry, Palacky Univer-ity, Czech Republic

Introduction: Circulating tumor cells (CTC) can create metas-ases, which are responsible for 90% causes of deaths at patientsith a cancer. CTC are located in a blood stream or bone marrow
nd they are most common cause of cancer recurrence after surgi-al resection of the primary tumor. Presence of CTC has becomen important prognostic and predictive factor, which is possibleo study by molecular methods such as polymerase chain reaction
drti



PCR). A correlation between recurrence of disease after surgerynd analgesics or anesthetics used during or after surgery was alsoound. Analgesia and anesthesia can affect amount of CTC becausef their immunomodulatory effects.

Methods and patients: Influence of morphine and pir-tramide was studied in 121 patients with colorectal carcinoma inur research. Detection of CTC was based on real-time PCR in sam-les from peripheral blood and bone marrow with using epithelialenes as markers (carcinoembryonic antigen - CEA and cytokeratin0 - CK20). Afterward, the presence of CTC was evaluated depend-ng on the type of analgesia and disease-free and overall survival ofatients. The main objective was to optimize analgesic techniquesfter surgery to decrease risk of cancer recurrence.

Results: One month after surgery, morphine-based analge-ia usually induced higher level of CTC. Disease-free survival ofatients was also shorter in case of morphine.

Conclusion: Piritramide seems to be better analgesic tech-ique than morphine, which negatively influences prognosis ofatients with colorectal carcinoma after surgery.

Acknowledgement: This study was supported byrants IGA UP LF 2014 019, CZ.1.05/2.1.00/01.0030 andZ.1.07/2.3.00/30.0004.


C-08

ifferential Expression of Circulating miRNAs Fol-owing Resistance Exercise and Carbohydrate/Proteinupplementation

oued S. Espindola1,∗ , Olga Bocanegra2, Renata Teixeira2, Mariaiqueira2, Matheus Gomes2, Miguel Diaz2

Universidade Federal de UberlândiaInstitute of Genetics and Biochemistry - Universidade Federal de Uberlândia

We investigated the levels of expression of 12 circulating miR-As (c-miRNAs) involved in cell proliferation, differentiation,

ngiogenesis, inflammation and glycemic control in responseo resistance exercise (RE) and dietary supplementation. Twelveubjects performed 10 sets of 10 repetitions with 80% ofheir respective 1RM followed by either carbohydrate or carbo-ydrate/protein supplementation in a randomized single-blindounter-balanced design. Samples of blood were collected beforeE, 03 and 24 hours afterwards. The relative expression data of allf the genes were analyzed using a two-way analysis of varianceith repeated measures. The molecular response in the group that

upplemented with protein was more pronounced for c-miRNAsnvolved in the regulation of myogenesis, particularly hsa-miR-33a and 503. Both treatments revealed a differential expressionf the c-miRNAs hsa-miR-126 and 16, which are associated withngiogenesis. We argue that hsa-miR-133a might be associatedith satellite cell proliferation, and possibly partially responsible

or muscle hypertrophy following RE. Further, both the up- and
own-regulation of hsa-miR-126 and 16, respectively, are likely toeflect neovascularization. These findings support the hypothesishat circulating miRNAs bear paracrine-like functions and thus, arenvolved in cell-to-cell crosstalk.

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C-09

he relationship between fragile sites gene of FHIT inype II pneumocytes and idiopathic pulmonary fibrosis

ei Duan ∗ , Duan Fei, Gu Yu

Hebei University

Objective: The relationship between fragile sites gene of FHITn type II pneumocytes and idiopathic pulmonary fibrosis(IPF).

Methods: With the techniques of cellular biology, moleculariology and immunocytochemisty (ICC), changes in expression ofragile sites genes, FHIT and WWOX, were investigated to providefoundation for gene therapy against IPF.

Results: 1.WWOX and FHIT were found to be expressed highlyn type II pneumocytes of rat in sham-operated group. Com-ared with sham-operated group, no significant difference in therotein expression of WWOX and FHIT were found in modelroup for 7 days (P>0.05). After 14 and 28 days, expression ofWOX and FHIT at the protein level was reduced greatly inodel group (P<0.05). Nevertheless, tetramethylpyrazine signifi-

antly enhanced FHIT and WWOX expression, compared to modelroup (P<0.05).

2. After 7 days, no significant difference in the mRNA expres-ion of WWOX and FHIT were found between all groups. After4 days, the expression of WWOX and FHIT at the mRNA levelas reduced greatly in model group when compared with sham-perated group. Nevertheless, tetramethylpyrazine significantlynhanced mRNA expression of FHIT and WWOX, compared toodel group. Results obtained after 28 days of administration were

imilar to observation after 14 days.Conclusion: We demonstrate that the expression of fragile

ites gene of FHIT and WWOX is altered during IPF. Tetram-thylpyrazine suppresses the alteration in FHIT and WWOX,uggesting the medication could play a protective role for IPF.


C-10

hage display as a tool for rapid in vitro cell characteri-ation by fluorescence imaging and Raman spectroscopy

aura De Plano1,∗ , Federica Calabrese1, Germana Lentini 1, Marcoicolò1, Domenico Franco1, Enza Fazio2, Sebastiano Trusso3,lessandro Allegra4, Fortunato Neri 2, Salvatore Guglielmino1

Department of Biological and Environmental Sciences, University of MessinaDepartment of Physics and of Earth Sciences, University of MessinaC.N.R. Chemical-Physical Processes Institute (Messina)Department of General Surgery, Oncology and Pathological Anatomy, Uni-ersity of Messina

The discovery of new markers for the identification and dis-rimination of cell types such as neoplastic cells is one of the

bnn

STEM CELLS, GENE THERAPY, BIOMARKERS ANDDIAGNOSTIC TOOLS

rincipal objectives in diagnostics. Using random M13 phage dis-lay libraries on the whole U937 cells, we selected a clone, namedIII6, displaying a peptide RKIVHAQTP that preferentially recog-izes these cells. The human promonocytic cell line U937 is aodel for leukemia, cancer therapeutics and in vitro hematopoietic

ell differentiation.Therefore, we directly labeled the phage clone EIII6 with fluo-

escein isothiocyanate (FITC) for microscopy imaging application.his method allowed to identify fixed U937 cells, while preservinginding affinity of labeled phage clones. Microscopic observationshowed that U937 cells were fluorescently labeled.

In order to further investigate the interaction between U937ells and EIII6, we utilized Raman Spectroscopy. Spectral peaksbserved at about 980, 1008, 1185, 1215, 1320,1340,1455, 1589nd 1660 cm−1, are commonly assignable to proteins, saccharidesnd lipids components of U937. These Raman peaks in U937-EIII6omplex are shifted in position or reduced in intensity when com-ared to U937 alone, suggesting that phage-probe were selectively

ocalized at cell membrane. Thereafter, we realised networks con-isting of EIII6 phage clone and Ag-Nanoparticles (AgNPs) as signaleporters in Surface Enhanced Raman Spectroscopy (SERS) to bet-er discriminate U937 cells. With this approach, U937-EIII6 Ramanpectral features become sharper and more intense, confirming theelective phage-cell interaction.

Both methodological approaches, proposed in this work, allowso quickly and selectively identify U937 and could be extended tohe identification of other types of neoplastic cells.


C-11

lycosphingolipids as specific markers during the differ-ntiation of mini-pig bone marrow mesenchymal stemells toward neuronal cells

oung-Kug Choo1,∗ , Malg-Um Lim1, Dong Hoon Kwak1, Ju-Hyoungee1, Sung-Youn Heo1, Ji-Su Kim2, Sun-Uk Kim2, Kyu-Tae Chang2

Wonkwang UniversityKorea Research Institute of Bioscience and Biotechnology(KRIBB)

For development of specific markers during the differentiationf mini-pig bone marrow mesenchymal stem cells (mpBMSCs)oward neuronal cells, we studied their glycosphingolipid pattern,ith particular attention to gangliosides. mpBMSCs containedD3 etc as major gangliosides. In order study, their distribution

n mpBMSCs and its possible change during the differentia-ion of neuronal cells. When mpBMSCs were cultured undereural differentiation media contained BME/DMSO/BHA, mostf mpBMSCs acquired the distinctive morphological featuresike neural cells. In differentiated cells, expression of neural

arkers such as neural precursor marker (nestin), neuronal mark-rs (�-tubulin, neurofilament-M) and astrocyte marker (GFAP)ere further demonstrated by revered transcription-PCR, western
lotting and immunofluorescence. Specifically, we find that a sig-ificant increase in GM1 and GD3 expression was observed duringeural differentiation of mpBMSCs. These results suggest that GM1

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C-12

KT and ERK dependent differentiated Mesenchymaltem Cells survival promoted by Pulse Electromagneticield

ung-Keug Park , Enerelt Urnukhsaikhan, Hyunjin Cho

Dongguk University

Researchers have reported that BM-MSC have ability to differ-ntiate into neuron cells in induction media. Also some studieseported that magnetic field has a role in promoting BM-MSCs dif-erentiation into neural cells. However from in vitro and in vivotudies it is not clear how the methods used induce BMMSCso differentiate into neural cells. Successfully differentiated theM-MSCs would be useful for developing clinical stem cell trans-lantation strategies against central nervous system diseases. Afterifferentiation, cells are no longer live, especially after chemicalifferentiation. We focus on maintaining cellular survival throughhe AKT, ERK pathway. In this work, we show the effect of Pulsedlectromagnetic Field (PEMF) on cell survival and differentiationotential of hBM-MSC in vitro. hBM-MSC were cultured with

nduced medium, and they displayed neuron-like morphology.he cells viability was increased after exposure to 10 mT PEMFor 24 h. Differentiation medium treatment combined with PEMFxposition reduced the death cells rate. NeuroD1, NF-L proteinsre expressed those early stage markers of neuronal differentia-ion and p-ERK, p-AKT after induced by PEMF. Our result is shownhat PEMF could play a role in regulating ERK and AKT pathway.lso phosphorylation of AKT and ERK promote a survival effect by

nhibiting or regulating the pro-apoptotic, anti-apoptotic proteinsuch as BAX, BAD and Bcl-xL. Furthermore PEMF exposure coulde inducing differentiation of hBM-MSCs.


C-13

he dynamics of biofilm formation in clinical strainsf Staphylococcus epidermidis and Staphylococcusaemolyticus associated with neonatal infections

onika Grzebyk ∗ , Anna Piotrowska, Monika Brzychczy-Włoch,iotr Heczko

Jagiellonian University Medical College, Chair of Microbiology

Introduction: Staphylococcus epidermidis and Staphylococcusaemolyticus are major causes of device-related infections ineonatal Intensive Care Units. The main virulence factor in S. epi-
ermidis is biofilm formation and S. haemolyticus also form biofilm,ut this process haven’t been well decribed jet. Biofilm formations usually tested in 24- or 48-hour cultures, but the dynamics andhe early stages of biofilm formation are still unlcear.
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Aim: To test and analyse dynamics of biofilm formation inlinical strains of S. epidermidis and S. haemolyticus

Matherials and Methods: The study covered 10 strains ofach S. epidermidis and S. haemolyticus. Overnight cultures in Tryp-ic Soy Broth (TSB) were diluted 1:100 in fresh TSB and placedn sterile 24-well plates and incubated in 37 ◦C. After 2 h, 6 h,2 h, 24 h or 48 h of incubation, the contents of each well werespirated and the remain biofilm was washed three times with00 �l of phosphate-buffered saline. Biofilms were fixed, dried andhen stained with crystal violet. At the end, 96% ethanol wasdded to each weel and the optical density (OD) of the stainediofilms was measured at wavelength of 600 nm. The measurementas repeated two times and averaged. Fresh uninoculated TSB

reated with the same procedure as test samples was used as neg-tive control. Strong biofilm producing Staphylococcus epidermidistrain RP62A was used as positive control. Study was supported by/DSC/001393 grant.

Results and conclusions: Tested strains differed in dynam-cs and strength of biofilm formation. S. epidermidis strains weretronger biofilm-producers than S. haemolyticus.


C-14

simple pre-analytical tool to enrich bacterial DNA:mplications for blood sepsis diagnosis application

go Tat Trung , Le Huu Song

Tran Hung Dao University Hospital

Despite of improved modern medicine, blood sepsis is stillemaining as a high risk of mortality. One barrier to therapeuticdvancement of the disease is the challenges to accurately iden-ify causative pathogens in a limited time frame. In this content,lood culture is still widely accepted as routine detection approach.owever, this conventional method embeds various drawbacks.evertheless, the multiplex PCR based detection art, although is

onsidered to be a complimentary approach, it is still confrontedy many challenges; of that, the most important factor is thenhibitory effect of human DNA: Imagine that an optimized PCReaction using DNA extracted by common DNA extraction kitsan only sense pathogen’s ribosomal 16S pieces if the bacteriaload exceeds roughly 500 CFU/ml, whereas typical blood sepsisatients would have bacterial density of 100–10000 CFU/ml blood,herefore in many case, the PCR is unable to detect pathogen’senetic materials. To circumvent this problem, various techni-al prompts have been resorted to deplete human DNA prior tohe PCR reaction. A common DNAse-based bacterial DNA iso-ation Kit (MolYsis) can acquire an enrichment ratio of 40.000ence allowing to detect 50 S. aureus CFU/ml blood, however,

ts price is hardly to be accepted in lower income communityabout £10/extraction). In the recent study, we introduce a simplere-analytical protocol that combines the use of polar detergentolvent and basic pH to remove 99% human DNA from patients’
pecimens hence making our downstream PCR assays’ technicalensitivity of 10 to100 CFU bacteria/ml blood.

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iomedical materials

D-01

ole of clpP and tpi genes in bionanocellulose biosynte-esis by Gluconacetobacter xylinus

arzena Jedrzejczak-Krzepkowska ∗ , Malgorzata Parniewska,laudia Jadczak, Michal Rozanski, Katarzyna Kubiak, Przemys-aw Rytczak, Karolina Ludwicka, Marek Kolodziejczyk, Stanislawielecki

Institute of Technical Biochemistry, Lodz University of Technology

Among the microorganisms that produce bionanocellulose,cetic bacteria of the species Gluconacetobacter xylinus are the mostfficient producers. Bacterial cellulose produced by these orga-isms have unique properties such as high hydrophilicity, the

ack of cytotoxicity, excellent biocompatibility, is non-allergic, notutagenic nor teratogenic. Due to these properties and possibility

f modification the bionanocellulose has found wide applicationn the food, paper, textile and chemical industry as well as in

edicine, e.g. production of wound dressing. This biopolymeras been tested for usefulness as implants of the ears, cartilage

mplants, a mesh for hernia operation, as well as a material forracheal reconstruction [1]. Industrial-scale production of bio-anocellulose is still difficult and not sufficiently cost effective.hese bacteria are susceptible to phenotypic variation, which veryften decreases the ability for the biosynthesis of cellulose. Unfor-unately, current knowledge about the molecular mechanisms ofellulose biosynthesis and its regulation is insufficient to effec-ively affect the intensification of biosynthesis (increase efficiencyr accelerate the process of production) and change the propertiesnd structure of the obtained cellulose membranes.

The aim of current research is to verify the effect of genes suchs clpP (encoding ATP-dependent Clp protease proteolytic subunit)nd tpi (encoding triosephosphate isomerase) selected in previoustudies of transcripts differentiating the cells of G. xylinus synthe-izing cellulose (Cel + ) from these non producing cellulose (Cel-)n cellulose biosynthesis.

eference

].Kowalska-Ludwicka K, Cala J, Grobelski B, Sygut D, Jesionek-Kupnicka D, Kolodziejczyk M, Bielecki S, Pasieka Z. Arch Med Sci
2013;9(3):527–34.

BIOMEDICALMATERIALS

D-02

utative motility-related genes in Gluconacetobacterylinus. Initial verification of their influence on Bio-anoCellulose biosynthesis

atarzyna Kubiak ∗ , Bartłomiej Porebski, Marzena Jedrzejczak-

rzepkowska, Przemysław Rytczak, Karolina Ludwicka, Marekołodziejczyk, Stanisław Bielecki

Institute of Technical Biochemistry, Technical University of Lodz

BioNanoCellulose (BNC) synthesized by Gluconacetobacterylinus is intensively investigated as biomaterial for medical appli-ations (best known as wound healing and bone regenerationaterial) and as a drug delivery system [1,2] but also has potential

n various materials preservation (such as skin, paper and textile)nd in electronics. The best known commercial usage of BNC isata-de Coco dietary dessert popular in Asia. Scientifically BNCained an attention of tissue engineers, since its features make it auitable material for scaffolds production [1,3]. However, in somepplications porosity of BNC is not accurate for mammalian cellsngrowth (pores diameter < 20 �m). One approach to address thisssue is genetic modification of G. xylinus in order to obtain strainapable to synthesize BNC with desired features. The aim of thisroject is to influence the motility of the cells secreting cellulosicbers.

After sequencing the G. xylinus genome [4] we have searchedor genes involved in auxiliary motility mechanism and identi-ed two encoding proteins potentially related to it. Subsequently,dequate disruption mutants of G. xylinus ATCC53582 strainere obtained. Motility test, conducted on semi-solid agar plates,

howed less intense colony spreading. The initial analysis ofutants’ phenotype revealed inhibition and limited BNC pro-

uction. Furthermore structural changes in BNC membranes werenvestigated.

eferences

].Arch Med Sci 2013;9:527.].J Pharm Sci 2012;102:579.].Tissue Eng Part A 2010;15:1091.


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iopharmaceuticals

E-01

unctions of natural pigments on gastric ulcer and can-er

yo-Ihl Chang1,∗ , Sun-Joong Kim2, Jee Min Kim1

Korea UniversityUC burkeley

The Natural pigments have many applications in inflammatory,nd oxidative related damage as well as in cancer chemother-py. Recently, precise cellular roles of natural pigments, suchs modulator of key cellular signaling pathway on variety dis-ases, are elucidated. On based on antioxidant, anthocyaninseduced naproxen-induced gastric ulcer. Anthocyanins reducedhe level of lipid peroxidation and increased the level of thentioxidant enzymes. Anthocyanins increased the expression ofuclear factor E2-related factor 2 (Nrf2) which is transactivator

or cellular defense genes. Interestingly, anthocyanins inducedastrointestinal-glutathione peroxidase expression via Nrf2 thatind to regions of antioxidant response element (ARE) in GI-GPXromoter. Otherwise, Shikonin, and genipin stimulates produc-ion reactive oxygen species (ROS) in gastric cancer cells. Theynduced apoptotic cell death in gastric cancer cells in a caspaseependent manner. They also induced cell cycle arrest at G2/Mhase via regulation of p21 by early growth response1 (Egr1).he p21 contains promoter region of Egr1 binding motif. Tran-ient expression of Egr1 in AGS cells enhanced shikonin andenipin-induced p21 promoter activity, whereas suppression ofgr1 expression by small interfering RNA attenuated the abilityf shikonin and genipin induced p21 promoter activity. Antho-yanins improve gastric ulceration through Nrf2 associated withntioxidant enzymes, such as GI-Gpx pathways. And, shikoninnd genipin induced cell damage in AGS cells through thegr1/p21 pathways.


E-02

ffect of P-Protein Inhibition by si-RNA on Melanogene-is in Melan-A Cells

unki Kim ∗ , E. Noh

Inha University

Background; Hyperpigmentation diseases, especiallyelasma and lentigines, are major psychological diseases inost of the societies. The obsession for the clean, fair and

ealthy skin is one of the basic instincts of every individual. Foruch diseases, a number of melanogenesis inhibitors have beencreened. They were effective but their side effects cause manyomplications. Pink eye dilution protein (P-protein) is a structural
rotein in the melanosome that plays a critical role in cellularelanogenesis.


Objective: The aim of the present study was to investigate theffect of P-Protein inhibition, by using P-protein small interferingNA (siRNA), on melanogenesis in Melan-a melanocyte.

Methods: si-RNA for p Protein was introduced into Melan-Aells. Melanin content, cell viability, PCR and western blot foryrosinase were performed.

Results and Conclusions; It has been observed the both P-rotien and mRNA level were significantly lowered by the siRNAreatment. siRNA of P-Protein also suppressed melanin synthesisithout any cytotoxicity in the melan-a melanocyte cells. These

esults suggest that molecular approaches using siRNA targeting-protein may provide a novel approach for the control of theelanogenesis


E-03

ex hormones regulate gender-dimorphic hepatic fetuinxpression in rats

ongWon Yun ∗ , SangWoo Kim, Jae Heon Choi, JungWon Choi

Daegu University

To date, there are limited studies on the sex-specific relation-hip between fetuins (Ft-A and Ft-B) and metabolic diseases. Ourecent proteomic study has shown that fetuins may play sex-ependent roles in obesity and diabetes. In the present study, we

nvestigated the expression of hepatic fetuins with respect to theffects of sex hormones both in vivo and in vitro. A sex hormone-reated rat model was established in order to study the effects ofex hormones on hepatic fetuin expression. Animal experimentsevealed that 17�-estradiol (E2)- and dihydrotestosterone (DHT)-reated rats showed opposite effects in terms of body weight gainn both genders. Interestingly, Ft-A and Ft-B were sex-dependentlyxpressed in the livers of rats, responding to different regulatoryodes of sex hormone receptors (ER�, ER�, and AR). To validate

n vivo data, rat normal liver cells were treated with E2 or DHT atifferent concentrations, and similar expression patterns as those

n the animal-based experiments were confirmed. We found thathese changes were mediated via sex hormone receptors usingntagonist experiments. The results of the present study indicatehat sex hormones induce gender-dimorphic expression of hepaticetuins directly via sex hormone receptors. To the best of ournowledge, this is the first approach to address the effects of sexormones on hepatic fetuin expression as well as possible roles of

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E-04

mmunomodulatory activity of natural polyamines inurine macrophages

un Chul Kang ∗ , Anil Kumar Chauhan, Souren Paul, Rekha Jakhar

Daegu University

Macrophages are the key players of innate immunity, defend-ng the body from foreign invaders through phagocytosis. Inhe present study we investigated the potent role of naturalolyamines in modulation of macrophage activity by analysingll the sequential steps involved during phagocytosis. Initially,e treated splenocytes with three natural polyamines (PUT, SPDnd SPM) to test their ability in proliferation of cells and involve-ent in mitosis. Thereafter, we detected their role in promotingembrane fluidity on RAW 264.7 cells, which is essential during

ptake of particles. Phagolysosome fusion activity of macrophagesn the presence of polyamines was then evaluated by measuringcid phosphatase through p-nitro phenyl phosphate. Further, weetermined the capacity of polyamines in generation of super-xide anion to create respiratory burst inside the macrophages.e find decrease in the proliferation of splenocytes in the pres-

nce of polyamines which proved their potent role as strongmmunomodulator. Uptake capacity of macrophages was observedo be enhanced after treatment with polyamines and also increasedysosomal activity of macrophages was detected at a concentra-ion dependent manner. Percentage of NBT reduction calculatedor superoxide anion generation revealed that polyamines poten-iated this property of macrophages too. We also determined thenti-complementory activity of polyamines by detecting classicalathway of complement which showed to be effective, suggesting

ts command over unwanted activation of complement. This studyresents the potential role of polyamines as an immunostimula-ory drug, which could be effective to treat various immunologicalisorders.


E-05

hymol protects gastric ulcer induced by alcoholhrough regulation of matrix metalloprotein 9 activity

nil Kumar Chauhan ∗ , Sun Chul Kang

Daegu University

In gastrointestinal disorders, ulcer is a common disease withultiple etiologies and most of them are observed due to high

onsumption of alcohol. Matrix metalloproteinases (MMPs) arefamily of zinc-dependent enzymes capable of degradation of

xtracellular matrix and are key players in various inflamma-ory diseases and among them MMP-9 is found to play majorole during gastric inflammation. In this study, we aimed toetermine the roles of thymol in expression of MMP-9 during
thanol induced gastric ulcer model in vivo. Sprague-Dawleyats, pretreated with thymol (10 mg/kg) or normal saline as con-rol were subjected to intragastric administration of 95% ethanol
maca

BIOPHARMACEUTICALS

5 ml/kg). Morphological examination includes ulcer index foremorrhage and hematoxylin and eosin staining was performed tonalyze the severity of ulceration. Gelatin zymography was doneo determine the expression of MMP-9 and a number of biolog-cal and immunological tests were carried out to determine thentioxidant and cytokine levels. Rats challenged with alcohol,eveloped severe injury in gastric mucosa with increased levelf pro-inflammatory mediators like TNF-�, Prostaglandin E2 anditric oxide. Expression of MMP-9 was up-regulated and less pro-uction of antioxidant enzymes (SOD and GSH) was documentedfter treatment with alcohol. In thymol pretreated rats, a signifi-ant decrease in ulcer index, level of pro-inflammatory cytokinesnd nitric oxide was observed. Expression of MMP-9 was down-egulated and antioxidant enzymes production was increased inhymol pretreated group. In conclusion, study suggests that thy-

ol protects gastric mucosa injury from ethanol consumption byown-regulation of MMP-9 and inflammatory cytokines.


E-06

-Hydroxydehydronuciferine induces human melanoma375.S2 autophagy and apoptosis and inhibits metasta-

is in vitro and in vivo

ui MinWang

Kaohsiung Medical University

Melanoma is the deadliest cancer. We identified 7-ydroxydehydronuciferine (7-HDNF) isolated from the

eaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be aio-active agent against human melanoma A375.S2 cells. 7-ydroxydehydronuciferine (7-HDNF) was known to induceutophagy and apoptosis response mechanisms, and anti-igratory activity of melanoma in vitro and in vivo. Cell

roliferation assay was used to test cell viability. Acridine orangeAO) staining and flow analysiswere applied to observe cell

orphology. The apoptotic cell death ratio was measured viawo-dimensional flow cytometry by annexin V-fluorescein iso-hiocyanate (FITC)/propidium iodide (PI) double stained. Westernlot was applied to examine protein expressions whereas woundealing assay was to examine cell activity. Strong anticancerffects of 7-HDNF were exhibited in a dose-dependent man-erand displayed minor cytotoxicities on normal human skinells.7-HDNF induced the formation of intracellular vacuoles andhe augmentation of acidic vesicular organelles (AVO). 7-HDNFncreased the cellular arrest in cell cycle at G2/M phase. Cellular

embrane asymmetry loss was confirmed. Protein expressionsere discovered to verify autophagy and apoptosis responseechanisms sharing the associated pathways. 7-HDNF presented

he high-quality anti-migratory activity. 7-HDNF inhibitedelanoma tumor growth in mice xenograft model, accompaniedith a decrease of phosphorylation of AKT. We demonstrated the
echanism of this compound starting with the formation and
ccumulation of AVO leading to autophagy. 7-HDNF caused theellular membrane asymmetry loss, triggering the G2/M cell cyclerrest in caspase-dependent apoptosis. 7-HDNF presented high-


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uality anti-migratory bio-functions and inhibited melanomaumor growth in mice xenograft model.


E-07

leuropein attenuates visceral adiposity in high fat diet-nduced obese mice through the modulation of WNT10b-nd galanin-mediated signalings

aesun Park ∗ , Narae Kuem, Sujin Song

Yonsei University

The aim of the present study was to investigate the anti-besity effect of oleuropein on high-fat diet (HFD)-induced bodyeight gain and visceral adiposity in mice, and to explore thenderlying mechanisms involved. C57BL/6 N mice were fed withhe normal diet (ND), high-fat diet (HFD; 40% fat of totalnergy) and HFD-supplemented with 0.03% oleuropein (OD) for0 weeks. OD-fed mice significantly reduced HFD-induced bodyeight gain and visceral adiposity. Oleuropein also significantly

eversed the HFD-inducedelevations of adipogenic-related genexpression involved in WNT10b- and galanin-mediated signal-ngsin adipose tissue of mice. Consistent with in vivo findings,leuropein dose-dependently suppressed lipid accumulation inT3-L1 cells during preadipocyte diffentiation. Additionally, expo-ure of the 3T3-L1 preadipocytes to oleuropein resulted in aarked attenuation of the galnon (galanin receptor agonist) or

FRP2 (WNT inhibitor)-induced cellular lipid accumulation. Thistudy demonstrated that oleuropein reduced body weight gainnd visceral adiposity in HFD-fed mice. The protective effect ofleuropein against HFD-induced adiposity in mice appeared to beediated through the upregulation of genes involved in WNT10b-ediated signaling and downregulation of genes involved in

alanin-mediated signaling cascades.


E-08

iologically active triterpenoids from birch andycamore bark

ucie Borkova1,∗ , Milan Urban2, Marian Hajduch3, Jan Sarek1

Department of Organic Chemistry, Faculty of Science, Palacky University inlomouc, Czech RepublicDepartment of Organic Chemistry, Faculty of Science and Institute of Molec-lar and Translational Medicine, Faculty of Medicine and Dentistry, Palackyniversity in Olomouc, Czech RepublicInstitute of Molecular and Translational Medicine, Faculty of Medicine andentistry, Palacky University in Olomouc, Czech Republic

Triterpenes are natural compounds usually occurring in plantsr marine animals and are often used in natural medicine in Asia.
hey have various biological activities (e.g. anti-HIV).[1] A largeumber of triterpenes are cytotoxic against wide range of tumorell lines, their anticancer activity was often observed in preclinical
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nimal models.[2,3] Therefore, they become promising candidatesor new drug development.

The main interest of our group is to modify lupane andleanane derivatives in order to improve their pharmacologicalroperties and to increase their therapeutic index. Whereas des-E-ketoacid (des-E lupane derivative) is our most active semisyn-hetic triterpenoid (cytotoxic on a set of 30 cell lines including

DR phenotypes with IC50 0.32 �mol/L on K562) causing fastnd selective apoptosis, the A-ring modified betulin and betuliniccid derivatives that we prepared later have a completely differentechanism of action. In order to obtain another hit compounds,e synthesized a set of derivatives with various substituents in theosition 2 of the skeleton and found them to be highly cytotoxicn T-lymphoblastic leukemia CCRF-CEM cell line. The mechanizmf action is still being investigated; however, we are able to giveome basic assumption about the structure–activity relationships.

eferences

].Dzubak P, Hajduch M, Vydra D, Hustova A, Kvasnica M, BiedermannD, Markova L, Urban M. J Sarek Nat Prod Rep 2006;23:394–411.

].Salvador J-A-R. Pentacyclic Triterpenes as Promising Agents in Cancer.Hauppauge NY: Nova Science Publishers, Inc; 2010.

].Murph M. Research on Melanoma. InTech; 2011.


E-09

ntranasal Delivery of Nanoparticles Containing n-utylidenephthalide to Treat Malignant Brain Tumor

zyy-Wen Chiou1,∗ , Yu-Shuan Chen1, Yu-han Chiu1, Yuan-Shengi1, Dean-Kuo Hsieh2, Horng-Jyh Harn3

Graduate Institute of Biotechnology,National Dong Hwa University, Hualien,aiwanDepartment of Applied Chemistry, Chaoyang University of Technology,aichung, TaiwanDepartment of Medicine, China Medical University, Taichung, Taiwan

Malignant brain tumor is a highly invasive disease with a veryigh death rate. The effective treatment method for this disease istill an unmet medical need. Intranasal delivery method is a non-nvasive administration route, may bypass the blood brain barriernd reduce the required drug dosage. n-ButylidenephthalideBP) has previously been shown as a drug candidate for treat-ng glioblastoma multiforme (GBM) and temozolomide-resistantBM. However, its hydrophobic property may limit some of

he applications. Therefore, in this study, emulsification methodas used to prepare BP-containing nanoparticles in order torovide desirable features. The obtained nanoparticles showedigh permeation ability as tested by artificial cellulose membraner nasal septum squamous cells. The BP-containing nanoparti-les also exhibited cytotoxic effect in human brain glioblastomaultiforme (GBM 8401) with IC50 vaule of 85 �g/ml. Moreover,

he nanoparticles delivered by aerosol could reduce the tumorize in transgenic GBM mice model after the treatment for 30ays. Based on these results, it is suggested that the prepara-ion of BP-containing nanoparticles and its application in nasal

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dminstration to treat GBM may have the potential in furtherevelopment for clinical use.


E-10

he chemical synthesis of sulfur analogues of lysophos-holipids and cyclophospholipids

rzemyslaw Rytczak ∗ , Maria Koziołkiewicz, Andrzej Okruszek

Lodz University of Technology, Institute of Technical Biochemistry

Development of new synthetic methods for the preparationf biologically active phospholipid derivatives is a challengingroblem of membrane-chemistry and biochemistry today. In facttructural and dynamic studies of biomembranes for the establish-ent of structure-activity relationships, phospholipids-proteins

nteractions and mechanisms of action of phospholipids metab-lizing enzymes require the preparation of a great number ofhospholipids derivatives as the key step in advancing membraneiochemistry. Lysophospholipids have recently become the focusf special attention since it was discovered that in addition to theirole in phospholipid metabolism they function as second messen-ers, exhibiting a broad range of biological activities in their ownight.

The chemical synthesis of new sulfur analogues ofysophospholipids has been described, including phospho-othioate/phosphorodithioate derivatives of lysophosphatidiccids (LPA), phosphorothioate/phosphorodithioate derivativesf cyclic phosphatidic acids (cPA) and phosphoroth-oate/phosphorodithioate derivatives of lysophosphatidylcholineLPC). For the preparation of LPA, cPA and LPC derivativesoth oxathiaphospholane and dithiaphospholane approachesave been employed. Each lysophospholipid analogue has beenynthesized as a series of five compounds, bearing five differentatty acid residues, both saturated (12:0, 14:0, 16:0, 18:0) andnsaturated (18:1).

Acknowledgements: This work was supported by a grantPBZ-MNiSW-07/I/2007) from the Polish Ministry of Science andigher Education and by a grant (011/01/B/ST5/06383) from the
ational Science Center (NCN-Poland).
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1886s

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E-11

nvestigation on preventive measures of virus diseases

inaida Klestova1 , Marina Marchenko2, Viktor Tashuta2,∗, Allaoronina3

State scientific-control institute of biotechnology and strains of microorgan-smsthe Institute of veterinary medicineThe Institute of pharmacology and toxicology

Viruses that caused diseases worldwide spread and have pos-ibility for mutations. More dangerous viruses attack animal anduman organisms, causing economic losses. Many countries arereating biosafety systems for preventing viral infections, forxample, by developing new prophylaxis and antiviral methods.

We investigated the possibility of using some plants andosage forms in anti-viral therapy in sensitive model systems. Inhe investigation we used: continuous cell culture of versenisedwine embryonic kidney and BHK-21; test-model virus-member oforonaviridae family; propylenglycol extracts of plants- Aloe vera,amellia sinensis var. ɑssamica, Echinacea purpurea, Húmulus lúpulus.

Results of experimental research of cytotoxic and antiviralction of preparations of a different origin are presented. MTDs10−5mkl/ml. Index of CC50 -10−4 mkl/ml. In experiments onell cultures we obtained the data that showed antiviral activityo 1,8 ± 0,04lg TCID50/ml.


E-12

n vitro assay by bioengineering of new antiviral drugs

iktor Tashuta1,∗ , Zinaida Klestova2, Alla Voronina3, Shotagebuadze4

Institute of Veterinary InstituteThe State scientific control institute of microorganisms strainsThe Institute of pharmacology and toxicologyGeorgian Technical University

The viral diseases are still increasing. There is a wide spectrumf tested antiviral compounds, application of which inhibits ortops viral activity, but universal antiviral means are absent.

The investigation was aimed to finding the new approaches ofhe infection eradication.

Methods: test-model viruses–members of Herpesviridae andoronaviridae family; continuous animal cell cultures:–cell culturef versenised swine embryonic kidney (CCVSEK), BHK-21, Vero,K-6. For investigation of anti-virus properties we have applied aomplex of standard methods.

Results of experimental research of cytotoxic and antiviralctions of 10 new substances from indol derivates are presented.e show the data about all examined means that significantly

educed the infection activity of tested virus strains in vitro systemsn different ways. We tested different schemes of antivirus actions
f tested substances in creation of treatment by viral infection pro-ess, as preventive as treatment. But the highest antiviral activity

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mong tested substances was shown by decreased viral infectionctivity to 7,04 ± 0,04 lg TCID50/ml.

Conclusion: All tested means are suitable in using for clini-al research for testing in eradication virus infections. The resultsonfirm the perspective of tested means for production of newntiviral drugs.

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ioprocessing and engineering

F-01

AMOS: New applications of an established online tech-ology for shaken vessels

ibor Anderlei 1,∗ , Tim Bürgin2, Andreas Richter2, Cedric Bürki3

olf Klöckner4, Kristina Meier4

Forum Shaking TechnologyKühner AGExcellGene SAAVT Aachen

RAMOS determines the oxygen transfer rate (OTR), the carbonioxide transfer rate (CTR) and the respiratory quotient (RQ) oficrobial, plantal and cell cultures online. The respiration rates

OTR, CTR) are the most suitable measurable variables to quan-ify the physiological state of fermented cultures. RAMOS is theight tool to meet the PAT initiative of the FDA regarding shakenioreactors.

On the one hand RAMOS was made to measure in 250 mL glasshake flasks. Therefore the application field was limited. To over-ome this bottle neck Kühner developed three new add-ons:

MicrOTR: With this tool the user is able to measure OTR andCTR in microtiter plates. The main application of this tool is tofind the right fermentations conditions for microtiter plates.Adapter for disposable flasks: With this new development dis-posable flasks can be directly attached to the RAMOS-System.Therefore the RAMOS system can now easily be used for theapplication field cell cultivation.On the other hand the application field was enlarged by apply-

ng the RAMOS technology for testing of plastic components toetermine their impact on the cultivation.

All the new features and their advantages will be presented andesults from microbial, plant and mammalian cell cultures will behown.


F-02

nline Monitoring of pH, Oxygen and Backscatteredight during Heterologous Protein Production in Dispos-ble Shake Flasks

ernot Thomas John1,∗ , Christian Ude2, Thomas Scheper2, Saschaeutel2, Michael Findeis1, Damian Andrzejewski1

PreSens Precision Sensing GmbHInstitut für Technische Chemie, Leibnitz Universität Hannover

Keywords Single-Use shake flask, online backscattered light,hakes flask reader, protein production, inclusion bodies

Shake flask cultivation is one of the classical methods to per-orm proliferation of cells with low effort and cost. It provides
recultures for the scale up and is suitable for standard screeningoutines like the evaluation of new media or cultivation condi-ions.
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BIOPROCESSING AND ENGINEERING

Estimation of the optimal culture conditions needs a con-inuous analysis concerning biomass data, growth rate, oxygenoncentration and pH. In the context of this work a multisen-ory platform (shake flask reader) was evaluated by monitoringf three basic cultivation parameters (pH, pO2, and biomass) [1].he biomass sensor is detecting the cell growth by backscattered

ight through the flask wall at a wavelength of 625 nm. The mea-urement is fully non-invasive and the measuring interval can beeduced to a minimum of 7 s. The sensor signal is calibrated againstDW or OD600 by application of logarithmic functions. With theiven calibration it is used e. g. for screening procedures in order toetermine the adequate moment of induction. Backscattered light

s also sensitive to changes in particle size and opacity. Thus theres evidence that inclusion body formation within the cells can be

onitored online.[1] Ude C., Beutel S., Findeis M., Andrzejewski D., John G.

., Scheper T. (2013): Online biomass monitoring of shake flaskultures via an optical multisensory platform, Dechema Früh-ahrstagung 2013, Frankfurt am Main, Germany 2013


F-03

eveloping strategies to improve the recovery ofperiplasmicaly expressed recombinant protein by

anipulation of fermentation conditions

oannis Voulgaris1,∗ , Alex Chatel1, Gary Finka2, Mark Uden2, Mikeoare1

University College LondonGlaxoSmithKline

A significant problem for the clarification of E.coli broths is themount of cytoplasmic DNA which may have been released intohe extracellular space by cell lysis. This leads to large increasesn viscosity which for example can make removal of cell solidsifficult. E.coli is not a natural protein secretor; hence, a degreef cell lysis is required for the product to be released from theeriplasm to the extracellular space. Changes in the relation-hip between protein and DNA release favouring protein releasehould favour the performance of the subsequent separationtages. Therefore, the balance between product and DNA releaseust be carefully monitored and if possible controlled during the

ermentation.We present strategies to facilitate a better recovery of an anti-

ody fragment from recombinant E.coli whilst diminishing thextent of the effect of nucleic acid release. These include by: (i)he manipulation of the cell growth rate in order to increasehe outer cell membrane permeability (ii) the incorporation ofeagents within the broth designed to increase the cell membraneermeability, (iii) the incorporation of reagents within the brothesigned to remove selectively nucleic acids released during cell

ysis.
Using an ultra-scale-down approach we have determined the
aximum level of DNA release into the culture while still allowingatisfactory removal of cell solids by continuous flow pilot scaleentrifugation. In this way a strategy may be developed integrating


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ermentation operation and cell recovery to optimise the recoveryf protein product in a well-clarified broth suitable for subsequentrocessing.


F-04

queous Phase Partitioning–from Analytics to Process

onathan Huddleston ∗ , Rana Hameed, Derek Fisher, Svetlana Igna-ova

Brunel Institute for Bioengineering

The presentation will focus on aspects of our work on thepplication of Aqueous Phase Partitioning both to the develop-ent of liquid-liquid extraction based approaches to the recovery

f biopharmaceuticals and to the development of a simple bio-rocess analytical technology. We will consider the recovery ofonoclonal antibodies using Counter Current Chromatography

nd highlight important differences in the dynamic flow regimesf different configurations of equipment and their impact on thetructural integrity of biomolecules. In addition, consideration wille given to some of the different operational regimes available inhis type of equipment and how these can impact upon overallrocess efficiency.

In contrast we will also present some aspects of our worko understand the molecular basis of phase partitioning in theevelopment of partitioning based assays having a strong struc-urally related component. We will show from a theoretical andractical perspective how the technique of Analytical Phase Par-itioning may be applied to the quantitative determination ofrocess derived changes and post-translational modifications ofiopharmaceutical products.


F-05

imulation of butanol production by an integrated fer-entation process using supercritical fluid extractionith carbon dioxide

ernadete Delgado1,∗ , Fernando Luiz Pellegrini Pessoa2

Federal University of Rio de JaneiroUFRJ

Petroleum is becoming a scarce resource and alternative bio-uels has been studied as biobutanol. It can be blended directlyith standard oil-based fuels and has the advantage of been pro-uced from glycerol. Nowadays glycerol is an excellent sourcef raw material derived from biodiesel production. Clostridiumasteurianum uses glycerol as the main source of carbon but isroductivity and butanol concentration is low. The main prob-

em related to fermentation for butanol production is its toxicityo the microorganism Clostridium. Downstream process for recov-ring solvents in the diluted fermentation broth may increaseutanol productivity by reducing the toxicity of solvents to the

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ells and minimize the energy consumption in the process. In thisork a conceptual design of downstream process using supercriti-

al fluid extraction with carbon dioxide and solvent extraction wasompared with traditional distillation process for butanol recoveryn the broth by simulation. The raw material used in fermenta-ion was glycerol and the main products of the metabolism of. pasteurianum were butanol and 1,3 propanediol. Supercriticalxtraction with carbon dioxide was applied to remove 1,3 propane-iol and glycerol then solvent extraction using n-butyl-butyrateas applied for butanol recovery. An integrated process using

ingle supercritical extraction was also developed. Simulationsere performed using SuperPro Design®. An economic evaluationas carried out to compare these systems. The integrated processsing supercritical fluid extraction with carbon dioxide was anconomically attractive scenario, although, butanol has a lowerurity than this process followed by solvent extraction with butylutyrate.


F-06

ssessing the volumetric productivity of an ultrafil-ration membrane bioreactor during the synthesis ofalacto-oligosaccharides

ndres Córdova ∗ , Carolina Astudillo, Andres Illanes, Cecilia Guer-ero

Pontificia Universidad Católica de Valparaíso

Galacto-oligosaccharides (GOS) are a potent prebiotic whichllows the upgrade of underutilized lactose of the dairy industry.owever, this industry is somehow reluctant to use enzymes, due

o their high-cost owing to the high-volumes of lactose processinghat entails. Ultrafiltration membrane bioreactor (UF-MBR) allowshe bioconversion of lactose into GOS, removing the reactionroducts while retaining the enzyme for re-use in a single step.

The objective of this research was to evaluate the effect of oper-tional variables on volumetric productivity (�) of GOS synthesishen using an UF-MBR.

A tubular ceramic-membrane (50 kDa) and retentate recircula-ion mode was used. The reacting mixture was a lactose solution40%w/w, pH 4.5) containing Aspergillus oryzae �-galactosidaseosed at 50IU/glactose. The temperature (40 ◦C to 60 ◦C), the trans-embrane pressure (PT) (2.5 to 4 bar) and cross-flow velocity (CFV)

3.5 to 7 m/s) were varied according to a 2k factorial design. Alleactions were conducted for 240 min.

Lower conversions were obtained at the higher levels of CFVnd PT, and at 40 ◦C.This can be attributed to a greater shear-forcen the enzyme, and more compaction of the solute on the mem-rane which may adsorb the biocatalyst, decreasing its activity. At0 ◦C partial precipitation of lactose and a higher permeate viscos-

by 318% (compared to a conventional batch-synthesis) by usingnzyme in two reaction-cycles.

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Work funded by Grants 1130059 and 11110402 FONDECYT,nd CONICYT-PhD-Scholarship, Chile


F-07

ontinuous photofermentative production of bio-ydrogen with Rhodobacter sphaeroides DSM 158

arsten Helbig ∗ , Rico Hellwig, Felix Krujatz, Thomas Bley, Josteber

TU Dresden

Because of the coming lack of fossil energy sources and toitigate the global climate change there is a need for an environ-entally friendly fuel. Hydrogen could be produced from nearly

mnipresent water and its utilization does not cause the emissionf environmentally harmful pollutants (like CO2). Light is a highlyvailable source of energy. Thus, hydrogen production with pho-otrophic microorganisms has a potential to make a significantontribution to use this energy and produce a renewable fuel. Forndustrial application there is a need to establish a continuousrocess. The aim of this study was to examine conditions for aontinuous hydrogen production with purple-non-sulfur bacteria.

Rhodobacter sphaeroides is able to produce molecular hydrogenith the enzyme nitrogenase. For the study of production condi-

ions a 1-L stirred glas bioreactor was operated as a chemostat andquipped with 12 radial installed 50-w-tungsten lamps for lightupply. The dilution rate has been varied to determine its influencento the hydrogen production rate.

Throughout the experiments the influence of different dilutionates onto hydrogen production and the biomass concentrationas been determined. Dilution rates have been regulated from.024 to 0.216 h−1. At a dilution rate of 0.12 h−1 the highestroduction rate (152 mL h−1 L−1) and a biomass concentration of.15 g L−1 were observed. Neither higher nor lower dilution ratesmproved productivity. The higher the dilution rate the smalleras the concentration of biomass. The shown data show the poten-

ial of Rhodobacter sphaeroides for a continuous photo-biologicalydrogen production.


F-08

ptimization of medium composition for the novelectin lyase producer Rhizomucor pusillus DSM 1331hrough response surface methodology

mira Rizk1,∗ , Sonja Diercks-Horn2, Mahmoud Yousef2, Marceloernández-Lahore2

Jacobs university BremenDownstream Bioprocessing Laboratory, School of Engineering and Science,acobs University, Campus Ring 1, D-28759 Bremen, Germany

Pectin lyase is a member of pectinases that plays an impor-ant role in food processing industries (e.g. juice clarification). In

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his study, the influence of solid state fermentation medium com-ositions containing wheat bran, sugar cane bagasse and lemoneel powder as agricultural by-products for pectin lyase produc-ion using Rhizomucor pusillus DSM 1331 were accessed. Responseurface methodology (RSM) was used for medium optimization,ermentation conditions, and the interactions among all thexperimental parameters at flask level. Several physico-chemicalarameters: pH, temperature, moisture content and fermentationime of the production media were optimizedby using the D-ptimal Design. The results show that pectin lyase production wasignificantly affected by fermentation time and moisture contenthich stimulates themaximal enzyme production to 100 U/mLnd specific activity of 45.24 U/mg at 30 ◦C temperature; 6 daysf fermentation and moisture content of 120%. Under these opti-ized conditions, the predicted maximal activity was 107 U/mL.

he obtained activity was two times higher than some of theommon pectin lyase producers. Additionally, the fermentationrocess was scaled up from 10 g to 1 kg using a rotating drum typeolid-state bioreactor in order to evaluate the difference betweenectin lyase production in flask and at bioreactor level. In con-lusion, the application of response surface methodology had aoteworthy enhancement in pectin lyase production indicating

his method was a promising approach for enzyme productionnd cost reduction


F-09

dvanced clarification of cell culture supernatant byMTM EmphazeTM AEX Hybrid Purifier for fast and eco-omic bioprocessing of recombinant proteins

ichael Maurer1,∗ , Frederik Hoppe2, Harald Schillinger3, Melanieutter2, Renate Kunert4, Kurt Eyer3

FH Campus Wien/School of BioengineeringFH Campus Wien/School of Bioengineering/ACIB3 M Alpine/3 M PurificationUniversity of Applied Life Sciences an Natural Resources Vienna

Mammalian cells are the most important expression platformor the production of biopharmaceutical proteins. More than 50%f all registered products are produced in those expression systemsccording to Ferrer et al. During this work, we used a CHO celline secreting the fusion protein EPO-Fc for the simulation of aiopharmaceutical manufacturing.

For pharmaceutical products it is of particular interest to reducend eliminate biological contaminates, such as genomic DNA, hostell protein, endotoxins and viruses by efficient DSP operations.n the other hand, biopharmaceutical companies are in a harsh

conomic competition, what generates a strong need for shorter,ave and economical processes (reduction of unit operations andhe rise of single use systems).

In this study we evaluated the new 3MTM EmphazeTM AEXybrid Purifier, which combines depth filtration with an all-

ynthetic construction, substantial chromatographic capability,nd a defined 0.2 �m pore size in one single-use cartridge, for clar-fication and purification very early in the manufacturing process.


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urther we compare the performance to a state of the art depth fil-er, 3MTM ZetaPlusTM EXT, and show the scalability from lab scale25 cm2) to pilot scale (340cm2).

Data on a dramatic reduction of gDNA and a significant reduc-ion of HCP by the new 3MTM EmphazeTM AEX Hybrid Purifier wille presented. The advantage of early removal of biological con-aminates and the protection of chromatographic material will beiscussed.

Ferrer-Miralles N., Domingo-Espín J., Corchero J., Vázquez E.,illaverde A. Microbial factories for recombinant pharmaceuticals.

Microbial Cell Factories 2009, 8:17


F-10

dhesion of anaerobic microbial beer spoilers to stain-ess steel

ita Prochazkova ∗ , Milan Bittner, Martina Brozova, Tomas Branyik

Institute of Chemical Technology Prague

To reduce the public health threat posed by food/beverageathogens or spoilers many sources of contamination have to beonsidered. Increased focus is necessary in the case of anaerobicicroorganisms. In the case of breweries, the highest risks are asso-

iated with the genus Pectinatus, but also the genus Megasphaeraust not be neglected. Due to their ability to form or to be a part

f microbial biofilms that are present on the surfaces of pipelines,oors, machinery etc. these microorganisms pose a continuoushreat to beer contamination.

The focus of the presented work is help localize condi-ions/materials prone to colonization by anaerobic bacteria andhus to prevent undesirable biofilm formation in breweries. Theork can be divided into three parts: (i) characterization of thehysicochemical properties (contact angles and zeta potentials)f two anaerobic bacteria (Pectinatus frisingensis and Megasphaeraerevisiae) and of typical construction material’s surfaces (stainlessteel), (ii) prediction of the cell adhesion to stainless steel accord-ng to thermodynamic, classical and extended colloidal models,nd (iii) comparison of model predictions with experimental datarom real adhesion tests carried out in test tubes with model sur-aces (stainless steel microparticles) under model conditions. Thein)consistency between model predictions and adhesion exper-ments can help identify the crucial interactions for microbial
dhesion.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1898tfspBrtTflc

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F-11

hisky by-products: a valuable source of protein andotential applications in aquaculture

aneWhite ∗ , Julio Traub, DawnMaskell, Paul Hughes, AlanHarper,ik Willoughby

Heriot-Watt University

Scotland is famous for its whisky, a tradition stretching backver 500 years. There are over 100 malt distilleries in Scotland withhe capacity to produce 280 million litres of pure alcohol annu-lly. This results in the generation of by-products, one of whichs pot ale, the liquid residue remaining after the first distillationtage. At least 8 litres of pot ale is produced with every litre oflcohol. It contains over 30% protein on a dry matter basis, origi-ating from yeast and barley residues and its main value is as potle syrup, an animal feed produced by evaporation. Evaporation isnergy intensive, the high temperatures have a deleterious effectn protein quality, all components in addition to the protein areoncentrated and it is only an option for larger distilleries. Newarkets for pot ale could be realised if cost-effective methods to

eparate the protein components were available. These proteinsre a good match with the protein requirements of salmon feednd may offer a local, sustainable and secure supply of protein forcottish salmon. The work presented here demonstrates the devel-pment of novel processes for the energy-efficient extraction andecovery of valuable proteins. The composition of pot ale, meth-ds for producing novel by-products and potential applications inquaculture are discussed.


F-12

ilot-scale biotrickling filter for hydrogen sulfideemoval from biogas under continuous discharge flow

artín Ramírez ∗ , Fernando Almenglo, José Manuel Gómez,omingo Cantero

University of Cadiz

Biogas is a valuable renewable energy source. However, the aver-ge H2S concentration (0.1-2%) into the biogas stream limits manypplications. Biotrickling filters (BTFs) have been studied undererobic and anoxic (nitrate like electron acceptor) conditions. Inhe conventional mode operation, the liquid phase is recirculatedrom bottom to the top of the column and a portion volume ormall split of the flow is discharge to help to remove the oxidationroducts (sulfate and elemental sulfur). In this study, a pilot-scaleTF (diameter column 0.5 m, bed height 0.85 m, packing mate-ial: open-pore polyurethane foam) was feed with real biogas andreated water (TW) from a wastewater treatment plant (WWTP).he aim of this work was study the performance under dischargeow equal to the recirculation flow. Moreover, the mass transfer
oefficient was determined.
Nitrate concentrate solution was mixed with TW stream at theop of the BTF. The trickling flow rate was set to 1.7 m3 h−1, biogas

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ow rate were of 1, 2 and 3 Nm3 h−1 and nitrate mass flow rateere of 20 and 70 g N-NO3

2–h−1. The elimination capacities (EC)ere equal under both nitrate mass flow rate. The critical EC (H2S

emoval efficiency (RE) of 99%) was 37.9 gS m−3h−1 and the max-mum EC was 88.4 gS m−3h−1. However, the nitrate consumptionas superior at the highest nitrate mass flow rate, probably due to

he higher gradient concentration and therefore the higher massransfer to the biofilm.


F-13

hromatographic Separation of Mono-PEGylated Teri-aratide Isomers

en-Yih Chen1,∗ , Ching-Wei Tasi 1, Wei-Hung Kao1, Li-Chiaohang2, Ruoh-Chyu Ruaan1

National Central UniversityScinopharm Inc., Taiwan

Peptide drugs covalently conjugated with polyethylene gly-ol (PEG) polymers can effectively prevent protease digestion androlong the circulation half-life. Chromatographic purification ofEGylated peptide drugs is essentially critical in pharmaceuticalndustry, especially for the positional PEGylated peptide isomers.n this study, we aimed to interpret the separation mechanismsf mono-PEGylated Teriparatide isomers by reversed-phase chro-atography (RPC) and further to provide guidance for a better

hromatographic separation of PEGylated isomers. Two mono-EGylated Teriparatide isomers, N-terminal and Lys13 PEGylatederiparatide, were synthesized through the succinimidyl esterunctionalized methoxy PEG (5 KDa) by controlling the pH ofEGylated reaction buffer. Both two PEGylated Teriparatide iso-ers exhibit high protease stability against trypsin, but their

tructural helicity decrease dramatically. However, two PEGylatederiparatide isomers are co-eluted by acetonitrile/H2O containingf 0.1% (v/v) trifluoroacetic acid. To separate these isomers, twopproaches were carried out. One is to tune the pH value of mobilehase. The results showed that two positional isomers are grad-ally separated as the pH value increased from 2.0 to 9.0. Thether is to alter the eluent composition. Based on the solubilityarameters theory (thermodynamics theory), we changed the elu-nt composition from acetonitrile/H2O to tetrahydrofuran/H2O,esulting in baseline separation of PEGylated peptide isomers. Bothpproaches revealed that the difference of polarity term (Ddp)etween peptide isomers plays an important role for baseline sep-ration. Consequently, we suggested that the tuning of mobilehase composition based on solubility parameters theory makes

t possible to achieve the separation of PEGylated isomers in chro-
atographic operation.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1901R2

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F-14

comparative study of chromatographic matrices forffinity chromatography of the diphtheria toxin variantross-Reacting Material 197

lessandra Stefan ∗ , Mattia Boiani, Luca Longanesi, Alejandroochkoeppler

University of Bologna

Cross-Reacting Material 197 (CRM197) is a variant of the diph-heria toxin characterised by a single mutation, a glycine-glutamiccid substitution at position 52 [1]. This mutation reduces itsoxicity but maintains the same inflammatory and immunostim-lant properties. The conventional industrial-scale production ofRM197 is performed using cultures of Corynebacterium diphthe-

iae, but recently we have proposed an alternative process for itsver-expression in Escherichia coli [2]. Accordingly, the recombi-ant protein, bearing a short artificial histidine tag, was purifiedy a metal chelating affinity chromatography (IMAC), performedither under denaturing and native conditions.

In order to investigate possible non-specific interactionsetween CRM197 and the matrix employed for the IMAC, weompared three types of polymers: a matrix consisting of agaroseeads (GE Healthcare Life Sciences), a macroporous silica matrixMachery-Nagel) and the Profinity IMAC resin, based on UNO-phere beads (Bio-Rad). The agarose-based matrix showed theigher non-specific sorption of the CRM197 protein, leading tolow final recovery. On the contrary, the Profinity IMAC resin

roved the maximum yield, likely due to a low retention of the pro-ein in the column via non-specific interactions with the matrix.

oreover, the Profinity resin was found to be suitable for both theurification and the refolding of CRM197 and, accordingly, theecovered protein featured enzymatic activity.

eferences

].Uchida T, Pappenheimer Jr AM, Greany R. J Biol Chem1973;248:3838–44.

].Stefan A, Conti M, Rubboli D, Ravagli L, Presta E, Hochkoeppler A. JBiotechnol 2011;156:245–52.


F-15

two-step one-pot bioprocess for production of 11�-ydroxyandrost-4-ene-3,17-dione from phytosterol

mitry Dovbnya1 , Vyacheslav Kollerov2, Sergey Khomutov1,anila Malov1, Marina Donova2,∗

G.K. Skryabin’s Institute of Biochemistry and Physiology of MicroorganismsASPharmins Limited

11�-Hydroxyandrost-4-ene-3,17-dione (11�-HAD) is a primary
drenal steroid in mammalians and the key precursor in theyntheses of halogenated corticoids (pharmaceutically valuablenalogs of natural corticosteroids). Conventional routes for itsbtaining are based on chemical synthesis, or microbial hydrox-

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lation of androst-4-ene-3,17-dione (AD). AD in turn is producedrimarily with microbial biotransformation of natural sterols byome actinobacteria.

The aim of this work was to develop a bioprocess for obtainingf 11�-hydroxyandrost-4-ene-3,17-dione from phytosterol.

Specific biochemical activities of two microbial strains weresed as a basis for the two-stage bioprocess. On the first stage phy-osterol was converted to AD by Mycobacterium sp. NRRL 3805B.he conditions of biotransformation were optimized to get approx.0% molar yield of AD from 12 g/l of the substrate. On the secondtage AD accumulated in the biotransformation broth was regio-nd stereo-specifically hydroxylated into C-11-alpha position byhe mycelial fungus Aspergillus ochraceus VKM F-830Y. Cultivationonditions for preparation of the fungal biocatalyst with higherpecific activity and mode of the biocatalyst application were opti-ized.Both biotransformations were carried out in a single laboratory-

cale bioreactor thus allowing exclude AD isolation andurification procedures. The two-stage bioprocess provided 65-8% molar yield of 11�HAD from phytosterol for 65-72 h. Theroduct was separated and purified to 95% by step-wise crystalliza-ion and re-crystallizations from a system of polar organic solvents.

For our knowledge, microbial production of 11�-HAD fromhytosterol was not so far reported.


F-16

lasmid DNA purification by integrating membraneechnology with arginine affinity chromatography

oão Queiroz ∗ , Catherine Nunes, Ângela Sousa, José Nunes,ntónio Morão, Fani Sousa

University of Beira Interior

The implementation of clarification and purification processeso isolate the supercoiled (sc) plasmid isoform at industrial scaleecomes crucial. In the present study, membrane filtration tech-ology was performed to isolate and clarify the sc plasmid DNA

pDNA) from lysates. Microfiltration process was implemented toliminate the suspended solids and to perform a diafiltration of theolution, followed by an ultrafiltration technique to concentratehe plasmid and to remove the different types of RNA [1]. Finally,suitable chromatographic strategy is essential to remove residual

mpurities and to obtain the sc pDNA as a highly pure prod-ct. Affinity chromatography with amino acids as ligands, suchs arginine, has been employed for this objective due to its highelectivity for the sc isoform and also because of the mild elutiononditions required to achieve its purification [2]. Thereby, theample resultant from the ultrafiltration process was applied in therginine chromatographic matrix, to attain an adequate strategyor sc pDNA purification. The nature of the arginine support andhe use of moderate salt concentrations render this operation more
conomically sustainable and viable to be used in large scale sys-ems. The separation of sc isoform was proved by electrophoreticnd HPLC analysis. Overall, the integration of membrane technol- p
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gy with affinity chromatography to efficiently purify the pDNAesults in a powerful tool for industrial manufacturing.

eferences

].Nunes JC, et al. Journal of Membrane Science 2012;415-416:24–35.].Sousa A, et al. Journal of Separation Science 2009;32:1665–72.


F-17

stablishment of Efficient Microalgal Harvesting Tech-ique by the Concomitant Application of Red or Blueight Wavelength with Chitosan

ae Geun Kim1,∗ , Yoon-E. Choi2

Department of Bioprocess Engineering, Chonbuk National UniversityLED Agri-bio Fusion Technology Research Center, Chonbuk National Univer-ity

Microalgae are considered to be one of the most promisingeedstocks for biodiesel, due to their rapid growth and high lipidontent. However, microalgal harvesting is one of indispensabletep claiming almost 20-30% of total biomass production cost. Chi-osan is a natural, non-toxic, polycationic polymer with multiplepplications in pharmaceuticals, food, agricultural, and chemicalndustries. There are multiple lines of reports that chitosan can alsoe a promising alternative flocculants for microalgal harvesting. Inur previous study, light-emitting diodes(LEDs) especially red andlue color were demonstrated to govern the specific microalgalell biology. Blue light illumination led to significantly increasedell size, whereas red light resulted in small-sized cell with activeivisions. Based on that, in this study, we attempted to establish aovel harvesting strategy of microalgal biomass by the concomi-

ant applications of both blue light illumination and chitosan. Weuccessfully proved that microalgal cells cultivated under blue lightettled more rapidly than those of biomass cultivated under redight illumination. Next, the combinationary effects of differentight wavelengths and chitosan concentration were thoroughlyested. The data suggested that the optimal harvest conditionnder the blue light with chitosan were significantly deviated fromhose under the red light with chitosan. Our strategy, based onhe illumination of blue light in conjunction with chitosan, willontribute to setting up the future biomass harvest process usingicroalgae.


F-18

ptimization of an industrial primary protein recoveryrocess by Design of Experiment

anja Buch ∗ , Ian Marison

Dublin City University

The primary recovery of proteins at the end of a fermentationrocess can be considered as a keystone for the overall protein lossuring purification. Recombinant proteins are widely produced in

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atch and fed-batch fermentations at industrial scale since thearly 1980’s using Escherichia coli or other microbial cells. Theain challenge remains in the isolation and recovery of the pro-

ein from the fermentation broth at the end of the fermentation,hich leads to increase loss in the protein of interest, especially atanufacturing scale.The primary protein recovery process of an industrial E.coli fer-

entation at large scale was characterised based on the proteinoncentration and protein mass balances were set up to iden-ify critical process parameters. A loss of more than 80% of theecombinant protein was observed occurring at two main criti-al process steps (CPS). A major problem at the critical processteps was identified to be the solubility levels of the recombinantrotein. The protein solubility at the CPS was optimized usingesign of Experiment (DoE) with a central composite face-centredesign. Statistical analysis was applied to determine significantactor interactions and to identify optimal process conditions.

The optimization of the solubility of the recombinant proteined to a four-fold improvement in the recombinant protein recov-ry and an enhancement of the overall process yield.


F-19

roduction of ethanol and biomass from thin stillagesing edible Neurospora intermedia

orge Ferreira ∗ , Patrik R. Lennartsson, Mohammad J. Taherzadeh

University of Borås

Thin stillage is a prime candidate for improvement ofhe industrial ethanol process. Production of biogas, cell oil,icosapentaenoic acid and biomass for animal feed has been inves-igated using thin stillage. In this work, the edible ascomyceteungus Neurospora intermedia was investigated for production ofthanol and biomass from mostly wheat-derived thin stillage.eration rate influenced the production of ethanol and biomassuring cultivation in a 26 L capacity airlift reactor; the highestmount of ethanol (3.2 g/L) was obtained at lower aeration ratef 0.5 vvm, while the highest amount of biomass (9.2 g/L) wasbtained at 2 vvm. The reactor was also used as a bubble col-mn. Similar amounts of ethanol (3.5 g/L) and biomass (5.0 g/L)ere obtained. N. intermedia was also investigated in continuousode in the bubble column; dilution rates up to 0.2 h−1 could

e used without cell wash-out. At dilution rate of 0.1 h−1, 5 g/Lf ethanol and 4 g/L of biomass containing 50% protein werebtained. The solid content in the thin stillage was reduced by8%. The inclusion of this process using N. intermedia can lead to5.5% improvement on ethanol production considering a facilityroducing 200,000 m3 ethanol/year. The produced ethanol can beent to the beginning of the process and follow the main streamowards the distillation column. The high value biomass can besed for animal feed e.g. fish feed, while the reduction of solids can
ave positive effects on energy savings and water recirculation.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1907gst


F-20

ustainable Manufacture of Industrially Relevant Plat-orm Chemicals Using Microbial Bioprocesses

aura Jeffrey1,∗ , Alison Arnold2, BrianMcNeil 1, Linda Harvey1

University of StrathclydeIngenza Ltd

As petroleum stocks decrease on a global scale, it is essen-ial that industry decreases its dependency on oil and petroleumased materials in favour of more sustainable resources. Biologi-ally produced chemicals could provide a sustainable route for theanufacture of high value monomers. Such biological processes

hould have high productivities, use simple media, be operablet large scale, produce a final process fluid with a high concen-ration of suitable bio-product which can easily be recovered andasily integrated into further chemical conversions. At present, nouch system is in place within the industrial biotechnology sector.his presents the unique opportunity to develop a novel processherein amino acids can be converted into high value chemicals.

Corynebacterium glutamicum, a Gram positive, non-sporulatingacterium was chosen as a model organism. Since its discovery itas become an industrial workhorse in the production of aminocids, especially L-glutamate. For this process to be industriallyompetitive, efficiency is paramount. Therefore, production of-glutamate was examined in batch processes using standard L-lutamate induction conditions including; biotin limitation, heatnduction and ethambutol addition. As biotin limitation exhib-ted a 10 fold yield increase during batch conditions compared tother chosen methods a fed-batch process was developed wherealancing carbon and nitrogen was crucial. Strain screening underptimal production conditions was employed to increase produc-ivity further. Once a sufficient titre was achieved, downstreamrocessing on the culture broth could be examined, convertinghis relatively low value amino acid into a high value, desirablelatform chemical for industrial purposes.


F-21

verproduction of glutathione by recombinantscherichia coli expressing bifunctional glutathioneynthetase

himin Li

East China University of Science and Technology

Glutathione is an important bioactive substance being appliedn pharmaceutical and food industries widely. Traditionally, theroduction of glutathione was conducted in yeast system. Inhe present study, a recombinant Escherichia coli strain express-ng the gene gshF coding for bifunctional glutathione synthetaseas used as a producer and the fed-batch cultivation was investi-
ated. During this process, glucose was the sole carbon and energyource. Without extra addition of three amino acids, 0.79 g/l of glu-athione was obtained and the productivity was 56 mg/l/h. With

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he addition of 75 mM glutamic acid, cysteine and glycine, 11.3 g/llutathione was formed with a productivity of 628 mg/l/h, whichere 13 and 10 times respectively higher than those in the absencef amino acids addition.


F-22

solation of microorganisms for biosurfactant produc-ion

ranco Liporace1 , Carla Quevedo1,∗, Ana María Giulietti 2, Juanlivera1

Facultad Regional Delta-UTN-ArgentinaFacultad de Farmacia y Bioquímica-UBA-Argentina

Microorganisms with surfactant producing ability were iso-ated from hydrocarbon-contaminated soil and water in order toarry out processes for biosurfactant production.The samples werebtained from a petroleum distillery (RHASA) located at Cam-ana, (Argentina). Isolation was performed by enrichment cultures

n a mineral salt medium (MSM) containing 4,5% of a mixturef three different hydrocarbons (HC) or polluted lagoon waterAgLag) as both carbon and energy source. The strains isolated wereultured in 250 ml erlenmeyer flasks containing 25 ml of MSMupplemented with HC (pH 7.00) and incubated at 25 ◦C ± 2 ◦Cinrbital shaker at 150 rpm for 7 days. The biosurfactant producingbility of isolated microorganisms was estimated by emulsifica-ion capacity, surface tension measurement using Du Nouy ringensiometer, oil spreading techniques, drop collapse method andaemolytic activity. Only six of the isolated strains were able toeduce the growth media surface tension in more than 40%. Onef them, Ag.A.1HC strain, was cultured in a stirred tank bioreac-or (NewBrunswick BioFlo115) containing 2500 ml MSM mediumith a mixture of HC, operating at 200 rpm and 25 ◦C ± 2 ◦C.iomass concentration and surface tension were evaluated duringhe process. A decrease in the surface tension value of the cell-ree supernatant was observed. The initial and final values were6,85mN/m and 34,90mN/m, respectively. The highest cell con-entration was found between day 5 and 7 of culture. Accordingo these results the Ag.A.1 HC strain, isolated from hydrocarbon-ontaminated water would have a potential use in both productionf biosurfactant and bioremediation processes of environments
ontaminated with hydrocarbons.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1910Hf

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F-23

he use of enzymes in the coating industry–nvironmentally friendly strategies for curing andydrolysing coatings

atrin Greimel1,∗ , Veronika Perz1, Karolina Haernvall 1, Enriqueerrero Acero1, Georg Guebitz2

acib GmbhUniversity of Natural Resources and Life Sciences, Institute of Environmentaliotechnology

The use of enzymes in industrial processes has become morend more common. For the coating industry, it could be envisagedhat enzymes will replace siccatives, paint removers or acticides.

e investigated the potential use of laccases as siccatives foroatings containing alkyd resins as well as the potential use ofydrolases as paint removers.

Alkyd resins are binders for coating formulations containingnsaturated fatty acids. During the hardening process the fattycids are cross-linked. Cobalt complexes are the most widely usediccative systems, but because they are suspected to be carcino-enic, they need to be replaced. The potential of a laccase fromrametes hirsuta in combination with two mediators was evaluatedegarding its capability to crosslink the unsaturated fatty acids [1].he drying reaction was evaluated using different methods, likeTIR spectroscopy, oxygen measurements, GC chromatographynd drying time recorder measurements.

The removal of coatings is usually performed using harsh chem-cals or mechanical strength. Also in this case the development ofnvironmentally friendlier methods will sooner or later be of greatmportance. We investigated the possibility of using enzymes forhe hydrolysis of polyester based coatings [2]. Two enzymes - aipase from Thermomyces lanuginosus and a cutinase from Humicolansolens–showed different activities in hydrolysing two different

odel substrates for polyester based coatings.

eferences

].Greimel, et al. Green Chemistry 2013.].Greimel, et al. Reactive and Functional Polymers 2013.


F-24

ollow fibre membranes as a high mass transfer gas dif-using system for microbial CO fermentation

uhammad Yasin ∗ , Shinyoung Park, Yeseul Jeong, In Seop Chang

Gwangju Institute of Science and Technology (GIST)

Recent research on the biological conversion of carbon monox-de (CO) and hydrogen (H2) into multi-carbon compoundsave revealed that syngas fermentation is one of the potentiallternatives for the production of more sustainable fuels and chem-
cals. However, poor mass transfer of the sparingly soluble gaseousubstrates (CO and H2) and low cell density in the fermentationedia are the big hurdles in the commercialization of the tech-
ology. These issues can be resolved by using the membrane based

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eactors (MBR) instead of most widely employed stirred tank reac-ors (STR) and less common bubble column reactors (BCR). Theurpose of this study was to develop a simple lab scale hollow fiberembrane bioreactor (HFMBR) for addressing the issues of mass

ransfer and kinetic limitations in syngas fermentation. The firsthase of the research has been completed and a hydrophobic PVDFPolyvinylidene fluoride) membrane has been successfully utilizedo achieve high mass transfer. The performance of the system haseen examined by measuring the gas-liquid volumetric mass trans-er coefficient (kLa). Pressure and membrane surface area have beensed as two controllable factors to achieve high mass transfer.e have found a kLa of 135.72 h−1 under 13.6psi transmem-

rane pressure at AS/VL (membrane surface area/working volumef the liquid) = 0.27 cm−1. High kLa of 155.16 h−1 was achieved byncreasing AS/VL to 0.62 cm−1 under lower transmembrane pres-ure of 5.4psi.


F-25

valuation of dynamic microbial communities in atyrene-degrading biotrickling filter using 16S rDNAag pyrosequencing and denaturing gradient gel elec-rophoresis

evin Portune ∗ , María Carmen Pérez, Francisco Javier Álvarez-

ornos, Carmen Gabaldón

University of Valencia

Accurately characterizing microbial communities within biore-ctors undergoing dynamic operating conditions is an essentialrst step towards understanding the relationship between micro-ial community structure and bioreactor performance. A detailedssessment of the changes in microbial populations within atyrene-degrading biotrickling filter was carried out using samplesollected at multiple time points ranging from 21 to 155 days ofiotrickling filter operation. Examination of microbial populationsas conducted by 16S rDNA tag pyrosequencing and denaturingradient gel electrophoresis (DGGE). Validation of pyrosequencingesults was performed by quantitative polymerase chain reactionqPCR) in order to examine the relative changes in percentages ofelected taxonomic groups. Pyrosequencing results revealed a pre-ominance of bacteria assigned to the phylum Proteobacteria forll sampling time points in the bioreactor. Relative fluctuationsn percentages of total bacterial sequences assigned to selectedaxonomic groups detected by pyrosequencing during biotrick-ing filter operation were confirmed by qPCR. Pyrosequencingevealed substantial changes in the community structure betweenampling time points, with observed differences in microbialiversity indices and operational taxonomic units (OTUs) amongertain samples. DGGE further revealed shifts in the dominanticrobial species during changes in biotrickling filter operational

arameters. The application of several different molecular tools
o examine changes within microbial populations from bioreac-ors allows a more detailed view of the community structure asompared to using only one molecular method. This study high-ights both the complementary as well as contrasting information

hat can be obtained in characterizing microbial populations usingultiple molecular methods.


F-26

nline estimation of metabolic state in industrial-scaleioreactors through the dissolved oxygen response toeed rate perturbations

la Johnsson1,∗ , Jonas Andersson2, Gunnar Lidén3, Toreägglund1

Lund University, Department of Automatic ControlNovozymes A/SLund University, Department of Chemical Engineering

Overflow metabolism is a significant problem in many indus-rial bioprocesses, which can lead to decreased productivity as wells total process failure. Avoiding excessive overflow metabolismhile maintaining a high feed rate and hence productivity is

herefore highly desirable. This is complicated by difficulties inodelling and online sensing in industrial processes using com-

lex media.The current study employs a method for online estimation

f the metabolic state in relation to overflow metabolism, basedn the response to sinusoidal perturbations in the feed rate. Thisethod requires only measurement of dissolved oxygen, for which

obust and precise measurement devices are universally availablen industrial bioprocesses. The method is based on directly study-ng the effects of the saturation in oxidative metabolism causingverflow, meaning that it allows estimation of metabolic stateegardless of which substrates are used.

The study includes a number of experiments in industrialroduction-scale fermentations (>100 m3), using an industrialacillus licheniformis strain and a complex starting medium. Firstly,hese have shown that for feed rate perturbations in a certainrequency interval fermentor mixing dynamics can be approxi-

ated by a simple model and that the perturbations do not have aegative impact on growth and productivity. Secondly, they havehown that the method’s estimation of the current metabolic states consistent with offline measurements of main substrate and by-roduct. The method therefore provides a useful measure of theetabolic state, which can be used for both online diagnosis and


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F-27

mproved scale down of industrial fermentations bypplication of oxygen enrichment on lab scale

ogier Meulenberg ∗ , Rogier Meulenberg, Jeroen Van Santen, Erikan der Lucht, Wouter VanWinden

DSM Biotechnology Center

Lab scale improvement programs of industrial fermentationsequire a good scale down of the process. Only then it is possibleo test process adaptations under representative conditions and,

oreover, scale up of the improved process will be faster and moreuccessful.

In the absence of headspace overpressure (in case of glassermenters) and negligible hydrostatic pressure, oxygen transferapacity in lab scale fermenters is often lower than on industrialcale. Therefore, process intensity needs to be decreased for propercale down. For such a scale down approach it is crucial that theimiting factor on industrial scale is the same as on lab scale. Ifot, production strain physiology may be different and represen-

ativeness of the lab scale process is lost. However, in some cases iturned out to be difficult to confirm that the same limiting factoras maintained after process intensity scaling.

In contrast to this, lab scale oxygen transfer capacity can alsoe increased to industrial levels by application of oxygen enrich-ent of the sparged air. With this approach, the saturating oxygen

olubility on lab scale can be increased to similar levels as on indus-rial scale at overpressure. In some cases this prevents the need forecreasing process intensity for a proper downscale.

Examples of different scale down approaches will be discussed.


F-28

apid prototyping meets bioreactor–a novel small-scalerganic light emitting diode based photobioreactor withntegrated sensor systems

elix Krujatz1,∗ , Karsten Fehse2, Matthias Jahnel2, Thomas Bley1,ost Weber1

TU DresdenFraunhofer COMEDD

Microalgae represent a promising raw material for variousndustries due to their wide range of valuable ingredients. By pho-osynthetic processes carbon dioxid is fixed under the influencef light and converted into products like proteins, fats, oils or pig-ents. Despite the great potential of algae research only 150 of the

stimated 400,000 algae strains are used for industrial applications.State of the art of microalgal cultivation is the illumination

ith inorganic semiconductor elements known as light emittingiodes (LEDs). However, this technology has disadvantages like an

nhomogeneous ligth distribution (point light source), a strong
elf-heating and thus the need to integrate cooling elements.ecause of these limitations no phototrophic screening system


or the small-scale cultivation under defined conditions could beeveloped so far.

Rapid prototyping allows the design and manufacturing of fine-tructured reactor components. Here, we present the first rapidanufactured cultivation system for phototrophic microorgan-

sms with a working volume of 12 milliliters equipped with organicight emitting diodes (OLED) as lighting source and an opticalrocess monitoring sensor system.

OLED light technology is characterized by a very homogenousight distribution (area light source) and low self-heating. Impor-ant process parameters of microalgal cultivation like pH, pO2 orCO2 can be monitored online by integrated optical sensor spots.oreover, cell-specific parameters likeoptical density and fluores-

ence signals (e.g. marker proteins or chlorophyll fluorescence) areetected online by optical readout approaches (LED-excitation andpectral sensitive photodiodes).

Thus, this reactor-setup represents an ideal screening and pro-ess optimization tool for photo-biotechnological processes.


F-29

acterial growth modeling for rapid fermentation pro-ess development using a parallel mini-bioreactor system

arron Erbas2,∗ , Tony Allman1, Frank Baganz2

Infors AGUCL

In order to establish a generic framework for the rapid develop-ent and optimisation of scalable fermentation processes a novelethodology will be explored which integrates small scale fer-entations with model-based experimental design and predictive

ontrol strategies. In the first instance mathematical models will beeveloped to predict microbial growth kinetics. Most commonlyhe first order kinetics logistic and Gompertz models are compara-ively used to assess the model fit to empirical data. Optical density

easurements may provide a quick off-line analysis of the growthurve of microbial populations, as compared to cell plate countsr dry weights that require more time. Here we propose a mod-fied four parameter logistical model for batch growth of E. coli3110 in a minimal medium. The mean square error (MSE) wassed to measure the fit of the empirical model to the experimentalata that were obtained using a quadruple parallel mini-bioreactorystem (Multifors). The average optical density after 23 hours was.46 + /- 0.09 (n = 4) demonstrating excellent reproducibility. Theest MSE for the model was MSE = 0.034 showing specifically aood fit to the log and stationary phases of bacterial growth butlso enabling fitting to the lag phase with a best MSE = 0.0028. Suchodels have the potential to predict typical microbial cell growth

nd thereby aiding the development of advanced fermentation

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F-30

novel lab-scale two-stage reactor for biogas productionhrough the use of efficient and stable microbial consor-ia

artyna Ciezkowska1,∗ , Krzysztof Poszytek1, Otton Roubinek2,acek Palige2, Aleksandra Sklodowska1, Lukasz Drewniak1

Laboratory of Environmental Pollution Analysis, Faculty of Biology, Universityf WarsawInstitute of Nuclear Chemistry and Technology

The anaerobic digestion is very fragile and sensitive process,hat requires to maintain the balance among different microbialopulations. To achieve stable biogas production different sourcef microorganism were tested and a variety and numerous techni-al solutions were developed.

The main aim of this work was verification and optimizationf a novel anaerobic digesters for laboratory studies of two-phaseiogas production with the use of efficient and stable microbialonsortia. The developed biogas production system consists of twoeactors, one dedicated to hydrolysis process (2L tank operated in0 ◦C) and second designed to fermentation (25L tank operatedn 37 ◦C). Both reactors use hydraulic agitation and operate in auasi-continuous mode.

The optimization of biogas production with the constructedeactors was carried out with the use of stable microbial consortiasolated from fermentation tank of biogas plant and was conductedn four phase until the stabilization of methane production. Forach phase the bioreactors were supplied with maize silage from 1o 5% DM increasing during process. During methane fermenta-ion the following parameters were analyzed: CH4 content; biogasield, concentration of VFAs and COD, and microbial communitytructure and activity changes.

The conducted analyses showed that the microbial consortiaave effective and stable production of biogas after 20 days of pro-esses and constructed reactors are perfect for the analyses of eachtep (hydrolysis/acetogenesis and methanogenesis) of two phaseiogas production process.


F-31

ethods for removing endotoxin contaminants fromiotechnological chondroitin

ariacarmela Marseglia ∗ , Paola Diana, Alberto Alfano, Katia Dellaorte, Rosaria Di Nuzzo, Chiara Schiraldi

seconda università degli studi di Napoli

Endotoxins released from the cellular membranes of gram nega-ive bacteria during their growth constitute dangerous pyrogens toe removed in case of biotechnological production of pharmaceut-cals, expecially in case of parenteral drugs. Polysaccharide-based
harmas have wide biomedical applications but also these prod-cts have to be endotoxin free to become suitable for specificses. Purification of mannans, for example, was performed

ith size separation techniques (gel filtration or ultrafiltration).earing in mind the importance of biotechnological produc-ion of glycosaminoglycans the aim of this research was toevelop new methods for purifying the capsular polysaccharide ofscherichia coli K4 (chondroitin-like) in order to obtain a pyrogen-ree product with high recovery yields and purity grade. Thisroduct may than be suitable for preclinical in vitro evaluationf potential biological activity. During fermentation these bacte-ia release into the culture media, together with the K4 capsularolysaccharide, the lipopolysaccharide molecules that result theain contaminant during the downstream process. The removal

f the lipopolysaccharides is difficult because of their amphiphilicature and because of structural similarities between its oligosac-haridic portion and the capsular polysaccharide chain. Strategiesased on activated charcoal either in powder form and/or packedisks where compared to newly developed solvent phase separa-ion processes.

In both case pyrogen free chondroitin of 30 KDa, with a purity98% was recovered.


F-32

ingle-use bioreactors for microbial application

ico Oosterhuis1,∗ , Stefan Junne2, Peter Neubauer2

CELLution BiotechChair of Bioprocess Engineering, Institute of Biotechnology, Technische Uni-ersität Berlin, Germany

Nowadays single-use bioreactors are fully accepted in the bio-harmaceutical industry. Reactors up to 2000 L working volumere commonly used. However, these bioreactors are limited inerms of mass-transfer and mixing capabilities and thereforenly suited for application in mammalian cell culture. Single-userocessing offers same benefits for microbial processes as for mam-alian processes. As microbial expression systems are widely use

n the biopharma industry, there is a strong need for single-useioreactors applicable for microbial processes as well.

The CELL-tainer® technology, based on a 2-dimensional rock-ng motion is available now with a working volume from 0,15–25 Ln one and the same bag as well as from 10 – 150 L in one bag size.

La values of 300 hr−1 and above have been reported for both sizesf reactors. Recently achieved culture data of E.coli and of a Rho-utorula yeast show that the CELL-tainer® single-use bioreactor isomparable to stirred fermenters and thus suitable for microbialultivations. Application in both the seed train as well as culti-ation system for small batches in GMP-production is possible


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F-33

utanol production by fermentation of Clostridium ace-obutylicum: Solventogenic kinetics

lessandra Procentese1 , Francesca Raganati 1,∗, GiuseppeOlivieri 1,aria Elena Russo2, Piero Salatino1, Antonio Marzocchella1

Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Indus-riali - Università degli Studi di Napoli Federico IIIstituto di Ricerche sulla Combustione, Consiglio Nazionale delle Ricerche

The awareness of fossil supply depletion and the impact ofetroleum fuel emissions have increased the research for alterna-ive fuel sources. Particular relevant are the fuels produced fromenewable resources such as rapid growth biomasses. Among bio-uels, butanol is particularly valuable because it possesses manyavourable physical properties [1].

Acetone–butanol–ethanol (ABE) fermentation from renewableesources is one of the biotechnological routes to butanol pro-uction. The potential of cheese-whey as feedstock for butanolroduction has been pointed out by several authors (e.g. [2]).

The traditional batch fermentation process for butanol pro-uction suffers from two major issues: i) low butanol specificroductivity, which means large fermentors and long fermen-ation periods; ii) severe product inhibition, which limits theutanol concentration and increases the industrial cost for solventecovery. Continuous operation mode provides several advantagesver batch processes. Increased productivity was also achieved byncreasing cell concentration by cell recycling or immobilization2].

The objective of this study was to characterize a continuousutanol production system by means of Clostridium acetobutylicumdopting lactose as the sole carbon source. A cell recycling systemCSTR equipped with a microfiltration unit) has been adopted.ontinuous cultures were carried out under a wide interval ofperating conditions (dilution rate and recycle flux) in ordero characterize the fermentation process under solventogenesishases.

eferences

].Cascone. Chem Eng Prog 2008 Aug:S4–9.].Raganati, et al. Bioresour Technol 2013;138:259–65.


F-34

valuation & optimization of yield, productivity, andustainability of the EBA process; A surface energeticspproach

rasad Babu Kakarla ∗ , Roy D’Souza, Marcelo Fernández Lahore

Jacobs University Bremen

Expanded bed adsorption (EBA) chromatography is an
dvanced integrated unit operation for the downstream processingf biomolecules. Biomass-adsorbent interactions not only lead tonstable hydrodynamics in these systems, but also to the elutria-ion of adsorbent beads, and the deposition of intact cell particles,
dss



ell debris, and suspended materials on the stationary phase. Weave employed extended Derjaguin– Landau–Verwey–Overbeek

xDLVO) theory, based on colloid theory principles, as an effec-ive tool in predicting these unfavourable interactions. We havedapted capillary rise method to characterize the surface thermo-ynamics of EBA adsorbents, and have used streaming potentialeasurements to describe surface electrostatics. Both these meth-

ds now avoid mechano-chemical alterations in surface propertiesssociated with the bead disruption, which was required for earlierharacterization methods. The corresponding xDLVO interactionsnergies calculated were used to generate a minimum adsorbent-iomass interaction energy surface as a function of pH and ionictrength, which led to the predictive optimization of EBA processonditions in terms of solution chemistry. Consequently, productecovery was shown to increase without compromising on purity99%).


F-35

igh throughput systems and mathematical models forrediction of protein renaturation processes

ernhard Mißbichler1,∗ , Cornelia Walther2, Sabrina Mayer3,orota Antos4, Alois Jungbauer3, Astrid Dürauer3

ACIB GmbHUniversity of Natural Resources and Life Sciences Vienna, Department ofiotechnology, Muthgasse 18, 1190 Vienna, Austria1ACIB Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190ienna, AustriaRzeszow University of Technology, Chemical and Process Engineering Depart-ent, a. Powstancow warszawy 6, 35-959 Rzeszow, Poland

Heterologous recombinant expression of biopharmaceuticalroducts in E. coli often leads to high density protein aggregates

n inclusion bodies. In contrast to empirical strategies for processevelopment we developed high throughput screening methodsor inclusion body solubilization and subsequent refolding on �-cale. Thereof parameters were extracted to predict this crucialrocess steps in a laboratory scale stirred tank reactor controlledy inline monitoring.

Mathematical modelling using solubility curves and dissolu-ions kinetics was carried out. The solubilization process could beescribed by a homogeneous layer model with a high order of reac-ion which enables evaluation of the process on a numerical level.ence, solubilization conditions and process parameters can be

elated to the rate and yield of the solubilization. Approximationf the resistances potentially affecting the IB solubilization showedhat the process is controlled predominantly by pore diffusion.

aintaining the homogeneity of the IB suspension is sufficient forfficient solubilization, and further power input will not improvehe process. The HTS on �-scale can predict solubilization in STRver a range of 500 and can be used to determine optimal solubi-ization conditions for laboratory and industrial scale.

Furthermore, kinetics of the refolding process determined inifferent scales – from static vial over beaker stirred by magnetictirrer to laboratory scale STR were equivalent keeping differentcale-up criteria such as phase number and Reynolds number

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ithin a certain range. The established models and predictionnable the engineering based and thus material and cost efficientrocess development of IB solubilization and refolding steps.


F-36

amma ray-mediated functionalization of monolithicryogels for macro-biomolecule purification

aveenkumar Singh1,∗ , Roy N. Dsouza1, Mariano Grasselli 2,arcelo Fernández-Lahore1

Jacobs University Bremen gGmbHUniversidad Nacional de Quilmes, Bernal, Argentina

Recently introduced megaporous cryogels are relatively newnd have not been fully exploited for the downstream processingf macro-biomolecules. Weak anion-exchange functionality wasncorporated by gamma irradiation-induced grafting. The totalonic capacity obtained for DEAE-functionalized cryogel was 0.59

eq/g. The resulting weak anion-exchange cryogels showed a poreize distribution of up to 100 �m with dynamic binding capaci-ies of ca. 27 ± 3 mg/mL. The adsorbents presented in here showedetter column efficiency when compared to packed-bed adsor-ents. The dynamic binding capacities of monolithic cryogels wereound to be independent of the applied flow rate. Scanning elec-ron microscopy confirmed that the porous nature of the cryogelsemained unaffected by the irradiation-grafting procedure. Due tohe large pores, high permeability, disposability, and cost-efficientature, megaporous cryogels are an attractive alternative for theurification of macro-biomolecules like mAbs, mRNA, pDNA andiruses.


F-37

igh throughput systems and mathematical models forrediction of protein renaturation processes

ernhard Mißbichler1,∗ , Cornelia Walther2, Sabrina Mayer1,orota Antos3, Alois Jungbauer1, Astrid Dürauer1

ACIB GmbHBOKURzeszow University of Technology

Heterologous recombinant expression of biopharmaceuticalroducts in E. coli often leads to high density protein aggregates

n inclusion bodies. In contrast to empirical strategies for processevelopment we developed high throughput screening methodsor inclusion body solubilization and subsequent refolding on �-cale. Thereof parameters were extracted to predict this crucialrocess steps in a laboratory scale stirred tank reactor controlledy inline monitoring.

Mathematical modelling using solubility curves and dissolu-ions kinetics was carried out.The solubilization process could beescribed by a homogeneous layer model with a high order of reac-

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ion which enables evaluation of the process on a numerical level.ence, solubilization conditions and process parameters can be

elated to the rate and yield of the solubilization. Approximationf the resistances potentially affecting the IB solubilization showedhat the process is controlled predominantly by pore diffusion.

aintaining the homogeneity of the IB suspension is sufficient forfficient solubilization, and further power input will not improvehe process. The HTS on �-scale can predict solubilization in STRver a range of 500 and can be used to determine optimal solubi-ization conditions for laboratory and industrial scale.

Furthermore, kinetics of the refolding process determined inifferent scales – from static vial over beaker stirred by magnetictirrer to laboratory scale STR were equivalent keeping differentcale-up criteria such as phase number and Reynolds numberithin a certain range. The established models and prediction

nable the engineering based and thus material and cost efficientrocess development of IB solubilization and refolding steps.


F-38

recipitation: A powerful tool for continuous purifica-ion of monoclonal antibodies

alf Sommer1,∗ , Anne Tscheliessnig1, Henk Schulz2, Bernhardelk2, Alois Jungbauer3

University of Natural Resources and Life Sciences Vienna/Department ofiotechnologyNovartis Pharma AGUniversity of Natural Resources and Life Sciences Vienna

Currently, recombinant biopharmaceutical protein productionperates discontinuous, in batch processes. Till 2012, the num-er of approved mAbs increased to 30. These trends require aore economical mAb production processes which only can be

chieved with a change from batch to continuous production.ost promising way for improvement of mAb production is

he implementation of novel continuous downstream processes.or mAb purification a combination of caprylic acid (CA) andolyethylene glycol (PEG) precipitation was developed. Target ofovel continuous precipitation purification was to reach the high-st possible purity values. Caprylic acid precipitation reduced bothMWI and HCP contents. For IgG capturing a PEG precipitationas performed, which further reduces HCP content. Adequateield and purity of the final product showed that CA/PEG pre-ipitation was competitive with affinity chromatography.

Furthermore, additional combination of precipitation methodsuch as of CA, PEG, CaCl2, and cold ethanol precipitation wereested to determine whether biopharmaceutical purity could bechieved with a continuous performable process. The most promis-ng combination provided a low HCP content (300 ppm), withoutggregates, and a yield of approximately 70%. That purity is nearlyuitable for a biopharmaceutical agent and the solitary use of pre-ipitation methods lead to a continuous performable process.



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F-39

evelopment of a rapid and low-cost tool for biophar-aceutical formulation screening

manda Quigley1,∗ , Stuart Hassard1, Dan Bracewell 2

deltaDOT LtdUniversity College London

One of the most significant challenges in protein formulations the tendency of proteins to aggregate. Aggregation can haveevere implications for the safety and effectiveness of biophar-aceuticals therefore methods to predict solution conditions that

imit aggregation would be of great industrial value. However, veryittle progress has been made in this area in recent years and cur-ent formulation screening methodologies are lengthy, outdatednd empirical and often incompatible with high protein concen-rations.

This has generated great interest in the measurement of theeak protein-protein interactions (quantified by the osmotic sec-nd virial coefficient, B22) involved in protein aggregation asmethod for rapidly predicting protein stability. Measurement

f protein-protein interactions also has a potential method forredicting other factors of importance to formulation such as sol-bility and viscosity.

This experimental study reports on the development of aovel screening platform to measure B22. The advantages of thisethodology over other techniques include significantly reduced

xperimental time and consumption of materials including valu-ble protein. In addition to increased resolution of peak shapeue to deltaDOT’s multipixel detection and algorithms, providing

mportant extra information on protein stability.B22 data is presented on a formulation screen of varying buffer

ype, pH and NaCl concentration for lysozyme as a model pro-ein, as well as 2 industrial monoclonal antibodies. This data isompared to that obtained via benchmark techniques for stabilitycreening; size exclusion chromatography (SEC) and dynamic lightcattering (DLS). This work establishes the decisive importance of

22 as a predictor of protein aggregation.


F-40

xtraction of compounds from the cultivation ofrthrospira (Spirulina) platensis using ion exchange

ernando Lisboa1,∗ , Luana Agassi 2, Evelin Brandão2, Marleiarbosa2, Mônica Okura2, Lúcia Pelizer2

Instituto Federal de Educacão, Ciência e Tecnologia do Triângulo Mineiro -ampus Uberlândia - Brazil/Universidade Federal do Triângulo Mineiro - BrazilUniversidade Federal do Triângulo Mineiro - Brazil

Arthrospira (Spirulina) platensis is known because of its highoncentration of compounds, so separating them is necessary to
romote their use, increasing the viability of the process. In thisontext, the aim of this study was to analyze the extraction of phy-ocyanin, chlorophyll, amino acids and beta carotene in the liquidhase of this cyanobacterium’s cultivation using ion exchange.


or this, inoculants were prepared in Erlenmeyer flasks, grownn ‘shaker’ for 7 days, with temperatures of 30 ◦C, 6 Klux illumi-ance and agitation of 150 rpm, with an initial concentration of0 mg L−1 in standard mineral medium. After that, the microor-anism was separated by filtration. The permeate was adsorbedn an ion exchange column using the resin IRA 402 (Rohm andaas), washed with distilled water and eluted with a NaCl gradi-

nt of 0.5 to 5.0% (w/v). The content of phycocyanin, chlorophyll,eta-carotene and amino acid present in the solution were evalu-ted by spectrophotometry UV. As a result, it was observed that thereatest concentration values were obtained for elution with 5%aCl. It was possible to obtain high concentrations of amino acids3 times, chlorophyll 16 times, phycocyanin 5 times and beta-arotene in 4 times at the exit of the column. Therefore, accordingo the experimental conditions under which this work was per-ormed, the ion exchange showed promise in the extraction of theompounds analyzed in Arthrospira (Spirulina) platensis cultivation.


F-41

evelopment of microfluidic system for screening andeaction optimization for lipase

siang-YuWang ∗ , Chong-Yi Ho

National Cheng Kung University

This study presents microfluidic platforms for the rapidcreening of lipase and the corresponding optimization of lipidydrolysis reaction. The conventional screening methods of lipase

nvolve time-consuming processes and high-end instrument, mak-ng the analysis not accessible. The proposed microfluidic systemstilize microdroplet (either dynamic or stationary) to enable rapidnalysis of the hydrolysis reaction and use instrument which iseadily accessible in common laboratories. Due to the merit ofigh surface area per volume (SAV) ratio of microdroplet, whichan be as high as 2.5 × 106 m−1 in this study, the lipid hydroly-is reaction occurring on the interface of droplet is extremely fast.he analysis for hydrolysis of soybean oil by the Burkhoderia sp.

ipase can be accomplished within 5 min. Reaction temperaturend pH can be easily adjusted via hydrodynamic control for reac-ion optimization. In the dynamic microdroplet system, dropletsontaining lipase and fluorogenic reagents that react with glyc-rol are generated at T-junction and flow through a serpentineicrochannel to allow lipase to react with the continuous phase

soybean oil). The retention time of the droplet inside the devicean be adjusted from less than 1 sec to 10 min; therefore, this sys-em is suitable for analyzing the initial rate to obtain the maximumeaction rate and Michaelis constant. The stationary droplet sys-em traps the droplet in an indentation for observation as long as0 days; therefore, it is applied on long-term examinations such

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F-42

rom small synthetic ligands to small protein scaffolds:ffinity reagents for biologics purification

na Roque1,∗ , Ana Pina1, Cláudia Fernandes1, Ricardo Branco1,na Dias1, Iris Batalha1, Olga Iranzo2, Christopher Lowe3

FCT-UNLUniversity of MarseilleUniversity of Cambridge

Over the past 40 years monoclonal antibodies and derivedtructures became the standard binding proteins representingowerful tools in biotechnology and biomedicine, namely on pro-ein purification, biocatalysis, diagnostic imaging and targetedherapy. Other protein binding scaffolds, with the robustness andersatility required, are recently being explored. We employediological and chemical combinatorial libraries supported by com-utational design tools to develop robust peptidomimetics basedn different scaffold molecules. The scaffold molecules rangedrom small synthetic ligands based on the triazine and Ugi reac-ions, to peptide-based �-hairpin engineered scaffolds and largeratural scaffolds. We studied the potential of these scaffold affin-

ty reagents to find binding partners against recombinant proteinsde novo designed fusion proteins), phosphorylated peptides andiral particles. These affinity reagents were employed as ligands forhe purification of the target biomolecules using standard affinityhromatography and magnetic fishing methodologies. The per-ormance of the new affinity adsorbents was benchmarked withurrently available purification and enrichment methodologies,howing high-performance and re-usability potential at low costs.


F-43

ptimization of lipase extraction/recovery produced byhe psychrotrophic yeast Leucosporidium scottii L117sing aqueous two-phase micellar systems

lysson Duarte1,∗ , André Lopes2, João Molino2, Adalbertoessoa2, Lara Sette3

UNICAMPUSPUNESP

This study aims to evaluate the lipase extraction by micellarystem using nonionic detergent Triton X-114 (TX-114)/McIlvaineuffer systems. Additionally, the parameters affecting the extrac-ion/recovery of lipase by ATPMS were optimized by Centralomposite Design (CCD23). The micellar system extraction wasrepared with TX-114 at 25 ◦C and 28 ◦C, both with 2.0% TX-14/McIlvaine buffer systems (pH 7.0). The variables optimizedere pH (3.5–7.5), temperature (19–31 ◦C) and TX-114 (2–14%

%w/w). The volume of the top and bottom phases were recov-
red (with sterile disposable syringes) and evaluated. The lipasectivity was measured using the synthetic substrate p-nitrophenylalmitate (p-NPP). The three variables were statistically signifi-ant (p < 0.05) when the response variable was of lipase recovery

%RECbot) in bottom phase (r2 = 0.93). Similar result was obtainedhen the variable was partition coefficient (K). For the parti-

ion coefficient of lipase and recovery lipase (%RECbot) in bottomhase, the influence of pH and temperature was negative and the

nfluence of concentration of TX-114 was positive. The majorityf lipase partition was in the micellar-rich phase (bottom phase)K = 7.7 and 4.7; 93.8 and 73.5% recovery in bottom phase andurification factor PF = 1.2 and 1.97, respectively). The greaterxtraction of lipase was in the bottom phase, this result was proba-ly influenced by the hydrophobic residues present on lipases. Theicellar-rich phase showed to be very clear and transparent. Thus,

his micellar ATPMS presents an alternative to the conventionalurification/extraction of lipase from L. scottii L117.


F-44

isk assessment of feed additives of microbial origin inhe European Union

aime Aguilera ∗ , Montserrat Anguita, Rosella Brozzi, Jaumealobart, Paola Manini, Jordi Tarrés-Call, Claudia Roncancio Pena

European Food Safety Authority, Italy

Microorganisms can be used in animal nutrition as probioticsr as silage agents, and are the source of other feed additives likenzymes, amino acids and vitamins. In the European Union, alleed additives need, by law, to undergo a risk assessment, which isonducted by the European Food Safety Authority. A proper char-cterisation of the microorganism is fundamental for its safetyssessment. This includes an unequivocal identification of thepecies, consideration of its pathogenic or toxigenic potential andhe presence of resistance to antimicrobials, the genetic basis ofhich may need to be established. Genetically modified strains

equire particular attention, including a full molecular characteri-ation of the modification. If the microorganism is the source of andditive which is purified after fermentation, it is necessary to testhether the final product is free from the production organismnd from its DNA if it encodes antimicrobial resistances.

The characterisation of the microorganism determines theature and extent of further tests to be done to establish the safetyf the product. EFSA grants a qualified presumption of safety (QPS)tatus to certain species having well documented safety. Strainsulfilling the requirements for QPS are considered safe for targetpecies, consumers and the environment without the need of fur-her testing.

EFSA has published a complete set of guidance stating the dataeeded for the safety evaluation. The conclusions of the assess-ents are published in form of scientific opinions, and allow the

uropean regulatory authorities to make scientifically sound deci-


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ell factories

G-01

ine-tuning of defined media for chemical production byactic acid bacteria

adri Aller ∗ , Kaarel Adamberg, Veronica Timarova, Andrus Seiman,aivo Vilu

The Competence Center of Food and Fermentation Technologies

The lactic acid bacterium Lactococcus lactis has emerged asn effective cell factory for recombinant protein production andecretion [1] and for the synthesis of various biochemicals [2]. Inrder to produce chemicals of interest, the growth physiology ofhe expression host has to be well characterized [3]. Chemicallyefined media (CDM) are required for studying the metabolism ofells as well as producing recombinant proteins [3]. However, if theedium contains an excess of nutrients, analytical measurements

re rendered imprecise and the interpretation of metabolic data isggravated.

We have previously elucidated the growth requirements of L.actis IL1403 and developed several CDMs where the utilization ofach amino acid is larger than the measurement error (5%). Nowe compare the growth of IL1403 on 3 of those media in continu-us cultivation experiments and show the differences in metabolicehavior. Naturally, decrease in the concentrations of componentseads to lower maximum specific growth rate values. Nevertheless,iomass yield and productivity were higher and energy spillingas lower in media with reduced concentrations of nutrients, e.g.

he metabolism of IL1403 is more efficient in minimalized media.hus, the importance of fine-tuning a growth medium for a specifictrain and process cannot be underestimated.

eferences

].Morello, et al. J Mol Microbiol Biotechnol 2008;14(1–3):48–58.].Gaspar, et al. Biotechnol Adv 2013;31(6):764–88.].Marreddy, et al. PLoS One 2010;5(4):e10317.


G-02

evelopment of a yeast cell factory for production ofromatic products

ngelica Rodriguez Prado1,∗ , Kanchana Kildegaard1, Mingji Li 1,rina Borodina1, Jens Nielsen2

Novo Nordisk Foundation Center for Biosustainability, Technical University ofenmarkDepartment of Chemical and Biological Engineering, Chalmers University ofechnology

There is much interest in aromatic chemicals in the chemicalndustry as these can be used for production of dyes, anti-oxidants,
utraceuticals and food ingredients. Yeast is a widely used cell
actory and it is particularly well suited for production of aro-atic chemicals via complex biosynthetic routes involving P450

nzymes. In Saccharomyces cerevisiae the fluxes towards aromatic

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cids (L-tryptophan, L-tyrosine and L-phenylalanine) are strictlyontrolled on transcriptional and kinetic levels and therefore areifficult to manipulate.

We engineered S. cerevisiae for increased production of aro-atic compounds by eliminating degradation, up-regulating the

ey enzyme encoding genes, and removing feed-back inhibitionn the pathway. In order to test the strain performance we over-xpressed heterologous pathway for coumaric acid production.e obtained 4-fold higher concentrations of coumaric acid in the

ngineered strain compared with the reference strain. Overexpress-on of the enzymes prephenate dehydrogenase and transketolasead a negative effect on production of coumaric acid.

In summary, we developed a S. cerevisiae platform strain suitableor production of aromatic amino acids-derived compounds.


G-03

omologous and heterologous expression of dehydroge-ases and oxidoreductases of Ralstonia eutropha H16

teffen Gruber ∗ , Petra Koefinger, Helmut Schwab

Graz University of Technology

Ralstonia eutropha is a Gram-negative, strictly respiratory fac-ltative chemolithoautotrophic bacterium which can use H2 andO2 as sole sources of energy and carbon in the absence of organic

ubstrates. It has attracted great interest for its ability to degradelarge list of chloroaromatic compounds and chemically relatedollutants. Furthermore it was already applied for the productionf biodegradable polymer polyhydroxyalkanoates on an industrialcale. R. eutropha serves as a model organism for the mechanismsnvolved in the control of autotrophic carbon dioxide fixation,ydrogen oxidation and denitrification.

In our project we are interested in establishing specialized R.utropha based cell factories by genetic engineering. The particularnterest is constructing cells efficiently performing oxidoreductaseeactions by overexpression of homologous and/or heterologousnzymes.

One of the main types of oxidoreductase reactions is per-ormed by dehydrogenases, particularly alcohol dehydrogenases,hich have a wide range of possible biotechnological applications.iotransformations involving the interconversion of alcohols,ldehydes and ketones have great potential for the commercialroduction of pure optically active compounds and also for otherrocesses such as the treatment of industrial effluents.

The genome of R. eutropha H16 contains a remarkable diver-ity of oxidoreducteases. A selection of alcohol dehydrogenases asell as short chain dehydrogenases of R. eutropha H16 was clonednd expressed in native versions in Escherichia coli. Their activityas analyzed by NAD/NADH dependent enzyme activity assaysith different substrates. Currently we are working on the homol-gous expression of these enzymes in R. eutropha H16 and their
unctional analysis.

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G-04

pplicability of a mechanosensitive channel inorynebacterium glutamicum as a versatile exporter

en-ichi Hashimoto ∗ , Tomoyuki Konichi, Isamu Yabe, Tsuyoshiakamatsu, Hisashi Kawasaki

Tokyo Denki University

Corynebacterium glutamicum is used worldwide in the industrialermentative production of glutamic acid.

In 2007, the involvement of the NCgl1221 gene, which encodeshomolog of the mechanosensitive channel of small conduc-

ance protein, in glutamic acid overproduction was reported [1].e used electrophysiological methods to obtain direct evidence

f the excretion of glutamic acid through the NCgl1221 proteinhannel. We showed that the NCgl1221 protein functions as aechanosensitive channel [2], and we found direct evidence of

lutamic acid excretion through this channel by passive diffusion3]. Furthermore we found that aspartic acid, phenyl propioniccid and lysine were also transported across the cytoplasmic mem-rane via NCgl1221 by passive diffusion. We further evaluatedhe possibility of the effective application of this mechanosensi-ive channel as a versatile exporter, for the creation of microbialell factories for the production of valuable chemicals other thanlutamate. If successful, this would be a substantial developmentn exporter engineering (ExE). A gain-of-function mutation inCgl1221 was expressed in a phenylalanine producer (Escherichia

oli AJ12741) and an inosine producer (Escherichia coli FADR adddd AJ13473). These strains displayed remarkably high productiv-ty compared with a control strain. These data suggest that utilizinghe mechanosensitive channel as a versatile exporter could be anffective method for improving fermentation yield.

eferences

].Nakamura J, et al. Appl Environ Microbiol 2007;73:4491–8.].Hashimoto K, et al. Biosci Biotechnol Biochem 2010;74:2546–9.].Hashimoto K, et al. Biosci Biotechnol Biochem 2012;76:1422–4.


G-05

esearch on the Relationship between Pyruvate Kinasend the Biosynthesis of Epothilones in Sorangium cellu-osum 2161

inli Liu ∗ , Lin Zhao, XiaonaWang

Qilu University of Technology

Pyruvate kinase (PK) catalyzes the formation of pyruvate andTP from phosphoenolpyruvate and ADP, and it is a key enzymef glycolysis. Pyruvate has been one of most important substratesor the biosynthesis of epothilones. There are two coding genesf PK in Sorangium cellulosum 2161: pyk sce5197 and pyk sce4540.
n this paper, we analyzed the relationship between PK activ-ty, epothilone production, and the gene expression quantity ofyk sce5197 and pyk sce4540 in S. cellulosum 2161 incubated withitamin K3, which is a specific inhibitor of PK. Results showed
CELL FACTORIES

hat PK activity significantly influenced epothilone production.n addition, the expression of pyk sce4540 and putative oxidaseene (OX) in S. cellulosum 2161 treated with vitamin K3 wasemarkably inhibited, while the response of pyk sce5197 was notensitive. These results indicate that PK is one of the key enzymesn epothilone biosynthesis across the metabolic network in S. cellu-osum 2161. It might also play an important role in the biosynthesisf epothilones.

Keywords: Epothilones, pyruvate kinase, inhibitor, Sorangiumellulosum 2161, qRT-PCR


G-06

ngineering of the UDP-precursor biosynthesis pathwayo enhance the production of capsular polysaccharide in. coli K4

lisabetta Carlino ∗ , Chiara Schiraldi, Ottavia Argenzio, Ileana Delloacono, Donatella Cimini

Seconda Università degli Studi di Napoli

Chondroitin sulphate is a linear polysaccharide chain made oflternating units of glucuronic acid and N-acetyl-galactosamine,odified by sulfation in various positions depending on both the

nimal source and tissue of origin [1]. Besides its established use inhe treatment of osteoarthritis, potential applications as an anti-nflammatory drug and surprising antiviral properties have beenuggested.

E. coli K4 produces a capsule that belongs to the class ofAG-like polymers; in fact the molecule can be described as a

hondroitin backbone decorated with fructose side branches onhe GlcA residues.

The synthesis of the necessary sugar precursors for chain elon-ation is performed by the cells through two different metabolicathways. The first starts from glucose 6-phosphate and ends withhe production of UDP-GlcA. The second one starts from fructose-phosphate and ends with the synthesis of UDP-GalNAc.

In the present work two E. coli K4 recombinant strains werengineered to improve the productive yields of K4 capsularolysaccharide (CPS) by overexpressing genes involved in theiosynthesis of one of the UDP-sugar precursor. The E. coli wildype strain 05:K4:H4 and EcK4r3 strain (previously obtained inur laboratories) were both transformed with the construct.

In order to evaluate the production of K4 CPS by each strain,hakeflask and microbioreactor experiments were performed.

Pathway modifications were also analysed to correlate the pro-uctive yields of K4 CPS to gene overexpressions.

eference

].Cimini D, et al. Homologous overexpression of RfaH in E. coli K4improves the production of chondroitin-like capsular polysaccha-


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G-07

he isolation of novel marine yeasts; a new procedure

bdelrahman Saleh Zaky ∗ , Chenyu Du

University of Nottingham

The recent research on marine yeasts highlighted that they areble to produce many bioactive substances including enzymes,mino acids, killer toxins, and vitamin C with many potentialpplications in food, pharmaceutical, fuel, cosmetics and chem-cal industries [1]. However, the employment of marine yeast inesearch and industry is limited due to the lack of suitable isola-
ion methods. Current methods suffer from fungus interferencend/or low number of yeast isolates. In this report, a novel 3-stepsolation method has been developed. Samples were taken fromeawater, sea sand or seaweed which could potentially contain
[1

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arine yeasts. These samples were enriched using a nutrient richedia with antibiotics for three cycles as the first step. In the

econd step, single colonies with different morphologies weresolated on plates of agar medium lacking antibiotic after 24–48 hf incubation at 30 ◦C. These isolates were further confirmed toe pure cultures of yeast by streaking them on agar plates andhecking their purity and morphology under a microscope. Inhis study, 14 samples from different marine habitats in Egypt, UKnd USA were used for the isolation. 122 isolates were obtainednd confirmed to be pure yeast cultures. Our 3-step isolationethod for marine yeast has demonstrated high level of efficiency

omparing with two reference methods.

eference

].Zaky, et al. Marine Yeast Isolation and Industrial Application FEMS Yeast
Research 2014 (accepted).

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nvironmental biotechnology

H-01

ormate oxidation-driven calcium carbonate precipita-ion by Methylocystis parvus OBBP for a concomitantuilding material surface protection and atmosphericethane removal

iovanni Ganendra1,∗ , Giovanni Ganendra2, Adrian Ho3, Nicooon2

UgentGhent universityNetherlands Institute of Ecology

Microbially Induced Carbonate Precipitation (MICP) is the basisor several biotechnological applications in the construction sec-or e.g., concrete surface protection. The typically used urea based

ICP poses several disadvantages, such as ammonia release to their. Therefore, an alternative MICP for the application on buildingaterials should be investigated. In this study, MICP was driven by

ormate oxidation by Methylocystis parvus OBBP, a methanotrophicacteria.

Up to 91.4 ± 1.6% of the initial calcium was precipitated in theethane amended cultures, but only a maximum of 35.1 ± 11.9%hen methane was not added. Because the bacteria could only uti-

ize methane but not formate for growth, a higher culture densitynd subsequently a higher calcium removal was exhibited by theacteria when methane was added. The methane oxidation rateMOR) of the bacteria decreased from 3.15 ± 0.32 �g CH4 (ml h)−1

hen formate was not added to 0.52 ± 0.01 �g CH4 (ml h)−1 when.88 g L−1 of formate was added (i.e., the maximum formate addi-ion). An optimum 0.67 ± 0.03 g CaCO3 g Ca(CHOOH)2

−1 calciumarbonate precipitate yield was obtained when 109 cells ml−1 and.5 g L−1 of calcium formate was used.

Compared to the currently used biogenic urea degradation ashe basis for MICP, several advantages are presented here: the pre-ention of ammonia release to the air and nitric acid productionnd the atmospheric methane removal.


H-02

ctivity of Bdellovibrio on Sludge bacteria and its poten-ial use for cleaning of Membrane Bioreactors

elek Özkan ∗ , Merve Akay Celik, Pınar Karagöz, Hilal Yılmaz, Ciseengezer

Gebze Institute of Technology, Environmental Engineering Department, 41400ebze Kocaeli, Turkey

Bdlelovibrio bacteriovirus is a gram negative predator bacteriumeeding on other gram negative bacteria. Its activity on biofilmsormed by different bacterial species especially pathogenic ones
as been investigated in recent years. One of the recent tech-ologies for wastewater treatment is Membrane bioreactors(MBRs)hich have high effluent capacity and good disinfection capa-
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ENVIRONMENTAL BIOTECHNOLOGY

ility. One important problem of MBR technology is membraneouling. In this study, Bdellovibrio activity was analysed for reduc-ng membrane fouling caused by sludge bacteria, and potentialf B. bacteriovirus as a biological cleaning method for MBR sys-ems was investigated. For this aim, nineteen bacteria were isolatedrom wastewater sludge and characterized by 16SrRNA analysis.acteria were mostly found to be a member of Gammaproteobacte-ia, Betaproteobacteria and Actinobacteria families. B. bacteriovirushowed high lysis activity on Aeromonas, Proteus, Alcaligenesnd Bordatella species. B. bacteriovirus activity was also tested onembranes plugged after filtration of wastewater. Pore size of theembranes was found to be important for B. bacteriovirus activity.

lux through Poly(ether)sulphone (PES) membrane with a poreize of 0.05 micrometer was improved after cleaning by predatoracteria. B. bacteriovirus successfully degraded the biofilm formedy sludge bacteria on the membrane surface.

cknowledgement

We thank Scientific and Technological Research Council ofurkey for supporting this study (Project no: 112Y156).


H-03

omparison of inoculums in the removal of 2-utoxyethanol from air emissions by biotricklinglter: Performance and microbial monitoring

aria del Carmen Perez1,∗ , Francisco Javier Alvarez-Hornos1,aniel Dobslaw2, Karl-H. Engesser2, Carmen Gabaldon1

University of ValenciaUniversity of Stuttgart

2-butoxyethanol is one of the most used glycol ether in indus-rial activities and the treatment of air 2-butoxyethanol-emissionsecome necessary. Biotechnologies are potential treatment tech-ologies due to their low operational costs. The use of two

noculums in the treatment of 2-butoxyethanol by biotrick-ing filters (BTFs) packed with polyurethane-foam was studied.

pure culture of Pseudomonas putida, previously adapted to 2-utoxyethanol, was used as inocula in a BTF operated in theniversity of Stuttgart. Fresh activated sludge from a munici-al waste water treatment plant was used as inocula in a BTFperated in the University of Valencia. An empty bed residenceime of 12.5 s and inlet concentrations of 400 and 800 mg/Nm3

ere applied. After 40 days of operation at 400 mg/Nm3, the BTFnoculated with Pseudomonas putida reached removal efficienciesREs) ∼ 80%, whereas the BTF inoculated with activated sludgeresented REs ∼ 60%. At 800 mg/Nm3, the BTF inoculated withseudomonas putida reached REs ∼ 60%. Microbial community wasonitored in both BTFs by using denaturing gradient gel elec-

rophoresis analysis (DGGE) with subsequent 16S sequencing and
lating methods using 2-butoxyethanol as sole carbon source.
AcknowledgementsThe research leading to these results has received funding from

he People Programme (Marie Curie Actions) of the European


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nion’s Seventh Framework Programme FP7/2007-2013/underEA grant agreement n◦ 284949. Financial support from Ministerioe Ciencia e Innovación (CTM2010-15031/TECNO) and General-

tat Valenciana (PROMETEO/2013/053), is also acknowledged. Ma

armen Pérez acknowledges her FPU contract


H-04

iversity of fungi present in the Sossego copper mine inará State, Brazil

uciana Jandelli Gimenes ∗ , Bruno Karolski, Claudio Nascimento,len Perpetuo, Ingrid Avanzi, Louise Gracioso, Marcela Baltazar,arcela Veiga, Tatiana Reis, Benedito Correa

Universidade de São Paulo

Industrial growth, which results from technological develop-ent and activities considered essential to human life, has caused

erious environmental problems. For some time, fungi have beensed in organic waste bioremediation. Recently, a good alterna-ive in the bioremediation of heavy metals has been discovered.his work aims to isolate, identify and characterize fungi andvaluates mechanisms and strategies for remediating areas con-aminated by copper. Soil and water samples have been collectedrom Sossego Mine (Pará State, Brazil) and have been grown onDA culture medium, incubated at 28 ◦C for 5 days. After thiseriod, the isolates were identified by morphologic and molecularethods. Since the project started, 49 fungi were isolated, and vari-

us species among 12 different genders were identified: Aspergillus,ureobasidium, Bionectria, Eupenicillium, Eurotium, Fusarium, Lenti-us, Microspheropsis, Nigrospora, Penicillium, Purpureocillium andhizopus. This work has great importance due to the low cost ofepair systems compared to conventional ones. It also allows aetter use of copper wastes and, consequently, a better miningconomic return. Moreover, the main advantage of this technol-gy is further reduction of environmental impact by the miningctivity.


H-05

uO nanoparticles toxicity against bacteria strains iso-ated from agricultural soil

andra I. Concha-Guerreroa , Elcia M.S. Britob, Hilda A. Pinón-

astilloa, M. Antonia Luna-Velascoa, Erasmo Orrantia-Borundaa,∗

Centro de Investigación en Materiales Avanzados, SC, Chihuahua, MéxicoUniversidad de Guanajuato, Guanajuato, México

The intensified use of nanoparticles by human society bringsut the risk of exposure to these particles. In fact, some kinds
f nanoparticles were found to effects on the organisms or onheir cells [1–3]. In the present work, we studied the interactionnd effects produced by oxide copper nanoparticles (CuONP) toative bacterial strains. Selected strains (Chryseobacterium indo-
h



heticum, Brevibacillus laterosporus and Pantoea ananatis) weresolated from an agricultural soil located on Chihuahua state,

exico. The most intense effects were observed on C. indotheticumnd on B. laterosporus, e.g., on C. indotheticum superficial damagescavities and pits) were observed on its membrane, and on B. lat-rosporus severe oxidative stress and the presence of Cu in the outerell membrane were detected.

eferences

].Gajjar P, Pettee B, Britt DW, Huang W, Johnson WP, AndersonAJ. Antimicrobial activities of commercial nanoparticles against anenvironmental soil microbe, Pseudomonas putida KT2440. Journal ofBiological Engineering 2009;3(9).

].Fent K. Ecotoxicology of engineered nanoparticles. F.F.H. Berlin: Springer-Verlag; 2010.

].Das M, S.K.H., An SSA, Yi DK. Review on gold nanoparticles and theirapplications. Toxicol Environ HealthSci 2011;3(4):193–205.


H-06

he use of dairy processing waste as a media for growthf recombinant microorganisms

ichael Ryan ∗ , GaryWalsh

University of Limerick

A laboratory-based study was undertaken to assess the potentialf whey waste as a media for the growth of recombinant Escherichiaoli, which remain a preferred choice for process-scale manufacturef many recombinant proteins.

Growth characteristics of a recombinant strain of Escherichiaoli (MC1061) was assessed on 2 whey-based media, and com-ared to growth on standard LB (Luria broth) media, known tochieve high cell densities. Whey-based media were: whey onlyWM), and whey mixed with LB media (9:1 ratio; W:LB). MediaH was adjusted to 7 prior to autoclaving.

All experiments entailed media inoculation (100 ml) with.0 ml of the recombinant strain (grown in Luria-Bertani broth [LB]o OD600 of 1.5) at 37 ◦C in a shaking incubator (250 rpm). Growthinetics and maximum biomass yield was followed by absorbancet 600 nm and dry cell weight determination, respectively.

E. coli growth rates (A600, 6 h cultures, n = 3)ere: WM (0.257 ± 0.0448) < W/LB (0.348 ± 0.0246) + LB

1.178 ± 0.0872). Dry cell weights attained after 24 hrs were:M (4.73 mg ± 0.001387) < W/LB (19.8 mg ± 0.00593) + LB

31.9 mg ± 0.00699).Un-supplemented whey waste is a poor media for E. coli growth.

owever, optimally nutrient supplemented whey may yet prove aiable and inexpensive media for E. coli fermentation to high cellensities, converting a potentially waste product into a valuableommodity.

This work is funded by the Irish EPA under the Science, Tech-ology, Research & Innovation for the Environment (STRIVE)


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H-07

ffects on Tomato Growth and Soil Bacterial Communityy Application of Arthrobacter woluwensis ED Immobi-ized in Alginate Beads

ong-Gyu Song ∗ , Seung-Tak Kwon

Kangwon National University

For the promotion of plant growth and increase of persistencef plant growth promoting rhizobacteria (PGPR) in rhizpsphere,omato growth was examined after application of PGPR Arthrobac-er woluwensis ED immobilized in alginate bead. When tomatoeedlings were treated with A. woluwensis ED of 1 × 106 cells goil−1 and incubated for 30 days in a plant growth chamber,hoot and root length, fresh weight and dry weight of the grownomato plants treated with the suspended inoculants significantlyncreased by 36.2, 59, 51.1 and 37.5%, respectively comparedo the uninoculated control. The treatment of the immobilizedacteria increased those by 42, 67.4, 62.5 and 60.4%, respectivelyompared to the uninoculated control. Therefore, the enhance-ent of tomato growth by the treatment of the immobilized

acteria was higher than those by the suspended inoculants. Theffects of the inoculation on soil bacterial community and the fatef the inoculated bacteria were monitored by DGGE analysis. TheNA band intensity of A. woluwensis ED in the tomato rhizosphere

reated with the suspended inoculants continuously decreasedfter inoculation, but the band intensity in the tomato rhizosphereoils treated with the immobilized inoculants showed the max-mum at 1 week after inoculation and the decreasing rate wasess than that of the suspended inoculants, which indicated theonger maintenance of the immobilized bacteria at rhizosphere.herefore, encapsulation of PGPR in alginate beads may be moreffective than liquid inoculant for the plant growth promotionnd survival of PGPR at plant rhizosphere.


H-08

ioremediation of heavy metal contaminated soil usinglant extract as biomaterial

n-Hyun Nam ∗ , Chul-Min Chon, Jae-Gon Kim

Korea Institute of Geoscience and Mineral Resources (KIGAM)

An indigenous plant extract was used to produce calcite fromanavalia ensiformis as effective biomaterial, and its ability to

orm under stable conditions was compared to that of purifiedrease. X-ray diffraction and scanning electron microscopy weremployed to elucidate the mechanism of calcite formation fromhe crude plant extracts. The results revealed that urease in thelant extracts catalyzed the hydrolysis of urea in liquid state cul-ures and decreased heavy metal amounts in the contaminatedoil. The heavy metal amounts were decreased in the leachate from
he treated mine soil; 55.4% of Pb, 35.6% of Cu, 33.6% of Mn,2.0% of As, and 25.6% of Fe, respectively. The procedure describederein is a simple and beneficial method of calcite biomineraliza-

ion without cultivation of microorganisms or further purificationf crude extracts. This study suggests that crude plant extractsf Canavalia ensiformis have the potential to be used in place ofurified forms of the enzyme during remediation of heavy metalontaminated soil. Thus, we report the molecular characterizationf the microbial diversity and composition change of the minempacted soil or waste ore taken from the site during bioremedi-tion processes are presented. To evaluate the stability of DGGEatterns in remediated mine soils in comparison with before orfter plant extract treatment, a number of soil samples with timenterval in the mine impacted soil were compared using PCR-GGE of 16S rDNA.


H-09

nvestigation of organic wastes from Mediterraneanlants to produce biogas by anaerobic digestion

amille Menard ∗ , Anais Fantoni, Pascale Bradesi, Eric Leoni,ominique Cancellieri

Universite de Corse

The University of Corsica is contributing to the research ofoth efficiency and integration of renewable energy in the mainlectrical grid. Since 2013, a scientific program concerning thealorization of biomass energy has been developed to investigatehe methane potential through the anaerobic digestion processf lignocellulosic resources. In Corsica, due to the clearing brusholicy to prevent forest fires, cellulosic wastes are generated.esides, Corsica is an important producer of essential oil fromediterranean species thanks to a process generating an important

mount of dried vegetation as waste every year.The aim of this preliminary study on biomass as renewable

nergy was to characterize and to select the most appropriateubstrates for the anaerobic digestion process. Fiber contents andiochemical Methane Potential (BMP) were performed on fiveubstrates. Heather, rockrose and strawberry tree were chosen asepresentative of forest fuels. Water distillation residues of laurelnd immortelle were considered as natural resource wastes.

On this work, we focus on the three species which had theest BMP. Rockrose, dry residue of immortelle and strawberry treeroduced 139, 124 and 87 Nm3 CH4 per grams of volatile solids,espectively. The ratio holocellulose: lignin, thanks to the Vanoest method of fiber determination, was determined for thosepecies: 3.8, 3.4 and 1.7. This parameter is a key factor to takento account for the correlation of the BMP with the composi-ional characteristics. Further work will be performed on thesehosen substrates to optimize the anaerobic digestion process in


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H-10

iomolecules for Environmental Application: Direct Usef Biorecovered Precious Metal Catalyst from Artificialastewater for Chlorinated Environmental Pollutantegradation

an Sofian Yunus ∗ , Shen-Long Tsai

National Taiwan University of Science and Technology

In the last decades, precious metals have been extensivelytudied for their potential use as catalysts for chlorinated pol-utant degradations. These pollutants are resulted from variousndustrial processes, groundwater, pharmaceuticals wastewaterreatment plant (WWTP) effluents, and many other sources. Inhis study, a new recovery method of precious metal catalyst forechlorination of environmental pollutant was examined. Therecious metal catalyst was produced by reduction of its ionic state,hich was recovered from artificial wastewater, using biomoleculesased adsorbent. This study demonstrates that the newly discov-red biorecovery concept of precious metal catalyst from artificalastewater using biomolecules based adsorbent and its direct uses catalyst for chorinated pollutant degradation are both feasiblend applicable.


H-11

egradation of recalcitrant polymers and synthetic dyesy microrganisms isolated from polluted soils

shutosh Kumar Verma1,∗ , Dornakova Veronika2, Hana Kotulova2,aterina Malachova2, Cenek Novotny3

University of OstravaOstrava UniversityAcademy of Sciences of the Czech Republic

An attempt was made to isolate microorganisms which mayave potential to degrade polymer plastics and recalcitrantrganopollutants. The prescreening was carried out on agar platesontaining 100 ppm of a model dye, Azure B, whose decolorizationas considered to be a tool indicating an enhanced degrada-

ion capacity towards recalcitrant polymers and xenobiotics. Theompost, sludge and polymer-polluted soils had been used for iso-ation. More than 50 monocultures including bacteria, yeast andungi belonging to various genera showed efficient decolorizationf Azure B. Identification of the isolates was carried out using ribo-omal molecular markers. A few isolates had been used for theegradation of virgin and pre-treated plastic polymers with limiteduccess. Culture-independent approach was applied to assess theiversity of microbial communities in polymer-polluted soil(s). Aungal isolate affiliated with Trametes sp. had been further used forhe decolorization of an anthraquinone dye, Remazol Brilliant Blue

(RBBR) at a concentration of 50 ppm. Our aim was to evaluate
he efficiency and behaviour of the fungus under the conditionsf rotating biological contactor reactor in both batch and contin-ous mode. The results indicated that the selected fungal species


an decolorize RBBR more efficiently in continous mode than inatch mode.

Acknowledgements:The research wassupported by the following projects: OPVK

Z 1.07/2.3.00/30.0019, FP7-KBBE-2012-6-singlestage no. 312100IOCLEAN, CZ.1.05/2.100/03.0100 (IET) and CR National Feasi-ility Programme I no.LO1208.


H-12

omposting of creosote-impregnated wood via com-osting with green wastes: Ecotoxicity and microbialommunity dynamics during polycyclic aromatic hydro-arbons degradation process

tefano Covino ∗ , Zdena Kresinová, Monika Cvancarová, Alenailipová, Tomas Cajthaml

Institute of Microbiology AS CR, v.v.i

Composting has been shown to be a suitable bioremediationethod for the clean-up of polluted matrices. In our pilot-scale

est, creosote-impregnated wood with an overall polycyclic aro-atic hydrocarbons (PAH) contamination of 26498 mg kg-1 was

he target material and two different substrates were used as bulk-ng agents, namely grass cuttings and pre-treated broiler litter.ncubation took place in 400 l static composters over a periodf 240 days (40 days active composting followed by 200 daysaturation). The effectiveness of the two composting processesas comparatively evaluated throughout the whole incubationeriod by means of contaminant degradation analyses and tox-

cological testing whereas shifts in microbial community structureere assessed via phosholipid fatty acid analysis (PLFA) and 454-yrosequencing. The grass substrate promoted an almost completeemoval (i.e. 97%) of the total PAH content in creosote wood, whileverall PAH depletion using broiler litter as bulking agent was 81%f the original concentration. The acute toxicity test towards theuminescent bacterium Vibrio fischeri and the phytotoxicity testased on germinability of barley seeds (Hordeum vulgare L.) showedhat the PAH degradation process was accompanied by a significantrop in toxicity. Regardless of the treatment typology, PLFA pro-ling highlighted an increased incidence of Gram- bacteria andungi in the crucial phases of PAH dissipation. Morevoer, fungippeared to be dominant also in the maturation phase, especiallyhen broiler litter was the substrate. Pyrosequencing analyses of6S rRNA and ITS gene sequences for bacteria and fungi respec-

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H-13

election and characterization of indigenousydrocarbon-degrading bacteria from tourist ports

n the Mediterranean Sea Basin

nrica Bullita1 , Claudio Ruggeri 1, Simona Sergi1, Laura Serreli 1,iovannimatteo Erby2, Alessio Nieddu2, Alessandra Carucci 2, Elenaamburini 1,∗

University of Cagliari - Department of Biomedical SciencesUniversity of Cagliari - Department of Civil-Environmental Engineering andrchitecture

Pollution by petroleum hydrocarbons is one of the major envi-onmental problems in ports and it is mainly associated withhip/boat traffic and related facilities. Ports are not closed systemsnd their pollution may impact adjacent coastal areas. Hydrocar-on degraders and particularly the obligate hydrocarbonoclasticacteria carry out a fundamental and global activity in biologicalemoval of hydrocarbons in marine habitats.

This study was carried out within MAPMED, a multidisciplinaryroject aimed to improve the environmental sustainability ofourist ports in the Mediterranean Sea with regard to monitoringnd reduction of hydrocarbon pollution.

Three tourist ports were selected as case study sites: CagliariItaly), El Kantaoui (Tunisia), and Heraklion (Greece). The degra-ation potential of the autochthonous bacterial communities wasvaluated enumerating heterotrophs and hydrocarbon degradersy MPN in the surface seawater. Heterotrophs were significantlyore abundant in seawater from Cagliari port as compared to

l Kantaoui and Heraklion ports. On the contrary, higher viableitles of diesel- and phenanthrene-degraders were found in sea-ater from El Kantaoui port compared to the other two areas.ydrocarbon-degrading bacteria were isolated and characterized

egarding their phylogenetic position and catabolic abilities. Theydrocarbon degradation activities were evaluated by GC-MS inerobic batch reactors on diesel as carbon source. The majorityf degraders from Cagliari were assigned to Pseudomonas whereastrains from El Kantaoui and Heraklion were assigned to Alcanivo-ax and Marinobacter.

The selection of the most appropriate methodologies for theco-efficient remediation of petroleum-hydrocarbon contamina-
ion of selected sites is currently in progress.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.1952h


H-14

haracterization of sulphate reducing bacteria commu-ities in sediments from tourist ports in the Mediter-anean Sea Basin

laudio Ruggeri 1,∗ , Paolo La Colla2, Enrica Bullita2, Simona Sergi2,rancesco Vitali 3, Giorgio Mastromei3, Elena Tamburini 2

University of CagliariUniversity of Cagliari - Department of Biomedical SciencesUniversity of Florence - Department of Biology

Ports receive pollution from land, ships and port facilities.urthermore, tourist ports are subject to seasonal anthropogenicmpacts. Hydrocarbon contamination associated with port activ-ties poses major concerns for human health and coastalcosystems.

This study was carried out within MAPMED, a multidisciplinaryroject aimed to improve the environmental sustainability ofourist ports in the Mediterranean Sea with regard to monitoringnd reduction of hydrocarbon pollution.

Three tourist ports were selected as case study sites: CagliariItaly), El Kantaoui (Tunisia), and Heraklion (Greece). In each port,ampling stations were located in different sectors according toheir different uses. Three sampling campaigns were carried out ininter, spring at the beginning of tourist season and late summer at

he end of tourist season. Samples of surface and anoxic sedimentsere collected at different stations.

The dsrAB gene was chosen as genetic marker to specifi-ally characterise sulphate reducing bacteria (SRB). It codes forhe dissimilatory sulphite reductase catalysing the last step inhe sulphate reduction pathway. Hydrocarbon degradation underulphate-reducing conditions is an important process in marineediments in anoxic environments. T-RFLP analysis of dsrAB geneas employed to elucidate the community structure of SRB. Thisork provides a spatial comparison of SRB at two scales, differentort sectors within each port area and different port areas acrosshe Mediterranean Sea, as well as a temporal comparison amongifferent seasons.

The selection of the most appropriate methodologies for theco-efficient remediation of petroleum-hydrocarbon contamina-



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H-15

xperimental design in the degradation of pyrene byarine-derived fungi Chaunopycnis alba CBMAI 1346

nd Xylaria sp. CBMAI 1464

aria Vasconcelos1,∗ , Rafaella Bonugli-Santos1, Marili Rodrigues2,inésio Boaventura2, Lara Sette3

Center of Chemical, Biology and Agricultural ResearchOrganic Chemical and Pharmaceutical DivisionSão Paulo State University

Marine-derived filamentous fungi are considered strategic foriotechnological applications, including bioremediation of envi-onmental pollutants under saline conditions. Chaunopycnis albaBMAI 1346 and Xylaria sp. CBMAI 1464 isolated from marine

ponges were subjected to experimental design in order to opti-ize their ability to degrade pyrene. At first a Plackett-Burman (PB)odel was used, with nine variables, providing 20 assays in total.

he matrix was analyzed statistically using STATISTICA 7.0. Afterdays C. alba CBMAI 1346 and Xylaria sp. CBMAI 1464 reached

9.39% and 46.00% of pyrene degradation, respectively. For theungus C. alba CBMAI 1346, salinity, concentration of malt extract,eptone and yeast extract and the presence of MnSO4 presentedpositive effect on the pyrene degradation, being significant at

0% (p < 0.1). For Xylaria sp. CBMAI 1464, pH, salinity, concen-ration of malt extract, peptone and yeast extract and inoculumresented a positive effect on the pyrene degradation, however,one of them was significant at 90% (p < 0.1). In the second PBomposed by 5 variables (total of 16 assays) the fungus C. albaBMAI 1346 reached 58.28% of pyrene degradation. The variables

iboflavin and bibasic potassium phosphate (KH2PO4) presented aegative effect on the process, suggesting that the inducers mightork as inhibitors. C. alba CBMAI 1346 was subjected to frac-

ional factorial (FF) using four variables, reaching 94.13% of pyreneegradation. Based on these results a central composite rotationalesign (CCRD) was performed. However, a higher percentage ofegradation was not obtained and the result from the FF was vali-ated.


H-16

eterologous expression of three laccases from differentrigin in Saccharomyces cerevisiae and their use for envi-onmental applications

lara Richterova1,∗ , Zuzana Antosova1, Jiri Dostal 2, Iva Pichova2,ana Sychrova1

Institute of Physiology, Academy of Sciences of the Czech Republic v.v.iDepartment of Biochemistry, Institute of Organic Chemistry and Biochemistry,cademy of Sciences of the Czech Republic v.v.i

Laccases are “eco-friendly” oxidoreductases with a wide range
f biotechnological applications [1]. Three laccase genes wereloned into the S. cerevisiae expression vectors under variousonstitutive promoters. The genes originated from Myceliophthorahermophila [2], Trametes versicolor [3], and Trametes trogii [4]. Func-


ional expression of laccases in yeast was detected as an ability toonvert ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphoniccid) to a green product. The level of expression and the com-osition of the expression media were optimized. All recombinant

accases were produced as secreted proteins due to their native N-erminal signal sequences, and thus they were easily isolated fromhe medium by ion-exchange and gel chromatography. The high-st specific activity was found for the laccase from Trametes trogii,hich, moreover, was the only laccase showing the ability of dyeecolorization.

This work was supported by TA CR grant TA0101 1461.

eferences

].Riva S. Trends in Biotechnology 2006;24(5):219–26.].Bulter T, et al. Applied and Environmental Microbiology2003;69(2):987–95.

].Cassland P, Jonsson LJ. Applied Microbiology and Biotechnology1999;52(3):393–400.

].Colao MC, et al. Microbial Cell Factories 2006;5(31).


H-17

xpression analysis of APX and CAT genes in eggplantsubjected to Cu + 2 and Zn + 2 heavy metals

lker Büyük , Semra Soydam Aydın, Demet Cansaran Duman, Sumeras ∗

Ankara University

Stress could be called as any change in unfavorable growingonditions that disrupts homeostasis in plants and can lead toower yields and possible crop failure. Biotic and abiotic stress fac-ors are the sources of environmental stress in plants and heavy

etal toxicity is one of the global environmental problems. Tovercome negative effects of heavy metal contaminations on plantealth, evaluation of the genes responsible for stress tolerance is

he first stage of producing stress-tolerant plants.For this purpose, Solanum melongena L. plants were exposed var-

ous concentrations of Cu+2 and Zn+2 heavy metals (blank, 80 �M,60 �M, 320 �M, 640 �M, 1280 �M) for 24 h. Gene expressionnalysis of catalase (CAT) and ascorbate peroxidase (APX) genesere performed using Light Cycler Nano Real-Time PCR instru-ent with cDNAs which were synthesized from mRNAs isolated

rom the leaf tissues of eggplant samples. Results indicated that alloncentrations of Cu+2 and Zn+2 triggered the expression changesf the CAT and APX genes. Almost all concentrations of both metalontaminations led to increases in mRNA levels of CAT and APXenes and this increment pointed to the importance of these genesor stress defence in eggplant. The overexpression and silencing ofhe APX and CAT genes in eggplants under Cu+2 and Zn+2 stresses

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H-18

xidation of low density polyethylene by a laccase-ediator system

uillermo Huerta ∗ , Marcela Ayala

Instituto de Biotecnología, Universidad Nacional Autónoma de México

Synthetic polymers such as low density polyethylene (LDPE)epresent a big ecological problem in today′s environment. Scien-ists have explored and proposed a variety of possible solutions tolleviate this problem [1]. Biological degradation is an eco-friendlyrocess in plastic waste management, first, the polymer must sufferhysical and chemical changes to facilitate the mineralization oregradation process by microorganism [2]. Such changes becomeossible when the plastics are oxidized by abiotic factors or byatalysts.

Laccases are oxidoreductases catalyzing the oxidation ofrganic substrates. Laccases have been described as efficient cata-ysts for many applications, from paper bleaching, decolourizationf textile effluents, biosensors and bioremediation [3]. In this worke investigated the use of a laccase and mediators, to modify LDPElms. We observed changes in chemical composition of enzyme-reated LDPE films, mostly the presence of carbonyl groups inhe infrared region of 1720 cm−1; along with these oxidation sig-als, there was a loss of surface hydrophobicity (measured byontact angle) and changes in mechanical properties, such as rela-ive elongation. The enzyme-treated LDPE films were incubated inhe presence of selected microorganisms in order to evaluate theirotential biodegradability.

Acknowledgments. Authors acknowledge the financial sup-ort of Conacyt 179241.

eferences

].Panda AK, Singh RK, Mishra DK. Renewable and Sustainable EnergyReviews 2010;14:233–48.

].Bonhomme S, Cuer A, Delort AM, Lemaire J, Sancelme M, Scott G.Polymer Degradation and Stability 2003;81:441–52.

].Rodriguez-Cuoto S, Toca-Herrera JL. Biotechnology Advances2006;24:500–13.


H-19

robing the formation of secondary minerals in theioleaching reactions of chalcopyrite by Raman andTIR microspectroscopy

onstantinos Varotsis1,∗ , Chrystaleni Giangou1, Sotirisapadatos1, Elena Xenofontos1, Ioannis Vyrides1, GiorgosManos2,icolas Messios2, Constantinos Xydas2

Cyprus University of TechnologyHellenic Copper Mines

Chalcopyrite passivation reduces the yields from leaching andioleaching. Despite the great efforts the problem has not beenuccessfully resolved. Passivation involves the formation of a layerf secondary minerals on chalcopyrite surface which becomes


diffusion barrier to fluxes of reactants and products. We willresent Raman and FTIR microspectroscopic evidence toward the

dentification of secondary minerals formed during chalcopyriteassivation in the presence of iron and sulphur-oxidizing bacteria

solated from the mines of HCM. Potassium jarosite is the initialroduct followed by the formation of ammonia-jarosite. Covellitend elemental sulphur are detected in the passivation layer. Theechanism of passivation and testing strategies to minimize it will

e presented.


H-20

haracterization of a bacterial collection isolated fromextreme environment with potentiality of use in

iotechnology industry

ose Manuel Gomez1,∗ , Arelys Diaz2, Jeannette Marrero2, Gemaabrera1, Orquidea Coto2

University of CadizUniversity of Havana

Heavy metal pollution is still a worldwide problem. The metal-ic elements can not be degraded but only could be transformedrom an oxidation state or organic compound to another. There-ore, bioremediation is a attractive option to be applied forleaning up the environment and recovery of soils. Microbiotesolated from extreme environmemts is considered as a sourcef enzymes and active metabolites which could be used in theiotechnology industry. In this work bioactive compounds andemotion capacity were evaluated in seven strains ofSerratia mar-encensisolated from nickel lateritic ores located in Moa, Cuba.00% of the strains produced extracellular enzymes includingNases, proteases, lipases, lecitinases and caseine hydrolases asell as were able to solubilize inorganic phosphorus. The strainsere classified as highly resistant strains based on the minimal

nhibitory concentration (MIC) of Ni(II) (>25 mM) and Co(II)>12 mM). Biomasses of the strainsS. marcescensC-1, 16 and 19howed removal capacity Ni(II) and Co(II) from solution untilalues higher than 100 ppm in two hours of biomass - metal con-act. Removal capacity of strain 16 in bioreactor reached valuesf 36% of Co(II) and higher than 50% of Ni(II), Cu(II) and Zn(II)rom multimetallic solutions. The results presented here suggesthe potentiality of the use of theses strains in the medical and


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H-21

arnessing microbial communities in tropical peatlandss resources for novel biocatalysts for plant biomasseconstruction

icole Chua1,∗ , Pui Yi Yung1, Shivshankar Umashankar1, I. Madeudiana2, Sanjay Swarup1

National University of SingaporeLIPI

Large amounts of plant biomass are generated annually, usuallyrom factory and municipal wastes, forest- and agricultural-esidues. Plant-derived lignocellulosics represents a major source ofenewable organic matter as raw material for fuel, macromoleculesnd aromatics. However, lack of efficient technologies to con-ert these “waste” into useful product resulted a great deal ofhem being burnt for heat generation, or merely to reduce theolume of biomass. Common rate limiting steps during biolog-cal degradation of biomass include accumulation of microbialrowth inhibitors during treatment process, and the lack of diversenzymes available for degradation of the heterogenic lignin- andignin-bounded chains.

Microbial enzymes are well known for their vast functionaliversity, which have increasing recognitions from industries. We

nvestigated the functional capabilities of the microbial communi-ies in tropical peatlands for their ability to degrade lignocellulosicompounds. Using both culturing and culture-independent tech-iques, enzymes capable of lignocellolytic activities were screened.

To date, we have found microbial isolates capable of hydrolyz-ng hemicelluloses such as xylan and arabinoxylan. The samesolates are also capable of growth in high concentration of growthnhibitors such as syringaldehyde, hydroquinone and furfural.ulture-independent, sequence-guided metagenomic approachesave revealed the microbial communities in tropical peatlandsontain several classes of multi-copper oxidases (e.g. laccases), dye-ecolorizing peroxidases, and feruloyl esterases. Sequence analysisevealed these enzymes are distinct from existing enzymes in theatabase.


H-22

unctional and genomic analysis of the plant growthromoting bacterium I

ui Yi Maria Yung ∗ , Wei Ling Ng, Yong Jian Lee, Shao Bing Johananow, Boon Kiat Lennon Lim, Nicole Chua, Shivshankar Umashankar,anjay Swarup

National University of Singapore

Plant growth promoting Pseudomonas species are of great agri-ultural interest, especially in food crops. Using biological agento enhance plant growth is both environmentally friendly (reduce
se of chemicals and fertilizers) and economical in a long term.e studied both the physiological and genomic properties of
ur model plant growth promoting bacterium Pseudomonas putida

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NL-MK25. The genome of P. putida PNL-MK25 strain has beenequenced and annotated, coupling with biological assays relatingo plant growth promoting properties.

Results indicated that the estimated genome size of the bac-erium is around 5.8Mb, encoding 5313 putative open readingrames. Whole genome sequence comparison between strain

K25 to other sequenced genomes placed it closest to Pseudomonasuorescens Pf01, sharing ∼82% of ORFs. Substrate utilizationevealed that the strain is capable of utilizing a range of car-oxylic acids, esters and fatty acids. It is also resistant to severallasses of known antibiotics. Plant growth promoting assays haveevealed that strain MK25 promotes lateral root formation inodel plant Arabidopsis. Genes potentially responsible for plant

rowth promotion, for example the production of antimicro-ial compounds and auxin, has been found and/or functionallyonfirmed.


H-23

Biodesalination’: a synthetic biology approach for these of photosynthetic bacteria in water treatment

nnegret Honsbein1,∗ , Mary Ann Madsen2, Jaime M. Amezaga3,atherine A. Biggs4, Tom Bond5, Catherine J. Gandy3, Estherarunakaran4, Linda Lawton6, Konstantinos Minas6, Michael R.empleton5, Anna Amtmann2

University of GlasgowInstitute of Molecular, Cell and Systems Biology, University of GlasgowSchool of Civil Engineering and Geosciences, Newcastle UniversityDepartment of Chemical and Biological Engineering, University of SheffieldDepartment of Civil and Environmental Engineering, Imperial College LondonInstitute for Innovation, Design and Sustainability, Robert Gordon University,berdeen

Shortage of freshwater is a serious global problem, and expectedo become even more urgent over the next decades. Many ofhe driest regions worldwide are close to the sea, but irrigationf fields with seawater–even if diluted–leads to the build-up ofalt levels in the soil that are toxic to all common food cropshttp://www.unwater.org). Current desalination technologies suchs membrane-based reverse osmosis, are successfully used inarge-scale desalination plants, however, they are expensive andnergy inefficient [1]. Our multi-disciplinary team of biologistsnd engineers from 5 UK universities is working on an innova-ive desalination technology based on biological processes [2].he “Biodesalination” strategy envisions the use of photosyntheticyanobacteria modified with light-driven ion transport proteins tounction as ion exchangers that selectively remove sodium chlo-ide from seawater. This process would harness solar energy torovide a more cost effective and energetically sustainable desali-ation process.

eferences

].Plappally AK, Lienhard JH. Costs for water supply, treatment, end-use and reclamation. Desalination and Water Treatment 2013;51:200–32.

].Amezaga JM, Amtmann A, Biggs CA, Bond T, Gandy CJ, Hons-bein A, Karunakaran E, Lawton L, Madsen MA, Minas K, Templeton

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MR. ’Biodesalination’: a case study for applications of photo-synthetic bacteria in water treatment. Plant Physiology 2014;164:1661–76.


H-24

haracterisation of Metal Transport Proteins for provid-ng metal stress tolerance in green microalgae

niefon Ibuot ∗ , Andrew Dean, Jon Pittman

University of Manchester

Characterisation of Metal Transport Proteins for providingetal stress tolerance in green microalgaeThe Cation Diffusion Facilitator (CDF) family is known for pro-

iding significant tolerance in metals such as Mn2+ and Zn2+. Theseroteins are well characterised in organisms such as human, yeastnd plants but not in unicellular green algae. There are five CDFenes in Chlamydomonas reinhardtii. MTP1 to MTP4 were clonednd functionally characterised by yeast heterologous expressionsing a Zn2+ and Co2+ sensitive mutant yeast strain zrc1cot1 and an2+ sensitive yeast strain pmr1. The MTP-expressing yeast strainsere screened for suppression of metal sensitivity to ascertain the

ransport function each MTP protein. MTP1 was able to stronglyescue the Zn2+ and Co2+ sensitivity of the zrc1cot1 strain, while

TP3 could weakly mediate Zn2+ and Co2+ growth. MTP2 andTP4 appeared to have no Zn2+ or Co2+ transport activity. MTP2

o MTP4 but not MTP1 could strongly rescue the Mn2+ sensitivityf the pmr1 strain. This clearly confirms the metal transport func-ions of the MTP family in Chlamydomonas. Further studies arever-expressing these MTP proteins in Chlamydomonas to deter-ine whether enhanced metal uptake and tolerance of specificetals can be achieved. This research could increase our under-

tanding of how yeast and algae could be utilized as a potentialnvironmentally sustainable tool for the remediation and decon-amination of metal-polluted wastewater.

Keywords: Metal transport proteins, metals, yeast, Chlamy-omonas reinhardtii


H-25

ngineering high-redox potential laccases in the lab toid biomass conversion into chemicals, materials andiofuels

usana Camarero1,∗ , Ana I. Vicente1, Miguel Alcalde2, Isabelardo1

Centro de Investigaciones Biológicas, CSICInstituto de Catálisis y Petroleoquímica, CSIC

Laccases catalyze the oxidation of a variety of aromatic com-
ounds without any other requirement than oxygen from air. Inrevious studies we have highlighted the biotechnological poten-ial of fungal laccases to improve the utilization of plant biomass in
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he modern biorefineries [1]. Lignin derived compounds releaseduring lignocellulose processing can be used as redox mediatorsf laccases or as bioactive precursors for the enzymatic synthesisf natural products. However, the industrial implementation ofungal laccases is hampered by the lack of powerful expression sys-ems and the need for active and stable enzymes under the harshperational conditions.

Recently we developed fungal laccases of high-redox poten-ial functionally expressed in S. cerevisiae by directed evolution2,3]. Using these platforms as the starting points and ad-hocigh-throughput screening methods [4,5] we developed a moreobust laccase with improved catalytic activity towards phenolicompounds under preferred pH conditions, which represents atep forward to aid the conversion of lignocellulosic biomass intohemicals, materials and biofuels.

This work has been funded by the Spanish Project BIO2010-9697.

eferences

].Canas AI, Camarero S. Biotechnol Adv 2010;28:694–705.].Maté D, García-Burgos C, García-Ruiz E, Ballesteros A, Camarero S,Alcalde M. Chem Biol 2010;17:1030–41.

].Camarero S, Pardo I, Canas AI, Molina P, Record E, Martínez AT,Martínez MJ, Alcalde M. Appl Environ Microbiol 2012;78:1370–84.

].Pardo I, Chanaga X, Vicente AI, Alcalde M, Camarero S. BMC Bio-technol 2013;13:90.

].Pardo I, Vicente AI, Alcalde M, Camarero S. Biotechnol Bioeng2012;109:2978–86.


H-26

nvestigation of polyvinylchloride biodegradation byicrobial consortia enriched from digested sludges

abio Fava ∗ , Noura Raddadi, Lucia Giacomucci, Nadia Lotti

University of Bologna

During last decades, production of synthetic plastics hasncreased dramatically to reach approximately 280 million tonnesn 2011. The accumulation of plastic waste in the environment isaising concerns about its effects both on human and the environ-ent. In EU, about 40% of plastic waste is currently disposed of in

andfills, where partially undergoes photodegradation, producingicroplastics which can absorb toxins and toxic chemicals and

ogether with plasticizers enter the marine environment and thushe food chain, where they exert toxic effects. Furthermore, colo-ization of plastics by sessile organisms may permit transport oflien species in the ocean environment and may threaten marineiodiversity. Therefore it is necessary to find new eco-friendly tech-iques for safe handling and degradation of plastic wastes.

In this work, ten microbial communities enriched from wastelastics from digested sludges were screened for their capability ofegrading non-pretreated films of polyvynil chloride (PVC) and
olypropylene (PP). After six months of anaerobic incubation inhe presence of the plastic films as main carbon source, growthf microbial community was recorded in all enriched consortia.hermogravimetric analysis (TGA) performed on PP and PVC films

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Acknowledgments. The support of the FP7 EU project BIO-LEAN (GA-312100) is acknowledged.


H-27

lectrical conductivity in granular biomass. Standard-zation and evaluation

iego Andres Suarez Zuluaga ∗ , Sebastian Canizales, Annemiek tereijne, JanWeijma, Cees J.N. Buisman

Wageningen University

Biologically produced oxidation-reduction reactions in anaero-ic granules require electron transfer between bacteria and archaeaells. Anaerobic types of biomass perform this electron transfer byean of different mechanisms. Understanding how this process

ccurs would help in the optimization and development of pro-esses for anaerobic wastewater treatment. So far, electron transferia outer-surface c-type cytochromes, long-range electron trans-er via nanowires, electron flow through a conductive biofilm

atrix containing cytochromes and soluble electron shuttles arehe mechanisms proposed for explaining such process. A simple

ethod, based on the one developed by Morita et al. (2011) [1], forhe measurement and analysis of electrical conductive of biomassranules is assessed in this study. By means of electrochemicalnterface equipment and a probe consistent of two electrodes sep-rated by a non-conductive gap, voltages were applied to granulariomass and current was measured. Different physical and micro-iological factors that could affect the measurement were studiednd their influence determined. A methodology was standard-zed and voltage vs. current graphs were generated. From them,onductance (and conductivity) was calculated. Biomass granulesriginated on Emmtec (sulphate reducing sludge) and Eerbeekmethanogenic sludge) were used for the standardization of thearameters of the tests and presenting conductance up to 625.86nd 46.94 nS respectively. By relating the measured conductanceith the origin, composition and specific activity of each gran-le a further understanding of the mechanisms used to transferlectrons was obtained.

eference

].Morita M, et al. mBio 2011;2(4):1–6.


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H-28

nalysis of the effectiveness of a cereal milling byroduct monocomponent medium for the low cost pro-uction of Bacillus thuringiensis

ihane Rahbany1,∗ , Dominique Salameh1, Cedric Brandam2,oger Lteif 1

Université Saint-Joseph LibanUniversité de Toulouse; INPT, UPS; Laboratoire de Génie Chimique France

Bacillus thuringiensis is a facultative anaerobe, gram posi-ive, spore forming bacterium. The biotechnological importancef this bacterium resides in its ability to produce, during sporu-ation, crystal proteins known as �-endotoxins which expresspecific insecticidal activity. At industrial scale, the culture mediaepresents an important part of B. thuringiensis based biopes-icides production cost. According to the literature, differentgro-industrial residues and byproducts were used as sources ofroteins in order to reduce the cost of B. thuringiensis cultureedium, but carbohydrates (glucose, starch or molasses) and/orineral sources were added.In this work, a cereal milling by-product (CMB) as a mono-

omponent medium was investigated and compared to syntheticediums in terms of �-endotoxin yield and productivity in sub-erged fermentation of different strains of B. thuringiensis. TheMB was shown efficient to be used as a complete substrate (sourcef proteins, carbohydrates and minerals) for B. thuringiensisroduction. The optimal CMB ratio in the culture medium wasound to be 6% in shake flasks experiments. The consumptionf the CMB sugars by the bacteria was analyzed. Production ofhe bio-insecticide in lab-bioreactor in controlled conditions wasqually performed to give basic elements for extrapolation inndustrial conditions.


H-29

aCl addition for increased polyhydroxyalkanoate pro-uction by Cupriavidus necator

earl Passanha ∗ , Gopal Kedia, Richard Dinsdale, Alan Guwy, San-ra Esteves

University of South Wales

The effect of five different NaCl concentrations, namely 3.5,.5, 9, 12 and 15 g/l NaCl on PHA productivity using Cupriavidusecator has been investigated alongside a control (no added NaCl)hen acetic acid was used as the sole carbon source. A dielectric

pectroscopy probe was used to measure real-time PHA accumula-ion online in conjunction with the chemical offline analysis ofHA. The highest PHA production was obtained with the additionf 9 g/l NaCl, which yielded 30% higher PHA than the control.ncreasing the addition of NaCl to 15 g/l was found however to
nhibit the production of PHA. NaCl addition can be used as aimple, low cost, sustainable, non toxic and non reactive exter-al stress strategy for increasing PHA productivity when compared

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o previous increased temperature and other types of chemicaltress such as ethanol, hydrogen peroxide or metal stress. Therder of PHA accumulation for the above salt concentrations wereg/l > 6.5 g/l > 3.5 g/l > control > 12 g/l > 15 g/l NaCl, which clearly

ndicates that addition of NaCl, which up to 9 g/l enhanced PHAroduction.


H-30

nhancement of treatment of poly aromatic hydro-arbon contaminated water using new biosurfactantroducing marine bacterium

arek Taha1,∗ , ELsayed Hafez1, HeshamMahdy2, Saad Alamri3

City for Scientific Research and Technology ApplicationsAlazhar UniversityKing Khalid University

Poly Aromatic Hydrocarbons (PAH) are well known for theirlight solubility in water which considered one of the most obsta-les that face the bacterial isolates to degrade hydrocarbons. Ourtudy succeed to isolate nine bacterial isolates able to consume


henanthrene as sole carbon and energy source. The bacterialsolates were grouped and classified using ISSR-PCR techniquehrough four different primers. The best phenanthrene degraderas chosen and identified using 16S rRNA gene sequencing asysinibacillus fusiformis EHTH1 and was able to degrade up to50 mg/l phenanthrene. The bacterial isolate was able to degradenother single, double and triple ring containing hydrocarbonss sole carbon and energy source. The strain was able to producehamnolipid biosurfactant which hasn’t been detected for thisenus before. The 800 bp amplified fragment of RhlA gene wasuccessfully cloned into TOPO vector with subsequent transfor-ation into E. coli (Top10 strain). The results showed successful

loning and transformation and showed the ability of the clonedtrain to degrade phenanthrene at higher concentrations as sole


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ENERAL BIOTECHNOLOGY

eneral biotechnology

I-01

-protein alpha subunit gene, CGA1, is involved in theesistance against heat and osmotic stress in Chlamy-omonas reinhardtii

hangsu Lee1,∗ , Yoon-E Choi2

Department of Bioprocess Engineering, Chonbuk National UniversityLED Agri-bio Fusion Technology Research Center, Chonbuk National Univer-ity, 79 Gobong-ro, Iksan-si, Jeollabuk-do

The function of heterotrimeric GTPase (G-protein) has beenharacterized in eukaryotic cells. G-proteins are composed of threeubunits, the alpha (�), beta (�), gamma (�) subunits. The gen-ral GTPase activity of G� induces the hydrolysis of the boundTP, thereby playing pivotal roles in relaying the signals. How-

ver, microalgal G-proteins including G-protein alpha subunitave remained largely unknown. In this study, we characterizedgene, CGA1, encoding G-protein alpha subunit in Chlamy-

omonas reinhardtii. Independent knock-down mutants of CGA1ere generated via the RNA interference (RNAi) approach. CGA1

xpressions consistently reduced significantly in all of the CGA1utants (cga1). Interestingly, all of the cga1 displayed higher sur-

ival rate at 35 ◦C, compared to wild type. In addition, all ofhe cga1 has an enhanced survival rate at high osmotic stress,ompared to that of the wild type. To further understand mecha-isms of CGA1-mediated resistance, the extent of reactive oxygenpecies (ROS), the expressions of several putative downstreamenes including heat shock proteins(HSPs) and MAP kinases werehoroughly analyzed. Our data indicated that CGA1 is associatedith the regulation of resistance against heat or osmotic stress

n C. reinhardtii by affecting multiple downstream genes. SinceG-protein alpha subunit is highly conserved across microal-

al species, our results will shed light on future biotechnologicalpplications of microalgae under the extreme environmental con-itions.


I-02

daptive laboratory evolution study of Corynebacteriumlutamicum resistant to oxidative stress and their appli-ation to develop an artificial oxidative stress-resistanttrain

oo-Young Lee1,∗ , Jiyoon Seo1, Eung-Soo Kim2, Heung-Shick Lee3,il Kim1

The Catholic University of KoreaInha UniversityKorea University

In this study, we performed adaptive laboratory evolution
ALE) study of Corynebacterium gluiamicum in chemostat for,900 hours with gradual increase of oxidative stress-intensityo understand the oxidative stress response and develop C. glu-amicum as a platform microbe. Through ALE experiment, C.
icr



lutamicum acquired an ability to grow under stress of 10 mM

2O2. In the transcriptome results of the adapted strain, aotal of 1,180 genes (38.6% of the genome) were up-regulated

ore than 2-fold, and 126 genes (4.1% of the genome) wereown-regulated less than 2-fold under oxidative stress conditionompared with those of wild-type (WT) strain under non-stressondition. Especially the genes encoding enzymes in the �-etoadipate pathway (pca genes) were up-regulated more than-fold. To develop an artificial oxidative stress-resistant strain, theca gene clusters in the �-ketoadipate pathway were expressedn the WT strain. WT strain was unable to grow under 2 mM

2O2, while the strains expressing pca gene clusters restoredrowth. The expressions of pca gene clusters also enabled theT strain to increase its resistance against various oxidative

tressors including H2O2. The oxidative stress resistance of thosetrain was correlated to the reactive oxygen species (ROS)-cavenging activity of the cytosol. These findings would be usefulo develop an oxidative stress-resistant synthetic strain for indus-rial applications.

[This study was financially supported by the Korean Ministryf Science, ICT & Future Planning (Intelligent Synthetic Biologyenter of Global Frontier Project 2012M3A6A8054887)]


I-03

ymbiosis Mechanisms of Lactic Acid Bacteria and Yeastsn Inner Mongolian Traditional Fermentative Milk Prod-cts

infeng He ∗ , Jianjun Tian, Minmin Liu, Bin Yan

Inner Mongolia University of Agriculture

The majority of the traditional dairy products in Innerongolia, China, are co-fermented with lactic acid bacteria and

easts. So far, forty-seven strains of lactic acid bacteria and nine-een strains of yeast have been isolated and identified fromoumisses, ripened creams, and yogurts. A systematic studyas performed to understand the co-culture properties and co-

ermentation characteristics between them. The results show: (1)he lactic acid bacteria and the yeasts found in the traditionalairy products in Inner Mongolia have symbiotic effects that can

ncrease active strain numbers and enhance acid productivity;2) The metabolites of the yeasts can act as pH buffer in theroth of the lactic acid bacteria, reduce acid inhibition, enhancehe growth of the strains, and increase titration acidity. In the

eanwhile, the metabolites of the lactic acid bacteria can alsoromote the growth of the yeasts, increase titration acidity, andecrease the pH value to an optimum growth range; (3) Whenhe lactic acid bacteria and the yeasts are co-cultured in skimmed

ilks, both small molecular acidic substances, such as the lacticcid and propionic acid, and flavor components that can formromatic substances such as ethyl acetate can be produced eas-
ly; and (4) The metabolites produced from the yeasts after aulture time of 60 hours are the best for the lactic acid bacte-ia, and the optimal amount of the metabolites in the culture

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edium is 40 wt%. Details of this study will be discussed in theresentation.


I-04

onstruction, expression and renaturation ofBI::human insulin analog single chains

iana Mikiewicz ∗ , Anna Wojtowicz-Krawiec, Natalia Lukasiewicz,wona Sokolowska, Agata Jagiello, Piotr Borowicz, Grazynalucienniczak, Andrzej Plucienniczak

Institute Biotechnology and Antibiotics

Insulin is a hormone secreted by the pancreas in response toigh blood sugar levels that induces hypoglycemia. Insulin reg-lates the body’s use of glucose and the levels of glucose in thelood. Insulin and its various derivatives are used in large amountsn treatments of diabetes.

The aim of this work was using a deubiquitinating proteasenalogue - UBP1 to obtain purified single chain A and B of humannsulin analog in E. coli. We constructed fusion genes - Ub::ins androtease analogue - UBP1 in one vector together. The region thatodes for the recombinant fusion gene is under control deoP1P2romoter. The protease analogue - UBP1 gene which is under theontrol of pms promoter(WO05066344 A2) was discovered andescribed in our Institute. The recombinant human insulin chainenes present downstream of ubiquitin gene was used as a signalrotein. Ubiquitin is composed of 76 amino-acid residues with aotal molecular mass of 8.6 kDa. This protein is an element of theniversal protein modification in eukaryotes called ubiquitinat-

on, a phenomenon which does not occur in bacteria. In spite ofhat, it has been shown that proteins fused to ubiquitin undergoreater expression in E coli and are easier to purify and renaturatehan nonhybrid foreign proteins.

We obtained a high level of expression of fusion proteinsb::ins analog A chain and Ub::ins analog B chain in E. coli. After

leavage of the ubiquitin by UBP1 analog, good yields of thepuri-ed insulin chains were produced.


I-05

he crude lecithin gum as a substrate for lipase produc-ion by Pseudomonas sp

aria Antonia Celligoi1,∗ , Cristiani Baldo1, Marcos Oliveira1, Lilianaggio1, Fabiana Gasparin1, Marcelo Melo1, Dionisio Borsato2

State University of Londrina - Department of Biochemistry and BiotechnologyState University of Londrina - Department of Chemistry

The refining of soybean oil involves the degumming process
hich results in byproducts with variety of quality grades. The
rude lecithin gum (CLG) contains a mixture of phospholipids andil, and may be submitted to a series of solvent extraction and pre-ipitation processes to produce the commercial lecithin. The aim

GENERAL BIOTECHNOLOGY

f this study was to optimize the production of lipase by a strainsolated from a slaughterhouse effluent, identified as Pseudomonasp, using the CLG as a carbon source. The lipase activity at 233,44/mL was obtained by growing the microorganism on minimumedium containing 1% (w/v) of CLG, after 24 hours. The highest

nzymatic activity (400,22 U/mL) was observed when 2% (w/v)f CLG was used. The optimization of the temperature levels, pHnd agitation rate, by Box-Benhken design, resulted in the maxi-um enzyme activity of 517,00 U/mL, obtained at 32 ◦C, pH 9.0

nd 200 rpm. These data showed the possibility of using the CLGs substrate for lipase production by Pseudomonas sp and can con-ribute in the reduction of production cost of lipases in industrialcale, and increase the market value of soybean oil byproducts.

Financial support: CAPES/PNPD


I-06

roduction of anti-cancer compound by microbial pro-ess

hanghyun Roh

KAERI

In this study, we elucidated that the production of anti-cancergent from microbial process for regio-specific hydroxylation ofesveratrol. Among the strains examined, a strain showed highegio-specific hydroxylation activity to produce Piceatannol. In a

L (w.v. 3 L) jar fermentation, the wild type Streptomyces sp. inatch system produced 205 mg of Piceatannol (i.e., 60% yields)rom 342 mg of resveratrol in 20 h. This biotransformation resultemonstrates that regio-specific hydroxylated resveratrol exhibit-

ng more potent anti-cancer activity could be produced on a largecale using microbial biotransformation. Using biotransformediceatannol, the in vitro anti-cancer study was performed againstuman cancer cell line (HeLa) and MTT assay was used to analyze

he cell growth inhibition. The results showed that the biotrans-ormed piceatannol possessed a significant anticancer activity.his is first report elucidating production of anti-cancer com-ound using microbial process for regio-specific hydroxylation ofesveratrol. This study has significant scope that a biotransforma-ion result provides one initiative example such that regio-specificydroxylated compound from resveratrol showing more potentiological activity could be produced in large scale using micro-ial biotransformation. Thus, microbial processes become a goododel system to develop an industrial biotransformation system


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I-07

nduced differential metaproteomics: identification ofellulases in a methanogenic microbial community athermophilic conditions

utta Speda1,∗ , Bengt-Harald Jonsson1, Martin Karlsson2

Linköping UniversityRational Enzyme Mining AB

The identification of novel enzymes for use in industrialiotechnology is an important goal in enzyme discovery. Mostndustrially relevant enzymes to date have been isolated from pureultured microorganisms. For future discovery of novel enzymeshis is however a major bottleneck since it is well established thatnly a small fraction of all microorganisms can be obtained inure cultures. The possibility to identify enzymes directly fromomplete microbial communities would therefore give access to auge number of novel enzyme candidates.

Metaproteomics has hitherto mainly been used to understandcosystem functions. We have instead used the dynamics of pro-eomics to develop a method based on “induced differential

etaproteomics”, by which a desired enzyme activity is inducedn a full microbial population and compared to a non-induced ref-rence of the very same population. In a first example the goalas to induce, select and identify cellulases from a thermophilicethanogenic community.Out of several hundred detectable proteins in a 2D-DIGE

xperiment, 24 proteins could be identified as at least two-foldp-regulated upon induction. For some proteins spots, the cel-

ulolytic activity was further validated by activity staining usingD-zymography. Mass spectrometry analysis revealed that 21 outf the 24 up-regulated proteins are cellulases or associated toellulolytic activity giving a remarkable hit-rate of 88%. Thisemonstrates the high efficiency and precision of the method, byhich a much wider span of the microbial world can be scanned

or novel and targeted enzymes.


I-08

ffect of lemongrass oil and powder on growth andflatoxin production by Aspergillus flavus IMI242648 inaize

usanee Thanaboripat1,∗ , Yaowapa Suvathi2, Manatsanunurdhom1, Suchada Rakphung1

King Mongkut’s Institute of Technology LadkrabangGovernment Pharmaceutical Organization

Aflatoxin is one of the most important mycotoxins producingn nature and poses health hazard to human and animals. Controlf aflatoxin producing fungi and aflatoxin production in variousommodities by various means have been investigated. Natural
lant extracts may provide an alternative way to prevent food oreed from fungal contamination. The objective of this study iso compare the effect of lemongrass (Cymbopogon citratus (DC.)


taph), an edible herb, in the forms of oil and powder on therowth and aflatoxin production of Aspergillus flavus IMI 242684n maize. Lemongrass oil at concentrations of 1, 2, 3, 4 and 5%nd lemongrass powder at 1, 2, 3, 4, 5, 10, 20, 30, 40 and 50%ere applied in maize with aflatoxin producing fungus for 7 dayst ambient temperature. The results show that lemongrass oil at alloncentrations inhibited fungal growth whereas lemongrass pow-er at 40 and 50% could inhibit fungal growth. For the ability to

nhibit aflatoxin production, it was found that lemongrass powdert 10-30% and at 40-50% could inhibit aflatoxin production for 3nd 7 days, respectively.


I-09

ioconversion of Ginsenosides from Red Ginseng Extractsing Candida allociferrii JNO301 Isolated from Meju

ulhee Lee1,∗ , Yong-Hun Lee1, Jung-Min Park2, Dong-Hoon Bai3,oung-Seo Park1

Gachon UniversityKorean Culture Center of MicroorganismsDankook University

Red ginseng (Panax ginseng), a Korean traditional medicinallant, contains a variety of ginsenosides as major functional com-onents. It is necessary to remove sugar moiety from the majorinsenosides to make them aglycone form due to their low absorp-ion rate into the intestine. To screen the microorganisms whichhow the bioconversion activity on the ginsenosides from red gin-eng, total 50 yeast strains were isolated from Korean traditionaleju (a starter made with soybean and wheat flour for the fermen-

ation of soybean paste). Twenty strains which form the black zoneround the colony on the esculin-YM agar plate were screenedrst, and among them 5 strains which have high β-glucosidasectivity on p-nitrophenyl-β-D-glucopyranoside as a substrate werehen selected. Strain JNO301 was finally chosen as a bioconver-ion strain in this study on the basis of high bioconversion activitygainst red ginseng extract by TLC analysis. The selected biocon-ersion strain was identified as Candida allociferrii JNO301 by theucleotide sequence analysis of 18S rRNA gene. The optimum

emperature and pH for the cell growth were 20-30� and pH 5-, respectively. By the analysis of TLC, it was confirmed that C.llociferrii JNO301 converted ginsenoside Rb1 into Rd and thennto F2, Rb2 into compound O, Rc into compound Mc1, Rf intoh1. Quantitative analysis using HPLC showed that bioconversionf red ginseng extract resulted in the increase in the concentrationf Rd, F2, compound O, compound Mc1, and Rh1 with 2.73-, 3.32-,

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I-10

onstruction of a genetically encoded binary countingodule in Escherichia coli

ia Zhao1,∗ , Sean Colloms2, Susan Rosser3

Institute of Molecular, Cell and Systems Biology, University of GlasgowUniversity of GlasgowUniversity of Edinburgh

Binary systems have been applied extensively in computer sci-nce because they allow large numbers to be encoded. Introducingsimilar counting system into living cells will be a fundamen-

al step towards building cellular computers. One way to do thiss to encode information in the DNA sequence using site-specificecombination to invert the orientation of a DNA segment, makinghe information heritable and easily detectable. Serine phage inte-rases catalyse recombination between specific DNA sites, attP andttB, producing sites attL and attR which are no longer substratesor integrases alone. However, in the presence of a recombinationirectionality factor (RDF) this directionality is reversed so thatttL and attR recombine to recreate attP and attB. If two att sitesre placed in inverted repeat, recombination flips the orientationf the intervening sequence between two possible states, whichan represent a single binary digit (0 or 1) heritably stored in theNA. Based on this principle, a toggle switch has been constructed

n E.coli. By first expressing the integrase, and then the integrasend the RDF together, its state can be changed between 0 and 1nder the control of different signals. This switch will be developedo a binary counting module by placing the RDF under the controlf the sequence state and using a single chemical signal to controlhe expression of the integrase. By linking N units together, eachsing a different phage integrase, a binary counter can be made toelp cells count to 2N-1.


I-11

valuation of gamma-oryzanol in Thai rice bran oilxtracted by conventional solvent extraction methodnd its antioxidant activities

ujitra Sukonthamut ∗ , Chitti Thawai, Duangkamol Ruen-ngam

King Mongkut’s Institute of Technology Ladkrabang

Rice bran oil was extracted by solvent-assisted extraction withexane, ethylacetate, acetone, isopropanol and ethanol using aolvent-to-rice bran ratio of 4:1 (w/w). The experiments were donen triplicate at room temperature (30 ◦C) with a total extractionime of 2 hours/sample. The oil components were separated byeverse-phase HPLC and quantified with a fluorescence detector.he radical scavenging capability of the oil was tested with DPPHnd was expressed as �mol Trolox Equivalent Antioxidant Activity.mong these solvents, ethanol was the best solvent for the extrac-

ion of gamma-oryzanol as compared with the others solvents foronventional solvent extraction while isopropanol was better foril yield extraction at room temperatures. No difference in oil yield

atep


as noticed between the extraction with isopropanol and acetone.amples extracted with ethanol had higher antioxidant activityhan the others and exhibited the rice fragrance.

Keywords: Gamma-oryzanol, Antioxidant activity; Rice branil; conventional solvent extraction


I-12

solation and characterization of antibiotic producingicromonospora from Thai Jasmine rice

hitti Thawai


Thirty eight actimonycete strains were isolated from tissue ofoot and stem of Thai jasmine rice (Oryza sativa). These strainsere grouped using phenotypic, chemotypic and genotypic char-cteristics into 5 groups. Phylogenetic position, chemotaxonomicnalyses including some phenotypic characterisation revealed thathe representative strains in each group belonged to the membersf the genus Micromonospora. The fermentation broths of thesesolates were extracted with ethyl acetate and were tested for anti-

icrobial activity. The results showed that more than 60% oficromonospora strains inhibited the growth of pathogenic agents

f leaf blight disease (Xanthomonas oryzae) and blast disease (Pyric-laria grisea). Based on these results, we conclude that endophyticicromonospora strains are great and should represent an excellent

ource for discovery of the bioactive compounds.Keywords: Actinomycetes, Micromonospora, Antibiotic,

light and Blast disease.


I-13

ndrogen’s effect on MCP-1 and monocyte attraction torostate cancer cells

un-Kyung Kim ∗ , Eun-Ju Choi

Konkuk University

Trigger of inflammatory monocytes to the tumor site is medi-ted by monocyte chemoattractant protein-1 (MCP-1) throughinding to its receptor. We assumed that androgen couldodulate MCP-1 expression in hormone-responsive prostate

ancer cells and then promote recruitment of monocytes. Dihy-rotestosterone (DHT) stimulated a time-dependent (0–72 hours)nd concentration-dependent (0–1 nmol/L) increase in MCP-mRNA levels in androgen-responsive human prostate cancer

ells (LNCaP). This increase in MCP-1 mRNA corresponded withncreased secretion of MCP-1 protein. The effect of DHT was

ediated through an androgen receptor (AR)-dependent pathway
s small inhibitor RNA (siRNA) against AR negated the induc-ion of MCP-1. Although DHT also induced TWIST1 mRNA, anpithelial–mesenchymal transition (EMT)–related factor, and pur-orted inducer of MCP-1, blocking its expression with siRNA

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id not inhibit DHT induction of MCP-1 mRNA. Moreover, con-itioned media from androgen-treated cells promoted humanonocyte THP-1 cell migration and this effect was inhibited by

ntibody against MCP-1. These results indicate that androgen mayegulate MCP-1 and promote inflammatory microenvironment inrostate cancer.


I-14

uppression of dust mite extract and 2,4-initrochlorobenzene-induced atopic dermatitis byhe water extract of DA-9601

hoi Eun-Ju ∗ , Kim Eun-Kyung

Konkuk University

DA-9601 is a novel anti-peptic formulation prepared from thethanol extracts of Artemisia asiatica possessing anti-oxidative,nti-allergic and anti-inflammatory activities. However, theirffect on atopic dermatitis (AD) has not been studied yet. In thistudy, we report that topical application of DA-9601 suppressedouse dust mite extract (Dermatophagoides farinae extract, DFE) and, 4-dinitrochlorobenzene (CDNB)-induced AD-like skin lesionsn BALB/c mice model. We established atopic dermatitis modeln BALB/c mice by repeated local exposure of DFE/CDNB to thears. Repeated alternative treatment of DFE/CDNB caused AD-ike lesions. DA-9601 reduced AD-like skin lesions based on earhickness and histopathological analysis, and serum IgE levels. DA-601 inhibited mast cell infiltration into the ear and elevation oferum histamine in AD model. In addition, DA-9601 suppressedFE/CDNB-induced expression of IL-4, IL-13, IL-31, and TNF-� in

he ears. Taken together, our results showed that topical applica-ion of DA-9601 exerts beneficial effects in animal model of AD,uggesting that DA-9601 might be a candidate for the treatmentf AD.


I-15

iotechnological application of Bacillus subtilis sporeisplay system

une Hyung Kim

Dong-A University

Bacterial surface display finds its important biotechnologicalpplication in the fields of screening tools of evolved enzyme,ioremediation, whole cell bioconversion and tool for live vac-ine production. For the functional bacterial surface display ofctive enzyme of multimeric form, which is generally impossibleue to molecular assembly of the monomer subunit subsequent to
he secretion of displayed target protein outside the cell, a newurface display system based on Bacillus subtilis spore is devel-ped. Here, we tried to develop a new bacterial surface displayormat for the efficient expression of multi-subunit enzyme. We


sed CotE, CotG, CotY spore coat protein as anchoring motivesor the successful display of beta-galactosidase, spreptavidin, andther interesting target protein with biotechnological applicationuch as laccase for the environmental usages. Surface localizationsing flow cytometry and enzymatic activity on the Bacillus sub-ilis spore was examined for the successful confirmation of surfaceocalization of target proteins. Further usage and other applicationill be discussed.


I-16

roduction of 3-hydroxybutyrate by E. coli: Applicationf Nitrogen and Phosphorous limitation to steer fluxeso product formation

onica Guevara1,∗ , Johan Jarmander1, Mariel Perez-Zabaleta1,orge Quillaguamán2, Gen Larsson1

Industrial biotechnology, School of biotechnology, KTH, StockholmCenter of Biotechnology, Faculty of Science and Technology, San Simón Uni-ersity, Cochabamba Bolivia

Polyhydroxyalcanoates are polyesters produced in largemounts by different microorganisms, but not by E.coli. Here wexpressed the bacterial polyhydroxybutarate (PHB) pathway ofalomonas boliviensis in E. coli using phbA and phbB genes toroduce the monomer 3-HB. Acetyl coenzyme A (AcCoA) is the

ntermediate of the carbon metabolism and precursor of the PHBathway. The objective is to design a process to produce 3-HB in.coli with high productivity which is a first step to further produceifferent quality polyesters.

Production of the wild type microorganisms takes place underutrient deficient conditions and excess of a carbon source. Theypothesis is that directing the flux to the desired product (3-HB)y cultivations with controlled feed of nitrogen or phosphorousill increase the flux to the precursor (AcCoA) and minimize the

ormation of byproductsThis will be done by:Fed-Batch cultivations with an excess of glucose and nitrogen

imitation. Excess glucose will give a surplus of NADH that willnhibit citrate synthase and the AcCoA produced will instead besed for 3-HB production

Fed-Batch cultivations with an excess of glucose and phospho-ous limitation. Excess glucose will give a surplus of NADH that willnhibit citrate synthase and the AcCoA produced will instead besed for 3-HB production. Also the limitation of phosphorous acti-ates the methylglyoxal pathway also leading to the more energy

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I-18

evelopment of a flow cytometer-based in vitro compart-entalization screening platform for directed protein

volution

eorgette Wirtz ∗ , Christian Pitzler, Ljubica Vojcic, Ronny Martinez,lrich Schwaneberg

Institute of Biotechnology, RWTH Aachen

We describe the development of an ultra high throughputcreening (uHTS) flow cytometer based in vitro compartmental-zation (IVC) screening platformfor directed enzyme evolution.he system combines IVC (water-oil-water emulsion) and theetection of enzyme activity by fluorescence-activated cell sor-ing (FACS),offering an advanced uHTS screening platform (∼108

vents/day) for the discovery and reengineering of industriallyttractive enzymes [1].uHTS offers a qualitative differentiationetween positive or negative enzymatic activity, enabling thenrichment of active variants by screening thousands of events perecond. In IVC, single cellulase gene variants are transcribed andranslated within a double emulsion compartment and upon trans-ation, a fluorogenic substrate is converted by active variants andorted using FACS [2,3]. IVC systems enable linking phenotype andenotype, which is a key requirement in directed evolution experi-ents. Additionally, IVC allows the expression of larger libraries (>

010) by overcoming challenges such as loss of gene diversity due toow cloning and transformation efficiency and protein productionvoiding technical limitations (e.g. inclusion bodies, toxicity). IVClso dramatically reduces the time required for screening enzymeibraries, allowing a greater number of directed evolution roundser campaign, increasing the chance of generating and identifying

mproved enzyme variants.

eferences

].Ruff AJ, Dennig A, Wirtz G, Blanusa M, Schwaneberg U. ACS Catalysis2012;2:2724–8.

].Mastrobattista E, Taly V, Chanudet E, Treacy P, Kelly BT, Griffiths AD.Chem Biol 2005;12:1291–300.

].Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U. J BiomolScreen 2011;16:285–94.


I-18

evelopment of a flow cytometer-based in vitro compart-entalization screening platform for directed protein

volution

eorgette Wirtz ∗ , Christian Pitzler, Ljubica Vojcic, Ronny Martinez,lrich Schwaneberg

Institute of Biotechnology, RWTH Aachen

We describe the development of an ultra high throughput
creening (uHTS) flow cytometer based in vitro compartmental-zation (IVC) screening platformfor directed enzyme evolution.he system combines IVC (water-oil-water emulsion) and theetection of enzyme activity by fluorescence-activated cell sor-
reoU


ing (FACS),offering an advanced uHTS screening platform (∼108

vents/day) for the discovery and reengineering of industriallyttractive enzymes [1].uHTS offers a qualitative differentiationetween positive or negative enzymatic activity, enabling thenrichment of active variants by screening thousands of events perecond. In IVC, single cellulase gene variants are transcribed andranslated within a double emulsion compartment and upon trans-ation, a fluorogenic substrate is converted by active variants andorted using FACS [2,3]. IVC systems enable linking phenotype andenotype, which is a key requirement in directed evolution experi-ents. Additionally, IVC allows the expression of larger libraries (>

010) by overcoming challenges such as loss of gene diversity due toow cloning and transformation efficiency and protein productionvoiding technical limitations (e.g. inclusion bodies, toxicity). IVClso dramatically reduces the time required for screening enzymeibraries, allowing a greater number of directed evolution roundser campaign, increasing the chance of generating and identifying

mproved enzyme variants.

eferences

].Ruff AJ, Dennig A, Wirtz G, Blanusa M, Schwaneberg U. ACS Catalysis2012;2:2724–8.

].Mastrobattista E, Taly V, Chanudet E, Treacy P, Kelly BT, Griffiths AD.Chem Biol 2005;12:1291–300.

].Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U. J BiomolScreen 2011;16:285–94.


I-19

fluorescent polymer shell-based enzyme screening plat-orm for hydrolases using flow cytometry

hristian Pitzler1,∗ , Georgette Wirtz1, Ljubica Vojcic1, Stephanieiltl 2, Alexander Böker2, RonnyMartinez1, Ulrich Schwaneberg1

RWTH Aachen University, department of biotechnologyDWI-Leibniz Institut für Interaktive Materialien

A novel whole-cell enzyme screening platform based on aoupled reaction of glucose-oxidase and a hydrolase (Yersinia mol-aretii phytase, YmPh) was developed and validated in a directedhytase evolution experiment. The coupled reaction producesydroxyl radicals through Fenton’s reaction (hydrogen perox-

de and Fe2+) which initiate poly(ethyleneglycol) diacrylate-basedydrogel polymerization, including the fluorescent Polyfluor-570-crylate. As consequence, fluorescent hydrogel is formed around. coli cells that express active YmPh variants. Formation of theuorescent hydrogel was confirmed by confocal microscopy. Inddition, scanning force microscopy was used to visualize theydrogel on the cell surface, revealing a distinct structure enclos-

ng the cells. Labeled cells were analyzed and sorted by flowytometry with a throughput of 1.8 × 107 events/h. Further sor-ing of mixed populations with a defined content of active/inactiveells yielded an enrichment factor of up to 5. Finally, the fluo-
escent polymer shell technology was validated by analyzing anrror-prone PCR mutant library (4.8 mutations/gene). Screeningf 107 events by flow cytometry yielded variant M1 with 97/mg increased specific activity compared to YmPh wild type

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315 U/mg). This screening platform is a promising approach forcreening mutant and metagenome libraries for novel or improvedydrolases, by using a variety of glucose-derived substrates.


I-20

ye as substrate for L-lactic acid production

iotr Walczak ∗ , Anna Rygala, Katarzyna Dybka, Patrycjaietraszek, Agata Czyzowska, Anna Otlewska, Elzbieta Oltuszak-

alczak

Faculty of Biotechnology and Food Sciences

The aim of work was L-lactic acid (LA) fermentation processsing enzymatic rye hydrolyzates as an inexpensive carbon sourcend nutrients supporting growth of lactic acid bacteria. Rye gritsr rye flour was enzymatically hydrolyzed in the low temperaturerocess (90 - 50 ◦C) with thermostable �–amylase Aquazym ATL,lucoamylase Spritase GA 14400L and malt prepared from nakedat Avena nuda as a source of pullulanase. Obtained hydrolyzatesere diluted to the glucose concentration of about 120 g/l, supple-ented with mineral salts (K+, Na+, Mg2+, Mn2+, PO4

3−) and yeastxtract (3 g/l) and used as the fermentation medium. L-lactic acidermentation processes were carried out in BioFlo 415 bioreactor5 l working volume, 42 ◦C, 44 h) with Lactobacillus rhamnosus asroducing organism. pH was controlled at 5,5 by automatic addi-ion of 12.5% ammonia water. At the end of fermentation (44 h),09.2 g × l−1 of glucose was utilized and 88.5 g × l−1 of lactic acidontaining 97.5% of L-isomer was produced. Dry mass of bacterialells reached value of 2.06 g × l−1 and average productivity was 2× l−1 × h−1. It was proved that rye may replace corn and be com-etitive substrate for microbial production of L-lactic acid in ryeelt countries with cold climate. The work was supported by theOIG 01.01.02-10-123/09 Project partially financed by the Euro-ean Union within the European Regional Development Fund,rants for Innovation.


I-21

ynamics of calcium L-lactate fermentation by Lacto-acillus rhamnosus in sugar beet thick juice and glucoseased media

lzbietaOltuszak-Walczak ∗ , PiotrWalczak, Agata Czyzowska, Annaygala, Patrycja Pietraszek, Katarzyna Dybka, Anna Otlewska

Faculty of Biotechnology and Food Sciences

Production of pure L-lactate stereoisomer from renewablearbon sources is required for the manufacturing of greeniodegradable tactic polymer poly-L-lactide (PLLA).The aim of
ork was evaluation of process dynamics of calcium L-lactate fer-entation using sugar beet thick juice or glucose as substrates.
ermentation processes with industrial strain of Lactobacillus rham-osus were carried out at 42 ◦C for 48-72 h in the juice based

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edium containing 93.8 g × l−1 of reducing sugars, or glucoseedium containing 120 g × l−1 of this saccharide. Biomass, reduc-

ng sugars and lactic acid concentrations were estimated duringermentation process. In the first 24 h of fermentation, specificroduction rate of lactic acid was 5.18 g × l−1 × h−1 for juice basededium and 4.77 g × l−1 × h−1 for glucose medium. Biomass con-

entration at the stationary phase reached value of 3.9 g × l−1

rrespectively of used medium. Specific substrate uptake rate was.98 g × l−1 × h−1 for juice and 2.80 g × l−1 × h−1 for glucoseedium. Reducing sugars were completely utilized after 31 h of

rocess in juice medium. In glucose based medium after 72 h ofermentation 29.5 g × l−1 of sugar was left unused. Final concentra-ion of L-lactic acid after 31 h of fermentation reached value of 92× l−1 for juice and 62 g × l−1 for the glucose medium respectively.he optical purity of produced L-lactate was 99.13%. The workas supported by the POIG 01.01.02-10-123/09 Project partiallynanced by the European Union within the European Regionalevelopment Fund, Grants for Innovation.


I-22

synthetic biology approach to characterise cyanobac-erial promoters

ary AnnMadsen ∗ , Anna Amtmann

University of Glasgow

Cyanobacteria are a phylum of bacteria with the ability toerform oxygenic photosynthesis using minimal nutrient require-ents: mainly sunlight, water and CO2. Cyanobacteria thus

rovide a cost-effective and energetically sustainable chassis foriotechnological applications. Cyanobacteria are relatively new tohe field of biotechnology, however, and the ‘tools’ available toenetically modify them are currently very limited. We thereforeescribe an approach to identifying promoters activated in highensity cultures and characterization using a synthetic biologypproach.


I-23

ifferential Adaptive Fermentation of E. coli K-12 andStrains in the Absence of adhE Gene Under Anaerobic

ondition

Lee1,∗ , Hyun Ju Kim1, Haeyoung Jeong1, Dong-Woo Lee2

Korea Research Institute of Bioscience and BiotechnologyKyungpook National University

Alcohol dehydrogenase (AdhE) plays a key role in the main-enance of redox balance in E. coli under anaerobic condition.
owever, in the absence of AdhE, oxidized redox cofactors such
s NAD+ could not be efficiently recycled in cells. Therefore,ighly-reduced status could be alternatively oxidized by lactateehydrogenase in E. coli adhE mutant cells. Indeed, under anoxic

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ondition E. coli K-12 adhE mutant cells rapidly adapted to thetressed condition and grew with lactate fermentation, whereas. coli BL21(DE3) adhE mutant cells begun very slowly adapt-ng to anaerobic growth. It was observed that the recycling ofAD+ was very poor in E. coli BL21(DE3) adhE mutant cells duringnaerobic culture, which caused severe redox stress and in turnecreased cellular viability. In contrast, K-12 adhE mutant cellsaintained cellular redox balance (NAD+/NADH ratio) and via-

ility under anoxic condition. This difference allows K-12 and Bells to undergo differential adaptations to anaerobic stress.


I-24

ihydroxyacetone-catalyzed phosphorylation

oland Wohlgemuth1,∗ , Dominik Gauss1, Israel Sanchez-Moreno2,duardo García-Junceda2

Sigma-AldrichCSIC

Biocatalytic phosphorylations have been shown to providenumber of advantages over chemical phosphorylations like

ncreased selectivity and high step economy [1,2].The use of recombinant dihydroxyacetone kinase from Cit-

obacter freundii in biocatalytic asymmetric phosphorylation haseen analyzed by direct quantitative 31P-NMR analysis of theinetics. The phosphoenolpyruvate/pyruvatekinase-system washereby selected for the regeneration of the cofactor ATP. Thisystem looks promising for further exploration.

eferences

].D. Gauss, B. Schönenberger, R. Wohlgemuth, CarbohydrateResearch, in press (2014).

].Matsumi R, Hellriegel C, Schönenberger B, Milesi T, van der Oost J,Wohlgemuth R. RSC Advances 2014;4(25):12989–94.


I-25

dducin-1 is essential for mitotic spindle assemblyhrough its interaction with myosin-X

ong-Chen Chen ∗ , Po-Chao Chan

National Chung Hsing University

Mitotic spindles are microtubule-based structures, but increas-ng evidence indicates that F-actin and F-actin-based motors areomponents of these structures. Adducin-1 (ADD1) is an actin-inding protein that has been shown to play important roles inhe stabilization of the membrane cortical cytoskeleton and cell-ell adhesions. In this study, we show that ADD1 associates withitotic spindles and is crucial for proper spindle assembly and
itotic progression. Phosphorylation of ADD1 at Ser12 and Ser355
y cyclin-dependent kinase 1 enables ADD1 to bind to myosin-XMyo10) and therefore to associate with mitotic spindles. ADD1epletion resulted in distorted, elongated, and multipolar spindles,

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ccompanied by aberrant chromosomal alignment. Remarkably,he mitotic defects caused by ADD1 depletion were rescued bye-expression of ADD1 but not of an ADD1 mutant defective in

yo10 binding. Together, our findings unveil a novel functionor ADD1 in mitotic spindle assembly through its interaction with

yo10.


I-26

egredable polymers of D-lactate from renewables

zlemOsmanagaoglu1,∗ , Haluk Hamamcı2, Onlu Harun3

Ankara UniversityMiddle East Technical University Department of Food EngineeringAnkara University Faculty of Sciences Department of Biology

The production of biodegradable plastics is of increasing inter-st because of environmental concerns. For the same reasons thetilization of renewable materials for the production of such mate-ials is also being supported. Lactic acid polymers are among theiodegradable materials that are being produced and whose pro-uction capacity is increasing. Lactic acid has two chiral forms,(-) and L (+); both of which can be produced by fermentation.

hese chiral forms, when polymerized may have distinct proper-ies; so both are important from the materials science point ofiew.

In this work we attempted to produce D (-) lactic acid fromenewable materials like sunflower seed hulls or wood shavingsy using Lactobacillus delbrueckii subsp. bulgaricus strains isolatedrom different home-made yogurts. The strains were tested for theirhiral lactate production and the better producers were chosen andubjected to fermentation tests. By several methods of neutraliza-ion, N-source supplementation and Na-Acetate addition so far weould reach a level of 80 g/L D (-) lactate production.


I-27

he Effect of Oral Administration of Pediococcus pen-osaceus OZF

arun Onlu1,∗ , Zhannatgul Sakyp2, Fadime Kıran2, Harun Onlu2,lker Büyük2, Sümer Aras2, ÖzlemOsmanagaoglu2

Ankara University Faculty of Science Department of BiologyAnkara University

The intestinal effects of Pediococcus pentosaceus OZF, a promis-ng probiotic strain and its encapsulated form was evaluatedn TNBS model of rat colitis. To increase the viability of OZFtrain under gastrointestinal conditions, encapsulation was car-ied out by whey proteins and calcium alginate. Female Wistarats (n = 6) were treated daily with oral administration of OZF
train (7 log cfu/ml) and its encapsulated form for 14 days, start-ng one week before the treatment of TNBS. The body weight,ater and food intake and the appearance of feces were recorded

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aily throughout the study. One week after induction of colitis,ll animals was killed and colonic damage was evaluated bothistologically and biochemically including the determination oflutathione, superoxide-dismutase, malondialdehyde (MDA) andatalase contents. MDA level, as a marker of oxidative stress wasignificantly increased in the colitis group. However, OZF admin-stration markedly decreased the MDA contents in colonic tissue.n addition, pro-inflammatory cytokines (IL-1beta and IL-6) inerum and colonic tissue were assessed by Real-Time PCR and a sig-ificant reduction was detected when compared to TNBS controlnimals. In conclusion, P. pentosaceus OZF could be a beneficialdjuvant/agent in the treatment of inflammatory bowel diseasesue to its intestinal anti-inflammatory activity.


I-28

pplication of confocal microscopy methods for esti-ation of lipid and proteins dynamics in thylakoidembranes

arketa Husakova1,∗ , Karolina Ditrychova2

University of Palackiana in Olomouc, Faculty of ScienceCharles University in Prague

The mobility of photosynthetic proteins is an important factoror light-energy conversion in photosynthesis. It has an importantole in regulation of photosynthesis and in protein transport afterts synthesis or repair. Mobility of photosynthetic proteins outsidehe thylakoid membrane is less understood. Cyanobacterial phyco-ilisomes attached to the thylakoid membrane can move relativelyast. We can measure specific feature of photosynthetic proteins

obility in vivo using microscopic methods such as FRAP (Fluo-escence Recovery After Photobleaching) which allows to observeutofluorescence. In our study we used procaryotic photosyntheticyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803s model organisms. To measure mobility on membranes wesed confocal microscopy and FRAP method as mentioned. FRAPethod is based on laser technology, thus we used stronger laser to

leach spots on the membrane and observed recovery of fluores-ence caused by mobility of proteins in membrane. We used threeifferent sizes of bleaching spots to compare rate of proteins mobil-

ty. We also studied mobility of lipids, therefore we used fluorescentye BODYPI because lipids are not autofluorescent whereas phy-obilisomes are. Main question of this study was, whether is FRAP
imited by diffusion or phycobilisome binding to the photosystem.

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I-29

haracterization of Pichia pastoris Golgi and plasmaembrane

ndreas Grutsch1,∗ , Karlheinz Grillitsch1, Pablo Tarazona2, Ericheitner3, Ivo Feussner2, Günther Daum4

Austrian Centre of Industrial Biotechnology (ACIB)Department for Plant Biochemistry, University of GöttingenInstitute of Analytical Chemistry and Food Chemistry, TU GrazInstitute of Biochemistry,TU Graz/Austrian Centre of Industrial Biotechnology

ACIB)

Pichia pastoris has a prominent status regarding expression ofecombinant proteins and is worldwide used as a highly efficientell system for the production of heterologous proteins. Despitehe importance of Pichia pastoris in biotechnology, little informa-ion is available about the cell biology of this microorganism. Thisempted us to intensify our studies on Pichia pastoris organellesith special emphasis on subcellular fractions involved in the

lassical secretory pathway. The Golgi harbors many processes ofost-translational protein modifications and is a major branch-

ng point within the secretory pathway. The plasma membrane ismportant for protein secretion as it is the last barrier on the wayf polypeptides to be externalized. We have established protocolso isolate Pichia pastoris Golgi and plasma membrane at sufficientield and high purity. Lipid profiling of the isolated membranesighlighted characteristics of the specific organelles. Whereas thehospholipid patterns were organelle specific, the fatty acid com-osition was similar in all membranes. By contrast to evidencerom Saccharomyces cerevisiae, a sterol gradient along the secre-ory route was not found in Pichia pastoris. Sphingolipids of Pichiaastoris showed a clear organelle dependent distribution pattern.eramides and hexosyl-ceramides were predominantly found inolgi fractions, whereas inositol containing phosphorylceramidesere almost exclusively enriched in plasma membrane fractions.

imilarities of Pichia pastoris and Saccharomyces cerevisiae becamevident, although certain differences have to be kept in mind.omparison of the two yeasts is important for understanding dif-

erent properties and designing new strategies to improve Pichiaastoris strains for industrial applications.


I-30

omatic embryogenesis from cultured zygotic explantsf some olive cultivars (Olea europaea L.)

ara Oulbi1,∗ , Ibrahim Toufik2, Ilham Belkoura3

National School of Agriculture, Meknes, MoroccoDepartment of Biology, Faculty of Sciences, University Moulay Ismail, Meknès,oroccoDepartment of basic sciences, National school of Agriculture, Meknes,orocco

Mature zygotic embryos taken from three olive cultivarsDahbia cv, Moroccan Picholine (PM) cv and Arbequine cv)ere used to test the embryogenic capacity of their juvenile

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xplants (cotyledons and radicles). These explants were cul-ured in Olive Medium (OM) (Rugini, 1984) supplemented withollowing combinations of auxin and cytokinin: AIB and 2iP6-dimethylallylaminopurine), AIB (Indole-3-butyric acid) + TDZThidiazuron) and AIB + Zeatin for 3 weeks; they were then trans-erred on OM free hormone. Calluses formation began during therst subculture in the three media; in term of the 3 weeks of cul-ure in the first medium, the highest rates of callus were obtainedhen medium supplemented with 2iP for Arbequine cotyledons

100%) and Moroccan Picholine radicles (81.5%) and TDZ foradicles of Dahbia (92%). However, somatic embryogenesis wasbserved on Dahbia and Arbequine cotyledons and radicles culti-ars during the second week of culture on OM free hormone whenalluses were formed on AIB + 2iP, whereas Picholine explantsnduced nodular calluses without somatic expression. Maturationf somatic embryos was observed when OM medium was supple-ented with 1 g of activated charcoal; indeed, globular somatic

mbryos were transformed to cotyledonary embryos (16.7% and.8% for Dahbia and Arbequine respectively). On attempt to ger-ination, somatic embryos were cultured on various media in a

6/8 photoperiod. Neoformation of buds was observed on somaticotyledons (37.5%) whereas rooting of somatic embryos was moreifficult to obtain.


I-31

ffects of 4 essential oils on growth of afaltoxin produc-ng fungi

ittichai Chareonsettasilp1,∗ , Dusanee Thanaboripat1, Chanitaarutipaisan1, Chutima Puangtong1, Phurin Chatpongsatorn1,aowapa Suvati 2

King Mongkut’s Institute of Technology LadkrabangGovernment Pharmaceutical Organization

There have been records that many essential oils shown antimi-robial properties. Some essentials oils can inhibit the growth offlatoxin producing fungi and aflatoxin production. In this studye compare the ability of 4 plant essential oils. i.e. ginger oil, anise

tar oil, cajuput oil and cinnamon oil for controlling aflatoxin pro-ucing fungi. The oils at concentrations of 0, 0.5, 1, 2, 3, 4 and 5%ere tested against Aspergillus flavus IMI 242684 and A. parasiti-

us IMI 102566 on Potato Dextrose Agar (PDA). The fungi wereultured and incubated at 30 C for 7 days. The results show thatnise star oil at all concentrations had the most inhibitory effectn both Aspergillus flavus IMI 242684 and A. parasiticus IMI 102566ith significant difference followed by cinnamon oil and cajuput
ils. Ginger oil had the least inhibition effect.
ttp://dx.doi.org/10.1016/j.nbt.2014.05.2000itcarta


I-32

NA-Seq analysis of the degradation of haloacetate byurkholderia caribensis MBA4

immy Tsang ∗ , Yanling Pan, Nan Zheng

School of Biological Sciences, The University of Hong Kong

Burkholderia caribensis strain MBA4 was isolated for its abil-ty to utilize monobromoacetate as carbon and energy source.his bacterium produced an inducible haloacid dehalogenase thatransforms monohaloacetate to glycolate and to glyoxylate by gly-olate oxidase. Genomic analysis of the bacterium showed that itontains three glycolate oxidases: ETY79679-81, ETY80271-3 andTY84258-60. Transcriptomic analysis showed that ETY79679-81as expressed constitutively to a reads per kilobase transcripter million reads (RPKM) value of around 100 no matter theubstrate was pyruvate, glycolate or chloroacetate. ETY80271-3ave values of 7 in pyruvate-, 867 in chloroacetate- and 1260 inlycolate-grown cells. ETY84258-60 gave values of 20 in pyruvate-1880 in chloroacetate- and 178 in glycolate-grown cells. Aalate synthase G gene, ETY84261, was found downstream of

TY84258-60. Apparently, ETY80271-3 converted glycolate to gly-xylate and through the glycerate pathway to pyruvate. WhenBA4 was grown on chloroacetate, gene products of ETY84258-

1 were mainly used and malate was generated. Putative regulatorlcC genes, ETY80275 and ETY84257, can be found upstream ofTY80271-3 and ETY84258-60, respectively. The expression pro-les of these glycolate oxidases suggested that both GlcC werectivated by glycolate and chloroacetate with ETY80275 moreesponsive to glycolate and ETY84257 more reactive towardshloroacetate. While the relative transcript levels of ETY80275ere rather stable, expression of ETY84257 was enhanced inlycolate- and even more in chloroacetate-grown cells. The charac-erization of the degradation of haloacetate by B. caribensis MBA4s made possible with the use of RNA-seq analysis.


I-33

nalysis of the physiology of an Aspergillus nidulansutant lacking the aoxA (cyanide-resistant alternative

xidase encoding) gene

rzsébet Fekete ∗ , Ákos P. Molnár, Mojtaba Asadollahi, Szabinalázsi, Erzsébet Sándor, Levente Karaffa

University of Debrecen

One of the most characteristic features of fungal mitochondrias the presence of an additional terminal oxidase, the alterna-ive oxidase, the activity of which is insensitive to inhibitors ofytochrome c oxidase and the bc1 complex. Responsible for thisctivity in the model fungus Aspergillus nidulans is the cyanide-esistant enzyme alternative oxidase, a quinol oxidase localized in
he inner mitochondrial membrane encoded by the nuclear geneoxA.

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onsequences of the deletion of the aoxA gene in A. nidulans inomparison with the wild-type reference strain. Growth propertiesn various carbon sources at different concentrations were com-ared. Furthermore, osmotic, heat and stress tolerance of the twotrains were analysed.

cknowledgement

The research was supported by the EU and co-financedy the European Social Fund under the project ENVIKUTTÁMOP-4.2.2.A-11/1/KONV-2012-0043), and also by the Hungar-an Scientific Research Fund (OTKA Grant K1006600 to Dr. Erzsébetekete).


I-34

nsights into diversity and specificity of heavy metalesistance and efflux systems in Bacillus oceanisediminis691

yun Ju Kim1,∗ , Yong-Jik Lee2, Haeyoung Jeong3, Dong-Woo Lee2,ang Jun Lee4

Biosystems & Bioengineering Program, University of Science and TechnologyUST)School of Applied Biosciences, Kyungpook National University, Daegu,epublic of KoreaKorean Bioinformation Center, Korea Research Institute of Bioscience andechnology (KRIBB), Daejeon, Republic of KoreaKorea Research Institute Bioscience & Biotechnology

We previously determined the genome sequence of aerobic,ndospore-forming, Gram-positive Bacillus oceanisediminis 2691hat was isolated from marine sediment of the South Koreanoast (Lee YJ et al., 2012 J Bacteriol.). Many genes encodingeavy metal resistance and efflux systems were found in theenome. Genes encoding putative cadmium efflux pumps, arsenicfflux pumps, a chromate transporter, and lead-, cadmium-, zinc-,nd mercury-transporting ATPases were found. Putative resis-ance proteins of copper, cobalt-zinc cadmium, and telluriumere also identified. Apparently, transcriptions of those genesre controlled by CadC homologous metal responsive repres-ors. The transcription profiles of CadC-controlled genes wereonitored in the presence of various heavy metals in B. oceanised-

minis 2691. Furthermore, six cadC promoter-operator-structuralenes were transcriptionally fused with egfp gene in E. coli. Aariety of heavy metals were treated and specific fluorescencentensities were measured. Taken together, the results showedhat CadC proteins specifically respond to heavy metals and

ay play separate roles in heavy metal resistances, which haveeen evolved in the heavy metal abundant marine sedimentilieu. In addition, CadC-controlled transcriptional modules



ould be used in the development of harmful heavy metal bio-ensor.


I-35

igh cell density cultivation of the chemolithoau-otrophic bacterium Nitrosomonas europaea in a dialysisembrane bioreactor

evente Karaffa1,∗ , Tibor Török2, Antal Kökényesi 1, István Kolláthrzsébet Sándor1, Zoltán Németh1, Noémi Lipták1, Erzsébet Fekete

University of DebrecenTEVA Pharmaceutical Ltd., Safety and Environmental Department, Debrecen,ungary

Nitrosomonas europaea is a chemolithoautotrophic nitrifierram-negative bacterium that gains all of its energy for growth

rom the oxidation of ammonia into nitrite ions. A major pre-equisite to produce a high cell density N. europaea culture is tonsure that inhibitory metabolic by-products remain at minimaloncentrations. The principal inhibitory metabolite being con-inuously formed during N. europaea fermentation is nitrite. As aonsequence, N. europaea cannot grow into high cell density underonventional batch conditions.

A suitable method presented in this study to overcome thisroblem is the application of a single-vessel dialysis membraneioreactor system. Fermentations were performed in a reactorith 2 L total/1.5 L useful and 6 L total/5.5 L useful volumes ofedium in the inner and outer chambers, respectively. Growth

f N. europaea was monitored via cell density determinations andy the measurement of nitrite formation. Metabolic activity waslso visualized by Acridine Orange staining. Maximal cell den-ity and maximal calculated specific growth rate were over fiveimes of the value achieved under conventional batch conditions.

e concluded that dialysis fermentors are suitable tools to over-ome growth limitations observed in standard batch cultures of N.uropaea.

cknowledgement

The research was supported by the EU and co-financedy the European Social Fund under the project ENVIKUTTÁMOP-4.2.2.A-11/1/KONV-2012-0043), and also by the Hungar-an Scientific Research Fund (OTKA Grant K1006600 to Dr. Erzsébet

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I-36

ffer, demand, and needs in training and education: atudy focusing on microbial culture collections withinhe MIRRI Consortium

ndré Antunes1 , Veerle Piessens2, Nelson Lima1, The MIRRI Con-ortium

Micoteca da Universidade do Minho, Centre of Biological Engineering, Uni-ersity of Minho, Braga, PortugalBCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, B-9000hent, Belgium

The bioeconomy is fueled by Biological Resource Centers,hich play a vital role in harnessing and preserving the world’siodiversity [1]. MIRRI (the Microbial Resource Research Infra-tructure: www.mirri.org) is an EU-project involving a total of3 partners, aiming to provide facilitated access to microbialesources, associated data and expertise, and promote knowledgeransfer and foster innovation. One crucial step is to properlyefine our stakeholder community and identify their current anduture needs, and match them to our offer.

In order to achieve these goals, MIRRI surveyed both its part-ers, and current and potential users of microbial resources andervices. Here we present and analyze some of the results of thisurvey, focusing on training and education.


Despite current trends and benefits in increased use and produc-ion of contents in new formats (e.g. video, interactive), and usef new tools and technologies (e.g. e-learning, b-learning), train-ng within MIRRI still has an overwhelming dominance of classicontent types and delivery methodologies. Also, we identified auch wider untapped market for education and training within

ur customer base, and we estimate a spike in demand of trainingrom culture collections, particularly from the profit sector.

Additional efforts are clearly necessary in adjusting our offer,dapting contents and content delivery and focusing on cost-fficiency and proper advertising to increase visibility, and bettererve the needs of our customers.

eference

].OECD. Underpinning the future of life sciences and biotechnology. OECD,


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lycobiology

J-01

loning and characterization of novel β-glucosidasesrom Aspergillus and their functional expression inethylotrophic yeast Pichia pastoris

ichard Auta ∗ , Paul Hooley, Iza Radecka

University of Wolverhampton

The production of fermentable sugars from lignocellulosicaterial has attracted interest in the optimization of conditions

elated to conversion of cellulose to glucose for biofuel production.spergillus strains are known as efficient producers of β-glucosidasehich is a rate limiting factor during enzymatic hydrolysis of cellu-

ose.A bioinformatics based approach to characterize β-glucosidasencoding enzymes in the genus Aspergillus is described and thepplication of bioinformatics in the selection and expression ofarget genes is explained. Five Pichia clones (carrying A. nidu-ans AN2227.2, AN2612.2, AN0712.2, AN1551.2 and AN1804.2n pPICZ vectors) that exhibit satisfactory levels of expression ofecombinant β-glucosidase were obtained from the Fungal Genet-cs Stock Centre (FGSC). A study to compare their hydrolyticctivities and relate these to their growth profiles using differentedia was carried out. The characteristics of these enzymes and the

se of P pastoris in the expression of these proteins are discussedKeywords�-glucosidase; Aspergillus; Bioinformatics; Hydrolysis


J-02

inetic evaluation of carbohydrate-protein interactionsing SPR

eong Hyun Seo1,∗ , Chang Sup Kim2, Hyung Joon Cha2

Yeungnam universityPOSTECH, Korea

Surface plasmon resonance (SPR) can provide kinetic infor-ation about an interaction, and it can also be used to rapidlyonitor dynamic processes, such as adsorption and degrada-



ion, without the need for sample labeling. Here, we employedPR to analyze carbohydrate-protein interactions, particularlyM1-related carbohydrate-Vibrio cholera toxin interactions. The

nteraction between cholera toxin subunits A (ctxA) and B (ctxB)as similar to general ligand-receptor interactions. After the direct

mmobilization of thiol-containing GM1 pentasaccharide on aold surface, the GM1-ctxB interaction kinetics were evaluated,nd they showed a similar degree of kinetics as reported in previouseports. We found that ctxA had a high affinity for the GM1-txAB complex, although its equilibrium dissociation constantas 10-times lower than that of GM1-ctxB binding. Comparativenalyses for GM1-related carbohydrates-ctxAB interactions werelso conducted to determine the kinetic values of several GM1nalogues with different structures, although their kinetic valuesere one-order of magnitude lower than those of the GM1-ctxAB

nteraction. The kinetic analysis results for the interactions ofM1 analogues and ctxAB indicated that the sialic acid thumb

s important for recognition, and the terminal galactose and N-cetylgalactosamine finger are required to stabilize the GM1-ctxABnteraction. Taken together, our results indicate that the directmmobilization of carbohydrate in an SPR-based analytical systeman be used to evaluate the structural contribution of carbohydrateoieties in carbohydrate-protein interactions, as well as provide

aluable information that can be used to understand the interac-

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reenhouse gases and global warming

K-01

O2 gas conversion to oxaloacetate by Escherichia coliarboring codon-optimized carbonic anhydrase andhosphoenolpyruvate carboxylase derived from marineacteria

oohyun Park1,∗ , Soohye Hong1, Sangwoo Kim1, Seung Pil Pack2,inwon Lee1

Sogang universityKorea University

Increased emission of CO2 is a major problem which causeslobal warming throughout the world. Interest in bio-refinery pro-ess has increased remarkably because microbial conversion ofO2 to chemicals has large number of industrial applications,nd also it can alleviate the increasing residual CO2. Carbonicnhydrase (CA) is an enzyme, which plays a role in carbon seques-ration process. And, phosphoenolpyruvate carboxylase (PEPC) is

biocatalyst which converts phosphoenolpyruvate to oxaloac-tate. Oxaloacetate can be converted in to various valuable C4

hemicals (succinate, malate etc.) in TCA cycle. First, novel CAnd PEPC genes were screened and found in marine bacteria byasic local alignment using the Escherichia coli PEPC amino acidAA) sequence as a query. The CA gene from Hahella chejuensisnd PEPC gene from Photobacterium profundum SS9 were selected.he codons of the heterologous CA and PEPC genes were syn-hesized using codon-optimization technique. Codon-optimizedenes were cloned into pETDuet1 and transformed in E. coliL21(DE3). Due to the SDS-PAGE results, it was confirmed thathe codon-optimized genes were expressed as soluble proteinorms in E. coli. The specific activity value of the codon-optimizednzymes were relatively high compared to the previously knownA and PEPC activities (codon-optimized CA (HC-aCA) = 478 ± 63AU/mg protein in the presence of Zn2+, codon-optimized PEPC

OPPP) = 80.3 U/mg protein). In this study, a CO2 gas convert-ng platform technology based on the co-expression system of CAnd PEPC is developed and the potential of CO2 gas conversion
latform technology is shown.

GREENHOUSE GASES ANDGLOBALWARMING

K-02

merging Biotechnologies For Landfill Greenhouseases’ Efficiency And Development Of Useful Webrawing Utilities For Public Health Protection

ilemachos Koliopoulos ∗

Director Telegeco - Scient. Colaborator Technological Educational Institute ofthens -

This paper analyses the effects of different waste manage-ent landfill biotechnology techniques influencing on produced

reenhouse emissions, leachate emissions, acids and landfill massiodegradation stages. The biodegradation of Mid Auchencarrochxperimental landfill project is studied in four different cells. Theariations of the examining emissions are analysed in order toevelop an efficient project management for the right operationaleasures for food safety in areas adjacent to landfill boundaries

nd public health protection. An analysis is made for emergingandfill biotechnology designs and modern spatial monitoringystems are developed utilizing properly web drawing utilitiesnd associated information technologies (IT’s). Useful emergingandfill biotechnologies and simulation models are presented forroduced landfill greenhouse gases making useful conclusions.

Keywordslandfill biotechnology; greenhouse gases; landfill emissions;

xperimental landfill design; public health protection; wateresources; global warming; climate change; food security; emerg-


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igh value plant products

L-01

ffect of pH on chromium and nickel biosorption byitchi chinensis seeds in single and binary metal systems

iliana Morales Barrera ∗ , Liliana Morales-Barrera, Imelda Guerrero-oronilla, Víctor Zambrano-Pérez, Jessica Reyes-Ledezma, Griseldahávez-Camarillo, Eliseo Cristiani-Urbina

National Polytechnic Institute

The extensive industrial use of hexavalent chromium [Cr(VI)]nd divalent nickel [Ni(II)], as well as their improper disposal haveed to heavy metal contamination of water [1]. Biosorption is anfficient and low-cost alternative for removing toxic metals fromqueous solutions. The purpose of this work was to evaluate theffect of pH on chromium and Ni(II) removal by Litchi chinensiseeds (LCS) in single and binary metal systems.

LCS was able to remove chromium and Ni(II) from mono-etal solutions, and its removal capacity depended on the pH.

CS exhibited the highest removal capacity of Cr(VI) (86.79 mg/g),otal chromium (64.74 mg/g) and Ni(II) (20.0 mg/g) at pH valuesf 1.0, 2.0 and 7.5, respectively.

At a pH value of 1, a difference between Cr(VI) (86.79 mg/g) andotal chromium removal capacity (34.4 mg/g) was observed andhis was probably due to the fact that part of the Cr(VI) initiallyresent in the solution was reduced to trivalent chromium [Cr(III)]y LCS.

In the bimetal systems, Ni(II) did not have any effect onhromium removal at any pH value. Ni(II) biosorption capacityas not affected by the presence of chromium in acidic solutions;

n contrast, at neutral pH values the capacity improved signif-cantly. These results suggest that chromium and Ni(II) do notompete between them for the same LCS binding sites.

eference

].Park D, Yun YS, Yim KH, Park JM. Effect of Ni(II) on the reductionof Cr(VI) by Ecklonia biomass. Bioresource Technol 2006;97:1592–8.


L-02

reparation and Functional Characteristics of Peptiderom Naked Oat Globulin by Protease Hydrolysis

eili Zhang ∗ , Rui Lin

Inner Mongolia Agricultural University

Globulin of naked oat was prepared by the Osborne meth-ds and hydrolyzed by alkaline protease. The degree of proteinydrolysis and clearance rate of hydroxyl free radicals were used to

dentify the best enzyme hydrolysis process. Solubility, emulsify-
ng property, viscosity and foamability of hydrolysate were tested.he result showed that the optimum conditions were as follows:ose of enzyme, 10000U/g; concentration of substrate, 5%; theemperature, 60◦C; and pH 9.0. After 3 h, the degree of hydrolysis


eached 75% and the clearance rate was above 60%. The solubilityf the hydrolysate was as high as 93%. Emulsifying property andhe emulsifying stability decreased in the pH range from 3.0 to 5.0,ncreasing with increased pH. Compared with the measured glob-lin, the foamability of hydrolysate was significantly increased,

owest at pH 5.0. The viscosity of hydrolysate was reduced duringhe whole process.


L-03

ombinatorial optimization of synthetic operons for theicrobial production of monolignols in Escherichia coli

hilana van Summeren-Wesenhagen ∗ , Raphael Voges, Stephanoack, Michael Bott, JanMarienhagen

Forschungszentrum Jülich

The monolignol p-coumaryl alcohol is an important precursorf lignans and key building block of the plant polymer lignin,hich is widely recognized as cheap source of aromatic com-ounds. However, due to its complex and irregular structure theitilization of lignin is technically challenging. In contrast, micro-ial production of p-coumaryl alcohol and other monolignolsepresents a promising alternative. Recently, the first syntheticathway for the production of p-coumaryl alcohol from L-tyrosine

n E. coli was published [1]. Here we introduce a fast and robustethod to optimize the product titers of this four gene pathway,

ased on the Phosphorothioate based Ligase-Independent Gene CloningPLICing) method [2]. An operon library was generated in whichhe translation efficiency of every gene was systematically variedy different spacings between the ribosomal binding site and theTART-codon. Screening of this library yielded mutants producingp to 55 mg/L p-coumaryl alcohol, 7 times more compared to thetarting strain under similar cultivation conditions.

eferences

].Jansen F, Gillessen B, Mueller F, Commandeur U, FischerR, Kreuzaler F. Metabolic engineering for p-coumaryl alcoholproduction in Escherichia coli by introducing an artificial phenyl-propanoid pathway. Biotechnology and Applied Biochemistry 2014, doi:10.1002/bab.1222. (published ahead of print).

].Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U.Phosphorothioate based Ligase-Independent Gene Cloning - A methodfor cloning of mutant libraries in directed evolution experiments.

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L-04

n vivo and in vitro cultures of Lavandula angustifoliaill. for essential oils production

ana Lohasupthawee ∗ , Prangmas Srisurat


The aim of this research was to extract the essential oils ofavender (Lavandula angustifolia Mill.) from callus culture, multiplehoots culture and hydroponic culture. The chemical composi-ion of each extract was analyzed by gas chromatography and

ass spectrometry (GC-MS). In vitro culture of lavenders, multiplehoots were obtained in culture medium fortified with Murashigend Skoog (MS) nutrients and 1.5 mg/l benzyladenine which pro-uced 12 shoots per explant. Callus was achieved by using leafxplants and showed best growth in MS medium supplementedith 0.5 mg/l 2,4-dicholorophenoxyacetic acid. In vivo culture,ydroponic lavenders were achieved by using deep flow tech-ique system. The leaves and roots of hydroponic lavenders werextracted for essential oils separately. The GC-MS results showedhat the leaves of hydroponic lavenders and in vitro shoots demon-trated the components of lavender essential oils whereas callusnd roots of hydroponic lavenders showed no essential oils pro-uction.


L-05

ermentative Nisin Production in Tofu for its Preserva-ion

icole Illas ∗ , Raphael Cziskus, Caterina Hünniger, Myriam Bello,uan Zhu, Markus Fischer, Bernward Bisping

University of Hamburg

Tofu has a high nutritional content and serves as stable food inastern Asia. In developing countries pasteurization is not afford-ble because tofu production takes place mostly in small factories.cost-effective and natural preservation of tofu may be thenisin

roduction in tofu cubes (2*2 cm) submerged in water by fer-entation with Lactococcus lactis ssp. lactis DSM 20729. Optimal

ermentation was conducted by adding 3.9% soy peptone. Further-ore it could be shown that there is a non-uniform distribution

f nisin in tofu cubes. The nisin concentration decreases fromhe surface to the interior of tofu but it was sufficient to extendhe shelf-life of tofu. Two methods for the detection of nisinn fermented tofu were tested. The comparison of an inhibitionest (modified [1]), and LC-ESI-MS/MS method (based on ISO/TS7106:2009 [2]) illustrates that the detection limit of the inhibitionest was significantly lower. Concentrations of 0.19 mg/kg nisin inofu and 0.06 mg/L in the supernatant could be determined. Theetection limit for nisin using LC-ESI-MS/MS was 0.34 mg/kg inofu. Matrix calibration of the liquid substrate could not be car-
ied out by LC-ESI-MS/MS, because the background noise of theatrix was too high.
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HIGH VALUE PLANT PRODUCTS

It could be shown that the fermentation of tofu with L. lactis isn efficient and cost-effective alternative.

eferences

].Pongtharangkul T, Demirci A. Appl Microbiol Biotechnol:2004;65:268–72.

].International Organization for Standardization ISO/TS 27106Geneva Switzerland 2009.


L-06

ermentation-like incubations of Theobroma cacao L. -ood quality in shorter time

laudia Bahmann ∗ , Tumforde Thomas, Lieberei Reinhard


In the course of the post-harvest treatment of cocoa seeds (fer-entation) the pulp is microbially degraded. During fermentation

rst ethanol, then acetic acid is produced in combination with aemperature increase. The conditions in the fermentation massntail the acidification of the cotyledon tissue and an extensiveroteolysis of storage proteins. The latter is carried out by endoge-ous proteases that are activated by acidic conditions. The cleavagectivity of the proteases as well as the conservative amino acidequences of storage proteins provide a defined pattern of flavorrecursors of cocoa. In the study at hand seeds have been incu-ated under conditions analog to those of fermentation, becausef the ability to control different parameters separately. Media withifferent pH-values, all in the acid range, and different organiccids have been used. The basic effects of these factors on the seedith regard to the proteolysis are examined. In order to imitate the

emperature conditions of fermentation, incubations are carriedut under different temperature sequences.

In the course of the incubation process comparatively highermounts of the amino acids and phenolic compounds examinedre achieved already after three days. Comparable values in theermentation procedures are achieved not until after six days. Thisllustrates the efficiency of the incubation procedure. In this study,n endogenic bioconversion has been proven to take place inocoa seeds in the course of incubations. Furthermore, the strivenccumulation of characteristic chocolate aroma precursors can benfluenced by processing seeds under defined external conditions.


L-07

lucanocellulosic biomass: learning from marineiomass to optimize terrestrial biomass conversion

hristian Voigt ∗ , Claudia Zwikowics


The recalcitrance of the plant cell wall is one of the obsta-les in improving biomass conversion. We followed a strategy tonrich biomass with a polymer that is easily degradable and would


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IGH VALUE PLANT PRODUCTS

ot impair the physiology of the plant. In marine biomass fromhe brown algae Fucus vesiculosus, the polymer (1,3)-β-glucan is a

ajor biomass component showing these characteristics and islso present in terrestrial plants. We used this glucan-enriched,arine biomass to optimize hexose release and bioethanol produc-

ion. The addition of a bacterial (1,3)-β-glucanase to a commercialnzyme cocktail as well as the usage of an optimized Saccharomyceserevisiae strain that we engineered for fermenting glucan-enrichediomass resulted in a 50% increase in bioethanol production. To
est the optimized processing of glucan-enriched biomass on ter-estrial biomass, we screened for plants with a high (1,3)-β-glucanontent and identified leaves from the energy crop Miscanthus xiganteus with an exceptionally high (1,3)-β-glucan content of 5%.
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pplying the optimized processing for glucan-enriched biomassn M. giganteus leaf biomass, we increased bioethanol productiony 13% compared to non-adapted conversion and fermentationtrategies. To further improve bioethanol production with thisdapted processing, we overexpressed a (1,3)-β-glucan synthaserom Arabidopsis thaliana in M. giganteus, which further increased1,3)-β-glucan content in leaf biomass to 8.5% and improvedioethanol production by 20%. Our results suggest that generationf glucan-enriched biomass via synthetic biology approaches com-ined with optimized processing for glucanocellulosic bioethanolroduction is a promising alternative in increasing efficiency of
iomass conversion.

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etabolic engineering

M-01

ex-Dimorphic Expression Of Caveolin 1 Is Linked Tobesity Development In Rats

ae Heon Choi ∗ , Rajib Mukherjee, SangWoo Kim, JongWon Yun

Daegu University

Recently, it has been reported that CAV1 is an important targetrotein in sex hormone-dependent regulation of various metabolicathways, particularly in cancer and diabetes. To clarify distinctoles of CAV1 in sex-dependent obesity development, we inves-igated the effects of high fat diet and sex steroid hormones onAV1 expression in adipose tissues of male and female rats. Resultsf animal experiments revealed that estrogen (17-β-estradiol, E2)nd androgen (dihydrotestosterone, DHT) had opposite effectsn body weight gain as well as on the regulation of CAV1, hor-one sensitive lipase (HSL) and uncoupling protein 1 (UCP1)

n adipose tissues. Furthermore, sex hormone receptors and aro-atase were differentially expressed in a sex-dependent manner in

esponse to E2 and DHT treatments. In vivo data were confirmedsing 3T3-L1 and HIB1B cell lines, where Cav1 knock down stim-lated lipogenesis but suppressed sex hormone receptor signalingroteins. Most importantly, co-immunoprecipitation enabled the

dentification of previously unrecognized CAV1-interacting mito-hondrial or lipid oxidative pathway proteins in adipose tissues.aken together, current data showed that CAV1 may play impor-ant preventive role in the development of obesity, with morerominent effects in females, and proved to be an important targetrotein for the hormonal regulation of adipose tissue metabolismy manipulating sex hormone receptors and mitochondrial oxida-ive pathways. Therefore, we can report, for the first time, the

olecular mechanism underlying the effects of sex steroid hor-ones in the sex-dimorphic regulation of CAV1.


M-02

etabolic engineering of Clostridium acetobutylicum forighly selective butyric acid production

u-Sin Jang ∗ , Sang Yup Lee

Korean Advanced Institute of Science and Technology (KAIST)

Butyric acid, a saturated four-carbon carboxylic acid, has beenidely used in chemical, food, pharmaceutical, and animal feed

ndustries. The well-known clostridial native butyric acid produc-rs include C. butyricum, C. thermobutyricum, C. tyrobutyricum, C.cetobutylicum and C. pasteurianum. A typical characteristic of suchutyric acid-producing Clostridium is coproduction of both butyricnd acetic acids. Increasing the butyric acid selectivity importantor economical butyric acid production has been rather difficult in
lostridia due to their complex metabolic pathways. In this work,. acetobutylicum was metabolically engineered for highly selec-
ive butyric acid production. For this purpose, the second butyrate

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inase of C. acetobutylicum encoded by the bukII gene instead ofutyrate kinase I encoded by the buk gene was employed. Further-ore, metabolic pathways were engineered to further enhance theADH-driving force. Batch fermentation of the metabolically engi-eered C. acetobutylicum strain at pH 6.0 resulted in the productionf 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AAatio) of 31.3 g/g from 83.3 g/L of glucose. These results suggestedhat the buk gene knockout was essential to get a high butyric acidelectivity to acetic acid in C. acetobutylicum.

[This work was supported by the Technology Development Pro-ram to Solve Climate Changes on Systems Metabolic Engineeringor Biorefineries from the Ministry of Science, ICT and Future Plan-ing (MSIP) through the National Research Foundation (NRF) oforea (NRF-2012-C1AAA001-2012M1A2A2026556).]


M-03

he parologues pyruvate kinases in Streptomyces coeli-olor have distinct roles in growth and antibioticroduction

anaHiltner1,∗ , Pablo Cruz-Morales2, Lorena Fernandez-Martinez3,rovje Petkovic4, Iain S. Hunter1, FranciscoBarona-Gomez2, Paul A.oskisson1

University of StrathclydeLangebio CinvestavJohn Innes CentreAcies Bio Ltd

Streptomyces species are prolific producers of antibiotics, nev-rtheless analysis of complete genomes still shows that therere many biosynthetic clusters present that are silent under lab-ratory cultivation conditions. The current increase in clinicalntibiotic resistance requires the discovery of new antibiotics, butlso a greater understanding of antibiotic production for indus-rial exploitation. Our interest is in studying the transition ofrimary metabolites into secondary metabolism. We focus onhe Phosphoenolpyruvate-Pyruvate-Oxaloacetate (PEP-PYR-OAA)ode of central carbon metabolism using Streptomyces coelicolor asmodel and have identified pyruvate kinase influences antibi-

tic production. The genome encodes two parologue pyruvateinase genes - SCO2014 (pyk1) and SCO5423 (pyk2). Phenotypicnalysis of the two mutants revealed differences in their physi-logical role, �pyk2 exhibits altered growth on glucose, whereaspyk1 shows a difference in antibiotic production. We used cross-

pecies complementation experiments with E.coli �pykF, �pykAnd �pykA�pykF and complemented these with pyk1 and pyk2rom S. coelicolor on different media to clarify their physiologi-al roles. Furthermore Pyk1 and Pyk2 were overexpressed in E. colior a detailed characterisation of their biochemical properties. Ourata show that gene parologues in primary metabolism have dis-inct physiological roles in Streptomyces that impact significantlyn growth and the production of antibiotics, which could be used
or industrial strain improvement in the biotechnology industry.


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M-04

e-distribution of carbon flux toward 2,3-butanediolroduction in Klebsiella by metabolic engineering

orim Kim ∗ , Soojin Lee, Daun Jeong, Jeongmo Yang, Jinwon Lee

Sogang university

As shown in previous studies, Klebsiella pneumoniae showedigh potential as a 2,3-butanediol (2,3-BDO) producer, and exhib-

ted great productivity. However, the accumulation of substratesther than 2,3-BDO such as; lactate, ethanol, and acetate, in theog-phase of cell growth remained as an obstacle for efficient largecale 2,3-BDO production. Hereby, in order to re-distribute theubstrate-directed carbon flux to 2,3-BDO production metabolicngineering was done. Incorporation of the gene deletion methoddeleting competitive NADH consuming pathway by ldhA geneeletion) and gene over-expression method (re-directing carbon-ux toward 2,3-BDO biosynthesis by budA gene over-expression)ere conducted for efficient utilization of glucose conversion to,3-BDO under slightly acidic condition(pH 5.5). The engineeredtrain SGSB105 showed a 40% increased 2,3-BDO productionrom glucose, compared to the wild-type strain SGSB100. Also thelosely related genes in the 2,3-BDO biosynthesis pathway werebserved at the gene transcription level by cultivating mutanttrains under unify culture conditions. As a result, the gene expres-ion levels of the budB, budA, and budC genes showed a 10%ncreased transcription level at the log-phase of the cell growth,ompared to the SGSB100. Also the 2,3-BDO gene transcriptionevels of SGSB105 was maintained at a high level during the log-nd stationary-cell growth phase. By incorporating the gene dele-ion and over-expression method, the carbon flux was re-directedo a valuable biochemical producing process, and also by combin-ng the batch culture data with the gene transcription data it showsn insight for improving the 2,3-BDO biosynthesis metabolic net-ork for industrial application.


M-05

etabolic engineering of Corynebacteruim glutamicumor production of 5-aminovaleric acid and glutaric acids C5 platform chemicals

ae Ho Shin1,∗ , Si Jae Park2, Sang Yup Lee1

Korea advanced institute of science and technologyMyongji University

The amino acid, L-lysine can be naturally degraded via multipleonduits including the cadaverine and 5-aminovaleric acid path-ays. The degradative intermediates, cadaverine, 5-aminovalericcid and glutaric acid are C5 platform chemicals that can be usedor bio-polyamide production. Here we report the development oforynebacterium glutamicum strains overproducing 5-aminovaleric
cid and glutaric acid by metabolic engineering of a classical-lysine producer and introducing novel synthetic pathways.he first synthetic novel pathway consists of the heterologously


ntroduced davB and davA genes from Pseudomonas putida ATCC2633 together with the endogenous gabT and gabD genes in. glutamicum. DavBA function as metabolic “gap” filling forlutaric acid production in engineered C. glutamicum since theon-engineered strain does not degrade L-lysine. This engineeredtrain cultured in a laboratory-scale bioreactor produced 24.9 g/Lf 5-aminovaleric acid and 11.9 g/L of glutaric acid in 144.5 hsing glucose as a sole carbon source. The second synthetic path-ay comprises the codon-optimized davB and davA genes from P.utida ATCC 12633 and the codon-optimized davT and davD genesrom P. putida KT2440. The engineered C. glutamicum harboring theecond synthetic pathway produced 23.7 g/L 5-aminovaleric acidnd 10.6 g/L glutaric acid in 150.5 h. These results suggest that C5latform chemicals can be efficiently produced by metabolicallyngineered C. glutamicum. This study also presents a strategy forssembling and establishing synthetic pathways in C. glutamicumor the production of chemicals, which will be useful for designingther strains for the bio-based production of chemicals.


M-06

unctional Expression and Characterization of Codonptimized Proteorhodopsin in Escherichia coli

ong-Jik Lee1,∗ , Sang Jun Lee2, Dong-Woo Lee1

Kyungpook National UnivKorea Research Institute of Bioscience and Biotechnology

Proteorhodopsin (pR) as an integral membrane light-harvestingroton pump generates proton motive force across the cellularembrane. A wide range of pRs have been studied on the eco-

ogical distribution and function, but the expression level of pRnd its physiological role in nonphotosynthetic host strains stillemain unclear. Here, we chemically synthesized the SAR 86 geneith codon optimization (co SAR86) and expressed the pR gene inscherichia coli as a fusion protein containing a C-terminal hex-histidine sequence. The recombinant enzyme was purified toomogeneity by solubilization of E. coli membrane, Ni2+ affin-

ty chromatography followed by gel filtration chromatography.omparison of thephysicochemical properties of both membrane-mbedded and purified co SAR86 in the presence and absence ofll-trans retinals established that these enzymes expressed in E. coliere integrated properly. To investigate whether the expressed

o SAR86 can enhance the cellular energy production of host cells,e compared the growth phenotypes of co SAR86 expressing E. coli

train and the wild-type strain under various conditions. Here,e report successful production and initial characterization of a

unctional co SAR86 that supports extra energy production for therowth of E. coli cells under certain growth conditions, which mayacilitate the exploitation of pR for commercial biotechnological

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M-07

eneration of Oxalic Acid Hyperproducers by Overex-ressing the Oxaloacetate Hydrolase Gene in Aspergillusiger

eiichi Kobayashi ∗ , ShotaroWatanabe, Kohtaro Kirimura

Department of Applied Chemistry, Faculty of Science and Engineering, Wasedaniversity

The filamentous fungus Aspergillus niger is worldwide usedn the industrial production of citric acid. On the other hand,nder specific cultivation conditions, A. niger accumulates oxaliccid. Oxalic acid is one of the valuable chemicals used as ahelator, detergent, or tanning agent, and industrially producedy the chemical method, but not microbial one. In this study,o generate oxalic acid hyperproducers by metabolic engineer-ng, we constructed transformants overexpressing the gene oahAncoding oxaloacetate hydrolase (OAH; EC 3.7.1.1) in citric acid-roducing A. niger WU-2223L [1] as a host. The mRNA level ofahA and specific activity of OAH in strain EOAH-1, a represen-ative oahA-overexpressing transformant, were higher than thosen WU-2223L. [2] To examine the potentiality of oxalic acid pro-uction, EOAH-1 was cultivated in OAP30 medium containing0 g/L glucose as a carbon source, (NH4)2SO4 as a nitrogen source,nd 2-[N-morpholino] ethanesulfonic acid (MES) as a bufferingubstance, and produced 28.9 g/L oxalic acid during 12 days ofultivation. Moreover, by the use of NaNO3 and K2HPO4-KH2PO4

uffer instead of (NH4)2SO4 and MES in OAP30 medium, EOAH-produced 35.8 g/L oxalic acid during 9 days of cultivation. The

ield of oxalic acid for EOAH-1 reached 79.6% of the maximumheoretical yield. Therefore, we succeeded in generating oxalic acidyperproducers by overexpressing a single gene, i.e., oahA in citriccid-producing A. niger.

eferences

].Kobayashi K, et al. Biosci Biotechnol Biochem 2013;77:1492–8.].K. Kobayashi, et al., J. Ind. Microbiol. Biotechnol., in press (2014).


M-08

rotein engineering for strain engineering

ing Zheng ∗ , Jibin Sun

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

Engineering microbial strains to overproduce chemicals repre-ents a promising topic in the field of industrial biotechnology. Theroduction performance and even the production portfolio can beell reconfigured by the advanced metabolic engineering technol-gy. Successful examples can be found in the area of antibiotics,mino acids, organic acid, biofuels, biopolymers, and recombinantlant-originated drugs. The gene expression by overexpression or
isruption is often used tools. Further fine-tuning the activity ofhe enzymes in particular by structure-based approach is very pow-rful for global optimization of the metabolic pathway but notell-recognized in the literature. Here we discuss the toolboxes
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vailable for this purpose, and use several examples to demonstratehe application of protein engineering for metabolic engineeringn amino acid overproduction.


M-09

iffusion in crowded cytoplasm-like environment

vyatoslav Kondrat ∗ , Olav Zimmermann, Eric von Lieres

Forschungszentrum Jülich

We perform Brownian dynamics simulations to study short-nd long-time diffusion of macromolecules in crowded environ-ent of biological cells. We confirm that the diffusion slows downith increasing volume fraction, and focus on its dependencen macromolecular composition of cell’s cytoplasm. The effect ofomposition on different types of diffusion, and its importance forodelling metabolic pathways will be discussed.


M-10

pplication of a controllable degron strategy foretabolic engineering

hristoph Knuf ∗ , JérômeMaury, Simo Jacobsen, Jochen Forster

Novo Nordisk Foundation Center for Biosustainability, DTU

In numerous cases of metabolic engineering, metabolite poolsave to be increased in order to obtain flux into heterologous path-ays. A simple tool for this would be the deletion of genes thatould practically lead to a block of the natural pathway, so that

he carbon can flow into the heterologous pathway. Unfortunatelyome deletions are lethal, as end products of pathways are neededor cellular growth. One example of such a pathway is the meval-nate pathway in S. cerevisiae with ergosterol as one of the mostmportant end products. A great number of bioactive compounds,ike various terpenoids, can be produced from intermediates of thisathway.

Different strategies have been applied in order to down-regulatehe expression of enzymes involved in the mevalonate pathway.ll these strategies work on the transcriptional level. This leads

o a delay of the actual regulation, as the existing enzyme willtill be active. We present a strategy for down-regulation that actsn the protein level and which can therefore be controlled in aore precise manner than the hitherto reported strategies. As a

ase study we show the action of the degron strategy for control-ing the pools of intermediates of the mevalonate pathway around,3-oxidosqualene, which is the precursor for triterpenoids. Manyriterpenoids are pharmaceutically relevant compounds whichowadays need to be extracted from plant material through an

ntricate and resource consuming process.



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M-11

fficient GABA production system development viantroduction of synthetic protein scaffold

oonho Hong ∗

University of Ulsan

Gamma-aminobutyric acid (GABA) is a precursor of one ofhe most promising heat-resistant biopolymers, Nylon-4, and cane produced by the decarboxylation of monosodium glutamateMSG). In this study, a synthetic protein scaffold was appliedo improve the GABA conversion in engineered Escherichia coli.caffolds were constructed by assembling a single protein–proteinnteraction domain SH3 to the glutamate decarboxylase (GadAnd GadB) and attaching a cognate peptide ligand to the gluta-ate/GABA antiporter (GadC) at the N-terminus, C-terminus, and

he 233rd amino acid residue. When GadA and GadC were co-verexpressed via the C-terminus scaffold, a GABA concentrationf 5.65 g/L was obtained from 10 g/L MSG, which corresponds to aABA yield of 93%. A significant increase of the GABA productiv-

ty was also observed where the GABA productivity increased 2.5old in the early culture period due to the introduction of the syn-hetic protein scaffold. The GABA pathway efficiency and GABAroductivity were enhanced by the introduction of the scaffoldetween glutamate decarboxylase and glutamate/GABA antiporter.his work was supported by a grant from the Next-GenerationioGreen 21 Program (SSAC, grant number: PJ00954904) by RDA,nd Basic Science Research Program by the MEST (2011-0022392).


M-12

elationship between amino acid properties and cat-lytic function in bacterial flavin-containing monooxy-enase using deep mutational scanning approach

amil Lee ∗ , Jongoh Shin, Byung-Kwan Cho

KAIST

Flavin-containing monooxygenases (FMOs) are the promisingnzymes that catalyze oxidation reactions of a wide array of sub-trates, including indole compounds. Thus, the engineered FMOsith broad substrate specificity, enhanced catalytic efficiency

kcat/Km), and increased thermal stability have great potential toroduce value-added bio-chemicals. However, their rational designor obtaining desired changes in the functional parameters is ham-ered by the lack of information about functions of amino acidesidues of the enzymes. In order to understand a relationshipetween the amino acid residues and function of the bacterialavin-containing monooxygenase (bFMO), we performed a satu-ated mutagenesis that all amino acid residues of the enzyme wereeplaced by other amino acids, followed by deep sequencing. Welassified amino acid residues of the enzyme into function-retained
nd function-loss subgroups, which are subsequently interpretedased on the three-dimensional structure. The functional relation-hip between amino acid residues and catalytic function was used


or the rational design of bFMP. This strategy can be applied to var-ous enzymes for the identification of the potential target sites forhe rational design, independent on the three-dimensional struc-ure of target protein.


M-13

tatistical Optimization for Simultaneous Production ofLA Degrading and Raw Starch Degrading Enzymes byhermophilic Filamentous Bacterium, Laceyella sacchariP175 Using Agricultural Crops as Substrates

ichien Kitpreechavanich ∗ , Thanasak Lomthong, Srisuda Han-

hakphoom

Kasetsart University

Optimization of medium using low cost agricultural productsy statistical mixture design for simultaneous production of PLAegrading and raw starch degrading enzymes by thermophilic fila-entous bacterium Laceyella sacchari LP175 was investigated to

ncrease biodegradation of polylactide/starch blend bioplastics.otal 5 g of different amounts of between cassava chip, soy beaneal and corncob were used as substrates in 0.035% PLA basaledium with 7 experiment runs with triplicate in shaking flask

t 50 ◦C. The highest enzymes productions were obtained fromhe mixed substrates of cassava chip and soy bean meal in ratio:1 at 24 h cultivation. Cassava starch as the additional carbonource was found to be the best for both enzymes productions.he response surface methodology with central composite designas used for enhanced both enzymes production consisted of PLAowder and cassava starch in the mixture of cassava chip and soyean meal as the basal medium. The maximum predicted activityf PLA degrading enzyme was 69.7 U/mL with 92.1 U/mL RSDEraw starch digesting enzyme) activity from the basal mediumonsisted of 0.52 g/L PLA powder and 3.34 g/L cassava starch, whilehe maximum predicted activity of RSDE was 92.9 U/mL with 69.2/mL PLA degrading activity was obtained from the basal medium

onsisted of 0.52 g/L PLA powder and 3.04 g/L cassava starch. Thealidation results of both enzymes were 68.8 and 86.1U/mL PLA

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M-14

tudy of the role of Escherichia coli central metabolismathways related genes in the synthesis of hydrogen andthanol by using glycerol as carbon source

orge Bolivar1,∗ , Antonio Valle2, Gema Cabrera3, Domingoantero3

University of CadizUniversity of Cadiz-Department of Biomedicine, Biotechnology and Publicealth-Biochemistry and Molecular Biology; Chemical Engineering and FoodechnologyUniversity of Cadiz-Department of Chemical Engineering and Food Technol-gy

Earth’s climate warming as a result of anthropogenic emis-ions of greenhouse gases, particularly carbon dioxide (CO2) fromossil fuel combustion, has provoked an urgent need to developlean and renewable energy sources. Bioenergy has emerged as anlternative source of fuel, which includes biodiesel, hydrogen andioethanol. However, the biodiesel industry currently generates auge amount of glycerol as a by-product in such a magnitude that

t has become an environmental problem. An achievable solutiono this problem is the use of waste glycerol as the main carbonource for microbial transformation to hydrogen and ethanol. Thiss an environmentally safe process that may lead to the produc-ion of renewable energy resources, which could contribute to theeduction of the CO2 emissions. Escherichia coli is a very promis-ng alternative for glycerol utilization and it has the advantage thats commonly used for metabolic engineering in many biotechno-ogical applications. In this work we study how the blockage ofome enzymes by using E. coli single knock out strains affect tohe H2 and ethanol productions as well as glycerol consumptionhen the cells grew in a glycerol-based medium. Due to the role

hat the central carbon metabolism plays in the hydrogen andthanol synthesis, several mutants of these important pathwaysave been analysed. We describe here several novel mutant back-rounds that could be useful in order to enhance the ethanol and

2 productions in E. coli.


M-15

etabolic Engineering an ATP-neutral EMP pathway in. glutamicum: adaptive point mutation in NADH dehy-rogenase restores growth

ajendar Komati Reddy1,∗ , Steffen N Lindner2, Volker FWendisch1

Chair of Genetics of Prokaryotes, Faculty of Biology and CeBiTec, University ofielefeldChair of Genetics of Prokaryotes, Faculty of Biology University of Bielefeld

Microorganisms produce ATP by substrate level phos-horylation in Embden–Meyerhof–Parnas pathway and/or bylectron transport phosphorylation. By bypassing substrate-
evel phosphorylation via phosphorylating NAD+-dependentlyceraldehyde-3-phosphate dehydrogenase(s) and phosphoglyc-ratekinase, we engineered a strain with ATP-neutral gly-
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olysis pathway in the industrially important and naturallutamate producer Corynebacterium glutamicum. To this end,

double deletion mutant devoid of the genes for phos-horylating NAD+ and NADP+-dependent glyceraldehyde-3-hosphate dehydrogenase gapA and gapB was constructed. Non-hosphorylating NADP+-dependent glyceraldehyde-3-phosphateehydrogenase from Clostridium acetobutylicum (encoded byapNCac) irreversibly oxidizes glyceraldehyde-3-phosphate (GAP)o 3-phosphoglycerate (3-PG) in an NADP+-dependent man-er without addition of Pi, thus, bypassing ATP generationia phosphoglyceratekinase. The resulting recombinant strain C.lutamicumΔgapAΔgapB (pEKEx3-gapNCac) was expected to oxi-ize glucose to pyruvate without net ATP yield whereas 2 molf NADPH are formed. However, this strain did not grow inlucose minimal medium. Upon prolonged incubation suppres-or mutants could be isolated. Further analysis of the suppressorutants by genome sequencing analysis revealed a SNP shared by

he four suppressor mutants. The SNP in the ndh gene (cg1656)ncoding the non-proton pumping NADH dehydrogenase causedn amino acid exchange. This mutation increased NADPH oxida-ion by NDH and, thus, provides a unique solution to re-oxidizeADPH generated in the engineered ATP-neutral glycolytic path-ay in C. glutamicum.


M-16

etabolic Engineering of Saccharomyces cerevisiae forsoprenoid Production

tefan Tippmann ∗ , Sakda Khoomrung, Verena Siewers, Jensielsen

Chalmers University of Technology

This project attempts to establish a yeast cell factory for theroduction of isoprenoids, which were attributed a key function

n the search for alternative transportation fuels. For this purpose,accharomyces cerevisiae was chosen as a host organism, whereashe main focus is set on sesquiterpenes such as farnesene, whichan be used as diesel alternative in its hydrogenated form far-esane. In order to enable for efficient production of farnesene,

wo central aspects are being addressed, i.e. metabolic engineer-ng for enhanced synthesis and analytical method developmentor accurate quantification of intra- and extracellular metabolitesrom two-liquid phase fermentations. In the first part, an exist-ng platform optimized for sesquiterpene production was recentlysed for the integration of farnesene synthase genes from differ-nt plant sources to enable the one-step conversion from farnesylyrophosphate to farnesene. As a result, maximal titers of ∼1 g/Lere attained in a comparative evaluation in fed-batch cultivationsith exponential feeding. Enhanced synthesis, however, will notnly involve heterologous expression of these enzymes, but it willlso include further engineering of the endogenous mevalonate
athway as well as the integration of different ‘omics’ analysis toupport the cycle of metabolic engineering.


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M-17

dentification of a new putative regulatory proteinnvolved in morphological differentiation and eryth-omycin production in Saccharopolyspora erythraea bymics approaches

rvoje Petkovic1,∗ , Vasilka Magdevska2, Benjamin Kirm2, Mihaome2, Marinka Horvat2, Katarina Karnicar2, Robert Vidmar3,ˇpela Baebler4, Polona Jamnik5, Stefan Fujs2, Jaka Horvat2, Markoonovic 3, Boris Turk3, Kristina Gruden4, Gregor Kosec2

University of Ljubljana, Biotechnical FacultyAcies Bio d.o.oInstitut Jozef StefanNacionalni Institut za BiologijoBiotechnical Faculty, University of Ljubljana

Erythromycin is a medically important antibiotic, biosynthe-ized by the actinomycete Saccharopolyspora erythraea. A significantncrease in erythromycin yields has been achieved, compared tohe wild type strain over decades of intensive strain improvement.onsidering the annual world production and commercial impor-

ance of erythromycin and its semi-synthetic derivatives, currentields remain relatively low. Therefore, there is a clear commercialncentive to further improve erythromycin production technol-gy. Genes encoding erythromycin biosynthesis are organized ingene cluster, spanning over 60 kbp of DNA. However, improving

he understanding of regulatory elements involved in erythromy-in biosynthesis in S. erythraea remains a challenging task becauseo regulatory genes are present inside the erythromycin geneluster. The difficulty of identifying key regulatory genes, crucialor improvement of erythromycin yield, is reflected by the facthat among about 7000 predicted ORFs in S. erythraea genome5.5% are putative regulatory genes. To address this issue, weave carried out a comprehensive comparative omics approach,omparing genome, transcriprome and proteome of erythromy-in high-producing ABE1441 and WT S. erythraea strains duringhe bioprocess closely resembling industrial large-scale productionrocess. Among others, we have identified a new putative regula-ory gene, profoundly overexpressed in the industrial strain, whichimultaneously influences sporulation during the life cycle of thisctinomycete and importantly, significantly affects erythromycinield of the WT and high-producing strains. Importantly, we havehown that “omics” approaches are valuable tools for identifica-
ion of industrially relevant genes/pathways.



M-18

dentification of engineering targets for improvingutrescine production by Corynebacterium glutamicum

nh Do Quynh Nguyen1,∗ , Jens Schneider2, Volker Wendisch3

Cebitec & Genetics of Prokaryotes, University of BielefeldEvonic IndustryChair of Genetics of Prokaryotes, University of Bielefeld

Corynebacterium glutamicum shows great potential for the pro-uction of the polyamide monomer putrescine. Previously, weave constructed the putrescine producing strain PUT21 by dele-

ion of argF, the gene for ornithine transcarbamoylase (OTC), andrgR, encoding the L-arginine repressor combined with heterol-gous expression of the Escherichia coli gene for the L-ornithineecarboxylase SpeC and low-level argF expression from a plasmidddiction plasmid system.

Acetylputrescine was detected as by-product in fermentationsith PUT21 (39% of putrescine formed). Mutation analysis of 18

putative) acetyltransferase genes revealed cg1722 to be responsi-le for putrescine acetylation. Subsequently, PUT21�cg1722 washown not only to produce acteylputrescine, but also 54% moreutrescine than PUT21.

To improve provision of L-glutamate as precursor, the activityf 2-oxoglutarate dehydrogenase complex (ODHC) was reducedy promoter exchange, which resulted in 27% higher putrescineroduction than PUT21. Similarly, replacing the translational startodon of proB encoding the first enzyme of L-proline biosynthesisrom ATG to TTG resulted in 58% higher putrescine productionhan the parental strain.

A transcriptome analysis revealed increased expression of theenes cgmR and cgmA during putrescine production. Overproduc-ion of the exporter CgmA in PUT21 improved putrescine yield by8% and productivity by 64%. Transcirional fusions and microar-ay analysis confirmed increased cgmA expression in the absencef the transcriptional repressor CgmR. Binding of CgmR to cgmONA was confirmed and shown to be counteracted by the putative

nducers putrescine and cadaverine. The current work focusses onombining all identified targets to improve putrescine production

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etabolic modeling

N-01

ffective Fast Filtration Method for Metabolomics Stud-es of Mammalian Cells

inoth Shanmukam1,∗ , Juan-Antonio Hernandez-Bort2,ichael Hanscho2, Christian Leitner3, Stephan Hann3, Gundaöllensperger3, Denise Sonntag4, Christine Heel5, Nicole Borth3

Austrian Centre of Industrial BiotechnologyAustrian Centre of Industrial Biotechnology, Graz, AustriaUniversity of Natural Resources and Life Sciences, Vienna, AustriaBiocrates Life Sciences AG, Innsbruck, Tirol, AustriaSandoz GmbH, Schaftenau, Austria

During the last two decades, the advent of metabolomics signif-cantly increased the investigation of cellular processes. Detection,uantification and determination of intracellular metabolitesighly rely on rapid inhibition of metabolic activity. Thus a quicknd effective quenching process is essential to corroborate in vivoonditions. To address this challenge the conventional centrifuga-ion method is replaced by a modified fast filtration protocol whichses commercially available components. This optimized protocolulfills the requirement of quenching by avoiding cell leakage andontamination with extracellular metabolites, by preserving cellembrane integrity and by rapid inhibition of enzymatic activ-

ty without metabolite degradation. An added advantage of thisethod is that the whole quenching process can be performed in

ess than 15 seconds.To understand growth behavior, viability and metabolic activ-

ty, two batch fermentations were run with protein-free suspensionultures of CHO-K1 cells grown in medium containing 8 mM glu-amine, and the same cell line adapted to growth in glutamine free

edium. Samples were quenched with 13 C labelled internal stan-ards and stored at -80 ◦C. Metabolite extractions were performedith 80% cold methanol and samples analyzed by a targetedpproach using LC/MS (HILIC and Atlantis) for the identificationf metabolites. A comparison of a wide range of metabolites of thewo fermentation setups will be presented.


N-02

dentification of metabolic characteristics of liver can-er stem cells by integrative systems analysis

ae Yong Ryu ∗ , Hyun Uk Kim, Sang Yup Lee


Liver cancer stem cells are known to be responsible for cancerecurrence, metastasis, and various types of resistances. Espe-ially, understanding mechanisms of their resistance to severalancer treatments are critical in combating cancers. To this end,
ntegrative systems analysis involving constraint-based modelingnd simulation was conducted to better understand metabolicharacteristics of liver cancer stem cells and potential cues forheir anticancer treatment resistances. Transcriptomic profiles of
METABOLICMODELING

D133-expressing liver cancer cells and exo-metabolic profilesf 60 cancer cell lines were integrated with a human genericetabolic model, Recon 2, to generate CD133+/--specificliver can-

er metabolic models; these two models were employed to simulateheir metabolic states. In particular, we paid attention to: 1)

etabolites differently consumed or secreted by each cell, 2)etabolic pathways whose overall fluxes appear to be different byore than 1.5-fold in the two cancer cells, and 3) potential roles

f microRNA on the metabolic states of liver cancer stem cells.xperimental validation of the prediction results from the inte-rative systems metabolic analysis conducted herein will furtherontribute to elucidating metabolic aspects of cancer cell resis-ances.


N-03

esign and flux modelling for recombinant productionf 3-Hydroxybutyrate in Escherichia coli

ariel Perez-Zabaleta1,∗ , Johan Jarmander1, Mónica Guevara1,orge Quillaguamán2, Gen Larsson1

KTHUMSS

Poly (3-hydroxybutyrate) (PHB) is accumulated intracellularlyy microorganisms, usually under nutrient deficient conditionsnd excess of carbon source, is known to possess plastic proper-ies, biodegradable and biocompatible. 3-hydroxybutyrate (3HB)s the monomer of PHB. It is believe that increasing the produc-ion of 3HB consequently can achieve high concentrations of PHB,ecause this monomer can be polymerized outside the cell by dif-erent methods.

H. boliviensis, a native strain of Laguna Colorada, Bolivia, isnown for produce large amounts of PHB. The PHB is synthe-ized by the successive action of b-ketoacyl-CoA thiolase (phbA),cetoacetyl-CoA reductase (phbB) and PHB polymerase (phbC). Theenes phbA and phbB of the phbCAB operon from H. bolivien-is were introduced into E. coli and the production of 3HB waschieved. The phbC gene was not cloned in E. coli because it hasnspecified thioesterases that can remove the CoA part of 3HB-oA and excrete 3HB to the medium.

Previous studies found that H. boliviensis posses 7 differentenes involved in the formation acetoacetyl-CoA (phbA) and 3enes able to produce 3HB-CoA (phbB). We want to determine theffect of the different combinations of these genes on the yield ofHB attained by the recombinant E. coli.

Wild type E. coli does not have the capacity to synthesize 3HB-oA but grows fast, at a higher temperature than H. boliviensis and

t is easy to clone because is one best understood microorganisms.ecombinant E. coli metabolism will be modeled in order to obtain


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ETABOLICMODELING

N-04

otton Breeding Research Progress in China

uwei Ye ∗

Institute of cotton research, CAAS, China

Cotton production plays a significant role to the economy ofhina, because china is one of the largest cotton producers world-
ide. In addition to these, china is also the largest consumer
ountry of cotton. Therefore, a healthy and stable developmentrend of cotton production is important to promote the efficiencyf china’s agriculture, the incomes of Chinese farmers, as well as

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he stability of china’s rural economy. Due to the reduction of cul-ivated area, damages by pests and diseases, food safety issues, theatio of grain to cotton prices, and other factors, the planting areaf cotton in china cannot be increased substantially. How to coor-inately develop the national cotton production to achieve highotton quality, and production efficiency is the major and urgentssue for the healthy development of china’s cotton industry. Inesponse to this issue, the overall situation of China’s cotton pro-uction, the cotton molecular assisted breeding progress in China,nd the development directions of China’s cotton industry will be
ntroduced.

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etagenomic applications

P-01

he impact of the compostable packaging materialoly(lactic) acid on fungal communities in compost

ehlika Karamanlioglu ∗ , Geoff Robson

The University of Manchester

The compostable biopolymer, polylactic acid (PLA), is mostlyerived from a renewable source, starch, and increasingly beingsed as an alternative to conventional plastics for short shelf-

ife products, disposable bags and packaging materials as itecomposes at elevated temperatures during composting. Abioticydrolysis in the presence of water leads to a progressive decrease

n the polymer molecular weight and ultimately the release ofactic acid. Despite the increase in the amount of PLA enteringomposting systems, few studies have examined the potentialmpact of PLA degradation on the compost microbial community.hermophilic fungi play an import role in the composting processnd in this study, the impact of PLA hydrolysis on the compostungal community was examined by incubating PLA films andLA granules in compost at different concentrations, 0-50% (w/w),y terminal restriction fragment length polymorphism (TRFLP)nd 454-pyrosequencing. When PLA was incubated at 50 ◦C, aiscernible PLA disintegration occurred and TRFLP revealed thathe fungal community profile initially changed but shifted backoward the initial compost community profile over time whenLA concentration was less than 50% (w/w) in compost. However,hen PLA was at a concentration of 50% (w/w), fungal commu-ity profile did not shift back toward to the initial profile and

he fungal diversity decreased due to a marked acidification of theompost. 454-pyrosequencing revealed that the presence of PLAnriched the thermophilic fungus, Thermomyces sp. in the compostopulation over time.


P-02

xploring biomass degrading communities for lignocel-ulolytic activities

enta Blanquet1 , Agnès Hébert1, Francoise Fayolle-Guichard1,edro Coutinho2, Bernard Henrissat2

IFP Energies nouvellesAFMB CNRS/Université Aix-Marseille UMR6098

Present schemes for bioethanol production from lignocellu-osic biomass, and especially the enzymatic hydrolysis step, areurrently still too costly. An interesting alternative is the “con-olidated bioprocessing” production scheme (CBP), which uses aingle organism to catalyse both biomass hydrolysis and fermen-ation of the liberated sugars, and which therefore represents an
mportant potential for cost reduction. The aim of the presentroject was therefore to identify new microorganisms and enzymesmployable in such a process. Compost samples were collected
cmt

METAGENOMIC APPLICATIONS

nd enriched for several months on lignocellulosic materials, suchs wheat straw, poplar and Miscanthus. The most active cul-ures, as evidenced by CO2 production, were selected for furthernalysis. 16S sequence data showed that significant shifts in theicrobial community composition occurred with time. Unexpect-

dly, important differences were noticed in submerged culturesompared to solid medium setups. Bacteriodetes and Actinobacteriarevailed in the solid medium, whereas Firmicutes and Proteobacte-ia were the dominating phyla in the submerged culture.

In parallel, metagenomic DNA was extracted from two ofhe enrichments and sequenced. Analysis of Carbohydrate activenzymes (CAZymes) revealed that members of glycoside hydro-ase (GH) families GH6, GH9 and GH48 (cellulases) as well asamilies GH27, GH29 and GH36 (xyloglucan side chain digestion)ad accumulated after enrichment. Families of CAZymes for hemi-elluloses and pectin breakdown, especially GH28, GH30, GH53,H106 and polysaccharide lyase (PL) families PL1, PL4 and PL22ere also enriched. Thus, the difference in taxonomical composi-

ion between submerged and solid medium was also mirrored inhe CAZyme family composition.


P-03

dentification and functional characterization of Est16,n esterase isolated from a metagenomic library oficrobe consortium specialized in diesel oil degradation

ariana Rangel Pereira1,∗ , Gustavo Fernando Mercaldi 1, Thaís Car-alho Maester2, Andrea Balan Fernandes3, Eliana G. de Macedoemos2

Laboratório Nacional de BiociênciasUNESPUSP

Lipolytic enzymes have been attracting global market atten-ion because they show enormous biotechnological potentialuch as production of detergents, processing of leather, produc-ion of cosmetics, perfumes and biodiesel. To search for novelipolytic enzymes, a DNA metagenomic library was constructedrom a microbe consortium isolated from oil contaminated soilt Ribeirão Preto, Brazil. After functional screenings using try-utirin, one clone referred to as Est16, was used for furthernalysis. The sequence analysis revealed that Est16 shares 87%ith lipases/esterases (ADM63076.1) from uncultured bacterium

n the database. After an amino acid sequence alignment of thest16 with 34 sequences of members of the eight lipolytic familiesemonstrated that Est16 is a new member of family V. The catalyticriad (Ser, Asp and His), is highly conserved and the serine is locatedn the conserved motif GXSMGG. The est16 gene was cloned intohe pET28a vector and expressed as a N-terminal fused His6tagrotein in Escherichia coli BL21(DE3) cells. The recombinant pro-ein was purified as active soluble form and used for activity assays.st16 showed wide range of substrates and highest catalytic effi-
iency against p-nitrophenyl valerate (C5), optimum activities atesophilic temperature ranges and the optimum pH of 9.0. In par-
icular, Est16 showed an increase in reaction rate using up to 5%


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f DMSO, suggesting tolerance to the presence of organic solvents.ere, we demonstrated that metagenomic approach can be used

s a DNA source to expand the lipolytic enzymes diversity andur results indicate that Est16 has potential for use in industrialrocess.


P-04

NASTASIA a versatile web platform for metagenomicnalysis

fthymios Ladoukakis 1 , Eleftherios Pilalis 2, Aristotelishatziioannou2, Fragiskos Kolisis 1

National Technical University of AthensNational Hellenic Research Foundation

In this work, ANASTASIA (Automated Nucleotide Aminoacidequences Translational plAtform for Systemic Interpretation andnalysis) web repository is presented. ANASTASIA enables auto-ated massive metagenomic sequence assembly, analysis and

nterpretation tasks under the same workflow. It does so by provid-ng a rich suite of computational tools integrated within numerouslgorithmic pipelines implementing and integrating versatile datarocessing tasks for (meta)genomic sequencing, assembly and pro-ein sequence datasets. The modules of these pipelines incorporatestablished bioinformatic algorithms like HMMER and BLAST asell as Python and Perl scripts, which perform annotation and

lassification tasks in synergy with stand-alone programs, whileeing integrated in an all-in-one inclusive solution exploiting thealaxy workflow engine. ANASTASIA supports the labor-free de-ovo creation and operation of workflows able to handle thenalytical challenge of large datasets (e.g. from metagenomicxperiments) and store the generated annotation results auto-atically into a MySQL database running in the background. In

ddition it promotes workflows and large datasets sharing throughts transparent shell which a user-friendly graphical interface. Theurrent configuration of the ANASTASIA installation comprises aluster of 36 CPU cores, 256 GB RAM and a fast storage machineith 32 TB capacity provided by the University of Copenhagen.NASTASIA represents the bioinformatics core of the FP7 projectotZyme which targets to the exhaustive analysis of metagenomes

n thermal springs, with the scope of tracing proteins with inter-sting enzymatic properties which will be indispensable in a wideange of biotechnological applications; from paper pulp bleaching
o production of biofuel from biomass.

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P-05

iscovery of thermostable hydrolytic enzymes of indus-rial interest by metagenomic screening

imitra Zarafeta1,∗ , Georgios Skretas2, Fragiskos N Kolisis 3

National Technical University of AthensInstitute of Biology, Medicinal Chemistry & Biotechnology, National Hellenicesearch Foundation, Athens, GreeceLaboratory of Biotechnology, School of Chemical Engineering, National Tech-ical University of Athens, Athens, Greece

Enzymes are biocatalysts used in a wide range of industrialpplications and provide a “green” alternative to chemical con-ersions. Hydrolases are a class of enzymes that exhibit highelectivity and potential synthetic ability when used in non-onventional media. These characteristics render this subcategoryf enzymes very appealing to the industry, especially for theroduction of fine chemicals and pharmaceuticals. Despite theirdvantages, a very limited amount of hydrolases is currentlyeing used in biotechnological applications, as many industriallyelevant processes require high temperatures where conven-ional biocatalysts perform poorly. For such processes, thermo-r hyperthermostable enzymes are required. Thermophilic orga-isms remain until today a largely unexplored source of suchnzymes since the vast majority of those organisms (>99%) can-ot be cultured using standard laboratory techniques. To addressome of these issues, the international consortium HotZymeas formed with the aim of applying systematic metagenomic

creening approaches in order to identify novel hydrolases fromot environments. Within the framework of this project, metage-omic libraries were constructed from samples collected fromiverse high-temperature ecosystems around the world (Russia,celand, Italy, China, New Zealand, Japan and U.S.A). Theseibraries were then analyzed using bioinformatic tools and high-hroughput functional screens in Escherichia coli cells to identifypen reading frames with desired hydrolytic activities and stabil-ty at elevated temperatures. These approaches have led to theiscovery of novel hydrolases of biotechnological interest. Theharacterization of these novel enzymes will be presented andheir potential use for the synthesis of fine chemicals will beiscussed.


P-06

unctional metagenomic and proteomic characteriza-ion of soil microbial community associated withecomposing reeds

aetano Perrotta ∗ , Linda Bianco, Fabrizio Carbone, Lorettaaddiego, Paolo Facella, Loredana Lopez

ENEA

Recent demands for the production of biofuels from lig-ocellulose biomass led to an increased interest in soilicrobial communities. Ligninolytic microbes have developedunique strategy to handle lignin degradation based on

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pletora of synergistically acting enzymes. Besides, specificiomasses can be characteristically affected by these networksf collaborative enzymes, deriving from typical soil microbialommunity.

In order to unravel the microbial environment of plant lit-er, we collected soil samples from sites characterized by theresence of different decomposing plants, Arundo donax andhragmites australis, DNA extracted from soil was used foreta-genomic analyses using Roche 454 platform. A total of

17,604 high quality reads have been collected, correspondingo more than 2000 microorganisms. Among them, a bacterial
onsortium potentially involved into biomass deconstructionas identified. A large number of bacteria belonging to this
onsortium are enriched in genes coding for ligno-celluloliticnzymes.

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METAGENOMIC APPLICATIONS

In parallel, decomposing leaves and stems of Arundo donaxnd Phragmites australis were collected and used for the isolationf cultivatable fungi. Molecular characterization were performedn the isolated fungi, leading to the identification of 7 fungalpecies. Among them, we isolated a white-rot fungus, belongingo the Polyporales order, showing outstanding metabolic activi-ies in ligno-cellulose degradation. A proteomic characterizationf this fungal secretome is currently under investigation.

The integration of the data coming from these analyses isxpected to point out a number of microorganisms, genes androteins likely involved into lignocellulose degradation pathwayshat could be used to improve biomass deconstruction in industrial
pplications.


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ANOBIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014

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anobiotechnology

Q-01

he use of chimeric virus-like particles with inserted tar-et peptides for tailored antibody production

urelija Zvirbliene ∗, Indre Kucinskaite-Kodze, Alma Gedvilaite

Vilnius University, Lithuania

Protein engineering provides an opportunity to generateew immunogens with desired features. Viral structural proteinsith their intrinsic capacity to self-assemble to highly-organizedirus-like particles (VLPs) have been shown to possess highmmunogenicity and were exploited as potential vaccines. Weave demonstrated that major capsid protein VP1 derivatives ofamster polyomavirus (HaPyV) harboring foreign sequences at cer-

ain surface-exposed regions allowed the formation of chimericLPs. The chimeric VLPs meet the requirements for a strong

mmunogen being able to activate both B cells recognizing theurface-located epitopes and T-helper cells providing the necessaryignals for Ig class switching and affinity maturation. Moreover,he immunogenicity of inserted peptides is enhanced due tohe repetitive multimeric structure of chimeric VLPs. We havexploited the VLP approach for generation of monoclonal antibod-es against short non-immunogenic peptides of human proteinss well as difficult-to-express antigens such as viral glycoproteins.he chimeric HaPyV-VP1 VLPs have been shown to induce in micetrong insert-specific B- and T-cell responses. The generated anti-odies were reactive with native full-length target proteins thusemonstrating the surface localization and proper folding of the

nserted sequences.


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Q-02

pH-responsive high density lipoprotein-like nanopar-icle of epothilone B

oo-Jong Lee1,∗, Ji-Chun Lee2, Byoung-Jae Kong2, Jonghyeokhin2, Sung-Gun Kim3, Heekyung An1, Chi-Heung Cho1, Dae-yuk Kweon2

Korea Institute of Industrial Technology, South KoreaSungkyunkwan University, South KoreaYoungdong University, South Korea

Epothilone B (EpoB) is a paclitaxel (PTX)-like microtubule-tabilizing agent; it induces mitotic arrest and apoptosis in cells.poB is a promising anti-tumor agent, and is thought to havehe potential to overcome well-known PTX resistance. However,poB is barely soluble in water, and is fatal to normal cells dueo extremely potent cytotoxicity. To reduce the unwanted cyto-oxicity of EpoB to normal cells, a reconstituted high-densityipoprotein (rHDL) of EpoB (EpoB-rHDL) was generated usingpolipoprotein A-I (apoA-I). The EpoB-rHDL, as well as PTX-rHDLa HDL-like nanoparticle of paclitaxel), was indeed mild (non-oxic) to certain cell lines such as MCF7, MDA-MB-231, andK-OV-3, while free EpoB and PTX were very toxic to these sameells. In contrast, the EpoB-rHDL and PTX-rHDL were very effec-ive in killing the Caco-2 and ZR-75-1 while free drugs were lessoxic to these cells. The susceptibility of cell lines to rHDLs wasependent on the expression of scavenger receptor class B type(SR-BI), indicating that EpoB-rHDL selectively and efficiently

ills only SR-BI-overexpressing cells. Furthermore, the EpoB-rHDLeleased EpoB only at acidic pH, which may facilitate the escape ofrugs from acidic endosome. Thus, EpoB-rHDL shown in this studynables safe and targeted delivery of the potent EpoB to cancer cells
n SR-BI-dependent manner.


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Q-03

nanovesicle-based olfactory biosensor and its applica-ion to disease diagnosis and grain quality assessment

ai Hyun Park ∗, Jong Hyun Lim, Jung Ho Ahn

Seoul National University, South Korea

We integrated the olfactory system to carbon nanotube plat-orms for biosensing applications. Human olfactory receptorOR)-containing nanovesicles were produced from human embry-nic kidney (HEK)-293 cells. The nanovesicles, which generatelfactory signals through a cAMP pathway, were integrated intoingle-walled carbon nanotubes field-effect transistors (SWNT-ETs). The nanovesicles and SWNT-FETs play roles in perceivingpecific odorants, and in amplifying cellular signals, respectively.n particular, this system can be used for the diagnosis of diseaseuch as lung cancer and also for the real-time monitoring of fungalontamination in grain. Specific olfactory receptors recognizinghe chemical biomarkers were first selected through screening aibrary of human olfactory receptors. The nanovesicle-integratedevice was able to detect a lung cancer biomarker (heptanal) and apecific compound generated from contaminated grain (1-octen-3-l) with excellent sensitivity and selectivity, similar to the originallfactory system.


Q-04

iological synthesis of silver nanoparticles using planteaf extracts and their specific antimicrobial activity

eom Soo Kim ∗, Bipinchandra Salunke, Shailesh Sawant, Bassamlkotaini

Chungbuk National University, South Korea

Several plant leaf extracts (Kalopanax, Magnolia, Persimmon,ine, Ginkgo, Platanus, etc.) were used for extracellular syn-hesis of silver nanoparticles. Stable silver nanoparticles wereormed by treating aqueous solution of AgNO3 with the plant leafxtracts as reducing agent. The synthesized silver nanoparticlesere characterized by UV-vis spectroscopy, FT-IR, inductively cou-led plasma spectrometry, energy dispersive X-ray spectroscopy,-ray photoelectron spectroscopy, high-resolution transmissionlectron microscopy, etc. Antimicrobial susceptibility tests of sil-er nanoparticle treatments revealed variability in sensitivity ofacillus cereus and Saccharophagus degradans. Minimum inhibitoryoncentration (MIC) values of the silver nanoparticles for B. cereusnd S. degradans were found to be 30 �g/mL and 10 �g/mL, respec-ively. The mixed culture of B. cereus and S. degradans treated withilver nanoparticles at 10 �g/mL after 24 h showed presence of only. cereus colonies. This study suggests that plant leaf extract syn-hesized silver nanoparticles can selectively inhibit growth of theram negative S. degradans and retain the Gram positive B. cereus

t MIC values of S. degradans.


CSF

NANOBIOTECHNOLOGY

Q-05

iosynthesis of single nanoparticles using various metalinding proteins

oojin Choi ∗, Sang Yup Lee, Doh Chang Lee

Korea Advanced Institute of Science and Technology, South Korea

Recently nanotechnology has attracted attention worldwideecause of the interesting physicochemical properties of these par-icles. However, most nanoparticles are chemically synthesizednd involve the use of expensive catalysts for reactions at highemperature and pressure. The environmental issues related tohe synthesis of nanoparticles have motivated research towardreener methods that utilize microorganisms such as bacteria,east, and fungi for their ability to reduce metal ions. We syn-hesized various single nanoparticles using metal binding proteinsn recombinant Escherichia coli (E. coli). The morphology andize of the synthesized nanoparticles was observed by low toigh resolution transmission electron microscopy (TEM) at 200 kVnd energy-dispersive X-ray (EDX) spectra. Finally, we suggest aossible mechanism of the biosynthesis process that might pro-ide a guide for conditions required for the synthesis of variousanoparticles.


Q-06

lectro-triggered, spatioselective, quantitative geneelivery into a single cell nucleus by Au nanowireanoinjector

eung Min Yoo ∗, Sang Yup Lee

KAIST, South Korea

Delivery of bioactive materials into a cell is highly impor-ant in the study of cell biology and medical treatments.deal nanoinjectors should be able to deliver biomaterials withigh spatial resolution while causing minimum cell damage.e developed a Au nanowire (NW) nanoinjector that has the

hinnest diameter among the DNA delivering devices as wells optimum mechanical properties, minimizing cell damage.ell-defined single-crystalline Au surface and high electric con-

uctivity of a Au NW nanoinjector allow precisely timed andfficient electrochemical release of DNA molecules attachedn a Au NW surface. Both linear DNA and plasmid DNAere delivered separately, and showed successful expression.he Au NW nanoinjector would find important biomedicalpplications in the fields such as gene therapy, DNA vaccina-ion, targeted drug delivery, and probe/control of cell signalingvents [1].

Acknowledgements: This work was supported by theechnology Development Program to Solve Climate Changes

1AAA001-2012M1A2A2026556) of the Ministry of Education,cience and Technology (MEST) through the National Researchoundation of Korea.


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ANOBIOTECHNOLOGY

eference

].Yoo SM, Kang M, Kang T, Kim DM, Lee SY, Kim B. Nano Lett2013;13(6):2431–5.


Q-07

ano-pattern integrated biomimetic system for woundealing assay

un Min Kim ∗, Insu Lee, Galahm Park, Tae-Joon Jeon

Inha University, South Korea

Wound healing process of damaged skin involves various stepsf cellular behavior and complex combinations of signaling path-ay. In this research, we simply fabricated nano-patterned surfacesith biocompatible PDMS (Polydimethylsiloxane) polymer and

ntegrated a patterned surface with a microfluidic system whichan mimic wound healing rad processes. To form wound damageo 3T3 fibroblast cell layer cultured on the surface, we gener-ted layered flows of cell culture media and trypsin/EDTA in aicrochannel. We monitored cell migration on the pattern dur-

ng wound healing process and found that the patterned surfaceuided the migration of cells as well as the intercellular cytoskele-on structure. The results demonstrate that cellular behavior cane controlled for wound healing by mechanical stimuli. We expecthat the developed 2D skin model can be integrated with variousypes of surface and used as a standard assay platform for woundealing research.


Q-08

ater purification through cross-linked proteolipo-omes using a preconcentrator

ae-Joon Jeon1,∗, Hyunil Ryu2, Huisoo Jang2, Insu Lee2, Gaahm Park2, Sun Min Kim2

Inha University/Biological Engineering, South KoreaInha University, South Korea

Aquaporin is the most efficient filter in nature due to its highater selectivity and permeability. However, the manufacturalifficulties of efficient and large scaled aquaporin embedded mem-rane preclude its industrial applications. It is mainly attributed tohe fragility of a lipid bilayer or other biomimetic membrane. Weave created robust membranes by cross-linking liposomes with

inkers. In addition, we concentrated liposomes effectively usingmicrofluidic preconcentrator which has nanochannels formed

y the electrical breakdown of a polydimethylsiloxane (PDMS)embrane at a high electrical bias with no nano-lithography.

roteoliposomes were continuously concentrated at the targetosition by applying an electric field through the junction of
icro- and nanochannels. Amine-terminated proteoliposomes
mbedded with aquaporin were conjugated to the surface of theDMS device via EDC/NHS reaction. As a result, the durability of



roteoliposome was increased to withstand up to ∼9 atm and waterurification was demonstrated using our device.


Q-09

comparative study of the effectiveness of �-glucosidasemmobilized on CNT-nanoparticles and Ca-alginateeads

hmad Jameel ∗, Labiba Mahmud, Faridah Yusof

International Islamic University Malaysia, Malaysia

Enzymes are extensively used in various industrial, biomedicalnd biopharmaceutical applications. However, enzymes in theirree form are unstable and expensive besides being characteris-ically susceptible to inhibition by high product concentrationsnd are highly sensitive to pH and temperature changes. Immo-ilization technology offers solutions to these challenges besidesnhancing operational stability, longevity and ease of separa-ion. �-Glucosidase has been widely employed as model enzymeor enzymatic studies. Ca-alginate beads provide a gentle envi-onment for immobilization, but have certain limitations suchs low stability, high porosity and limitations in biocompat-bility. Carbon nanotubes (CNTs) on the other hand havexcellent mechanical, thermal and electrical properties, as wells dimensional and chemical compatibility with biomoleculesike DNA and enzymes, suitable for biosensor design. Here,-glucosidase was immobilized in Ca-alginate gel and multi-alled carbon nanotubes (MWCNT) using standard techniquesnd their activity was compared with that of free enzyme. Thectivity was found highest (12.53 U/mL) for the free enzymend lowest (9.768 U/mL) for the immobilized Ca-alginate. Thectivity of immobilized MWCNT (12.20 U/mL) was close to theree enzyme activity. The enzyme reaction was found to fol-ow Michaelis–Menten kinetics. The Michaelis constants, Km and

max, determined using Langmuir linearized plots are, respectively,.09048 �mol/mL and 0.00989 �mol/mL min for immobilizeda-alginate; and 0.0985 �mol/mL and 0.01237 �mol/mL min for

mmobilized MWCNT. The corresponding values for the freenzyme are 0.0854 �mol/mL and 0.01263 �mol/mL min. Thus, theWCNT appears to be a promising support material for enzyme

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Q-10

mpacts of carbon nanotubes on biochemical reactions:nsight into interaction between carbon nanotubes andNA polymerase enzyme

bru Uysal1,∗, Meral Yuce2, Hasan Kurt1

Faculty of Engineering and Natural Sciences, Sabanci University,stanbul, TurkeyNanotechnology Research and Application Center, Sabanci Uni-ersity, Istanbul, Turkey

Recently, the Polymerase Chain Reaction technique has beguno benefit from nanotechnology. In this paper, effects of carbonanotubes in the Polymerase Chain Reaction were investigatedy Electrophoresis, Circular Dichroism Spectrometry and Dynamicight Scattering Techniques.

The unique ability to amplify low copy number DNA withininutes has made in vitro Polymerase Chain Reaction (PCR) one

f the most essential techniques in modern biology. In order toarness this technique to its full potential, certain obstacles, suchs nonspecific by-products, low yield, and complexity of GC richnd long genomic DNA amplification need to be surmounted.anomaterial-assisted PCR, so-called nanoPCR, is a new area iniotechnology that introduces nanostructured materials into PCReaction to obtain improved results.

Nanomaterials have unique physical and chemical properties,uch as high thermal conductivity, stability and high surface toolume ratios. The effects of nanomaterials in PCR depend onheir size, shape, concentration, heat conductivity, electron trans-er properties and surface modifications.

Carbon nanotubes are predicted to bind major PCR compo-ents, such as primers, template and polymerase enzyme, viapecific or non-specific interactions. In this paper, we demonstratehe interaction of carbon nanotubes with wild type DNA poly-

erase enzyme, and the effect of this interaction in PCR for therst time.

According to the results, chiral properties of the wild typeNA polymerase enzyme has changed after incubation with amine

unctionalized multiwall carbon nanotubes, which confirms directnteraction between the enzyme and tubes. Furthermore, thisnteraction has been found to be temperature dependent via
ynamic light scattering spectroscopy.

NANOBIOTECHNOLOGY

Q-11

ron-based nano-particles for Lipolase immobilizationnd stabilization

urabhi Mehra ∗, Shamsher S. Kanwar

Himachal Pradesh University, Shimla, India

In our study, a commercial Lipolase 100L (Novozymes, Banga-ore, India) was covalently immobilized on silane coated modified

agnetic nano-particles. These magnetic (Fe3O4 or �-Fe2O3) NPsere in the size range of 25–30 nm and their surface modifica-

ion was carried out by coating with Tetra Ethoxy Silane (TEOS)y sol–gel reaction and these silane-coated magnetic NPs werehen used for immobilization. Thereafter, Lipolase immobilizedano-particles [30 mg] were separated by magnetic decantationnd suspended in 6 ml of phosphate buffered saline (pH 7.4).hese Lipolase-bound NPs were kept in a homogeneous form20 �l] and were assessed [∼2 mg = 118.29 U of enzyme in ml]or enzymatic activity, stability and reusability using 3 ml reac-ion system containing p-nitrophenyl palmitate as a substraten 0.05 M Tris buffer pH 8.2. NPs-immobilized Lipolase showednhanced activity (59.2 U/mg NPs) and stability. Effect of C-hain length/substrate specificity of NPs-bound Lipolase was alsossessed and found maximum towards p-nitrophenyl palmitate139.1 U/ml] followed by p-nitrophenyl myristate [87.8 U/ml],-nitrophenyl caprylate [77.7 U/ml] and p-nitrophenyl laureate75.3 U/ml]. The temperature, pH and reusability measurementshowed that Lipolase immobilized on NPs were capable of work-ng at broader pH and higher temperature ranges and was reusable


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ATURAL AND SYNTHETIC POLYMERS

atural and synthetic polymers

R-01

mathematical model for polyhydroxybutyrate pro-uction by a wild type Bacillus megaterium using rawlycerol from biodiesel industry as sole carbon source

aalo Andrea Moreno Yanez1,∗, Débora Jung Luvizetto Faccin2,ilo Sérgio Medeiros Cardozo2, Humberto Escalante1, Marianny. Combariza1, Carolina Guzmán1

Universidad Industrial de Santander, ColombiaFederal University of Rio Grande do Sul, Brazil

Polyhydroxybutyrate (PHB) is a biodegradable, biocompat-ble and thermoplastic biopolymer, synthesized naturally asytoplasmic inclusions by various genera of Gram-positive andram-negative bacteria. PHB shares similar properties witholypropylene, and could potentially replace it, but its industrialroduction is limited by its high cost and low productivity. Weave previously reported [1] optimized growth conditions for aild type Bacillus megaterium with the ability to produce PHB from

aw glycerol byproduct of a Colombian biodiesel industry. Aimingor a better understanding of the PHB biosynthetic process withhe referred carbon source and wild type Bacillus megaterium, inhe present work we present a mathematical model that allows uso describe the kinetics of microbial growth, substrate consump-ion, product formation and also to simulate different cultivationtrategies. In this model, microbial growth and product formationere described by the Monod and the Luedeking–Piret equations,

espectively. Model implementation and parameter estimationere carried out in the EMSO process simulator. Key model param-

ters were estimated from experimental data obtained in batchultivation using a submerged bioreactor. Experimentally, underptimized bioreactor conditions, a maximum PHB concentrationf 0.96 g/L was reached at 12 h. The model provided excellenttting of the experimental data previously obtained, providingorrelation coefficients around 0.9.

eference

].Moreno P, Yanez C, Tarazona N, Cardozo NSM, Escalante H, Com-bariza MY, Guzmán C. Statistical optimization of PHB productionby a wild type Bacillus megaterium using raw glycerol as sole carbonsource. Int J Biol Macromol 2014 (submitted for publication).


R-02

cetobacter pasteurianus DSM 3509 produces cobalamin

lemens Bernhardt ∗, Xuan Zhu, Bernward Bisping

Uni Hamburg, Germany

A strain of acetic acid bacteria used in food applications wasound to have the ability to synthesize cobalamin. A prelimi-
ary genetic study of the gene of uroporphyrinogen-III synthasend a survival test indicated the ability to synthesize cobalamin.y a modified microbiological assay based on Lactobacillus del-rueckii spp. lactis DSM 20355, 4.57 ng/mL of real cobalamin and


.75 ng/mL of analogues were detected. The product extracted andsolated in its cyanide form had the similar UV spectrum as stan-ard cyanocobalamin and as cobalamin produced by Lactobacilluseuteri DSM 20016. No cobalamin was detected in the fermenta-ion broth containing 1% acetate, and less cobalamin was obtainedhen acetate started to be consumed.


R-03

ltrasonic-assisted production of active polysaccharidesrom Crassostrea hongkongensis

ingna Cai1,∗, Jianyu Pan2, Huili Sun3

No. 164, Xingangxi Rd, Haizhu District, P.O. Box 510301, ChinaSouth China Sea Institute of Oceanology, Chinese Academy ofciences, Guangzhou, ChinaChinese Academy of Sciences, Guangzhou, China

The beneficial effects of oyster extract against various disor-ers and diseases induced by oxidative stress have aroused greatoncern. Oyster polysaccharides, as immune nutrients, were con-ucted to provide nutrients for cell metabolism and prevent theide effects such as immunotoxicity and gastrointestinal toxic-ty caused by chemoradiotherapy. Ultrasonic-assisted enzymolysisas studied to increase the active polysaccharide yield and purity

rom Crassostrea hongkongensis, showing that it is more efficienthan ultrasonic extraction or enzymatic hydrolysis alone. Onhe basis of Box-Behnken design and ridge analysis, the opti-

um conditions were obtained as ultrasonic treatment time of4 min, power of 876 W, temperature of 49 ◦C and material-solventatio of 1:6 (w/v). Furthermore, polysaccharide fraction (CHP),hich was obtained by ultrasonic pretreatment and then alcalaseydrolysis at the conditions: 3000 U/g, 55 ◦C, pH 8.0 for 4 h, exhib-

ted obvious scavenging effect on DPPH and hydroxyl radical98.48 ± 0.55% and 99.20 ± 0.12%, respectively) and linoleic acideroxidation inhibition effect (85.48 ± 0.65%) at concentrationf 5.0 mg/mL. Then the CHP was separated into three fractionsy graded ethanol precipitation. The 30–60% ethanol precipita-ion fraction (C30–60%) from CHP showed the highest activities,ncluding promoting RAW 264.7 murine macrophage, tlympho-yte and IEC-6 cells proliferation (the highest cell proliferationate was 137.10% at 0.0391 mg/mL, 160.48% at 0.0781 mg/mLnd 153.70% at 0.0195 mg/mL, respectively). The activities of CHPight attribute to its uronic acid, sulfate composition and molec-

lar weight. These results reveal the potential application of CHP

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R-04

ew clues to design cell factories for tailor-madeiopolymer production: Bacillus cereus as a source ofolyhydroxyalkanoates biosynthetic proteins

arco Vastano ∗, Angela Casillo, Maria Michela Cosaro, Giovanniannia, Cinzia Pezzella

University of Naples Federico II, Italy

Polyhydroxyalkanoates (PHAs) are biopolymers, accumulateds intracellular storage-granules by a variety of bacteria. The mate-ial properties and the potential biotechnological use of PHAsre strictly dependent on their monomeric composition. In PHAsroducing Bacilli, the presence of a peculiar biosynthetic proteinluster, encoded by the operon phaRBC has been revealed [1].

In this work a functional study of the phaRBC operon from B.
ereus has been carried out through its heterologous expressionn Escherichia coli. Deletions mutants in phaR, phaB and phaCave been assayed for PHA accumulation in culture media boostedith a related (fatty acids) carbon source. PhaR function has been
h

NATURAL AND SYNTHETIC POLYMERS

valuated in its two potential active forms (LPhaR/sPhaR), whichiffer for the presence of an extra 16 aminoacids long N-terminalxtension. This N-terminal tail seems to play a crucial role in deter-ining PhaR function, promoting PhaR activity as transcriptional

egulator in its longer form (LPhaR), and switching its role to PHAynthase (PhaC) activator/stabilizer in its shorter form (sPhaR).haB role was found to be crucial in determining biopolymeromposition, influencing monomer chain length depending onts substrate specificity.

Functional information obtained from this study represents theroundwork to design new cell factories for low-cost productionf tailor-made PHAs.

eference

].Rehm B. Polyester synthases: natural catalysts for plastics. Biochem J



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NTIMICROBIALS, PATHOGENS ANDDISEASE

ntimicrobials, pathogens and disease

S-01

enomics-based discovery of macrolide glycosyltrans-erase from Bacillus sp.

on-Gon Kim ∗, Ji Yeong Park, Hyun Ju Kim

Korea Research Institute of Bioscience and Biotechnology, Southorea

Glycosylation of pharmacologically active secondary metabo-ites is an attractive method to improve their biological activitys well as pharmacokinetics. The availability of suitable glycosyl-ransferases for naturally unglycosylated compounds, however, isimited because of substrate specificity. Recently, Bacilli have beensed as a source for the isolation of GT enzymes involved in theodification of aromatic or bulky substrates. In an effort to dis-

over enzymes for the glycosylation of a macrolide compound, wedentified putative glycosyltransferases by the genomic analysisf Bacillus sp. that produces various glycosylated macrolides. Theelected glycosyltransferases were screened, leading to the iden-ification of a glycosyltransferase that could catalyze the transferf a glucose residue from UDP-�-D-glucose to the macrolide com-ound. Using NMR and MS analysis, we determined the chemicaltructures of the new products to be a new glucosylated macrolidehich exhibited improved water solubility. These data showed

hat genomics-based discovery of glycosyltransferase from Bacillusp. was an effective strategy for the isolation of a suitable glycosyl-ransferase for naturally unglycosylated compounds.


S-02

ntifungal protein of seed coat extracts of Theobromaacao L. during fermentation

ahrurrozi Fahrurrozi1,∗, Claudia Bahmann1, Nicolas Niemenak2,einhard Lieberei1, Bernward Bisping1

University of Hamburg, GermanyUniversity of Yaounde I, Cameroon

Seed coat is an important tissue for the regulation of imbibitionnd maintenance of the integrity of seed, and it is also the first seedarrier encountered by pests and pathogens. Seed cotyledons con-ain an array of proteins that may be involved in the protectionf quiescent seeds against fungi. In a previous study (Fahrurrozit al., 2013) we found that the seed coat from Theobroma cacao. seeds contains an antifungal activity. Seed coat extract cannhibit growth of fungi (e.g. Aspergillus niger, Penicillium citrinum,enicillium purpurogenum, Penicillium roquefortii) and yeasts (e.g.andida lipolytica, Candida krusei, Rhodotorula rubra, Rhodotorulaucilaginosa, Saccharomyces cerevisiae). 25 and 10 mg/mL of seed

oat extract can inhibit growth of fungi and yeasts respectively.y separation of seed coat proteins using SDS-PAGE 17 proteins
ands were found. Analysis of the bands using mass spectrometryhowed that 3 proteins have antifungal effect namely: glucanase,hitinase and osmotin (Bahmann, 2014). Further Niemenak (per-
h



onal communication) found 4 proteins that have antifungal effectamely: glucan endo-1,3-beta-glucosidase, chitinase, 2S albumintorage protein, nonspecific lipid-transfer protein. During the fer-entation process the seed coat proteins seem to be degraded,

robably caused by an increase in temperature and by activity ofroteases produced by microbes during the fermentation. To findut more details about the degradation of proteins during the fer-entation, we are currently analyzing 28 protein bands, which we

xtracted during the fermentation. Temperature characterizationhowed that the seed coat protein is not stable at 40 and 50 ◦C for4 h.


S-03

nactivation of microbial biofilms by visible light withporphyrinic photosensitizer

andra Beirão, Sara Fernandes, Joel Coelho, Adelaide Almeida,aria da Graca Neves, Maria do Amparo Faustino, João Tomé,ngela Cunha ∗

University of Aveiro, Portugal

Biofilms are aggregates of microbial cells imbedded in aatrix composed essentially by water and extracellular poly-eric substances (EPS). The matrix provides a first line of defense

gainst biological attack, environmental stress, and biocide diffu-ion, making biofilms a challenge to conventional antimicrobialpproaches.

The photodynamic inactivation (PDI) of microorganismselies on the interaction of a non-toxic photosensitizer, molec-lar oxygen and light. This study aimed the assessmentf the photodynamic effect on the matrix of model Pseu-omonas aeruginosa biofilms, using the tetra-cationic porphyrinerivative tetra-iodide 5,10,15,20-tetrakis(1-methylpyridinium-4-l)porphyrin (Tetra-Py+-Me) as photosensitizer (PS) and white light380–700 nm) at an irradiance of 40 W m−2. The photodynamicnactivation of imbedded cells, in single-species or mixed biofilmsf Staphylococcus aureus, Pseudomonas aeruginosa and Candida albi-ans was also determined.

A reduction of 81% in the polysaccharides content of matrix of. aeruginosa biofilms was observed after treatment with a light dosef 64.8 J cm−2 and 20 �M of Tetra-Py+-Me. The photosensitizationith Tetra-Py+-Me also caused inactivation of cells in all testediofilms. The maximum reduction factors were 3, 7 and 6 logs, foriofilms of P. aeruginosa, S. aureus and C. albicans, respectively. Inixed biofilms, the inactivation of S. aureus was as efficient as in

ingle-strain biofilms (7 log reduction in colony counts) but wasess efficient (5 log) for the yeast.

Photosensitization with Tetra-Py+-Me caused EPS destructionnd a significant inactivation of cells. PDI may, therefore, beegarded as a promising approach for biofilm control, even in cases


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S-04

ntibacterial activity of marine Bacillus spp. isolatedrom mangroves of Saudi Arabian Eastern Province

bdurahman Hirad1,∗, Ali Bahkali 2, Chandra Santhappa2

King Saud University, Saudi ArabiaCollege of Botany and Microbiology, King Saud University, Saudirabia

Bacteria that synthesize bioactive substances against knownuman pathogens were screened from water, sediment, andecomposing leaf litter samples collected from Tarut Island of eastoast of Saudi Arabia. Bacillus aquimaris KSAWD2 was selecteds a potential strain that showed considerable bioactivity frommongst 320 isolates. This isolate was identified based on mor-hological, biochemical and gene sequence of 16sRNA. Cultureltrate of this bacterium showed satisfactory inhibition activitygainst the ATCC bacterial reference strains and clinical pathogensbtained from Military Hospital laboratory in Riyadh. The targetedathogens included Salmonella enterica subsp. enterica serovaryphimurium (ATCC 13311), Shigella sonnei (ATCC 11060), E. coliATCC 25922) and Pseudomonas aeruginosa (ATCC 15442), Staphy-ococcus aureus subsp. aureus (ATCC 6538P, 25923, and 33591),treptococcus pneumoniae, Haemophilus influenzae, Campylobacterejuni and Streptococcus pyogens. Inhibitory activity was testedmploying cross streaking, agar well diffusion method, agar over-ay method and assay of growth inhibition in liquid medium. Thissolate showed a broad spectrum of bioactivity by recording inhi-ition of growth against more than one pathogen. It was observedhat the culture filtrate of Bacillus aquimaris KSAWD2 showedonsiderable inhibition against the tested pathogens better thanommercial antibiotic discs including chloramphenicol, gentam-cin, tetracycline, penicillin, neomycin and ampicillin. Resultsndicate a scope for deriving potential bioactive compounds fromhis marine Bacillus aquimaris KSAWD2 against well-known humanathogens.


S-05

evelopment of recombinant baculovirus for high yieldroduction of enterovirus 71 virus-like particle vaccine

u-Chen Hu ∗, Shi-Yeh Lin

National Tsing Hua University, Taiwan

Enterovirus 71 (EV71) is the major pathogen responsible forand, foot and mouth disease prevalent in East Asia and can causeerious neurological complications and even death. Thus EV71as posed a tremendous threat to children health. To develop theaccine, we previously constructed recombinant baculovirus Bac-1-3CD for the production of EV71 virus-like particle (VLP) thatonsists of EV71 capsid proteins. The VLP elicited potent humoral
nd cellular immune responses in mouse and monkey models.owever, the previous generations of baculoviruses resulted in rel-
tively low VLP yield and excessive degradation products uponroduction. In this study we constructed a new generation of

ANTIMICROBIALS, PATHOGENS ANDDISEASE

aculovirus using the flashBACÔ system to improve the quantitynd quality of EV71 VLPs. The VLP produced by this baculoviruseached a yield of about 200 mg/L (10-fold improvement) withower amounts of degradation products, putatively thanks to theelayed cell death and reduced cellular protease activity. The puri-ed VLP, which mainly consisted of VP0, VP1 and VP3 proteins,esembled the intact EV71 capsid in shape, size and composition.fter injection into Balb/c mice, the VLP elicited promising titersf binding antibody (�212) and neutralization antibody (�27)gainst EV71 in immune serum with broad spectrum to cross-eutralize the infection of different EV71 subtypes (C2, C4 and5 subtypes). In conclusion, the improvement of EV71 VLP pro-uction using the 3rd generation recombinant baculovirus moveshe EV71 vaccine development one step further towards commer-ialization and clinical applications.


S-06

uantitative analysis of protein orientation in mem-rane environments by kinase activity

hunshan Quan ∗, Wen Xiong, Wenzhong Hu, Aili Jiang,hengdi Fan

Dalian Nationalities University, China

AgrC is a membrane-embedded histidine kinase in Staphylococ-us aureus that is thought to act as a sensor for the recognitionf environmental signals and the transduction of signals intohe cytoplasm so as to regulate and control a series of relatedathogenic gene expressions. However, due to the complexity ofhe cell membrane, it turns to be difficult to study AgrC on bacte-ial cell membrane directly. Many researches try to take advantagef proteoliposome which could provide an approximate naturalembrane environment and keep the protein activity to study

he structure and function of membrane proteins. Given thatost membrane proteins have vectorial functions, both functional

tudies and applications require effective control over protein ori-ntation within a lipid bilayer. Many studies have been carriedut to determine membrane protein orientation, however, most ofhe methods are complicated and time consuming. In this study,grC orientation in liposomes was determined based on thiol-

eactive reagent labeling and kinase activity which showed thathe method based on kinase activity of AgrC could get an accurateercentage of protein orientation and only costs nearly one-sixthf the time compared with the method based on thiol-reactiveeagent labeling. The results showed that an efficient and rapidethod was established to determine the orientation of membrane


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NTIMICROBIALS, PATHOGENS ANDDISEASE

S-07

he impact of MDCK cell source on the production ofnfluenza and canine adenovirus

aulo Fernandes1, Rute Castro2, Tanja Laske3, Yvonne Genzel3,ric Kremer4, Paula Alves2,∗, Ana Coroadinha2

iBET, PortugaliBET/ITQB-UNL, PortugalMax Planck Institute for Dynamics of Complex Technical Sys-ems, GermanyInstitut de Genetique Moleculaire de Montpellier, France

MDCK cells are widely used for the production of viral-basedaccines, namely influenza virus. We have shown that MDCK arelso suitable for canine adenovirus type 2 vectors (CAV-2) manu-acturing. Apart of their suitability for virus production, there areeveral sources and subclones from the original cell line obtainedy Madin and Darby. Furthermore, given the well-documentedeterogeneity of MDCK cell-line population, understanding theifferences between MDCK cells currently available would help toetter select producer cells and develop more reproducible biopro-esses.

In this work, MDCK cell-lines from ECACC and ATCC suppliersere adapted to suspension and compared for the production ofAV-2 and Influenza.

Despite similar cell growth, ATCC cells were more easilydapted to suspension. However, ECACC cells showed to beetter CAV-2 producers, attaining productivities 2-9 fold higherhan ATCC cells. ECACC cells also showed to be more infected,ndicating that cells hold different susceptibilities to infection. Fur-hermore, the evaluation of infection progression and cell volumencrease due to virus production indicates that virus replicationinetics and cell response to infection was compromised in ATCCells. A more effective antiviral response negatively impactingAV-2 propagation in ATCC cells is currently under evaluation.imilarly to CAV-2, Influenza productivities were 2-3 fold higherith ECACC cells in adherent cultures.

To our knowledge, this is the first attempt to characterize MDCKell-lines from different suppliers with respect to their perfor-ance. Our findings show that the cell source/supplier represents a

ey issue to be considered when implementing a robust bioprocess.


S-08

esensitization of antibiotic-resistant bacteria usinglustered Regularly Interspaced Short Palindromicepeats (CRISPR)—–Cas9 system

a-Hyeong Cho ∗, Jonghyeok Shin, Myungseo Park, Younghunung, Byoung-jae Kong, Junghoon In, Dae-Hyuk Kweon

Sungkyunkwan University, South Korea

Antimicrobial drug development is increasingly lagging behindhe evolution of antibiotic resistance. As a result, there is pressingeed for new antibacterial therapies that can be readily designednd implemented. In this study, a new antibacterial therapy

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sing CRISPR-Cas9 system was developed. Clustered Regularlynterspaced Short Palindromic Repeat (CRISPR)/CRISPR-associatedCas) system are adaptive immune system that silences invadingucleic acid by using RNA-guide endonuclease activity in bacte-ia and archaea. Targeting of single guide RNA (sgRNA) with Cas9irects sequence-specific double strands DNA cleavage. Using L-rabinose induction of Cas9 and a constitutive sgRNAbla cassette, its shown that cleavage of targeted double-strands DNA resulted inegradation of plasmids containing bla gene. Ampicillin-resistantells could be re-sensitized by expressing Cas9/sgRNAbla, therebyeducing the number of resistant cells. The strategy demonstratedn this study enables the continuous use of old antibiotics, whichre already developed and the safety issue was cleared, by revertinghe resistant cells to sensitive cells.


S-09

iosurfactant produced by marine bacteria interactsith diffusible pyoverdine produced by Pseudomonaseruginosa

. Alejandro Dinamarca1,∗, Natalia Romo2, Claudia Ibacache-uiroga3

University of Valparaíso, United StatesUniversidad de Valparaíso, ChileCentro Nacional de Biotecnología CNB-CSIC/Micromarineiotech, Spain

Biosurfactants are surface-active molecules with a wide diversityf biological functions. Recently, we reported that specific surface-ctive compounds produced by marine bacteria are able to captureiffusible chemical signals from bacteria, affecting the cell-to-cellommunication system based on quorum sensing. In this context,he goal of the present work was to study the interaction between

arine surface-active molecules and pyoverdine, a diffusibleiderophore produced by Pseudomonas aeruginosa for the acquisi-ion of iron from the environment. In human hosts, pyoverdineroduction by this opportunistic pathogen promotes its viru-

ence and the cooperation with other microorganisms, affectingealth recovery in patients. In order to determine the interac-

ion between the biosurfactant and pyoverdine, P. aeruginosa wasxposed to different concentrations of the selected marine biosur-actant. At these conditions, pyoverdine presence was measuredy fluorescence spectrophotometry (460 nm). Also, the transcrip-ional levels of PvdS (transcriptional regulator of the pyoverdineynthesis), and PvdQ (periplasmic acylase) were measured by qRT-CR. Results show that when P. aeruginosa was exposed to thearine surface-active compound at the critical micellar concen-

ration, the fluorescence generated by pyoverdine was reduced in1,97%, respect to the condition without biosurfactant. On thether hand, gene expression of pvdS and pvdQ were increased 1.85-old and 1.0-fold, respectively. Since the assays were performed inon-limiting iron conditions, we propose that the marine surface-
ctive compound captures the extracellular pyoverdine, inducingr simulating an iron limiting condition in P. aeruginosa.

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S-10

nalysis of 16S rDNA sequences of endophytic bacteriassociated with Huanglong disease of citrus plants inuangning County, China

hongbi Li ∗, Feiyun Gao, Min Zhang, Yugan Hao

Center of Biopharmaceutical Engineering in Zhaoqing University

Citrus yellow shoot disease is an incurable disease of citruslants that has long been recognised. It is a serious threat to theevelopment of the local citrus industry. The reports a study ofhe Chinese citrus industry in Guangning County of Zhaoqing.ndophytes were isolated from the roots and stems. EndogenousNA was extracted and bacterial 16S rDNA was amplified by PCR.equences were analysed and compared with those isolated fromacteria on both healthy and diseased plants. The results revealedifferences between the endogenous dominant bacteria in phloemissue of healthy plants and plants with Huanglongbing disease.phingobacterium multivorum, Acinetobacter junii, and Pseudomonaspecies similar to those that infect the fish, perch, were more abun-ant in healthy plants. In contrast, Exiguobacterium acetylicum, thelant pathogen Pantoea agglomerans and Acinetobacter baumanniiere more abundant in infected plants. The discovery of bacteriassociated with found only in citrus plants with Huanglongbingisease of citrus plants should provide a theoretical and experi-ental basis for the prevention and control of citrus yellow shoot

isease.


S-11

he importance of a conserved residue of the H10 helixn �-lactamase

atma Gizem Avcı1,∗, Elif Özkırımlı Ölmez2, Berna Sarıyarkbulut3

Marmara University, Bioengineering Department, TurkeyBogazici University, TurkeyMarmara University, Turkey

The most common �-lactam resistance mechanism utilized byacteria is the production of �-lactamase enzymes that cleave the
mide bond in the �-lactam ring rendering the antibiotic inactive.-Lactam antibiotics in combination with �-lactamase inhibitorsre used to overcome bacterial resistance. Thus design of newnhibitors is a promising area.
ANTIMICROBIALS, PATHOGENS ANDDISEASE

The dynamic study of TEM-1 �-lactamase by our group hashown that binding of the �-lactamase inhibitory protein causeshanges in the flexibility of regions away from the �-lactam bind-ng site. In addition to the previously identified H10 helix, whichorms a lid over an allosteric inhibitor binding site, sequence con-ervation analysis has shown that Trp229 residue of H10 is highlyonserved and it has a stacking interaction with Pro226 and Pro251esidues.

In order to investigate the importance of Trp229, this residueas been mutated to alanine. To this end, R-TEM-1 �-lactamaseene of pUC18 was cloned into pET28a(+) with an C-terminalxHis tag. The mutation was performed using QuikChangeite-Directed Mutagenesis Kit (Agilent Technologies, USA).nzyme expression was achieved in Escherichia coli BL21 (DE3)ells at 30 ◦C with 0.1 mM IPTG. Using nickel affinity column chro-atography �-lactamase was purified and its activity was measuredith CENTA as the substrate. These measurements have shown

hat W229A mutation causes significant loss of activity, hence itould be an important residue that enables communication withhe active site.

Acknowledgements: This work was supported by TUBITAK


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LANT GENETIC ENGINEERING

lant genetic engineering

T-01

dentification and characterisation of key genesnvolved in fruit ripening of the Chilean strawberry

ichael Handford ∗, Analia Espinoza, Milagros Bracamonte,liosha Figueroa, Sara Zapata, Uri Aceituno, Lorena Norambuena

Universidad de Chile, Chile

The Chilean strawberry (Fragaria chiloensis L. (Duch.)) is aromising fruit product for Chile. The appeal of these whitish-ink fruits is associated with their higher sweetness and strongerroma compared to the red fruits of the commercial strawberryF. x ananassa). Strawberries are non-climacteric fruits and little isnown about the factors governing their ripening. Nevertheless,he ripening of these fruits is coupled with multiple changes in tex-ure, aroma, sweetness and colour, which are affected by hormonalnd environmental cues. Therefore, this work aims to generate areater knowledge of these processes in the Chilean strawberry, forncreasing our understanding of fruit ripening, for use in breed-ng programs and for maximising the commercial potential of thisroduct.

By contrasting four different developmental and ripeningtages of the Chilean strawberry, including small green, largereen, large white and ripe whitish-pink fruit (soft), six suppressionubtractive hybridization (SSH) libraries were generated. Of the809 differentially expressed cDNAs (ESTs), we identified poten-ial events which could be key in fruit development and ripening,ncluding genes which code for proteins putatively involved inhe resistance to oxidative and biotic stress, the perception andignalling of auxins and abscisic acid, vasculature developmentnd in anthocyanin synthesis. The expression patterns obtainedy quantitative real time PCR generally confirm the data obtainedrom the SSH libraries and phenotypic observations; for exampleranscripts of a potential anthocyanidin synthase gene peaked inhitish-pink fruits, consistent with pigment accumulation. Fur-

her findings will be discussed.Financial support: Anillo ACT-1110.


T-02

mprovement of Agrobacterium-mediated transforma-ion in Hi-II maize (Zea mays L.) by heat-shock treatmentith immature embryos

a Liu ∗, Jiuran Zhao

Beijing Academy of Agricultural and Forestry Science, China

Improving Agrobacterium-mediated transformation of Hi-IIaize (Zea mays L.) would be beneficial for transgenic breeding.lthough Hi-II maize is used in various maize transformation

esearch efforts, improvements in the transformation frequency
f this genotype are still needed. In the present study, themmature maize embryos were given heat shock treatmentrior to Agrobacterium tumefaciens mediated transformation. The


mmature embryos of maize were treated at different temperaturesor different time durations. Our results showed that the peak GUSxpression occurred when the embryos were subjected to a tem-erature of 42 ◦C for 3 min or at 38 ◦C for 9 min. A 9 percent (%) inhe transformation frequency was achieved in a number of experi-

ents. Heat shock pretreatment at 38 ◦C for 9 min also resulted in7% increase in the “embryo positive callus”. Besides, it was found

hat 100 mg/L Casein Hydrolysate (CH) could accelerate the redif-erentiation of somatic embryos; and 2 mg/L Multi-effect triazoleMET) promoted root elongation, lateral root formation and robusteedlings to improve the survival rate of the plantlets. Therefore,ur protocol can help advance the overall breeding of transgenicaize.


T-03

simple, novel and high efficiency transformationethod to introduce foreign DNA into corn plants medi-

ted by cell-penetrating peptides

hongyi Wu ∗, Jianhua Wei

Beijing Agro-Biotechnology Research Center, Beijing Academy ofgriculture and Forestry Sciences, China

Genetic engineering breeding and identification of novel genesn maize are very dependent on high efficiency of corn transfor-

ation. The major deficiencies in current plant transformationystems include but are not limited to the production efficiency ofhe system, transformation variability due to genotype or speciesiversity and explant limitations and a long labor-intensive pro-ess requiring much skill. In particular, there is a continuing needn the field of plant biotechnology to provide more efficient, sim-le and low cost transformation methods suitable for high capacityroduction of economically important plants, particularly eliteultivars. Cell-penetrating peptides (CPPs) were discovered to pro-ect DNA from degradation and deliver a variety of moleculesncluding DNA into cell. Here, we studied the function of CPPsTat2) in improving maize transgenic efficiency through the pollenube method. The transformation experiments in Jing 24 and 178orn inbred lines were conducted under the different conditionsf linear DNA concentration, ratio of Tat2, calcium, sucrose, ATP,nd GTP concentration. Screened with herbicide for T0 seedlings,he optimal transformation system was obtained by the media-ion of Tat2 through the pollen tube transformation method, itsransgenic efficiency was up to 1–1.5%. Screened with herbicideor T1 seedlings, genetic inheritance and segregation of T1 progenyas revealed under this genetic manipulation, supporting a genetic

atio 3:1 of T1 progeny was about 16–18% in all transgenic lines.his is a novel transgenic method for corn with high efficiency,

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T-04

mpact of non-edible transgenic plant on soil microbialommunities

ijong Lee ∗, Sung-Dug Oh, Tae-Hun Ryu, Soo-In Sohn, Jong-Bumim

NAAS, South Korea

Studies on the adverse effects of non-edible transgenic plantsn soil microbial colonies are scarce. Here, we evaluated the effectf virus-resistant trigonal cactus on nearby soil microbial commu-ities. We collected soil samples at the site of genetically modified

GM) and non-GM trigonal cactus cultivation. We collected soilamples during the vegetative growth period and the post-harvesteriod to provide the data for comparative evaluation. Ecoplateas used to evaluate the functional diversity of soil microbial

ommunities. We found no significant difference between theM and non-GM soil samples collected during the vegetativerowth period. However, the post-harvest soil sample exhibitedmarked difference in carbon substrate utilization between the

tudy group and the control group, but the observed change wasot permanent. Principal component analysis showed that simi-

ar soil sample groups were not formed based on GM or non-GMrigonal cactus cultivation, but based on the cultivation period.enaturing gradient gel electrophoresis fingerprinting revealed

hat virus-resistant trigonal cactus cultivation had negligible effectn soil microbial communities including dominant rhizosphereacteria, actinomycetes, and fungi. We found no clear evidence ofM trigonal cactus cultivation affecting the functional diversityf soil microbial communities.


T-05

evelopment of genetic resistance to plum pox virus inrunus

iming Wang

Agriculture and Agri-Food Canada, Canada

Plum pox virus (PPV) is the causal agent of the devastat-ng viral disease known as Sharka in Europe, on many stoneruit spp. To engineer genetic resistance against PPV throughhe hairpin-mediated RNA silencing (RNAi) approach, previouslywo plant transformation vectors, pAWp1 and pAWcp were con-tructed by cloning two highly conserved regions of the PPVenome corresponding to portions of viral RNA coding for P1 andP, respectively, into a Ti binary vector under the control of theouble Cauliflower mosaic virus 35S promoter as inverted repeatspanned by an intron from the peach endo-polygalacturonaseendo-PG) genomic DNA. The resulting transgenic plants express-ng hairpin RNAs either targeting the viral P1 or CP sequenceshowed resistance to PPV. Here we report construction of a new
onstruct pAWp1-cp by cloning the P1 and CP hairpin sequencesogether into the Ti vector and the inserted DNA contains ariple-intron double-hairpin sequence simultaneously targetinghe P1 and CP sequence of PPV. This vector was transformed into
PLANT GENETIC ENGINEERING

icotiana benthamiana and plum (Prunus domestica L.). Resistancessays showed the transgenic plants were efficiently resistant toPV.


T-06

nducible expression of Agrobacterium virulence geneirE2 for stringent regulation of T-DNA transfer iniosafe plant transient expression systems

rna Denkovskiene1,∗, Sarunas Paskevicius2, Stefan Werner3,natoli Giritch3, Ausra Razanskiene2

Vilnius University Institute of Biotechnology, UAB Nomads,ithuaniaUAB Nomads, LithuaniaNomad Bioscience GmbH, Germany

Agrobacterial delivery of viral expression vectors is a com-ortable solution for the transient production of biomoleculesn plants. Valuable proteins are successfully produced in plantsn contained facilities, avoiding release of transfecting agrobac-eria to the environment. However, field applications would beeeded to gain the full economical advantage in the productionf lower value and high-volume products, or transient molecu-ar reprogramming of plants to resist stresses or modulate timeo flower. The release of GM agrobacteria into open field envi-onment requires appropriate addressing of biosafety issues. Theafety of GM Agrobacterium could be increased by controlling its-DNA transfer. We constructed chemically regulated T-DNA trans-er based on inducible expression of the essential Agrobacteriumirulence gene VirE2. Looking for the strongest and stringentlyegulated promoters in Agrobacterium, we evaluated IPTG inducibleac, tac, T7/lac, T5/lac promoters in �-galactosidase assays and alsovaluated hybrid cumic acid inducible promoters lacUV5/CuO,ac/CuO, T5/CuO and VirE/CuO. For inducible T-DNA transferests, Nicotiana benthamiana plants were transfected with a VirE2-eficient A. tumefaciens strain containing transient expressionectors harboring inducible VirE2 expression cassettes in vectorackbone and a marker GFP gene in their T-DNA region. Thefficiency of T-DNA transfer was evaluated by counting GFP expres-ion foci on plant leaves. VirE2 expression regulated by tac/CuO,5/CuO and VirE/CuO promoters resulted in 50-73% of T-DNAransfer efficiency in comparison with the wild type Agrobacterium.

e present efficient and tightly regulated promoters for genexpression in A. tumefaciens and a very new approach to address


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T-07

uantitative determination of phytase activity and inor-anic phosphorus of transgenic barley and dihaploidines

omas Vlcko1,∗, Marie Hanakova2, Jana Vaskova3, Ludmilahnoutkova3

Centre of the Region Hana for Biotechnological and Agriculturalesearch, Czech RepublicDepartment of Plant Biology, Faculty of Agronomy, Mendelniversity in Brno, Czech RepublicInstitute of Experimental Botany AS CR, v. v. i. and Centre ofhe Region Hana for Biotechnological and Agricultural Research,aculty of Science, Palacky University Olomouc, Czech Republic

Barley is one of the most prominent crops in the world withhousands of square kilometers of sown fields. Harvested bar-ey grains are commonly utilized as livestock feeding and in therewing industry. Phytic acid (about 70%) is the main storageorm of phosphorus in plant seeds. Owing to the acidic naturef the phytic acid molecule it binds with cations forming annsoluble complex. This is considered the main antinutritionalactor for the availability of minerals (calcium, zinc and ironrincipally), because the salts of phytic acid are unfortunately

ndigestible for monogastric species such as swine, fish and fowl.hosphorus in feces ultimately leads to environmental pollution.hytase is an enzyme which catalyzes the degradation of phy-ate complexes thus providing necessary phosphate and cationsuring germination. This enzyme converts the indigestible formf phosphorus into a fully exploitable form. Transgenic barleyine SCLW-GP-PHYA developed using biolistic transformation ofmmature embryos exhibits stable expression of the microbialAspergillus niger] phytase enzyme. This line was used for a hydridrogramme, crossing with Czech barley cultivar Azit. The activityf phytase and content of digestible phosphorus in obtained linesere analyzed. The original transgenic line was also assessed inrewing trials.

Acknowledgement: This work was supported by project OPaVPI CZ.1.05/3.1.00/14.0327 – New biotechnological products of

EB ASCR.


T-08

enetic modification to enhance breeding for improvedostharvest quality of ornamental plants – barriers forommercial application

argrethe Serek

Leibniz University Hannover, Institute for Horticulture Productionystems, Germany

Development of novel varieties with improved postharvest
haracteristics is an important goal for the floriculture industry.mproved varieties can secure higher productivity for growers,ncreased marketing opportunities, reduced postharvest losses dur-ng storage and distribution and finally provide better products
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or consumers. While conventional breeding programs have beenuccessful in achieving such objectives, directed genetic modi-cation can provide additional ways to create new, improvedrnamental products. In the last two decades the postharvest per-ormance of a range of varieties has been successfully improved byenetic modification. In our laboratory we used the etr1-1 mutantene for improvement of display life of several ornamentals, suchs kalanchoe, campanula and orchid.

Although significant progress has been made in developingmproved new varieties using tools of plant biotechnology, most ofuch improved crops never reach consumers. While scientists areeveloping the products, they are not being released to the mar-et because of legal obstacles, including lack of interest or financialncentive of patent owners in negotiating a license. The result ishat such improved products never reach the market. Possibilitiesor ensuring that remarkable varieties do not become neglectednd forgotten somewhere in an experimental greenhouse or ultra-ow freezer must be discussed.


T-09

ubcellular localization of maize cytokinin dehydro-enases using heterologous expression in Arabidopsishaliana Ler cell suspension cultures

atricie Johnová1,∗, David Zalabák1, Ondrej Plíhal1, Olgaˇamajová2, Petr Galuszka1

Department of Molecular Biology, Palacky University inlomouc, CR Haná, Czech RepublicDepartment of Cell Biology, Palacky University in Olomouc, CRaná, Czech Republic

Cytokinins are plant hormones playing a key role during manylant developmental processes, such as shoot initiation, bud for-ation, apical dominance, delay of leaf senescence, etc. As the

ytokinins act at the nanomolar concentrations, its homeostasisust be precisely controlled on the tissue as well as cellular and

ubcellular levels. One of the ways to control the cytokinin levels its irreversible degradation. This process is mediated via a classf flavoproteins, cytokinin dehydrogenases (CKX; EC 1.5.99.12).

Plant genomes usually encode for small CKX gene families. Soar, best characterized is the AtCKX gene family from Arabidop-is thaliana, comprising seven members. Individual CKX isoformsxhibit various biochemical features e.g. substrate specificity, dis-inct spatial and temporal distribution. Interestingly, respectivetCKX’s differ in their subcellular localization too. While four oftCKX proteins are secreted to the apoplast, two are vacuolar and

he last one is cytosolic (Werner et al., 2003; Kowalska et al., 2010).In contrast to Arabidopsis as the representative dicot, the

nformation on the CKX subcellular localization in monocotss still not clear. So far, only two out of 13 maize (Zea mays)mCKX isoforms were studied in detail. It was shown that
mCKX1 is apoplastic, while ZmCKX10 is cytosolic (Smehilovát al., 2009).

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The goal of the presented research is to find out the localiza-ion of remainder ZmCKX isoforms, to complete the informationn cytokinin catabolism in maize model. For this purpose, themCKX-GFP fusions were prepared, heterologously expressed inrabidopsis thaliana Ler cell suspension cultures and studied using

ive cell confocal microscopy.


T-10

egulation of trichome density in Artemisia annua

iaofen Sun ∗, Fangyuan Zhang


Artemisia annua is the source of the most potent antimalarialrug, artemisinin, which is synthesized in multicellular glandular

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PLANT GENETIC ENGINEERING

ecretion trichomes of Artemisia leaf surface. To increase the tri-home number is one of the efficient ways to increase artemisininield. To investigate the development of multicellular glandularrichomes in A. annua, AaMYB1, a R3 MYB family gene was clonednd analyzed. Overexpression of AaMYB1 in Arabidopsis led tohe disappearance of trichome and seed mucilage, enhanced rootair number and reduced anthocyanin contents, showing thataMYB1 had a similar function with AtCPC. Overexpression ofaMYB1 in A. annua reduced the artemisinin content as well as the

richome number. On the contrary, the suppression of AaMYB1 in. annua increased the trichome number as well as the artemisininontent. This study indicates that AaMYB1 is a negative regula-or of multicellular glandular trichome development in A. annuand could be useful in engineering of A. annua for increasing
rtemisinin content.


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ECOMBINANT PROTEIN PRODUCTION

ecombinant protein production

U-01

rotein extraction by means of electroporation and bac-erial viability of E. coli

asa Haberl Meglic ∗, Tilen Marolt, Damijan Miklavcic

University of Ljubljana, Faculty of Electrical Engineering, Slovenia

Proteins extracted from recombinant bacteria have proved ofreat value in industry and medicine. Established processes toxtract proteins from bacteria often include use of mechanicalorces or chemicals, which cause complete disruption of the cellnd release of membrane contaminants (endotoxins). Thus, addi-ional purification steps are needed, which at large scales representsp to 80% of the production costs [1].

Extraction by means of electroporation is a quick, chemical freend cost efficient release of intracellular components from E. coli2]. In order to avoid cell disruption and by that the release of

embrane contaminants, and to obtain adequate quantity of pro-eins, electroporation pulse parameters need to be adjusted. Thus,e studied the influence of pulse strength, duration, number and

epetition frequency on extracted proteins from E. coli and onacterial viability.

Our results show that by increasing electric field strength, pulseepetition frequency and/or pulse duration, also concentration ofxtracted proteins increases (maximum 15.29 �g/ml), while E. coliiability decreases (minimum 2.2 – log reduction). The correla-ion was expected, since more bacteria were destroyed and moreroteins were released into surrounding media. The best choicender experimental conditions used and for our strain was a trainf eight pulses with 1 ms duration, 1 Hz pulse repetition frequencynd electric field strength of 5 kV/cm. Under these conditions theest ratio between extracted proteins and bacterial viability wasbserved.

eferences

].Assenberg R, et al. Curr Opin Struct Biol 2013;23:393.].Haberl S, et al. J Membr Biol 2013;246:861.


U-02

roduction of an antibody fragment by transient genexpression in insect cells

ideki Yamaji ∗, Tomohisa Katsuda, Hirotsugu Hamada, Keitaori

Department of Chemical Science and Engineering, Kobe Univer-ity, Japan

Monoclonal antibodies and their fragments have been usedn a variety of diagnostic and therapeutic applications. Novel
ntibody-based biologics are currently selected from a large pool ofead candidates. High-throughput production systems for rapidlyroviding a large number of recombinant antibody moleculesf high quality and in sufficient quantity are of major impor-
chr



ance. Insect cells have been shown to be an excellent platformor the production of functional recombinant antibodies [1]. Inhe present study, the production of an antibody Fab fragmenty transient gene expression in insect cells was investigated. TheDNA fragments encoding the Hc (Fd fragment) and Lc genesf a mouse anti-bovine ribonuclease A Fab fragment with therosophila BiP signal sequence were cloned separately into thelasmid vector pIHAneo [2], which contained the Bombyx moriucleopolyhedrovirus (BmNPV) IE-1 transactivator, the BmNPVR3 enhancer, and the B. mori actin promoter for high-level

xpression. After co-transfection with the resulting plasmids car-ying the Hc and Lc genes, Trichoplusia ni BTI-TN-5B1-4 (Highive) cells were incubated with a serum-free medium in staticr shake-flask cultures. Transfection conditions were optimizedith flow cytometric analysis using the green fluorescence pro-

ein. Western blot analysis and enzyme-linked immunosorbentssay (ELISA) of culture supernatants revealed that transfectedells secreted an Fab fragment with antigen-binding activity.roduction of over 30 mg/L of Fab fragment was achieved indays.

eferences

].Yamaji H, et al. Cell Eng 2011;7:53–76.].Yamaji H, et al. Biochem Eng J 2008;41:203.


U-03

MCE reference sites – A valuable tool for comparingntibody expression capabilities in CHO cells

atrick Mayrhofer ∗, Alexander Mader, Renate Kunert

University of Natural Resources and Life Sciences, Austria

Chinese hamster ovary (CHO) cells represent the major hostor recombinant monoclonal antibody production for biothera-eutic applications. Traditional methods for generation of stablentibody producing cell clones are based on random integra-ion of the gene of interest into unpredictable chromosomal loci.herefore, much effort is undertaken for tedious and time consum-

ng screening and selection procedures. Additionally, integrationvents into chromosomal loci with variable transcription ratesnd co-introduction of residual vector sequences triggers epige-etic mechanisms in various extent making prediction of expectedxpression levels and comparisons between cell lines a tediousask.

The development of site-directed gene integration methodsnables the integration of different transgenes into pre-determinedhromosomal positions.

Here we demonstrate that the application of recombinaseediated cassette exchange (RMCE) provides a valuable tool for

ntroducing a re-targetable gene cassette into a stable and tran-criptionally active chromosomal locus.

A RMCE competent cassette was introduced into DUKX-B11ells by two rounds of RMCE reactions leading to stable andomogenous gfp reporter gene expression. This RMCE cassette waseplaced by two different anti-HIV antibody variants leading to cell

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lones with reproducible expression behavior in T25 roux flasksnd spinner vessels during batch cultivation. Intracellular prod-ct formation analyzed by flow cytometry and qPCR indicated aniform antibody expression behavior of the established cell linesemonstrating the success of targeted integration by RMCE.

The established RMCE host cell line enables the comparisonf different cell clones and product specific expression patterns inuture projects based on an identical chromosomal environment.


U-04

evelopment of a method to quantify genomic rear-angements in Chinese Hamster Ovary cells

nmaculada Hernandez Lopez1,∗, Vaibhav Jadhav2, Martinaaumann1, Norbert Auer2, Angelika Zotter1, Nicole Borth3

Austrian Center of Industrial Biotechnology, AustriaUniversity of Natural Resources and Life Sciences, AustriaUniversity of Natural Resources and Life Sciences/Austrian Centerf Industrial Biotechnology, Austria

Chinese Hamster Ovary (CHO) cells are the preferred host cellsor the production of therapeutic proteins and the most commonlysed mammalian expression system due to properties such as anasy cultivation, fast growth, complex protein folding and human-ike post-translational modifications.

Nevertheless, development of recombinant CHO cell lines isslow and work intensive process that requires testing of thou-

ands of clones as these tend to be heterogeneous in behaviour.hile this is the source of easy adaption and the occurrence of

xtraordinarily high producers, it also means that frequently theost promising clones turn out to be unstable with respect to

roductivity and phenotype.Despite the high frequency of genomic rearrangements due to

enomic instability, no methods are currently available to rapidlyssess large scale chromosomal rearrangements, with the excep-ion of FISH (fluorescence in situ hybridization) and karyotyping.

The focus of the present work is to adapt a method for this pur-ose, Amplified Fragment Length Polymorphism (AFLP), whichllows simple detection and quantification of genomic changesver time using standard laboratory equipment. The approach,hich is already established in cancer research and plant breeding,as modified for use in CHO cells. It defines an initial pattern of

lectrophoretic bands that enables detection and quantification ofhanges over time and the determination of the degree of genomicifferences between CHO strains and subclones and therefore can
lso be used for cell line identification and characterization.

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U-05

stablishment of stable, high producing, recombinantHO cell lines using Rosa26 Bacterial Artificial Chromo-omes for transgene delivery

olfgang Sommeregger1,∗, Andreas Gili 2, Katalin Zboray3,hoams Sterovsky2, Emilio Casanova3, Renate Kunert1

VIBT/DBT/University of Natural Resources and Life Sciences,ienna, AustriaPolymun Scientific Immunbiologische Forschung GmbH,losterneuburg, AustriaLudwig Boltzmann Institute for Cancer Research (LBI-CR),ienna, Austria

Chinese Hamster Ovary (CHO) cells are the most frequentlysed mammalian host for the production of biopharmaceuti-als. The achieved volumetric titers using CHO cell factories havencreased significantly over the past two decades. However, thestablishment of well-producing cell lines remains tedious. Manyarameters like the host cell line, the genetic construct, the cellulture medium, the applied cultivation strategy and the producty itself are influencing volumetric titers.

Bacterial Artificial Chromosomes (BACs) harbouring the Rosa26ocus show improvements concerning transcriptional efficiencyhen used as shuttle vectors for the delivery of transgenes. Theigh transcription rate of the Rosa26 BACs enables to use thexpression system without gene amplification in auxotrophic andon-auxotrophic CHO hosts.

In this work we combined the transcriptional efficiency of theosa26 BAC with CHO host cell lines growing to high cell densities

n order to increase the volumetric product titers. The commonlysed CHO hosts CHO-K1, -DUKX-B11, -DG44, and CHO-S werecreened for their growth behaviour in batch experiments usingptimized serum-free cultivation strategies. CHO-K1 and CHO-performed best and were used for the establishment of stable

lones. The model proteins in this study were two anti–HIV-1 IgG-1ntibodies and the HIV-1 gp140 (CN54) envelope protein.

For all products we could show that the combination of cellsrowing to high cell densities and the transcriptional efficiency ofhe Rosa26 BAC system leads to the accumulation of high productoncentrations. Even for the highly glycosylated HIV-1 enveloperotein we achieved titers above 1 g/L.


U-06

odification of signal peptide for enhanced productionf recombinant erythropoietin from animal cells

uk Jae Oh1,∗, Ji Hye Park2

Sejong University, Department of Bioscience and Biotechnology,outh KoreaSejong University, South Korea

Background and novelty: Various secretory proteins areynthesized as precursors with additional N-terminal signal pep-ide. The signal peptide is cleaved off by signal peptidase once it has


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erved its purpose of targeting the protein to, and importing it into,he ER. Recently, recombinant DNA research was used to study sig-al peptide and made it possible to show the efficient activity of aroposed signal peptide by fusing it to another protein.

Experimental approach: In this study, signal peptide ofuman erythropoietin was replaced with the signal peptide ofuman IL-2, then the hydrophobic region of the signal peptideas modified and its effect on the production of the target protein

erythropoietin) was evaluated. The nucleotide sequence of mod-fied signal peptide was transported directly into the upstream ofhe 5’end of the human erythropoietin gene by performing oneound of amplification with pfu DNA polymerase. The gene of tar-et protein with modified signal peptide was transiently expressedn HEK293/CHO cells and quantification of protein secretion wasvaluated.

Results and discussion: As a result, we could observe sig-ificantly increased protein secretion up to 4 fold higher byodifying the native signal peptide, in particular the hydropho-

ic region. This observation is believed to be applicable to improvehe productivity of recombinant therapeutic proteins from animalells.


U-07

ffect of gene copy number on production yield ofecombinant ergopeptine hydrolase ErgA in Pichia pas-oris

ulia Panhölzl1,∗, Julia Lindenberger1, Patricia Menczik1, Markusleschko1, Irene Hahn2, Heidi Schwartz-Zimmermann2, Gerdchatzmayr1, Michaela Thamhesl1, Wulf-Dieter Moll1

BIOMIN Research Center, AustriaChristian Doppler Laboratory for Mycotoxin Metabolism andenter for Analytical Chemistry, Department for Agrobiotechnol-gy (IFA-Tulln), University of Natural Resources and Life Sciences,ienna, Austria

Our long term goal is to prevent ergot alkaloid poisoning inarm animals by providing enzymes for use as feed additives toegrade ergot alkaloids in the digestive tract. We isolated an ergotlkaloid degrading bacterial strain, Rhodococcus erythropolis MTHt3,rom a soil sample, cloned the genes for ergot alkaloid degradation,nd characterised the recombinant enzymes. In the present work,e attempted to optimise the production yield of the recombinant

rgopeptine hydrolase ErgA in Pichia pastoris by increasing copyumber of the heterologous gene.

We cloned plasmids with one, two, and three cassettes of ergAused to the secretion signal sequence of Saccharomyces cerevisiaelpha-mating factor under control of the GAP promoter. Plasmidsere transformed into Pichia pastorisNRRLY-11430 (CBS 7435),

ecombinant Pichia strains were cultivated in YPD medium inrlenmeyer shake flasks, and yield of secreted ErgA was measuredy SDS-PAGE analysis and enzyme activity assay based on ergo-
amine hydrolysis and HPLC.
We found that two copies of the ergopeptine hydrolase gaveigher ErgA yield than one copy, but a third copy ofergA showed



ittle or no further increase. Further strain optimisation will beequired to increase production yield of ergopeptine hydrolasergA to a level suitable for a technological enzyme productionrocess.


U-08

smolality as a determining factor in screening for higherforming clones of human hematopoietic expressionystems designed for monoclonal antibody production inhemically defined media

alina Kaseko ∗, Kim Lou, Derrick Theys, Tohsak Mahaworasilpa

The Stephen Sanig Research Institute, Australia

In cell line development for monoclonal antibody (mAb) pro-uction, the screening and selection of a highly productive andtable clone from transfectant population in a limited time frameemains a major challenge. The clone performance includingroduct quality, productivity, cell growth and cell metabolic pro-le is often dependent on cell culture conditions. With industryeing pushed towards chemically defined, non-animal compo-ents or low protein media, the selection of the best performinglone faces an additional challenge as not all antibody-producingell lines can achieve high yields in such conditions. In attempto evaluate a possibility of obtaining high performance clonesf an in-house generated host cell line derived from humanematopoietic cells, the cells were cloned in four either chemi-ally defined, low protein or animal component free media. Weound that a key factor in establishing commercially viable clonesas culture osmolality. Most cell culture media have an osmolal-

ty range 270–330 mOsm/kg. The impact of high osmolality seemso be cell line specific with reports of inhibition of cell growthnd specific productivity and an increase in specific productiv-ty balanced with reduction in cell growth to yield an increasedolumetric productivity. In our experiments, osmolality above00 mOsm/kg had deleterious effects on single cell cultures regard-ess of the types of culture media used. Reducing osmolality to50–290 mOsm/kg lead to an improved clone performance. Itemains to be determined whether these results are specific tour host cell line or have broader implications to other expres-

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U-09

evelopment of improved methanol dehydrogenasessing directed evolution and biological methanol sensorystem for the elimination of formaldehyde

ong Hyun Sung1,∗, Ji-Yeun Yi1, Seung-Goo Lee1, Sun Changim2, Jung-Hoon Sohn1

Korea Research Institute of Bioscience and Biotechnology, SouthoreaKAIST, South Korea

Formaldehyde is an important organic precursor to manyaterials and chemical compounds. Despite its widespread use,

xposure to formaldehyde is a significant consideration for humanealth due to its toxicity and volatility. Methanol dehydroge-ase (MDH) is an NAD+-dependent oxidoreductase that catalyzes

ormaldehyde to methanol reversibly. Reduction activity of MDHs noticeable in two aspects of the elimination of toxic formalde-yde and the production of methanol as an energy source. Herein,e screened improved mutants of Bacillus methanolicus MDH insehcichia coli that reduce formaldehyde to methanol effectivelyith directed evolution and biological methanol sensor system. To

xamine the resistance to formaldehyde, E. coli strains expressingach mutant were cultured in 3 mM formaldehyde, toxic concen-ration for E. coli. In the best mutant, three phenylalanine residuesere substituted to leucine, valine and serine respectively and all

he three substitutions were required to increase reduction activityor formaldehyde. Interestingly, the mutant which has F213V and289L increased the reduction relative to the wild type MDH, onhe other hand, the mutant that has F356S reduced the reduction.ased on these results, mutant MDH eliminates formaldehyde effi-iently, therefore it could help solve environmental problem suchs sick house syndrome.


U-10

ptimizing secretion efficiency of industrially relevantecombinant proteins in Pichia pastoris by means ofeast surface display and cytometric sorting

ars Toellner1,∗, Fabian Schneider1, Brigitte Gasser2, Diethardattanovich2

ACIB GmbH, AustriaBOKU Vienna/ACIB GmbH, Austria

Pichia pastoris has become a competitive host system for theroduction of industrially relevant enzymes and biopharmaceuti-al proteins. Strong constitutive and inducible promoter systemsnd efficient export signals enable production of authentic solubleroteins in the range of grams per litre. Still, similar to other hostystems, the expression and secretion of recombinant protein cane impaired drastically when the secretory capacity of the host isverloaded with the additional burden. Co-expression of proteins
hich support protein folding, ER transport or other relevant
teps in expression and secretion, has been shown to potentiallyncrease the yield of secreted recombinant protein. However,

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hese factors have to be evaluated case-by-case for any new targetrotein and without any guarantee for improvement. Here, weescribe the combination of a randomised library approach inonjunction with yeast surface display (YSD) and cytometricorting with the aim of selecting producer clones with enhancedecretory capacities. In order to link the specific secretion rate tondividual cells, the recombinant target protein is fused with aell wall anchor sequence. Thus, the avidity of the recombinantrotein on the cell surface reflects the secretory capacity of eachell clone. Using immunofluorescence staining, promising candi-ates can be sorted and enriched in consecutive rounds applyinguorescence-activated cell sorting (FACS). Finally, isolated clonesre subjected to DNA sequence analysis and the identified genes areupertransformed in host cells secreting soluble target protein inrder to achieve proof-of-principle.


U-11

urification of tag-free recombinant fumonisin esteraseumD from Pichia pastoris culture supernatant for uses calibration standard for enzyme quantification

orinna Kern ∗, Markus Aleschko, Wulf-Dieter Moll, Gerd Schatz-ayr

Biomin Holding GmbH, Austria

Fumonisin esterase FumD is used as feed additive for gastroin-estinal hydrolysis and detoxification of fumonisins, carcinogenic

ycotoxins produced by Fusarium verticillioides, which are nat-rally contained in maize from warm growing regions, in theastrointestinal tract of farm animals. The goal of our presentork was to provide a preparation of pure, stable and activeumD for use as calibration standard for enzyme quantifica-ion and characterization. Instead of purifying FumD-6x His viaffinity chromatography as before, we attempted purification ofag-free FumD from Pichia pastoris fermentation culture super-atant. We compared three different purification protocols: anionxchange (AIEX) versus cation exchange (CIEX) chromatographynd hydrophobic interaction chromatography (HIC). Analysis waserformed by SDS-PAGE, immunoblotting and/or gel filtrationGFC) on Superdex 200 resin. IEX led to higher purity of theumD fraction, and HIC showed higher yield. We tested proteintability by storing a FumD preparation in different buffers at◦C and -20 ◦C (including freeze-thaw cycles) for defined timeeriods. After quantification via ELISA and activity analysis, Trisuffer pH 8 without any additives was selected for FumD storage.he final FumD preparation was generated by two-step purifi-ation via AIEX-GFC using Tris as final buffer, and offered theame specific enzymatic activity as the starting material, suggest-ng no loss during the purification process. After quantificationia several spectrophotometrical methods (BCA-assay, Bradford,V-absorption), ELISA and Amino Acid Analysis, the FumD cal-

bration standard was available with well-defined concentrationnd specific activity.



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U-12

eveloping a versatile tool set for heterologous genexpression in Pichia pastoris

elmut Schwab1,∗, Mudassar Ahmad2, Melanie Hirz2, Markusolmbauer2, Ingund Rosales Rodriguez2

ACIB/Graz University of Technology, AustriaGraz University of Technology, Austria

Pichia pastoris has become an important microbial host forecombinant protein production. We have chosen as basis thetrain P. pastoris CBS 7435 (also NRRL Y-11430), which was orig-nally patented for single cell protein production via methanoltilization, and for which the patent protection has expired.

high quality genome sequence of this strain was recentlyetermined [1]. On the one hand proper integration vectorsased on different promoters and selection markers have beenewly designed and constructed. For engineering P. pastoristrains, an efficient vector construct was developed for marker-ree genome knock-out or integration of gene. Based thereon, aet of amino acid auxotrophic and protease deficient host strainsas constructed. In addition, an AOX1 promoter based methanol

nducible host system was developed allowing for efficient induc-ion with low amounts of methanol. Moreover, a methanol-freelternative has been developed for induction of the AOX1romoter.

eference

].Küberl A, Schneider J, Thallinger GG, Anderl I, Wibberg D, HajekT, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, Pühler A,Schwab H, Glieder A, Pichler H. High-quality genome sequence ofPichia pastoris CBS7435. J Biotechnol 2011;154(4):312–20.


U-13

emperature effect on recombinant protein productionn continuous cultures of methylotrophic yeast Pichiaastoris: a comparative study of sorbitol and glycerol asco-substrate

ulio Berrios ∗, Maria-Olga Flores, Alvaro Diaz-Barrera

Pontificia Universidad Catolica de Valparaiso, Chile

Pichia pastoris is a methylotrophic yeast that is widely useds an expression system of heterologous proteins. The use of co-ubstrates (e.g. glycerol, sorbitol) together with methanol, andulture temperatures below 30 ◦C are among the proposed strate-ies to improve recombinant protein productivity. Since thesepproaches normally affect the specific cell growth rate (μ), and its well known that μ also affect the protein productivity, we haveerformed an experimental design based on continuous culturesperating at a steady-state, thus fixing μ by a constant dilution rateD = μ = 0.05 h−1). Two sets of continuous cultures were run using
ither glycerol or sorbitol as co-substrates, exploring at 22 and0 ◦C, together with co-substrates and methanol mixtures with 30nd 60% of co-substrate in the mix. The recombinant protein Rhi-opus oryzae lipase (ROL) was used as model. Our results indicate


hat sorbitol as co-substrate leads to a higher specific productivityf ROL (qROL) whereas glycerol allowed higher volumetric produc-ivity of ROL (QROL). Besides, there is a synergic effect of glyceroleeding and lowering the temperature on QROL. Controversially, weave found that decreasing the cultivation temperature from 30 to2 ◦C at constant μ did not result in a qROL increase, suggesting thathe increase of specific productivity by decreasing the cultivationemperature previously reported elsewhere is actually a result of aecrease in μ rather than temperature itself. This may open a newpproach to optimise production of heterologous proteins basedn decreasing the cultivation temperature.


U-14

ydrophobins class I versus class II: Trichoderma virensFB9a and HFB9b (class I) are more effective as enhanc-

ng agents in enzymatic PET hydrolysis and surfaceodulators compared to HFB4 and HFB7 (class II)

gnieszka Przylucka1,∗, Doris Ribitsch1, Enrique Herrero-Acero1,eorg Gübitz1, Christian P. Kubicek1, Irina Druzhinina2

ACIB GmbH, AustriaTechnical University of Vienna, Austria

Poly(ethylene terephthalate) (PET) is a thermoplastic polyesterith excellent tensile and impact strength, transparency, andppropriate thermal stability. Because of its high production36 mio tons per year), PET constitutes a significant waste mate-ial, which – although not representing a direct hazard to thenvironment - is not readily decomposed in nature. Recently,icrobial cutinases were shown to be capable of partial or com-

lete hydrolysis of the synthetic polymer PET, therefore offeringew possibilities in the processing and recycling of this highly

nert material. We have demonstrated that the addition of class IIydrophobins of Trichoderma can enhance the rate of PET hydrol-sis by cutinases.

It is currently unknown whether this observed effect isproperty of some specific hydrophobins, and whether the

ydrophobins need to bind to the cutinases to enhanceheir action. In this work we therefore compared the class IIydrophobins (HFB4 and HFB7) to the Trichoderma-unique non-lass II hydrophobins (HFB9a and HFB9b) with respect to theirctivity as enhancers of PET hydrolysis by cutinase I (CutI) fromhermobifida cellulosilytica and surface modulators. Time scan anal-sis over 72 h revealed a hydrophobin-specific interaction withutinases. Whereas the non-class II hydrophobins HFB9a andFB9b where able to increase the rate of PET degradation by CutI,

lass II hydrophobins were less active: HFB4 showed an increasedctivity on the hydrolysis of PET at lower time points, an inhibition

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U-15

trategy for on-column refolding of human recom-inant His-tagged prethrombin-2 produced using thescherichia coli-based expression system

ichaela Osadska ∗, Martin Safranek, Hana Bonková, Jánrahulec, Stanislav Stuchlík, Ján Turna

Department of Molecular Biology, Faculty of Natural Sciences,omenius University in Bratislava, Slovakia

One way to obtain an active human thrombin for pharmaceuti-al purposes is to prepare recombinant human thrombin with these of an Escherichia coli expression system. Prethrombin-2, themallest single chain thrombin precursor, modified with His-tagt N-termini forms inactive intracellular inclusion bodies duringhe expression. Therefore the refolding process to obtain an activeonformation is necessary. Here we report two on-column refold-ng methods of His-tagged prethrombin-2. Immobilized metal ionffinity chromatography and hydroxyapatite chromatography forne-step protein purification and on-column refolding were used.olubilization of aggregated structures was carried out with vari-us buffers composition under the denaturing conditions, whereigher yield of solubilized prehrombin-2 was obtained in theresence of 6.0 M guanidine hydrochloride compared to 8.0 Mrea. After the solubilization step gradual refolding of the proteinas performed on-column using a linear guanidine gradient from.0 M to 0.0 M in the presence of 50 mM Tris–HCl and 0.5 M NaCln the case of IMAC and 1 mM KH2PO4 if hydroxyapatite chro-

atography was used. There was one protein peak in the A280rofile during the refolding process, which means that protein waseleased during reduction of chaotropic reagent concentration.inal yield of refolded protein was therefore in minimal concen-ration and on-column refolding requires next optimization.

Acknowledgements: This work is result of project imple-entation: “Production of biologically active agents based on

ecombinant proteins” (ITMS 26240220048) supported by theesearch and Development Operational Program funded by theRDF.


U-16

reparation of human growth hormone in Pichia pas-oris under a constitutive promoter

iana Hopkova ∗, Kristina Jirickova, Zdenko Levarski, Janrahulec, Stanislav Stuchlik, Jan Turna

Department of Molecular Biology, Faculty of Natural Sciences,omenius University, Slovakia

The human growth hormone (hGH) is the polypeptide usedn modern medicine for a treatment of different diseases suchs growth deficiency in children. Moreover, the marketed phar-
aceuticals based on hGH got the approval for the treatment of
urner’s syndrome or cachexia associated with AIDS.Pichia pastoris was chosen as the host organism because of its

bility to grow to high cell densities and its strong promoters capa-


le of the production of high yields of a recombinant protein. Theystem combines both advantages of prokaryotes – high expres-ion, simple scale-up, cheap production media–, and advantagesf eukaryotes – ability to perform various post-translation modifi-ations.

The hGH gene was integrated into genome of P. pastoris underhe control of a constitutive promoter, in order to produce nativeGH into the extracellular media. Then, the genome integrationas validated through a PCR and expression in flasks. The SDS-AGE results were confronted with a Western blot analyses. hGHas then successfully produced in 1L fermenter and partially puri-ed using anion-exchange chromatography.

Acknowledgements: This publication is the result of theroject implementation: “Production of biologically active agentsased on recombinant proteins” (ITMS 26240220048) supportedy the Research and Development Operational Program funded byhe ERDF.


U-17

solation of novel lignin degrading enzymes and ligninegradation products from bacteria and fungi

atai Bello ∗, Takanori Furukawa, Louise Horsfall


Lignocellulosic biomass offers an alternative to non-sustainableossil fuels for the production of useful chemical substances and isxpected to become one of the key renewable energy resourcesn the near future. Lignin makes up as much as 30% of ligno-ellulosic biomass and is considered one of the most importantimiting factors in the yield of bioethanol obtained from biomassermentation. Currently, industries regard lignin as a nuisance inhe utilization of cellulose and hemicellulose, which are the targettarting materials for these industries. However, lignin is a poten-ial source of a variety of aromatic chemicals, if a system for itsontrolled degradation can be developed. Several species of fungind bacteria possess lignin-degrading ability through the produc-ion of different types of ligninolytic enzymes. This project aims todentify ligninolytic enzyme gene sequences from fungi and bac-eria. The genes will then be cloned and expressed in bacterial andungal hosts. The optimum expression conditions will be deter-

ined and the activity of the resulting enzymes on lignin andignin model compounds will be determined. Structural analysisy X-ray crystallography will be performed on the best enzymesrom the activity studies. The lignin degradation products will benalysed and methods for quantitative estimation are in develop-ent. The enzymes discovered from this study are intended to


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U-18

usion protein and solubility enhancing strategies foreterologous expression of novel plant sesquiterpeneynthases

teffen Hartwig ∗, Thore Frister, Thomas Scheper, Sascha Beutel

Leibniz University of Hannover, Germany

Terpenes constitute the largest class of flavor and fragranceolecules in nature. Important representatives of this class are

sed widely in various foods, beverages, exclusive perfumes andersonal care products. Biocatalytic production of terpenes is aromising method, but requires the availability of large amountsf soluble and active terpene synthases. Plant terpene synthasesTPS) exhibit large variations in amino acid sequence and proteinold. The soluble yield when expressed in E. coli varies substantiallyrom enzyme to enzyme. In most cases, TPSs are considered hard toxpress in a heterologous host organism. We chose cDNAs of syn-hases, partially synthesized as well as extracted from native plants,hich all produce chemically interesting and industrial relevant

erpene products. We identified a cDNA variant of patchoulol syn-hase from Pogostemon cablin [1,2]. The active expression levelf the enzyme variant was increased substantially by fusion to ahioredoxin moiety [3].

A previously not characterized zizaene synthase from Vetiveriaizanoides was assembled from artificial DNA strings. Because nooluble expression could be detected by conventional strategies,nduction using a cold shock promoter as well as fusion to theighly soluble SUMO protein moiety were evaluated.

The presented results enable other researchers in the field toecide what expression strategy might be most suitable for newPSs, which have not been expressed in E. coli.

eferences

].Croteau, et al. Arch Biochem Biophys 1987;256:56–68.].Deguerry, et al. Arch Biochem Biophys 2006;454:123–36.].Hartwig, et al. Prot Expr Purif 2014;97:61–71.


U-19

xpression of soluble recombinant human growth hor-one in Escherichia coli cultures using shake-flask, batch

nd fed-batch cultivation

an Turna ∗, Zdenko Levarski, Jan Krahulec, Kristina Jirickova,iana Hopkova, Stanislav Stuchlik

Comenius University in Bratislava, Department Mol. Biol.,lovakia

Human growth hormone (hGH) was one of the first recom-inant proteins approved for the treatment of human growthisorders. Its small size (191 amino acids), possession of only 2
isulphide bonds and absence of posttranslational modificationsake Escherichia coli the host of choice for its production on any
cale. We have developed an efficient E. coli based expression sys-em for the production of high levels of soluble hGH fused to



hioredoxin (Trx). In this work, we compare the expression level,ortion of soluble product, efficiency and yield of Trx-hGH fusionrotein using three cultivation methods – shake-flask, batch anded-batch bioreactor runs. While more than 95% of the producedrx-hGH was in soluble form when cultivated in shake-flask orioreactor in batch mode, this number decreased to 47% whenroduced in fed-batch mode. We have been able to purify 1.045 gf soluble Trx-hGH from the liter of fed-batch culture and 0.8 grom the batch culture. These numbers represent a significant yieldhen compared to other published data and are of potential inter-

st to the biotech industry.Acknowledgements: This work is the result of the project

mplementation: “Production of biologically active agents basedn recombinant proteins” (ITMS 26240220048) supported by theesearch and Development Operational Program funded by theRDF.


U-20

ultiple chromosomal gene integration for productionf pharmaceutical proteins in S. cerevisiae

alene Jensen1,∗, Uffe Mortensen1, Nina Gunnarsson2, Tomastrucko1, Line Due Baron2

Technical University of Denmark, DenmarkNovo Nordisk, Denmark

When studying protein folding and secretion the general con-eption is that all cells in a population express an equal amount ofrotein. Recent work has shown that expression levels vary greatly

n cell populations which express proteins on plasmids. Hence aeast expression platform has been developed at the Departmentf Systems Biology, DTU. The platform offers the opportunity toxpress genes on the chromosome in 1 to 10 copies. A comparisonetween the expression of CFP and RFP by the platform and bylasmids reveals the problems of plasmid expression. FACS analy-es of two cell populations, expressing CFP and RFP on the separatelasmids or expressing CFP and RFP using the yeast expressionlatform shows expression varies greatly in a cell population basedn plasmid expression compared to the yeast expression plat-orm. When expressed on plasmids a few cells are high performersn both proteins but the largest fraction of cells is actually notxpressing either of the proteins. The yeast expression platform iseveloped to facilitate stable expression of integrated genes. The

ntegration sites are separated by essential genes which ensure thathe integrated genes are not lost by recombination. An amplifica-ion method has been developed for the platform which enablesast integration of genes. Future perspectives involve exploring theapabilities of the platform for recombinant protein production

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U-21

iosynthesis, purification and biointeraction of humanCOMT with Parkinson’s disease inhibitors

uis Passarinha ∗, Filipa Correia, Augusto Pedro, Diana Oliveira,oão Queiroz

CICS-UBI: Health Sciences Research Centre – Universidade da Beiranterior, Portugal

Catechol-O-methyltransferase (COMT) is an important targetn protein engineering due to its role in human neurological dis-rders such as Parkinson’s and Alzheimer’s disease [1]. Therefore,s relevant to study new COMT inhibitors since it will lead to anmprovement in Parkinson’s therapy. So, it is necessary a biosyn-hesis process to produce and stabilize the human SCOMT andubsequently, a suitable purification strategy to obtain pure frac-ions for protein-inhibitors interaction studies. The biosynthesisrocess was performed based on pPICZ A and Pichia pastoris ashost cell where hSCOMT was synthesized with a hexahisti-

ine tag. Moderate to high expression values were obtained forefined media at 24 h, 30 ◦C and 250 rpm. Since COMT losesapidly its activity during isolation and storage [2], stabilizationssays were performed in order to improve the main isolationtep by Immobilized-Metal Affinity Chromatography (IMAC) andurther storage. Through a neuronal network we studied the pro-ein stability in the presence of potential compounds, varyinghe time and temperature at which the protein is stored. Theest input was selected and applied. Then, to obtain a purifiedraction for inhibition trials, IMAC was applied and highly puri-ed hSCOMT fractions were obtained. Finally, biophysical studiesere performed by microcalorimetry in the presence of specific

nhibitors in order to assess kinetic profiles for human SCOMT.Acknowledgements: A.Q. Pedro acknowledges a doctoral

ellowship (SFRH/BD/81222/2011) from Fundacão para a Ciên-ia eTecnologia. This work was partially funded by FEDERunds through Programa Operacional Factores de Competitividade

COMPETE:FCOMP-01-0124-FEDER-027563 with the projectXPL/BBB478/BQB/0960/2012.


U-22

irected evolution to remove expression and secre-ion bottlenecks of heterologous enterokinase inscherichia coli

eiluo Lee ∗, Paul Dalby

University College London, Biochemical Engineering, Unitedingdom

Bovine enterokinase catalytic subunit (EKcat) has significantndustrial potential for selective modification of therapeuticroteins. The enzyme is typically expressed heterologously in
ukaryotic cell strains such as Pichia pastoris to ensure properlycosylation and folding into its soluble active form. EKcat produc-ion in E. coli offers several advantages (i.e. shorter fermentationimes, undemanding growth conditions, and favourable pro-
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essing economics) but is challenging due to the formation ofnclusion bodies, leading to inactive enzymes. Engineering EKcat

or periplasmic secretion generated a basal level of soluble andctive enzyme. Directed evolution was then applied to screen forariants with improved total activities attained during expres-ion. The generated library of variants contained mutations acrosshe following gene sequences: EKcat, secretion signal peptide, pro-

oter, lac operator, and ribosomal-binding site. The solubility,tability, and activity of the resultant EKcat variants were examinednd hypotheses given based on sequence, structural, and bio-hysical findings. Furthermore, variants were also shown to adaptifferently when the fermentation temperature was raised, provid-

ng another dimension for investigating the complex dynamicsetween expression and secretion. In addition to improving EKcat

xpression and activity in E. coli, the study presents insights intongineering proteins for high functional expression and periplas-ic secretion.


U-23

iRNA-mediated engineering of DNA recombination formproved transgene integration and expression

aja Kostyrko ∗, Nicolas Mermod

University of Lausanne, Switzerland

Achieving high levels of stable heterologous gene expressionn eukaryotic cells is limited in part by the efficiency of trans-ene integration into the genome. This likely relies on one ofhe cellular DNA repair pathways – non-homologous end-joiningNHEJ), homologous recombination (HR), or on less well charac-erized microhomology-mediated end-joining (MMEJ) pathways.owever, the exact molecular basis of this process is still not fullynderstood.

In the effort to identify the transgene genomic integra-ion mechanism we silenced important DNA recombinationepair components in Chinese hamster ovary (CHO) cells usingmall interfering RNA (siRNA), and analyzed the integrationnd expression of transgene-containing plasmid DNA in theseells. In addition, we used vectors bearing matrix attachmentegions (MARs), genetic elements commonly used to increase andtabilize recombinant gene expression, which were recently pro-osed to also play a role in DNA recombination.

We find that knock-down of the NHEJ pathway componentsoes not affect transgene integration or expression, while silencinghe HR pathway increases both. We also show that the use of a MARlement stimulates increased transgene integration and expres-ion. However, in the absence of the HR pathway, MAR inducesn even higher expression, without further increasing integration.nstead, we observe an increased expression per individual geneopy. Therefore, we propose that HR knock-down, as well as theAR element, stimulate an alternative repair pathway, most likelyMEJ, which has a beneficial influence on transgene integration

nd resulting expression. This knowledge should help engineerells for improved recombinant protein production.



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U-24

AR elements and transposons for improved transgenentegration and expression

éborah Ley1,∗, Valérie Le Fourn2, Pierre-Alain Girod2, Alexandreegamey2, Nicolas Mermod1

Institute of Biotechnology, University of Lausanne, SwitzerlandSelexis SA, Geneva, Switzerland

A critical step for protein production in biotechnology orxpression of a therapeutic gene in gene therapy is the abilityo achieve consistent gene expression into the target cells. Thismplies transgene integration in a locus which allows its efficientnd sustained expression. Among the wide variety of non-viral vec-ors used to deliver DNA, transposon systems have the advantageo provide increased frequencies of single-copy integration intohe host genome. However, as with other methods of transgeneelivery, the use of transposon-based systems are not immune tohe risks of silencing and insertional mutagenesis. These unwantedvents may be limited by the use of genetic elements, called matrixttachment region (MAR). Transgenes associated with MARs haveeen shown to have a higher and more stable expression and areess prone to silencing. Here, we assessed the effect a piggyBac (PB)ransposon containing the MAR-1-68 in CHO cells. Using this com-ination, it was possible to obtain an enriched transgene-positiveopulation harbouring few integrations sufficient for high trans-ene expression, without the need of selection pressure. As a prooff feasibility for biotechnology applications, we demonstrated thebility of the system to obtain significant amounts of therapeuticsroteins from unselected cell populations 2–3 weeks after trans-ection. Since antibiotic-enforced selection protocols often resultn a higher integrated copy number and mosaic expression pat-erns, this strategy could benefit many applications in which lowntegrated copy number and antibiotic-free conditions are desired.


U-25

xploration and characterization of novel lignin degrad-ng enzymes for a synthetic biology platform to aidnzymatic lignin disruption

akanori Furukawa1,∗, Aaron McKerracher2, Franck Escalettes2,euben Carr2, Loise Horsfall 1

The University of Edinburgh, United KingdomIngenza Ltd, United Kingdom

Lignin is the most abundant aromatic polymer on eartherived from photosynthesis and considered to be potentialeedstock for the production of renewable aromatic chemicals.owever, due to its structural nature, it exhibits high resistance

owards chemical and biological degradation, and this is a majorbstacle for achieving efficient conversion of lignin into value-
dded products. Many microorganisms including both fungind bacteria have been reported to produce a wide variety ofxidative enzymes that are involved in the degradation of lignin.hese include lignin peroxidases, manganese peroxidases, versatile
epo



eroxidases and laccases, which act cooperatively to degrade ligninacromolecules.In this research project, we aim to develop a synthetic biolog-

cal platform to optimize the degradation process and productsf lignin disruption. To this end, several nucleotide sequencesncoding a putative lignin-degrading enzyme were identified fromeneBank at the National Center for Biotechnology Informa-

ion (NCBI) based on its putative domain structure and sequenceimilarity to reported lignolytic enzymes. Codon usage of the iden-ified genes was optimized for Pichia pastoris and Saccharomyceserevisiae to enable high-level protein expression, and heterolo-ous production of the optimized genes was investigated in thewo different yeast expression platforms. Furthermore, enzymaticroperties of the expressed proteins were characterized in ordero select lignolytic enzymes with the desired properties for theuture development of synthetic microbes for controlled ligninegradation.


U-26

etabolic model-based prediction of engineering targetsor increased production of heterologous proteins

ustyna Nocon1,∗, Matthias G. Steiger2, Martin Pfeffer3,eung Bum Sohn4, Tae Yong Kim4, Hannes Rußmayer5, Ste-an Pflügl3, Christina Haberhauer-Troyer6, Karin Ortmayr6,unda Köllensperger6, Brigitte Gasser2, Sang Yup Lee7, Diethardattanovich5

University of Natural Resources and Life Sciences, AustriaAustrian Centre of Industrial Biotechnology, Vienna, AustriaDepartment of Biotechnology, University of Natural Resourcesnd Life Sciences Vienna, AustriaBioinformatics Research Center, Korea Advanced Institute of Sci-nce and Technology (KAIST), Daejeon, South KoreaCentre of Industrial Biotechnology, Vienna, AustriaDepartment of Chemistry, University of Natural Resources andife Sciences Vienna, AustriaKorea Advanced Institute of Science and Technology (KAIST),aejeon, South Korea

The production of recombinant proteins can be enhancedy influencing transcription, optimizing codon usage, increasingrotein folding and secretion. Overproduction of heterologousroteins, however, also directly affects the primary metabolismf the host organism. The incorporation of recombinant proteinroduction into the genome scale metabolic model of the yeastichia pastoris, allowed the simulation of the effects of overpro-uction. Gene targets for deletion or overexpression for enhancedroductivity were predicted. Overexpression targets were local-

zed in the pentose phosphate pathway and the TCA cycle, whilenockout targets were found in several branch points of glycoly-is. Five out of 9 tested targets led to an enhanced production ofytosolic human copper/zinc superoxide dismutase (hSOD). Ben-
ficial mutations were mainly related to reduction of the NADP/Hool and the deletion of fermentative pathways. Overexpressionf the hSOD gene itself had a strong impact on intracellular fluxes,

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hich were predicted with high accuracy by the model. Genomecale metabolic modeling is shown to predict overexpression andeletion mutants which enhance recombinant protein productionith high accuracy.


U-27

he production of recombinant cationic a-helicalntimicrobial peptides in plant cells induces the for-ation of protein bodies derived from the endoplasmic

eticulum

ristina Ruiz Ramirez1,∗, Nuri Company2, Anna Nadal2, Jose-uis La Paz3, Sílvia Martinez3, Stefan Rasche4, Stefan Schillberg4,aria Pla2

Institute for Food and Agricultural Technology (INTEA), Univer-ity of Girona, SpainInstitute for Food and Agricultural Technology (INTEA), SpainCenter for Research in Agricultural Genomics (CRAG), SpainFraunhofer Institute for Molecular Biology and Applied EcologyIME), Aachen, Germany

Synthetic linear antimicrobial peptides with cationic a-helicaltructures, such as BP100, are valuable as novel therapeutics andreservatives. However, they tend to be toxic when expressed atigh levels as recombinant peptides in plants, and they can be diffi-ult to detect and isolate from complex plant tissues because theyre strongly cationic and display low extinction coefficient andxtremely limited immunogenicity. We therefore expressed BP100ith a C-terminal tag which preserved its antimicrobial activitynd demonstrated significant accumulation in plant cells. We usedfluorescent tag to trace BP100 following transiently expression inicotiana benthamiana leaves and showed that it accumulated in

arge vesicles derived from the endoplasmic reticulum (ER) alongith typical ER luminal proteins. Interestingly, the formation of

hese vesicles was induced by BP100. Similar vesicles formed intably transformed Arabidopsis thaliana seedlings, but the recom-inant peptide was toxic to the host during latter developmentaltages. This was avoided by selecting active BP100 derivatives basedn their low haemolytic activity even though the selected peptidesemained toxic to plant cells when applied exogenously at highoses. Using this strategy, we generated transgenic rice lines pro-ucing active BP100 derivatives with a yield of up to 0.5% total
oluble protein.

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U-28

eneration and application of tools for increasingeriplasmic soluble product yields of recombinant ther-peutic proteins in Escherichia coli

lexander Büttner1,∗, Simon Stammen1, Mark Dürkop1, Arturchuller1, Lina Heistinger2

Boehringer Ingelheim RCV GmbH & Co KG, AustriaUniversity of Natural Resources and Life Sciences Vienna, Austria

Soluble production of properly folded target proteins inscherichia coli can avoid cumbersome protein refolding stepshen compared to inclusion body (IB) production processes. Due

o several advantages over the cytoplasm, the periplasmic space of. coli is the compartment of choice for production of soluble andorrectly folded disulfide-bond containing proteins.

To address bottlenecks that can prevent efficient production,ecretion and folding, a toolbox consisting of different host cellodifications, expression cassettes and process control strate-

ies was created and tested for applicability. Cell engineeringpproaches comprised, e.g., different bacterial strains, genomicntegration of the target gene expression cassette, its organiza-ion within the integration locus as well as the co-synthesis ofelper factors, which improve secretion or folding of the targetroteins. A range of inducible and constitutive promoter variantsere tested concerning their applicability to control helper fac-

or gene co-overexpression. Different fermentation processes withegard to temperature, feeding and induction mode and time werepplied.

For several therapeutically relevant proteins of interest, wehowed that application of one or a combination of several ofhe developed methods increased the overall yield of the desiredroduct. Additionally, the ratio of recombinant protein was shiftedrom insoluble (cytoplasmic and/or periplasmic aggregates) to sol-ble and correctly folded product in several cases.


U-29

nhanced periplasmic production of therapeutic pro-eins in Escherichia coli

imon Stammen1,∗, Alexander Buettner1, Lina Heistinger2, Markuerkop1, Arthur Schuller1, Franz Kollmann1

Boehringer Ingelheim RCV GmbH, AustriaUniversity of Natural Resources and Life Sciences, Austria

For manufacture of therapeutic proteins that do not requireranslational modification (e.g. glycosylation) for their biologicalctivity, the production in Escherichia coli is still the host system ofhoice. Its well-known biology, the ability to grow on inexpensiveedia, the availability of genetic tools for host cell modifications

nd last but not least its long history as pharmaceutical work
ost are only the most prominent benefits. However, pharma-eutical proteins became more and more complex and thereforehe requirements on the expression systems grew. Many modern,iologically active proteins contain multiple disulphide bridges,

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hat makes classical production routes (e.g. production as inclu-ions bodies and subsequent in vitro refolding) more and morehallenging. Even though recent advances in folding technologye.g. high pressure refolding) enable refolding in commercially rel-vant yields for many target proteins, Boehringer Ingelheim (BI)lso significantly increased its efforts in production of properlyolded target proteins in the E. coli periplasm.

We present several approaches to increase the amount ofoluble therapeutic protein within the periplasmic space ofhe bacterial cell. Application of BI’s expression system, which

akes use of a genomically integrated product gene signif-cantly increased the titre of properly folded target proteinhen compared to one of the typically used plasmid-based

xpression systems. Furthermore, the secretion and refoldingachinery was supported by co-overproduction of different

elper proteins and chaperones to enhance cellular productivity.he fermentation process was optimized with regard to pro-ess variables to enhance the yield of properly folded targetrotein.


U-30

dentification of a novel master regulator of cellu-ase and hemicellulase production in Trichoderma reeseising genome-wide approach

ari Häkkinen, Mari Valkonen ∗, Ann Westerholm-Parvinen,ina Aro, Marika Vitikainen, Merja Penttilä, Markku Saloheimo,iina Pakula

VTT Technical Research Centre of Finland, Finland

Trichoderma reesei (anamorph Hypocrea jecorina) is an efficientroducer of enzymes degrading lignocellulosic biomass. The cel-

ulases and hemicellulases produced by the fungus are widelymployed in industry, and this production system has a central rolen biorefinery applications. Various environmental and metabolicactors together with the physiological state of the cell affect thenzyme production in T. reesei. In previous studies, both positivelynd negatively acting regulatory factors for cellulase and hemicel-ulase genes have been characterised in T. reesei.

In this study, an expression microarray data on T. reesei cul-ivated in the presence of different carbon sources was analysedn order to identify additional regulatory genes for cellulasend hemicellulase production. In total, 28 putative regulatoryactors were chosen to be over-expressed in T. reesei based on theact that they were induced by lignocellulosic substrates. In therimary screening, over-expression of seven of these factors ledo increased production of cellulases and/or xylanases. Amonghese factors is a novel master regulator of the cellulose and hemi-ellulose genes designated ace3. Its over-expression increasedellulase production 2 to 4-fold and also enhanced hemicellulaseroduction. Its deletion abolished cellulase production totally,ecreased hemicellulase production, and in the ace3 deletion
trains the major cellulase gene transcripts were at extremelyow levels. Interestingly, the modifications of ace3 also affectedhe mRNA levels of the previously identified hemicellulase and
pdrc



ellulase master regulator xyr1, suggesting interplay between theseactors.


U-31

ffects of K87 mutations in the alpha-crystallin domainf Tpv sHSP 14.3 on oligomerization and in vitro chaper-ne activity

emra Kocabiyik ∗, Ilir Sharej

Middle East Technical University, Turkey

Small heat shock proteins (sHSPs) act as molecular chaperonesy binding denaturing proteins and protecting them from aggre-ation before refolding. Their level is up-regulated in particularvents, such as stress and pathological conditions. ACD is the sig-ature of the sHSP monomer which is flanked by N-terminal armf variable sequence and C-terminus with a conserved I/L-X-I/Lotif. Seven or eight �-strands in ACD arranged into antiparallel

heets constituting �-sandwich which mediates dimerization. TheHSPs form reversible homo- or hetero-oligomers indicating theynamic plasticity of the quartenary structure that modulate theecognition of substrate. Mutations in the ACD occur in positionshat would have impact on this dynamic behavior. One of theell studied mutations is R120G in human CRYAB that causes

ongenital cataract and desmin related myopathy. At the dimernterface R120 makes two interface ion pairs with D109. Theim of this study is to determine if the topological equivalentf this residue has the same impact on oligomeric structure ofon-metazoan sHSPs. We investigated the effect of substitutionst K87 position of Tpv HSP14.3 (an archaeal sHSP) on oligomer-zation and chaperone activity. The K87R and R87I mutationsesulted in increased chaperone activity when citrate synthaseas used as the client. Increased substrate binding capacity due to87I mutation can be explained by increased hydrophobicity. Theligomere size of all mutant sHSPs increased at the temperaturerom 25 to 60 ◦C. The notable increase was observed at 25 ◦C forhe K87I mutant particles.


U-32

evelopment of a “toolbox” approach for recombinantrotein production

ania Selas Castineiras1,∗, Steven G. Williams2, Jeff A. Cole1, Tim. Overton1, Anthony Hitchcock2

University of Birmingham, United KingdomCobra Biologics Ltd, United Kingdom

The high demand of recombinant proteins produced in micro-ial hosts, which accounts for more than 30% of the biologicalroducts in the market, have reinforced the necessity of the
evelopment of more efficient processes for the production ofecombinant proteins. Higher production efficiencies and lowerost have become essential pre-requisites for a commercially viable

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rocess. Optimisation of fermentation conditions to decrease celltress has been shown to favour the accumulation of soluble andctive recombinant proteins [1].

We have shown that shake-flask studies are an appropriateool for the optimisation of fermentation conditions, reducing theime and cost involved in process development. This approachas used for the production of tumour necrosis factor � (TNF�)

n E. coli using the arabinose-inducible T7 expression system. Aolumetric yield of 3.82 g L−1 of TNF� was achieved by fermen-ation, 92% being soluble and active. The proven success of thispproach can be now applied to a broader range of recombinantroteins.

We will discuss the selection of adequate expression systemsnd approaches for the optimisation of cultivation conditions asey factors for the production of recombinant proteins. The mainoal of this project is to test and integrate fermentation conditionsllowing the design of platforms following a “toolbox approach”or protein production.

eference

].Sevastsyanovich Y, Alfasi S, Overton T, et al. Exploitation of GFPfusion proteins and stress avoidance as a generic strategy for the pro-duction of high-quality recombinant proteins. FEMS Microbiol Lett2009;299:86–94.


U-33

n vivo reconstitution of membrane protein by caveolin1o-expression

onghyeok Shin ∗, Paul Heo, Joon-Bum Park, Myungseo Park,ounghun Jung, Da-Hyeong Cho, Byoung-jae Kong, Junghoonn, Jichun Lee, Dae-Hyuk Kweon

Sungkyunkwan University/Bioengineering Department, Southorea

Caveolae is a membrane-budding structure which exists inany animal vertebrate cells. One of the important functions

f caveolae is to form membrane curvature and endocytic vesi-le. Recently, It was shown that caveolae could be formed inscherichia coli by expressing caveolin-1. The heterologous cave-lae may host other membrane proteins overexpressed inside theell. We utilized this system for construction of proteo-liposomen Escherichia coli. SNARE proteins (Syntaxin1a, SNAP25, VAMP2)ere introduced to prove our in vivo reconstitution system. Here,e show that the purified heterologous caveolae indeed contain

he co-expressed membrane protein and the membrane proteinsere facing outward. The size of the purified caveolae with mem-rane protein reconstituted were measured by dynamic lightcattering. The presence of VAMP2 & Syntaxin1a on this proteo-ndosome was confirmed by Western blot analysis. Furthermore,embrane proteins (VAMP2 & Syntaxin1) embedded in caveolae

etained the ability to form SNARE complex. Our study proposes
n in vivo membrane protein reconstitution system.

pcit


U-34

enetic engineering of Saccharomyces cerevisiae for effi-ient IgG-assembly and secretion

ssi Koskela ∗, Alexander Frey

Aalto University, Finland

IgG-antibodies are complex molecules that require multiple ele-ents to assemble efficiently, including folding factors and an

xidative environment. The growing demand for biosimilars andlternative treatments makes the production process of humanntibodies an intriguing area of research. Although simple micro-ial expression platforms such as Saccharomyces cerevisiae are notntrinsically suited for IgG-production, the tools to genetically

odify the yeast cells for this purpose are versatile.To improve the secretion of produced protein, we focus on

odifying ER luminal environment and protein folding process.ne important approach is increasing the size of ER, which cane achieved with a single gene knock-out [1]. The extra spaces supplemented with additional folding factors important ingG-tetramer assembly including molecular chaperones, PDI andPIases. These factors are tested in different amounts and combi-ations and the strains producing the highest yields are identifiedith high-throughput screening.

Genetic engineering requires a substantial amount of labor-ntensive cloning work. To optimize this aspect, we recentlyiscovered a new approach to molecular cloning, which outcom-etes many of the existing cloning methods in simplicity andffordability. This novel protocol is presented along with pre-iminary results of genetic modifications on IgG-secretion. Withuitable high-throughput methods in hand, the microbial plat-orm for IgG-production is optimized at the level of the wholeystem.

eference

].Schuck S, a Prinz W, Thorn KS, Voss C, Walter P. Membrane expan-sion alleviates endoplasmic reticulum stress independently of theunfolded protein response. J Cell Biol 2009;187:525–36.


U-35

tudy on the domain function of Listeria monocytogenes60 protein

inliang Guo ∗, Hao Gu, Qian Xu, Jinrong Zuo

College of Bioscience and Biotechnology, Yangzhou University,hina

Listeria monocytogenes p60 protein is an autolysin that canydrolyze the peptidoglycans of bacterial cell walls. L. monocy-

ogenes p60 protein was required for L. monocytogenes virulence.esides the importance of p60 protein in bacterial pathogenesis,
60 protein can also be developed as a new proteinaceous antimi-robial if its activity to hydrolyze the peptidoglycans can be greatlymproved. It contains two independent structural domains, N-erminal LysM domain and C-terminal NlpC/P60 domain, which

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an be separated at the amino acid residue 270. However, the activeunctions of these two domains in p60 protein and their influencesn the substrate recognition and catalytic activity of p60 proteinre unknown. Here we identified both of the functional hot spotnd the mutational hot spot amino acid residues in these two struc-ural domains by means of the amino acid sequence alignment ofifferent p60 variants, including two p60 variants screened in our

ab. The functional hot spot and the mutational hot spot aminocid residues were substituted to alanine (A) by using site-directedutation to construct p60 variants. These p60 variants in combi-

ation with some truncated p60 proteins were used to unveil theolecular mechanism of substrate recognition and catalysis of p60

rotein in the domain level. Results confirmed that the N-terminalysM domain in p60 protein could bind to the bacterial cell wallightly, whereas the C-terminal NlpC/P60 domain showed slightbility to hydrolyze the cell walls. These fundamental studies on60 protein variants will provide strong support for engineeringhe p60 protein molecular.


U-36

lycoDelete technology: shortcutting mammalian cell N-lycosylation

rancis Santens ∗, Leander Meuris, Morgane Boone, Nico Calle-aert

VIB Ghent University, Belgium

Mammalian complex-type N-glycan synthesis is a multi-steprocess that results in heterogeneous glycosylation of proteins.eterogeneity in therapeutic glycoproteins causes difficulties forrotein purification and process reproducibility and can lead toariable therapeutic efficacy. Here we report engineered mam-alian cell lines that have a shortened Golgi N-glycosylation

athway, which leads to the expression of proteins with small,ialylated trisaccharide N-glycans. This glycoengineering strategy,hich we call GlycoDelete [1], results in proteins with substan-

ially reduced glycan heterogeneity. To assess the potential of theselycoDelete glycans and their influence on glycosylated phar-aceutical proteins, human GM-CSF and an anti-CD20 antibodyere produced in 293s and 293sGlycoDelete cells. Both proteinsere purified and thoroughly analysed. For hGM-CSF we did not

ee a significant influence of the GlycoDelete sugars on the activ-ty of the protein. GlycoDelete anti-CD20 on the other hand hassignificantly reduced Fc�R affinity and an increased circulation

imes in mice compared to 293S produced anti-CD20.

eference

].Meuris L, Santens F, Elson G, et al. GlycoDelete engineering of mam-malian cells simplifies N-glycosylation of recombinant proteins. Nat
Biotechnol 2014, advance online publication.

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U-37

xtracellular transaminases for biocatalysis

atrin Weinhandl1,∗, Margit Winkler1, Anton Glieder1, Andreaamattari 2

Austrian Center of Industrial Biotechnology (ACIB), AustriaTU Graz, Austria

Branched chain aminotransferase (BCAT, EC 2.6.1.42) ofscherichia coli is an intracellular protein and an interesting toolor the production of chiral amines or amino acids. Secretion ofCAT to the culture supernatant was the method of choice to facili-ate industrial enzyme applications and downstream processing byhole cell applications while counteracting limited cell permeabil-

ty for target substrates. Pichia pastoris was chosen as expressionost because of its positive characteristics, such as the ability toeach high biomass levels as well as the lack of background proteinsn the extracellular environment during expression.

Although secretion of intracellular proteins was reported to beroblematic in the past, we were able to secrete BCAT in Pichia pas-oris and obtained a maximum activity level in the supernatant of50 �mol/min/mg total protein (L-leucine conversion in a couplednzymatic assay [1]).

In order to improve the expression level, several approachesere investigated: on the one hand we examined different Pichia

trains. On the other hand, alternative signal peptides and dif-erent promoters were evaluated for improved expression andecretion of BCAT. In our hands methanol-induced expression leado a higher activity in the supernatant, compared to constitutivexpression which still allowed satisfying BCAT secretion.

eference

].Weinhandl, et al. Tetrahedron 2012;68(37):7586–90.


U-38

method to stably integrate multiple genetic elementsnto Chinese hamster ovary (CHO) cells

abine Vcelar1,∗, Martina Baumann1, Nicole Borth2

ACIB - Austrian Centre of Industrial Biotechnology, AustriaDepartment of Biotechnology, University of Natural Resourcesnd Life Sciences, Vienna, Austria

CHO cells are the preferred host cells for the productionf therapeutic proteins and the most commonly used mam-alian expression system. Advantages such as an easy cultivation,

ast growth, complex protein folding and human-like post-ranslational modifications are in part set of by slow cell linend process development. These constraints lead to an increasedequirement for CHO cell line modification tools.

The present work focuses on the integration of up to four
enetic elements into CHO cells. Two different approaches werestablished. The principle of both systems is the Recombinase-ediated cassette exchange (RMCE). Systems A is engineered on
he basis of transfection vectors comprised of different resistance

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arkers and two cloning sites. The incorporation of the geneticlements of choice is mediated by RMCE at two specific genomicocations. The second system established combines the RMCE sys-em with the R4 Integrase System to enable integration of up toour genes at a single specific genomic location.

The stable integration of DNA elements of choice at specificenomic locations and the testing of candidate genes for phe-otypic effects without disturbance by clone specific variation isetting feasible with these methods.


U-39

onstruction of pH-sensitive Her2 binding antibodyragment by directed evolution using yeast display

lisabeth Lobner ∗, Michael Traxlmayr, Florian Rüker, Christianbinger

University of Natural Resources and Life Sciences, Vienna, Austria

The half-life of therapeutic antibodies, which are internal-zed together with their antigenic receptor, can be increased byecreasing their affinity at acidic endosomal conditions. Here, airected evolution protocol was developed for construction of pH-ependent binding sites by using yeast display. The C-terminaltructural loops of an antigen binding crystallizable fragment ofmmunoglobulin G1 (Fcab) [1] have been engineered for reducedinding to the extracellular domain of human epidermal growthactor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. Aibrary based on a Her2-ECD binding lead Fcab was constructedy parsimonious mutagenesis and displayed on yeast. Alternatingelections for binding at pH 7.4 and non-binding at pH 6.0 wereerformed by FACS probing the binding to the antigen as well asstructurally specific ligand. The three best performing variants

P1, P2, P3) were selected. Displayed on yeast they showed clearH-dependent binding to soluble Her2-ECD (decrease in affinityt pH 6.0 compared to pH 7.4). Additionally, solubly expressed1, P2 and P3 exhibited pH-dependent interactions with Her2-ositive cells whereas their conformational and thermal stabilityas pH-independent. The interaction of P1, P2 and P3 with theeonatal Fc receptor remained wild-type like showing the inverseH-dependence compared to Her2-ECD binding. Interestingly,wo of the three Fcabs did not contain a single histidine muta-ion but all of them contained variations next to histidines thatlready occurred in loops of the lead Fcab.

eference

].Wozniak-Knopp, et al. Protein Eng Des Sel 2010;23:289–97.


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U-40

he employment of a heterologous yeast expression sys-em for production of VP1-derived virus-like particlesriginated from novel human polyomaviruses

lma Gedvilaite ∗, Milda Norkiene, Rita Lasickieke

Vilnius University Institute of Biotechnology, Lithuania

Virus-like particles (VLPs) resemble their parent virion in struc-ure, immunogenicity, tropism and transduction efficiency, buto not contain any viral genetic material. They can be used foriagnostic purposes, vaccination and gene therapy. Polyomaviri-ae is a growing family of naked, double-stranded DNA viruseshat infect birds and mammals. The major capsid protein VP1 ofll polyomaviruses (PyV) is sufficient for assembly of VLPs andepresents the major immunogenic protein of PyV. In the last fewears, the human polyomavirus (HPyV) family has expanded to 12embers. Serological studies are the primary tool to investigate the

revalence of various polyomaviruses in human populations. Theecombinant VP1 VLPs are particularly valuable for the serologicaletection of these viruses as many PyV cannot be easily cultured.

The earlier discovered PyV VP1-derived VLPs were successfullyroduced using different eukaryotic and prokaryotic expressionystems including yeast. Here, we report that the galactose-nducible yeast S. cerevisiae expression system is efficient forigh-level production and self assembly of VP1 derived from newPyV: KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7. The

ormation of empty VP1-derived VLPs was confirmed by cesiumhloride ultracentrifugation, agarose gel electrophoresis and elec-ron microscopy. Yeast-generated VP1 VLPs were free of toxins,ost cell DNA and proteins. The purified VP1 VLPs originating

rom KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7 were suc-essfully used for generation of monoclonal antibodies and mighte useful for the generation of new diagnostic tools, antiviral vac-ines or gene delivery systems.


U-41

urification by affinity chromatography of recombi-ant L-asparaginase I from Saccharomyces cerevisiaexpressed in Escherichia coli

dalberto Pessoa-Jr1,∗, Gisele Monteiro1, Joao Santos2, Johannases1, Albert Peixoto3, Juan Santos1, Joao Molino1, Lauraliveira4, Joao Coutinho2, Sonia Ventura2, Andre Lopes1

University of Sao Paulo, BrazilUniversity of Aveiro, BrazilUniversidade Estadual do Sudoeste da Bahia, BrazilUniversity of Campinas, Brazil

L-Asparaginase is known by its capacity to catalyse the hydroly-is of L-asparagine into L-aspartic acid and ammonia. This enzyme
as been clinically acceptable as an anti-tumour agent for treat-ent of acute lymphoblastic leukemia and lymphosarcoma. This
iopharmaceutical is produced by fermentation processes and sub-equently it needs to be correctly separated from the contaminants


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rom the fermentation broth. Here, Affinity Chromatography ispplied as a novel approach to purify (His)6-tagged recombi-ant L-asparaginase I from Saccharomyces cerevisiae expressed inscherichia coli. The Affinity Chromatography was performed on aast Protein Liquid Chromatography (FPLC) system using a Ni2+-harged, 5 mL HiTrap IMAC FF. A linear gradient of 0 mM to00 mM of imidazole at 5.0 mL min−1 was applied in order to iden-ify the lowest imidazole concentration of the elution buffer thatxtracts the higher amount of L-asparaginase. This concentrationange was further used in a step imidazole gradient. Two-step con-entrations of the initial imidazole concentration (32.0 and 54.4%)ere applied. Polyacrylamide gel electrophoresis combined with

ilver staining and Nessler activity assay were used to confirm theresence of the purified L-asparaginase (≈45 kDa). In the gradi-nt step, the eluted recombinant L-asparaginase showed a highpecific activity of 110.1 ± 0.3IU mg−1. Furthermore, a recovery of1.00 ± 0.01% was obtained resulting in a purification factor of 17.n this respect, the FPLC process has undoubtedly proved to be anfficient tool for the purification of this enzyme.

Acknowledgements: This research was supported byAPES, CNPq (301248/2010-9) and FAPESP (2013/08617-7),razil; FEDER funds through the program COMPETE/FCT (Pest-/CTM/LA0011/2013); SFRH/BPD/79263/2011; and Santander.


U-42

nalysis of modifications in the ER protein qualityontrol system on heterologous protein expression inaccharomyces cerevisiae

org de Ruijter ∗, Essi Koskela, Alexander Frey

Aalto University, Finland

The yeast Saccharomyces cerevisiae is a widely used host in theroduction of therapeutic glycoproteins. However, the expressionf high levels of heterologous proteins is known to interfere witharious processes in the cells and can trigger stress responses.

High volume production of heterologous glycoproteins is proneo overflow the ER with unfolded proteins. Glycoproteins thatemain unfolded in the ER for too long are predisposed to beargeted for degradation by components of the ER associated degra-ation (ERAD) pathway. In this present study, the ERAD pathwayas modified by deleting genes encoding various components,ith and without a blocking of the unfolded protein responseathway (UPR). In addition, overexpression of the folding chaper-nes KAR2, PDI1, ERO1 and CPR5 was introduced. These proteinsre expected to assist in the quick folding of proteins to decreasehe blocking of the ER by unfolded proteins.

The production levels of a human IgG1 molecule of theseutant strains were analyzed using ELISA. Since changing the

R quality control system might lead to a decreased quality ofroteins, the quality of the produced IgG1 was analyzed by PAGE.

The results at hand indicate that deletion of early ERAD fac-
ors is detrimental for protein production, as that removal of latertages leads to an increase. For the folding chaperones, control ofxpression levels is key, as high level expression tend to decrease
or



eterologous protein secretion, possibly by cluttering the ER withn overload of unfolded proteins.


U-43

xpression of C-terminal Fab fragment variants: the hostetermines the best variant

rigitte Gasser1, Rebecca Goengrich1,∗, Christoph Kiziak2,iethard Mattanovich1

Department of Biotechnology, BOKU University of Naturalesources and Life Sciences, Vienna, AustriaLonza AG, Visp, Switzerland

Expression of antibody fragments is routinely done in microbialost cells such as the bacterium Escherichia coli or the yeast Pichiaastoris. In yeast, Fabs are usually secreted to the cell supernatantfter passing the secretory pathway, while Fab expression in E. colis usually done in the periplasm. Different Fab variants have beeneported in literature, however, it has not yet been comprehen-ively assessed which work best in which host. Here we compareifferent Fab variants expressed in E. coli and P. pastoris, whichesulted in a preference for different variants in each host.


U-44

ell-free incorporation of unnatural amino acidsor cloning-independent engineering of elastin-likeolypeptides

ong-Myung Kim1,∗, Su-Jin Oh2, Young-A Son3

Department of Fine Chemical Engineering and Applied Chem-stry, Chungnam National University, South KoreaDepartment of Fine Chemical Engineering and Applied Chem-

stry, Chungnam National University of Korea, South KoreaDepartment of Advanced Organic Materials Engineering, Chung-am National University of Korea, South Korea

The physicochemical nature of a protein is determined by theollective properties of consisting amino acids and their sequentialrder in the protein molecule. Although natural proteins exhibitwide array of structure and biological functions with the concise

et of 20 canonical amino acids, the ability to introduce unnat-ral amino acids into protein molecules will further expand theiversity, creating protein species that have never been explored.n this study, we demonstrate the use of a cell-free protein syn-hesis system as a versatile platform for synthesis of engineeredio-materials through the incorporation of unnatural amino acidsnto recombinant proteins. Elastin-like-polypeptides (ELPs) werehosen as a model protein to investigate the effects of unnaturalmino acid incorporation. Herein, we discuss on the manipulation
f the transition temperature of ELPs through cell-free incorpo-ation of proline analogues. Through the replacement of proline

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U-45

nhancing recombinant protein production with anscherichia coli host strain lacking insertion sequences

un-Chang Kim ∗, Myung Keun Park, Jun Hyoung Lee, Kyungeok Yang

Korea Advanced Institute of Science and Technology, South Korea

The genomic stability and integrity of host strains are criti-al for the production of recombinant proteins in biotechnology.acterial genomes contain numerous jumping genetic elements,he insertion sequences (ISs) that cause a variety of genetic rear-angements, resulting in adverse effects such as genome andecombinant plasmid instability. To minimize the harmful effectsf ISs on the expression of recombinant proteins in Escherichia coli,e developed an IS-free, minimized E. coli strain (MS56) inhich about 23% of the genome, including all ISs and manynnecessary genes, was removed. Here, we compared the expres-ion profiles of recombinant proteins such as tumor necrosisactor-related apoptosis-inducing ligand (TRAIL) and bone mor-hogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping ofSs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred athe rate of ∼10−8/gene/h in MG1655 whereas such events were notbserved in MS56. Even though IS hopping occurred very rarely10−8/gene/h), cells containing the IS-inserted TRAIL and BMP2lasmids became dominant (∼52% of total population) 28 h afterermentation due to their growth advantage over cells containingntact plasmids, significantly reducing recombinant protein pro-uction in batch fermentation. Our findings clearly indicate thatS hopping is detrimental to the industrial production of recom-inant proteins, emphasizing the importance of the development
f IS-free host strains.
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U-46

nalysis of amino acid residues in the N-terminal regionf the beta-neurotoxin CssII and its specificity foroltage-gated Na+ channel subtypes

odrigo Ibarra Vega1,∗, Juana María Jiménez Vargas2, Rita Restanoassulini2, Georgina Estrada3, Lourival D. Possani2, Gerardoorzo2

Instituto de Biotecnología, Universidad Nacional Autónoma deéxico, UNAM, Mexico

Instituto de Biotecnología de la Universidad Nacional Autónomae México, UNAM, MexicoCentro de investigación Científica de Yucatán, AC. (CICY),érida, Yucatán, México, Mexico

The scorpion toxin CssII from the scorpion Centruroides suffususs a peptide that affects the voltage-gated sodium channel Nav 1.6.he CeII9 peptide toxin of Centruroides elegans is 86% identical tossII but selectively affects Nav 1.4. The most significant variationetween these two peptide toxins is observed at the N-terminus,pecifically in residues 7 and 8. Therefore, these two residues inssII were substituted for residues corresponding to the peptideeII9 to evaluate if the CssII variants keep or shift their selectiv-

ty for the Nav1.6 or Nav1.4, respectively. Three CssII variantsere generated by site-directed mutagenesis, named CssII K8H,ssII S7N and CssII S7N/K8H. Since the CssII native toxin has anmidation at the C-terminal and this amidation is important forinding the Navs, the aforementioned variants were constructedut including basic charges at the C-terminal (T64R/N66R). Theseubstitutions improve the affinity of the CssII recombinant toxinsor the Nav1.6. The synthetic genes were cloned into the pQE30ector and expressed in E. coli BL21 strain. The recombinant vari-nts had an N-terminal fusion peptide composed of a 6His-tagnd a FXa proteolytic cleavage region. The mutants were puri-ed, folded in vitro, digested with FXa and electrophysiologicalssayed on HEK293 cells stably expressing the Nav1.4 and Nav1.6hannels.


U-47

xpression of recombinant formate dehydrogenase inscherichia coli and its immobilization onto magneticano-particles

tanislav Stuchlik1,∗, Lukas Hason2, Zdenko Levarski2, Luciaocanova2, Kristina Jirickova2, Diana Hopkova2, Pavol Kois3,an Turna2

Comenius University in Bratislava, Department of Moleculariology, SlovakiaComenius University in Bratislava, Faculty of Natural Sciences,epartment of Molecular Biology, SlovakiaComenius University in Bratislava, Faculty of Natural Sciences,
epartment of Organic Chemistry, Slovakia
The enzymatic bioconversion of aromatic compounds in foodnd cosmetic industry is often accompanied by the limitation in


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erms of co-factor depletion or its insufficient regeneration. Toddress this issue, we have constructed an efficient E. coli basedxpression system capable of producing functional yeast formateehydrogenase (FDH), an enzyme used for the regeneration ofxidized NAD+, during conversion of trans-2-hexenal to trans--hexenol catalyzed by NAD-dependent alcohol dehydrogenaseADH). To further improve this reaction, we have immobilizedDH onto magnetic nano-particles and tested the ability of pro-uced enzyme to improve activity of ADH by NAD+/NADHonversion. The activity of FDH has been tested by applying var-ous physical and chemical condition changes. The results showhat although the activity of the immobilized FDH is decreasedompared to the free enzyme, its stability is increased signifi-antly allowing prolonged incubations or repeated use of a singleatch.

Acknowledgements: This work was supported by the Slo-ak Research and Development Agency grant APVV-0061-11 ands also result of the “Production of biologically active agents basedn recombinant proteins” (ITMS 26240220048) project implemen-ation supported by the Research and Development Operationalrogram funded by the ERDF.


U-48

roduction of selenoproteins in yeast via geneticallyncoded incorporation of a photocaged selenocysteine

asa Rakauskaite ∗, Viktoras Masevicius, Giedre Urbanaviciute,udrone Ruksenaite, Saulius Klimasauskas

Vilnius University, Institute of Biotechnology, Lithuania

Recent advancements in protein engineering materialized in aumber of valuable products for pharmaceutical, industrial, andesearch applications. One promising way to achieve advancedngineered protein products is based on targeted incorporationf rare and unnatural amino acids. For instance, selenocysteineSec) provides a selenium atom with unique chemical char-cteristics (higher nucleophilicity, lower pKa, and lower redoxotential) not attainable in common proteins. Although Sec isf significant technological importance as a component of bothatural proteins and designed biocatalysts, the availability ofuch proteins is hampered by technical limitations. Here weeport a universal method for production of recombinant seleno-roteins in Saccharomyces cerevisiae cells via genetically encodedECIS-independent incorporation of a photocaged unnaturalmino acid, 4,5-dimethoxy-2-nitrobenzyl selenocysteine (DMNB-ec). Photodeprotection of the incorporated residue in the targetelenoprotein can be performed under mild conditions. The newpproach was validated by inserting a Sec residue i) at a permissiveosition (Tyr39) of enhanced green fluorescent protein (EGFP) and

i) replacing an essential catalytic cysteine (Cys103) of a bacterialNA cytosine-5 methytransferase, M.HpaII. Photodeprotection of

he incorporated DMNB-Sec residue in the model EGFP protein
emonstrated light-controlled protein dimerization and efficientec-specific covalent labeling in vitro. This method has a potential


o simplify the synthesis of selenoproteins and therefore expandheir biotechnological and biopharmaceutical utilization.


U-49

evelop new tools for investigation of albumin trans-ortation

inh Khoi Nguyen Ly1,∗, Philip Poronnik2, David Nikolic-atterson3

HiRi RMIT AustraliaUniversity of Sydney AustraliaMonash Medical Center

Albumin is most abundant protein in blood and plays impor-ant homeostatic roles in the regulation of oncotic pressure ands a carrier of many small proteins, peptides and hormones. Albu-in in the circulation can be covalently modified at surface lysine

esidues and thus the circulation contains a mixture of nativelbumin (i.e. not modified) and modified albumin. A significantmount of albumin is lost from the blood each day as a result of theltration occurring in the capillaries of the renal glomeruli. Thisltered albumin in the early filtrate is thought to be mostly takenp by and degraded in the early renal proximal tubular epithe-

ial cells, while a small proportion is excreted in the final urine.his degradation of albumin by proximal tubules operates via theegalin scavenger receptor and an endocytic pathway.A major gap in our knowledge is whether the megalin-based

lbumin uptake is a non-selective process, or whether megalinas a significantly different affinity for modified versus nativelbumin. Using yeast expression system, we develop radioactiveative recombinant albumin. The uptake experiment using 14C-HA suggest the uptake pathway of native and modified albuminre different in opossum kidney cell line. The uptake of 14C-rHA is

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V-01

reliminary analysis of DNA isolated from flax grownn the radio-contaminated Chernobyl area for six gen-rations suggests the stability of fatty acid desaturaseenes

eronika Lancíková1,∗, Jana Ziarovská2, Maksym Danchenko3,alentyna Berezhna4, Milan Bezo2, Katarína Razná2, Namikashydov4, Martin Hajduch5

Institute of Plant Genetics and Biotechnology of Slovak Academyf Sciences, SlovakiaDepartment of Genetics and Plant Breeding, Faculty of Agrobiol-gy and Food Resources, Slovak University of Agriculture, SlovakiaInstitute of Virology, Slovak Academy of Sciences, Institute of Celliology and Genetic Engineering, National Academy of Sciencesf Ukraine, SlovakiaInstitute of Cell Biology and Genetic Engineering, Nationalcademy of Sciences of Ukraine, SlovakiaInstitute of Plant Genetics and Biotechnology, Slovak Academyf Sciences, Institute of Virology, Slovak Academy of Sciences, Slo-akia

Plants have the ability to survive and reproduce under changednvironmental conditions which were induced by biotic andbiotic stress. The area of the Chernobyl nuclear accident is aery interesting example of plant adaptation to abiotic stress inhe form of increased radiation. Two experimental fields werestablished in the Chernobyl region – radio-contaminated andon-radioactive. Flax (Linum usitatissimum L., variety Kyivskyi) wasultivated in these experimental fields since 2007, and during the012 the sixth generation of seeds was collected. The aim of theresented study was to analyze fatty acid desaturase (FAD) genes inhe sixth generation of flax, specifically FAD3A and FAD3B, usinghe restriction fragment length polymorphism technique. In aiosynthetic pathway of fatty acids, these genes perform an impor-ant function because they encode conversion of linoleic acid to-linolenic acid. This study analyzed three parts of FAD3A geneith following length of the nucleotide sequences 1201, 1500,924 base pairs. Also, FAD3B gene was divided into three partsith length of the nucleotide sequences 1246, 1330, 1279 baseairs. For restriction analysis four different restriction enzymesere applied – MnlI for restriction digestion of the first part of

AD3A and FAD3B genes, NlaIII for the second and third part ofAD3A gene, AciI for the second and Hpy188I for the third part ofAD3B gene. Based on these preliminary data, no polymorphismf FAD genes in sixth flax generation was detected. However, thetability of FAD genes will be further verified by more detailed
nalyses.

caldtetus

STRESS RESPONSES

V-02

verview of the biomedical research in the newly estab-ished Laboratory of Genome Integrity at the Palackyniversity, Olomouc

uzana Loubalová ∗, Kamila Jahodíková, Martin Liptay

Institute of Molecular and Translational Medicine Palacky Univer-ity Olomouc, Czech Republic

The Laboratory of Genome Integrity (LGI) is part of the recentlypened Institute of Molecular and Translational Medicine belong-ng to the Palacky University in Olomouc, Czech Republic. Thenstitute was built with the aim to conduct cutting edge researchn the field of drug design and discovery. The collaborative net-ork at the institute covers all crucial phases of the drug designrocess including search for novel drug targets.

The research in the LGI is focused mainly on various mechanis-ic aspects of the DNA damage response and DNA repair pathways.oth pathways play a key role in the onset and progress of cancernd thus represent ideal targets for novel anti-cancer drugs.

In addition, we have been trying to understand how elevatedeplication stress in rapidly growing pre-cancerous and cancerousells affects the progression of the disease. We would also like tonderstand in detail how a cell copes with the replication stress athe molecular level.

Our next long term goal is to set up specific DDR response basedigh throughput screens capable of assessing effect of differentompounds and environment on DNA integrity in human cells.

Apart from basic research, we have been also developing novelesearch and diagnostic tools, such as devices for more efficienthromosome spreading and non-laborious mammalian cell syn-hronization.


V-03

canthopanax sessiliflorus stem confers increased resis-ance to environmental stresses and lifespan extensionn Caenorhabditis elegans

ang-Kyu Park, Jin-Kook Park ∗, Chul-Kyu Kim, Sang-Ki Kong, A-eum Yu, Mi-Young Lee

Soonchunhyang University, South Korea

Acanthopanax sessiliflorus is a native Korean plant and used asraditional medicine or an ingredient in many Korean foods. Theree radical theory of aging suggests that cellular oxidative stressaused by free radicals is the main cause of aging. Free radicalsan be removed by cellular anti-oxidants. Here, we examined thenti-oxidant activity of Acanthopanax sessiliflorus extract and itsifespan-extending effect in Caenorhabditis elegans. Oxidative DNAamage was reduced and survival under oxidative-stress condi-ions was significantly enhanced by Acanthopanax sessiliflorus stem
xtract. In addition, Acanthopanax sessiliflorus stem increased resis-ance to other environmental stresses, including heat shock andltraviolet irradiation. Treatment with Acanthopanax sessiliflorustem extract significantly extended both mean and maximum

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TRESS RESPONSES

ifespan in C. elegans. However, fertility was not affected bycanthopanax sessiliflorus stem. In conclusion, Acanthopanax ses-iliflorus stem had strong anti-oxidant activity and conferred aongevity phenotype without reduced reproduction in C. elegans,hich provides conclusive evidence to support the free radical

heory of aging.


V-04

ranscriptome characteristic of echiuran worm Urechisnicinctus exposed to sulfide by digital gene expressionnalysis

hifeng Zhang ∗, Litao Zhang, Xiaolong Liu

Ocean University of China, China

Sulfide is a well-known toxicant. However, some organismsan tolerate and utilize sulfide. In this study, Urechis unicinctus,nhabiting coastal sediment and tolerating high concentration sul-de, was used to examine its transcriptional profile in responseo 50 �M sulfide for 24 h by digital gene expression analysis. Aotal of approximately 16 million cDNA tags were sequenced and,909,160 and 4,057,279 clean tags were obtained in the controlnd 24 h libraries. Compared with the 0 h library, 1181 tag-mappedenes were detected as differentially expressed genes (DEGs) in the4 h library. The DEGs were further subjected to GO and path-ay analysis. More than 80% pathways were rarely reported toe related to sulfide stress before. Three key physiological actionsnduced by sulfide involving in energy metabolism, DNA dam-ge response and inflammation were discussed in details. To ourest knowledge, this study is the first transcriptome-wide effort toeveal the transcriptional response to sulfide stress by digital genexpression analysis and the identified DEGs can serve as a basis forurther understanding of sulfide roles in organisms [1,2].

Acknowledgement: This work was supported by the Naturalcience Foundation of China (31372506).

eferences

].Tamizhselvi R, et al. Preprotachykinin-A gene deletion regulateshydrogen sulfide-induced toll-like receptor 4 signaling pathway incerulein-treated pancreatic acinar cells. Pancreas 2011;40:444–52.

].Janssens S, Tinel A. The PIDDosome, DNA-damage-induced apopto-
sis and beyond. Cell Death Differentiation 2012;19:13–20.

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V-05

etermination of reliable house keeping gene(s) forPCR in maize under different boron dosages

asan Can1,∗, Mehmet Hamurcu1, tijen Demiral2, Anamikaandey1, Mohd Kamran Khan1, Seyit Ali Kayıs1, Zuhal Zeynepvsaroglu1, Nimet Can2, Sait Gezgin1, Erdogan Esref Hakki1

Selcuk University, TurkeyHarran University, Turkey

Maize is one of the major cereal crops in the world. In Turkeyproduction of 5.9 million tons was recorded in the year 2013

nd became third most important crop of the country. Productionf maize has recently increased in Central and South Anato-ian regions of Turkey. However, boron toxicity has emerged as

major crisis of the Central Anatolian Soil affecting the maizeield and product quality. For solving this important issue, dif-erent approaches, including boron-tolerant variety developmentnd use, are implemented. Understanding the mechanism oforon tolerance in plants is an active research area. Differentenes are known to be involved in this mechanism. Trackingroperly the expression patherns of these genes requires normal-

zation of the expressions using constantly expressed referenceenes. The main aim of our research was to determine reli-ble house keeping genes for qPCR in maize under the effectf various boron concentrations. Initially, we focussed on theetermination of boron tolerant commercial hybrid varieties.urther, experiments have been made for the determinationf the gene/genes with constant expression level among the 6andidates reference genes namely, S18, alpha-tubulin, globu-in S, GAPDH, ubiquitin, beta2 tubulin. Results were computedsing efficient software programs like geNorm and NormFinder.wo genes, S18 and GAPDH, were found to be the mostonsistently expressed genes under implemented boron stressonditions.

Acknowledgement: This study was financially supported byhe Selcuk University Scientific Research Projects Funding UnitProject No: 13401089).


V-06

ene expression profiling of Thermoplasma volcaniumnder extreme stress conditions

ema Zabcı ∗, Semra Kocabiyik

Middle East Technical University, Turkey

Identifying the stress response mechanisms in Archaea mightave potential applications considering the industrial importancef their enzymes. In this study, as an extension of our researchn anti-stress mechanisms, we have carried out genome wideranscriptome analysis of Thermoplasma volcanium (grows opti-

◦
ally at pH 2.7 and 60 C) under extreme stress (i.e., heat- shockt 68 ◦C, hydrogen peroxide, 0.03 mM H2O2 and pH stress atH 5.0) using Agilent Custom Gene Expression Microarray.he data was analyzed by using GeneSpring Ver 12.6 Software.

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ith a pH shift from 2.7 to 5.0 most of the gene expressionas repressed: of 47 genes differentially expressed by ≥2-fold,6% were up-regulated and 34% were down-regulated. Among theotably induced genes, some code for membrane proteins, sugarnd amino acid transporters, cation transporter and antiportershich are crucial for maintaining cellular pH homeostasis. Oxida-

ive stress mostly resulted in decreased gene expression; out of 452enes expressions of which changed ≥2 fold, 75% were down regu-ated. Up regulation of ferrodoxins, oxidoreductases, thioredoxin,e-S oxidoreductases, aldo/keto reductases and dehydrogenases asresponse to H2O2 may be important for maintaining intracellular

edox potentials and detoxifications. Temperature up-shift favoredhe expression of heat-shock proteins (including GroEL, sHSP, Dna, DnaJ, GrpE) as well as genes related to carbohydrate metabolism

e.g., sugar permease).


V-07

tress responsive global regulatory mechanisms in theethylotrophic yeast Hansenula polymorpha

hsuk Kwon1,∗, Eun Hye Kim1, Doo-Byoung Oh1, Hyun Ahang2

Korea Research Institute of Bioscience and Biotechnology, SouthoreaChung-Ang University, South Korea

The thermotolerant methylotrophic yeast Hansenula polymor-ha has been regarded as an attractive model organism forundamental studies of methanol metabolism, peroxisome biogen-sis and function, nitrate assimilation, and protein glycosylation.. polymorpha has become one of the promising hosts for theroduction of recombinant proteins on an industrial scale owingo the availabilities of strong inducible promoters and a multi-opy integration system for target protein expression cassettesnto the genome. In addition, recently H. polymorpha is gainingncreasing interest for its several peculiar physiological charac-eristics, such as resistance to heavy metals and oxidative stress,nd thermotolerance since these properties are advantageous forarious biotechnological applications. However, only a limitedmount of information is available for the global regulatoryechanisms for stress response in this organism. In the present

tudy, we investigated the roles of representative signaling andegulatory proteins to understand the regulatory mechanismsoverning the osmotic and oxidative stress responses of H. poly-orpha. The hybrid histidine sensor kinases Sln1 and Nik1,istidine-containing phosphotransfer protein Ypd1, response reg-lator proteins Skn7 and Ssk1, high osmolarity glycerol pathwayegulator Hog1, and oxidative stress response regulator Yap1ere functionally characterized by mutant construction, growthhenotype comparison, in vitro protein phosphorylation, andomparative transcriptome analysis. Obtained results indicate thathe Skn7/NiK1-Ypd1-Skn7/Ssk2 two-component signal transduc-
ion pathway plays a critical role in oxidative, osmotic, and cellall stress responses in H. polymorpha.
STRESS RESPONSES

Acknowledgement: Supported by the Intelligent Syntheticiology Global Frontier Program, and the Next-GenerationioGreen 21 Program.


V-08

he role of S-nitrosoglutathione reductase in defenceesponse of tobacco plants and cells to elicitins

ereza Jendrisáková1,∗, Pavla Moricová1, Martina Zelezná1,enka Luhová1, Jan Lochman2, Tomás Kasparovsky2, Mareketrivalsky1

Palacky University Olomouc, Czech RepublicMasaryk University Brno, Czech Republic

Nitric oxide (NO) is an important signalling molecule whicharticipates in the plant immune responses. S-nitrosoglutathioneeductase (GSNOR) is known as key enzyme in NO metabolismhrough cysteine S-nitrosylation, the post-translational modifica-ion mediated by reactive nitrogen species (RNS). GSNOR belongso the family of alcohol dehydrogenases class III (ADH3; EC.1.1.1). GSNOR is highly specific for the S-nitrosoglutathione sub-trate and was demonstrated to indirectly control the total level ofrotein S-nitrosothiols.

Elicitins, extracellular proteins secreted by oomycete pathogensf Pythium and Phytophthora spp., represent effective elicitorshat trigger defence responses in plant-pathogen interactions.he aim of this work was to study a possible GSNOR role inefence response of tobacco plants and cell culture to selectedlicitins. We tested cryptogein and its mutated forms V84F and41F with different ability to trigger the formation of reactive oxy-en species (ROS) and cell necrosis. Oligandrin, another elicitinrom Pythium oligandrum, does not elicit hypersensitive reactionn plant tissue. The production of RNS and ROS was determinedfter elicitin application using specific fluorescent probes. Cryp-ogein and V84F mutant triggered significantly increased ROSevel, which correlated with decreased cell viability. On the con-rary, little effect of oligandrin and mutant L41F on growth wasbserved, whereas higher NO production was induced. Increasedctivity and expression of GSNOR were detected after applicationf cryptogein and V84F mutant. The results bring new insightnto the involvement of ROS and RNS in plant response to elic-tors and possible regulatory role of GSNOR in control of protein


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V-09

ensitivity of selected root hair mutants of Arabidopsishaliana to abiotic stress

enka Vaskebová1,∗, Barbora Satná1, Miroslav Ovecka2, Jozefˇamaj2

Palacky University in Olomouc, Czech RepublicCentre of the Region Haná, Olomouc, Czech Republic

Plant growth requires the continuous uptake of solutes andinerals from the rhizosphere by the root as well as mainte-

ance of ion homeostasis and avoidance of intracellular ionoxicity. Plants also possess effective molecular, physiological andevelopmental mechanisms to minimize harmful effects of exter-al abiotic stress factors. Root hairs are tubular extensions ofoot epidermal cells, and they are formed by specialized tiprowth. Since root hairs effectively extend surface area of theoot, they could play significant role in plant sensitivity to abi-tic stresses originating from the soil. Arabidopsis thaliana rootair mutants such as rhd2(root hair defective 2, lacking the activ-

ty of the NADPH oxidase AtRBOH C, Takeda et al., 2008) andip1 (tip growth defective 1, inactive in S-acyl transferase, Hems-ey et al., 2005) are defective in the tip growth. This suggestsheir possible involvement in root stress sensitivity. We studiedffects of abiotic stresses on root growth and root hair devel-pment in certain stages of stress perception in these mutants.n general, root growth of rhd2-1 mutant was well compara-le to Col-0 wild type, while root growth of tip1-1 mutant waseaker. Upon application of diverse abiotic stresses, rhd2-1 mutant

howed similar sensitivity as Col-0 and all lines showed a compa-able sensitivity to salt and oxidative stress. Stress responses wereested also in Arabidopsis plants stably overexpressing mitogen-ctivated protein kinase kinase SIMKK from Medicago sativa. Theseesults contribute to better elucidation of root hairs as integralarts of the root developmental program in changing environ-ent.


V-10

ources and repair of nitrosative stress in Escherichia coli

asema Balasiny ∗, Claire Vine, Jeff Cole

University of Birmingham, United Kingdom

Escherichia coli is a Gram negative, facultatively anaerobe. Insidehe mammalian gut, E. coli utilises various alternative electroncceptors to respire efficiently when oxygen is unavailable. Tourvive, it must defend itself against reactive nitrogen speciesRNS) generated as products of its own metabolism, or the innatemmune response of the host. Under anaerobic conditions, nitrateeductase NarGHI generates nitric oxide (NO) as a side product dur-ng reduction of nitrate via nitrite to ammonia. However, some
O is formed from nitrite even by mutants that lack NarGHI.itric oxide is an extremely toxic gas that damages metal centresf proteins, especially iron-sulphur proteins such as aconitase andumarase. Several studies have reported that the di-iron protein


tfE has a crucial role in repairing iron-sulphur centres damagedy nitrosative stress conditions [1–3]. This poster will show that aajor role of YtfE is to release nitric oxide that has become bound

o proteins such as aconitase and fumarase, thereby restoring theirctivity under conditions of nitrosative stress.

eferences

].Justino MC, Almeida CC, Goncalves VL, Teixeira M, Saraiva LM.Escherichia coli YtfE is a di-iron protein with an important func-tion in assembly of iron-sulphur clusters. FEMS Microbial Lett2006;257:278–84.

].Justino MC, Almeida CC, Teixeira M, Saraiva LM. Escherichia coli di-Iron YtfE protein is necessary for the repair of stress-damaged iron-sulphur clusters. J Biol Chem 2007;282:10352–9.

].Overton TW, Justino MC, Li Y, Baptista JM, Melo AMP, ColeJA, Saraiva LM. Widespread distribution in pathogenic bacteria ofdi-iron proteins that repair oxidative and nitrosative damage to iron-sulphur centers. J Bacteriol 2008;190:2004–13.


V-11

itrosative stress response in Escherichia coli

ing Wang ∗, Claire Vine, Jeff Cole

University of Birmingham, United Kingdom

Bacteria encounter various stress as the living environmenthanges. For instance, in anaerobic growth conditions when nitriter nitrate is used as electron acceptor, nitric oxide (NO) is generateduring the reduction process. Up to present, three NO reduc-ases, Nrf, Nor and Hmp have been reported in the bacteriumscherichia coli. A recent microarray study revealed several geneshat are highly up-regulated during nitrosative stress, one of whichas hcp, a gene that encodes a hybrid cluster protein (HCP) withunique structure.

We tested two E. coli strains, one lacking all the above men-ioned NO reductases and one with a further deletion of hcp. Thecp mutation decreased nitrosative stress tolerance of E. coli. Aecond gene, hcr, is co-transcribed with and located in the sameperon as hcp. We detected a positive protein interaction betweenCR and HCP in vivo using the bacterial two-hybrid system. This

upports the hypothesis that HCP might function as another NOetoxifying enzyme, with HCR being a reductase of HCP. We sub-

ected the two strains to NO stress and observed that in our controltrain NO was reduced to N2O, probably via a reaction catalysedy HCP.

Our data demonstrated that HCP is essential for the reduction

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V-12

olecular markers based approach for genetic improve-ent of tea (Camellia sinensis)

ahul Kumar1,∗, Himanshu Sharma2, Sanatsujat Singh2, Rakeshumar Sud2, Arvind Gulati 2, Paramvir Singh Ahuja2, Ram Kumarharma2

DAV University, IndiaCSIR-IHBT, Palampur, HP, India

Tea, due to multiple health benefits with various flavors andntioxidant properties is the most consumed beverage across thelobe and becomes an important agro-based revenue source forany countries in the world including India, which ranks first

n black tea production. Genetic improvement studies, however,ave been limited in tea due to non-availability of sequenceased co-dominant microsatellite markers. Novel microsatellitearkers were identified through construction and sequencing of

nriched genomic clones and mining of public expressed sequenceata in tea. These markers along with other dominant DNAarkers like AFLP and RAPD were utilized for genetic mapping

sing pseudo-test cross population comprising of 212 individualserived through crossing between parental clones SA6 (resistanto blister blight disease) and Asha (susceptible). Of one thou-and microsatellite markers identified, 576 recorded successfulmplification in selected tea accessions including parental lines.nformative 80 SSR markers along with 102 AFLP and 160 RAPDere used for construction of linkage map in tea. Following aseudo test cross approach, in total, 400 markers loci (292 AFLP,1 RAPD, 27 SSR) were found to be segregating in a test cross rationd 127 of these could be linked to 17 linkage groups coveringtotal length of 1659 cM. Novel SSRs enriched the limited reper-

oire of co-dominant markers in tea and will be further utilized inaturating the linkage map and subsequently in QTL analysis andAS in tea.


V-13

novel chitinase gene homologue from Chinese wildryenhances tolerance to biotic and abiotic stress in trans-enic tobacco

ongyun Hao1, Xiangguo Liu2,∗, Ying Yu3, Yang Liu2, Yinningu3, Shuo Yang3, Siping Han2, Shudan Feng4

Institute of Agricultural Biotechnology, IndiaJilin Academy of Agricultural Sciences (JAAS), IndiaJilin University, IndiaHarbin Normal University, India

Plant chitinase is a glycosyl hydrolase that is believed to beesponsible exclusively for pathogens stress. However, there haseen no report suggesting its function in relation to salinity stress

STRESS RESPONSES

olerance in monocot. Here, we report a new member of thehitinase family 19, designated as LcChi2, isolated from Chineseildrye (Leymus Chinensis). Sequence analysis demonstrated thatcChi2 belonged to chitinase class II subgroup containing twoonserved domains, i.e. Cys49Gly71 and Val161Met171. In compar-son with wild-type tobacco, the transgenic tobacco (Nicotianaabacum) overexpressing LcChi2 showed increased tolerance toungal and bacterial pathogens stresses. Semi-quantitative RT-PCRnalysis indicated that the expression of LcChi2 in Chinese wildryeas up-regulated with the treatment of 400 mM NaCl, 100 mMa2CO3 and 20% PEG, so was the activity of LcChi2 product

oincidently in similar treatments. Transgenic yeast (Saccharomyceserevisiae) overexpressing LcChi2 exhibited an enhanced toler-nce to 1.6 M NaCl, 10 mM Na2CO3, 1.5 M sorbitol, and 10 mMnSO4 stresses respectively. Interestingly, LcChi2-overexpressedransgenic tobacco presented increased tolerance to 200 mM NaCl,mM Na2CO3 and 500 mM sorbitol stresses during germinationnd to 600 mM NaCl stress during seedling development. This sug-ested that LcChi2 is likely an interesting candidate gene to besed in transgenic breeding to improve broad-spectrum tolerance

n crop. Current results added information to plant chitinase fam-ly in abiotic stress tolerance over its existing functions in biotic


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ynthetic Biology

W-01

esign of the novel catalytic units by key motif-directedomain recombination

an Feng1,∗, Xiaoli Zhou2, Yuan Xie3, Guangyu Yang3

State Key Lab of Microbial Metabolism, Shanghai Jiao Tong Uni-ersity, ChinaJilin University, ChinaShanghai Jiao Tong University, China

Diverse functional domains in proteins provide a resourceor designing novel biocatalysts. Recombination of more distantequences in evolution offer greater opportunity for functioneaps, but also introduces more disruptions in the chimeras. Here,e report two lipase-like chimeras engineered from a mesophilic

ipase and a hyperthermophilic peptidase/esterase with only 14%r 21% amino acid identity. Recombination was at the conservedey motif regions which are part of the hard cores of the proteinsnstead of the flexible loops between protein functional domains.he resulting chimeric lipases retained the desirable preferenceor long-chain acyl ester substrates of their mesophilic parents,ith pNP-C14 as the best substrate. Meanwhile, compared to

he mesophilic parent, the chimeras exhibited more than 100-old increased thermostability at 50 ◦C and the optimum catalyticemperature improved 40 ◦C. These results suggest that the key

otif-directed recombination (KMDR) approach promises for thefficient construction of robust hybrid enzymes.


W-02

ther-lipid membrane engineering of Escherichia coli

ntonella Caforio ∗, Samta Jain, Arnold Driessen

University of Groningen, The Netherlands

The membrane lipid composition of archaea differs from bacte-ia and eukarya in having an ether-linked, isoprenoid hydrocarbon



hain with an enantiomer sn-glycerol-1-phosphate backbone. Thisnique structure is believed to be vital for the adaptation of theserganisms to extreme conditions and the lipid divide is consideredignificant in a split of prokaryotes into archaea and bacteria. Theim of this project is to functionally introduce the archaeal etheripid biosynthetic pathway into E. coli to examine the propertiesf mixed membrane lipids, to study the archaeal lipid biosyntheticathway and to understand evolutionary aspects associated withhe lipid divide.

Using a synthetic biology approach, different modules of theathway were designed for the synthesis of the isoprenoid chainnd CDP archaeol, the precursor for polar head group attachment.he latter ether-lipid is produced by an uncharacterized enzyme,hose gene has not yet been identified in Archaea. Several in vivond in vitro assays were performed to access the enzymatic activitiesnd the hypothetical gene encoding for CDP-archaeol synthaseas expressed in E. coli and validated.

A membrane integrated precursor of the archaeal lipid biosyn-hetic pathway was produced in E. coli and formation of CDP

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X-01

ntegral exploitation of olive tree pruning in the paperndustry

uis Jiménez1,∗, Alejandro Rodríguez1, Juan Domínguez2, Anto-io Rosal2, Gustavo Cordero-Bueso2, Eva Valero2

University of Córdoba, SpainUniversity Pablo de Olavide, Spain

This work seeks to obtain valuable products through an integralxploitation of lignocellulosic residues generated by agriculture,uch as olive tree prunings. These sorts of residues are studied aslternative sources for lignocellulosic raw material in the paperndustry and the black liquors generated could be used for severalurposes, including the production of bioethanol.

To reach these goals, a central composite factorial design wassed to study the influence of operational variables [temperature155, 170 and 185 ◦C), cooking time (40, 65 and 90 min) and sodaoncentration (10, 14 and 18%)], on pulps and paper sheets prop-rties obtained from olive tree prunings, likewise black liquorsenerated in these pulping processes were set up to enable botheast strains (Saccharomyces cerevisiae CECT 1170 and Pichia stipitisECT 1922) to be capable of doing an alcoholic fermentation at0 and 26 ◦C respectively, 5.5 pH, and under 150 rpm shaking.

The results were similar to those obtained with other agricul-ural residues which are alternative sources for the paper industry.he best values of the physicochemical properties of celluloseulps, such as Kappa number (31.98) or viscosity (716.11 mL/g)nd the paper sheets, such as tensile index (608 m) or tear index1.655 mNm2/g), were reached operating to high values of sodaoncentration and temperature and slight cooking times. How-ver, the black liquors of this study were not an optimal mediumor the bioethanol production. It might be due to the importantresence of inhibitors in the fermentation processes, such as 5-ydroxymethyl-furfural.


X-02

tilization of agroindustrial residues for hydrolyticnzymes production

na Belen Diaz, Ana Blandino ∗, Ignacio de Ory, Ricardo Martin,aria Jose Munoz, Ildefonso Caro

University of Cadiz, Spain

Agricultural and agroindustrial residues are the most abun-ant resources on earth, which have significantly increased as aesult of industrialization. In recent years there has been a grow-ng trend towards efficient utilization and value-addition of theseesidues. Biotechnological processes, especially solid state fermen-ation (SSF), have contributed enormously to such reutilization as
t can convert these agro-feedstocks into a wide variety of valuablehemical products. Agroindustrial residues are generally consid-red the best substrate for the solid state processes. Given their
wc

USE OF ORGANICWASTES

omposition, rich in sugars such as cellulose, hemicellulose andectin, they can be easily assimilated by microorganisms.

Our research group has demonstrated that high levels ofydrolytic enzymes are produced by SSF of Aspergillus awamori onmixture of grape pomace and orange peels. Furthermore, these

nzymes could be safely added to food preparations for this funguss considered a GRAS (Generally Recognized as Safe) microorgan-sm. However, higher enzyme activities could be achieved by usinghe same methodology but growing different fungi. In fact, itould be interesting to be able to produce these enzymes from dif-

erent agroindustrial residues but in the same conditions, as theyould be processed in the same manufacturing facilities. More-ver, if possible and considering seasoning crops as raw material, itould be profitable for having the production plant operating forlonger time. Thus, the aim of this work is to evaluate the produc-

ion of hydrolytic enzymes by SSF of Botryotinia fuckeliana grownn several agroindustrial residues with different composition in theame conditions of fermentation.


X-03

iquid chromatography/mass spectrometry based iden-ification, cloning and characterization of thermostableacterial enzymes useful for the production of long-hain oligosaccharides from agro-industrial wastes

omeda Kuisiene ∗, Raimonda Petkauskaite, Dangiras Lukosius,ndrius Jasilionis

Vilnius University, Lithuania

Oligosaccharides are important ingredients of the functionaloods. The development of novel and highly functional oligosac-harides with physiological properties is now continuing. Overhe past few years, long-chain oligosaccharides have evoked areat interest. Such oligosaccharides are absorbed to a much loweregree and persist longer in the colon than the shorter ones. Con-equently, beneficial effect of the long-chain oligosaccharides isreater. Thermophilic microbial enzymes have potential to be usedor the production of the long-chain oligosaccharides.

We report identification of thermostable bacterial enzymesnvolved in the degradation of starch and pectin using liq-id chromatography/mass spectrometry (LC/MS) based analysis.ymographic analysis was combined with LC/MS. A range ofolysaccharide degradation associated enzymes was identifiedsing LC/MS of zymographic samples. The obtained peptidesere used for the construction of primers in order to clone

elected enzymes. Recombinant proteins were expressed, purifiednd characterized. The length of oligosaccharides produced byecombinant enzymes was determined. In order to determine con-itions to monitor the chain length of obtainable oligosaccharides,e also tested activity of recombinant enzymes at suboptimalH and temperature values. The potential of recombinant ther-ophilic bacterial enzymes in the degradation of agro-industrial
astes was also evaluated. It was shown, that long-chain oligosac-
harides can be produced from starch and pectin (including that


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Acknowledgement: The authors are thankful to the Lithua-ian Science Council for the financial support (project No.VE-08/2011).


X-04

olubilization of animal bone char by Yarrowia lipolytican medium containing glycerol

ikolay Vassilev1,∗, Bettina Eichler-Löbermann2, Vanessaartos1, Maria Vassileva1

University of Granada, SpainUniversity of Rostock, Spain

Phosphorus (P) is accepted as an essential element for all liv-ng organisms. P is mainly derived from mined rock phosphate,hich is a non-renewable resource. It has been suggested that agri-

ultural demand for P will outstrip mineable resources in 50–100ears and economically mineable resources will be depleted beforehe end of this century. Biotechnology offers a number of sustain-ble solutions that can mitigate these problems by using variousaste materials as a source of P and, on the other hand, their sol-bilization by selected microorganisms. In this work we presentesults on solubilization of animal bone char (HABO) with a highontent of phosphate by Yarrowia lipolytica. Free yeast cells wereultivated using different combinations of glycerol and HABO,he latter being simultaneously solubilized by the released citriccid. Biomass accumulation of 6.1 g/l, citric acid (8.7 g/l) and sol-ble phosphate (379 mg/l) concentrations were measured at 50 g/llycerol and 6 g/l HABO after 96 h of shake-flask cultivation. How-ver, the highest percentage of soluble phosphate of the total Pf 44.8% was obtained at 30 g/l glycerol and 2 g/l animal boneshar. Employing the latter combination, a separate experimentas carried out in fermenter where the solubilization efficiencyas enhanced to 345 mg soluble phosphate/l (61.6% of the totalhosphate) after 40 h of cultivation. The main advantage of HABOs an alternative P source is its abundance and low price. The EUroduces about 3 million tons of meat and bone residues.


X-05

lant growth enhancement by biotechnological tools

aria Vassileva1,∗, Massimiliano Fenice2, Antonia Galvez1, Niko-ay Vassilev1

University of Granada, SpainUniversity of Viterbo, Spain

An alternative, more environmentally friendly strategy for
lant growth promotion was developed which include variousypes of plant-soil improvers and low-cost P-bearing material.wo types of microbial P-solubilizers were employed: a lactic
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cid producing bacterium and the filamentous fungus Aspergul-us niger. The latter was introduced into a typical Mediterraneanegraded soil as a biotechnological product containing partially-olubilized phosphate (hydroxyapatite of animal bone origin,ABO), mineralized organic matter and mycelial mass. The lactic

cid bacterium was entrapped in alginate beads and further inoc-lated in the soil, before transplanting plant seedlings (Lavenderpica), to serve as a slow-release microbial formulation. Cultureltrate of Piriformospora indica was added to the above system

n the beginning of the experiment and after two weeks. Allreatments were supplemented with HABO. Results showed a sig-ificant plant growth promotion in treatments with immobilizedacterial cells (IC). Similar effect was observed when organic mat-er was used as an amendment. The highest plant growth wasowever registered in the treatment with both IC and microbially-

reated organic matter. This effect was more pronounced in theresence of filtrate of P. indica. Our results confirmed the ben-ficial effect of root-colonizing fungi and PGP microorganismsn plant growth. P. indica is a mycorrhiza-like root-colonizingungus which can be cultivated in fermentation systems. In ourtudy, the combination between P. indica filtrate, P-solubilizing IC,nd, on the other hand, organic matter/soluble P product resultedn a highly effective promotion of plant growth in degradedoil as an alternative of chemical-fertilizer-based conventionalcheme.


X-06

roduction of carotenoids, ergosterol and other lipidicompounds by red yeasts cultivated on lignocelluloseaste substrates

vana Marova ∗, Andrea Haronikova, Sinisa Petrik, Stanislavbruca, Iveta Kostovova

Brno University of Technology, Faculty of Chemistry, Czechepublic

Carotenogenic yeasts are a diverse group of unrelated organ-sms (mostly Basidiomycota), that can be found in soil, fresh and

arine water, on plants and also in foods. Due to its ubiquitous andorld-wide occurrence, these yeasts have been able to assimilatearious carbon sources, such as glucose, xylose, cellobiose, sucrose,lycerol, etc. Therefore, agro-industrial waste materials includingignocellulose materials can be used as cheap substrates.

Presented work is focused on growth and production activityf red yeast strains Rhodotorula, Sporobolomyces and Cystofilobasid-

um cultivated on some lignocellulose wastes: pre-treated wheytraw, pine hydrolyzates, SCG and rapeseed waste. The main aimf the current investigation was to assess the potentialities of redeasts to transform these waste substrates to high-value productss carotenoids (about 1–3 mg/g CDW), ergosterol (2–4 mg/g CDW),oenzyme Q (0.5–1 mg/g CDW), lipids (11–21% of dry mass) andatty acids as well as enriched red yeast biomass. Production prop-
rties of red yeasts were compared between tested strains andtudied also during scale-up process in 5-L laboratory fermentor.he yields of about 30 g per liter of biomass enriched by 30–50 mg

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or provitamins A, D and CoQ could serve as an additionalatural source of significant nutrition factors in feed and food

ndustry.Acknowledgement: This work was supported by project

Materials Research Centre at FCH BUT-Sustainability and Devel-pment, REG LO1211 NPS I MEYS CR”.


X-07

ruit residues: low cost substrates for development ofew food products

inna Granucci ∗, Silas G. Villas-Boas

Centre for Microbial Innovation, School of Biological Sciences,he University of Auckland, New Zealand

In recent years, there has been increasing interest in devel-ping alternative uses for agricultural residues for production ofigher added-value products. Bioconversion of these substratessing microbial fermentation could be an attractive option. There-ore, the main goal of my project is to optimise a fermentationrocess for bioconversion of fruit residues developed at labora-ory scale using apple pomace as model. In our previous work,andida utilis and Pleurotus ostreatus were employed for bioconver-

ion of apple pomace, resulting in a nutritious enriched whitishubstrate (flour-like) with attractive walnut/hazelnut aroma thatontained high protein and low sugar. We have data demon-trating that the sequential fermentation of apple pomace with. utilis and P. ostreatus resulted in 400% enrichment of pro-

ein content in the substrate in addition to increased availabilityf phosphorus, calcium and potassium as compared to non-ermented substrate. Moreover, the flavor and appearance ofhe processed substrate was very attractive, which made it idealor a human consumption product. Thus, we envisage the fol-owing main steps for further research: 1 Assess other fruitesidues to be bioconverted; 2 Scale up fermentation; and 3 Nutri-ional assessment of fermented product. This same strategy cane used for the bioconversion of different agriculture wastes,uch as residues of other fruit processing and/or dairy indus-
ries.
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X-08

OSHAN EU-project (Sustainable Production of Func-ional and Safe Feed from Food Waste): molecularharacterization of selected food waste materials

ullia Tedeschi ∗, Mariangela Bencivenni, Chiara Bottesini, Judithuller-Maatsh, Federica Meli, Arnaldo Dossena, Stefano Sforza

University of Parma, Italy

Food processing activities produce in Europe large amountsf by-products and waste. Such waste streams are only par-ially valorized at different value-added levels (spread on land,nimal feed, composting), whereas the main volumes are man-ged as waste of environmental concern, with relevant negativeffects on the overall sustainability of the food processingndustry.

The main focus of NOSHAN1 is to investigate the process andechnologies needed to use food waste for feed production at lowost, low energy consumption and with maximal valorisation oftarting wastes materials. Nutritional value and functionality asell as safety and quality issues are investigated and addressed asain leading factors for the feed production using food derived

fruit/plant and dairy). According to this not only wastes are char-cterized for their nutritional potential, but suitable technologieso stabilize them and convert them into suitable raw materials forulk feed are researched.

The topic of this communication is the characterisation, atolecular level, of the different waste streams selected. In par-

icular a detailed composition of the nitrogen fraction has beenerformed. Determination of amino acids was performed by chro-atography methods and racemisation was studied by GC–MS.

eptide and protein profiles were determined by LC/MS.Waste streams turned out to be quite promising to be trans-

ormed in feed or to be exploited as a source of feed additives, asar as their content in desirable compounds was concerned.


X-09

roduction of polyhydroxyalkanoates from anaerobicigested grape pomace by employing a pure culture ofupriavidus necator

onzalo Martinez1,∗, Lorenzo Bertin1, Joana Domingos1, Ger-art Braunegg2, Fabio Fava1

University of Bologna, ItalyTechnical University of Graz, Italy

The present work was dedicated to verify the possibility ofeplacing the carbon source commonly employed for the biotech-ological production of polyhydroxyalkanoates, i.e., glucose or

ructose, with grape pomace (GP), which is a solid organic wastef the winery industry. To this aim, a grown culture was fed withhe volatile fatty acids (VFAs)-rich effluent obtained by fermenting

1 Project full title: “Sustainable Production of Functional and Safeeed from Food Waste” Grant agreement no.: 312140.


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P under acidogenic conditions (GPAcid). GPAcid mainly containedg/L) acetic (14.69 ± 0.57), propionic (0.77 ± 0.04), iso-butyric0.83 ± 0.03), butyric (4.67 ± 0.21) and caproic (0.55 ± 0.02) acids.

Experiments were carried out in 500-mL Erlenmeyer flasksworking volume: 150-mL) at 30 ◦C and 180 rpm. The whole poly-ydroxyalkanoates production process was separated into twotages, namely: a balanced cell growth phase (by using DSMZ-1 mineral medium amended with 5 g/L of fructose) and anccumulation phase, where harvested cells were suspended inNH4 free-DSMZ-81 medium containing 20 or 40% of GPAcid

v/v). Higher VFAs concentrations were observed to inhibit poly-ydroxyalkanoates accumulation. A control experiment aimedt indirectly evaluate water matrix inhibition was performed byubstituting GPAcid with a VFAs water solution, wherein VFAsoncentrations were the same of GPAcid. First results showed aigher polymer production when employing 40% of GPAcid, with anal polyhydroxybutyrate content of 60% (cell dry weight bases).hermo-gravimetric analyses confirmed gas-chromatography onesnd identical results were obtained in the control experiment.

To our knowledge, this work represents the first attempt to pro-uce polyhydroxyalkanoates with a pure culture of Cupriavidusecator and a fermented GP as alternative carbon source.


X-10

omposition of pectins from food waste to be used inulk feed and as feed additives

tefania Baldassarre1,∗, Stefano Sforza1, Barbara Prandi1, Marcelaantarelli 1, Neha Babbar1, Kathy Elst2, Monica Gatti 1, Stefanoforza1

University of Parma, ItalyUniversity of Antwerp, Italy

Pectins are heterogeneous carbohydrate polymers found in theell wall of higher plants; they are formed by a backbone of (1->4)--D-galacturonic acid units interspersed with rhamnose residues

inked to neutral sugar side chains. They contribute to primary wallunctions such as cell strength, cell adhesion, stomatal functionnd defense response. Furthermore pectic oligosaccharides (POS)ave been proposed as a new class of prebiotics able to exert severalealth-promoting effects.

The main objective of this work is the characterization ofectins in vegetal food waste, in order to ensure their suitabil-

ty as raw material for feed production. Two raw materials wereonsidered: bulk feed prepared from vegetal food waste (rapeseednd malted barley mixed with dairy waste) and pectins extractedrom vegetal waste to be used as feed additive .In the first case,ectic substances are precipitated with ethanol and the total alco-ol insoluble residue is used for the determination of total pectinsy quantitatively measuring the total uronic acid through a colori-etric assay after acid hydrolysis. In this way it is possible to assess

he technological impact of the processing on the composition and
he extractability of pectins.
In the latter case, samples of pectic oligosaccharides (POS)xtracted by enzymatical means, are analyzed in order to deter-



ine the content of galacturonic acid, their composition in POSnd their prebiotic activity, in order to define structure–functionelationships.


X-11

ungal bioconversions of various lignocellulosic by-roducts to edible biomass

eorgios Koutrotsios ∗, Konstantinos Mountzouris, Georgios Zer-akis

Agricultural University of Athens, Greece

The world-wide trend for a continuous increase in the amountf food produced leads to the accumulation of huge quantities oflant residues. Controlled solid state fermentation of various agro-

ndustrial and forestry residues rich in lignocellulosics were treatedy selected strains of Agrocybe cylindracea, Ganoderma lucidum, Heri-ium erinaceus and Pleurotus ostreatus (phylum Basidiomycota), therocesses were monitored, and composition of end-products wasomparatively evaluated. Olive mill waste (composted or not),live prunings and grape marc were among the most promis-ng substrate ingredients for mushroom production since theirse contributed at obtaining significantly higher yields than theonventional cultivation media. In most cases, mushroom produc-ivity correlated with substrates nitrogen, lignin, hemicelluloses,nd residual mycelial content. Spent substrates exhibited higheductions in hemicelluloses and cellulose, and low in lignin;hese values in conjunction with elevated protein content advo-ated their use in animal feeds. Mushroom proximate analysishowed correlations of protein and crude fat with substrates nitro-en and lipids, whereas total carbohydrates, crude fiber and ashontent demonstrated relatively less variability among strainsnd cultivation media tested. Mushroom nutritional value coulde suitably enhanced by appropriate selection/modification ofhe growth substrates. Exploitation (incl. detoxification) of wastetreams could be combined with the generation of value-addedroducts and provide a viable solution for the sustainable use ofuch materials.

Acknowledgements: This research has been co-financed byuropean Union (ESF) and Greek national funds (NSRF) throughhe project titled “Metagenomics of ligninolytic microorganismsBioconversion of plant by-products into high-added value prod-

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X-12

nfluence of cell size on performance of microbial fuelells of single chamber using organic solid waste withmazonian and High Andeans soils from Ecuador

ashington Logrono, Celso Recalde, Magdy Echeverria

Escuela Superior Politécnica de Chimborazo, Ecuador

It was the first work done in Ecuador where compared threeolumes to produce bioelectricity in Microbial Fuel Cells of Singlehamber, employing organic solid waste as substrate with 50:50

elationships (vegetables and fruits) to enrich electrogenic bacte-ias, without renovation of microbial fuel and over a testing timef 171 days. The study used soils from Kaiptach Achuar Commu-ity – AMAZON to 1000 m.a.s.l. and the same procedure with theichan Central Community-ANDES to 4000 m.a.s.l. The samplesere taken among 20–40 cm depth of natural soils, each treatmentave used polyethylene containers as bioreactors and carbon fiberas used as both electrodes with different surface areas.

The initial physical chemical analysis indicated differences inarameters like: P, K, CaO, MgO, Rel.C/N, %H; while the initial het-rotrophs counted in the Andean soil showed higher amount, butmaller microbiological diversity, in accordance to morphologicalescription done under aerobic conditions. The means compari-on test has indicated that there are significant differences amongizes both studies cases, at the ANDES the best treatment was thentermediate, with a mean generation of 317 mV, moreover at theMAZON was the smallest size, with 270 mV. It was observed that

he cell size is a parameter influencing at performance and stabilityf Microbial Fuel Cells in accordance to the ecosystem.


X-13

ptimisation of scale-up of microbial fuel cell for sus-ainable wastewater treatment with positive net energyeneration

urania Dimou1,∗, John Andresen2, Veyacheslav Feodorovich3,gor Goryanin4, Alan Harper2, David Simpson5

Heriot-Watt University, UKDepartment of Chemical Engineering, Heriot-Watt University,dinburgh Campus, EH14 4AS, UKM Power World, Vavilova str. 5/3, Moscow, RussiaInformatics Life-Sciences Institute, Edinburgh University, Edin-urgh EH8 9AB, UKBiological Systems Unit, Okinawa Institute of Science and Tech-ology, Okinawa 1919-1, Japan

From eight to fifteen litres of liquid by-products are generatedor every litre of grain whisky produced. ‘Spent Wash’is the mainiquid stream. If discharged untreated into the environment it

ight contribute to pollution such as eutrophication [1].
Microbial Fuel Cells (MFCs) are a natural bio-technology solu-
ion to the issue working either independently or in conjunctionith established wastewater treatment technologies. Utilisingetabolic reactions of electrochemically active microorganisms,

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FCs provide a dual benefit: wastewater treatment and direct elec-ricity generation. For industrial scale-up, the approach in thisork is through plurality. Multiple units of relatively small scale

an be connected electrically and hydraulically to achieve the nec-ssary capacity [2].

Initially a single chamber open air cathode MFC of 170 mlolume treating spent wash subsequent to anaerobic digestionreatment of 2 g/l Chemical Oxygen Demand demonstrated anverage voltage of 0.4 V in open circuit and a COD reductionf 70%. Scaling up, two 100 lt units connected in parallel andperating in directly diluted spent wash at 26.4 g COD/l.d demon-trated 84% COD removal, maximum voltage 0.9 V and current60 mA. Ongoing experiments using this configuration and previ-usly anaerobically treated spent wash demonstrate encouragingesults, bringing practical industrial scale Microbial Fuel Cell treat-ent closer.

eferences

].Mohana S, Acharya K, Madamwar BD. Distillery spent wash:treatment technologies and potential applications. J Hazard Mater2009;163:12–25.

].Gálvez A, Greenman J, Ieropoulos I. Landfill leachate treatmentwith microbial fuel cells; scale-up through plurality. Bioresour Technol2009;(100):5085-509.


X-14

ew application of agro-industrial waste to preventelanosis of Mediterranean pink shrimp

iorgio Rizza ∗, Giuseppina Rosaria Antonella Alberio, Rosaalmeri, Aldo Todaro, Giovanni Spagna

University of Catania, Italy

Food byproducts represent an environmental and economicroblem because peel, pulp, pulp wash, and yellow water areifficult to digest. Additionally, citrus byproducts are relativelyesistant to microbial degradation (high COD and BOD5 indexes)ue to their high content of bioactive compounds with antimi-robial activity, e.g. ascorbic acid, limonoids, and polyphenols.n addition, agro-industrial wastes are potential sources of bio-henols that have proven antioxidant, anti-inflammatory andnticancer properties. Aims of this work is to test how naturalxtracts obtained from vegetable waste, in particular orange andemon peel, may inhibit the process of melanosis in pink shrimppecies (Parapeneus longirostris). The treatment with orange peel,xtracted in hot water, significantly reduced (p < 0.05) the enzy-atic activity of the PPO at the level of the cephalothorax of

hrimp. Ethanolic extract of lemon peels was also effective for inhi-ition of melanosis. The results were related to the evaluation ofhe polyphenol quality index (QI) that confirmed the efficacy ofreatment of the orange and lemon peel extracts. The addition ofhese natural extracts in pink shrimp samples can be considered a



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ETERINARY BIOTECHNOLOGY

eterinary biotechnology

Y-01

xpression and bioactivity analysis of the expression ofEDV COE gene in Pichia pastoris

umu Li ∗, Fazhi Xu, Qingsong Hu, Xiaoling Ding, Xiongyuan Si

Anhui Agricultural University, China

The purpose of this study was to express the porcine epidemiciarrhoea virus (PEDV) COE protein in Pichia pastoris, and eval-ate the neutralizing ability of mice serum after immunization.

pair of primers was designed based on the cloned PEDV Sucleotide sequences. Intestinal tissues were collected from pigletuffering from PEDV, and the mRNA of PEDV was extracted byhe TRIzol reagent. The PEDV COE gene was amplified by RT-CR. Furthermore, the PEDV COE gene was inserted into pPIC9K,nd the recombinant plasmid of pPIC9K-COE was transformednto P. pastoris GS115 by electroporation. High copy recombinanttrains were screened and then expression was induced by addi-ion of methanol. SDS-PAGE and Western blotting were used tonalyse the immunogenicity of recombinant protein. The neutral-zing ability of mice serum antibodies by PEDV COE recombinantas analyzed by virus neutralization test. PEDV COE gene wasmplified by RT-PCR, and it was 530 bp in length .The PEDV COErotein was expressed in P. pastoris. The MW of the proteins wasbout 30 kDa as analyzed by SDS-PAGE, and the concentration was0 mg/L. Western blotting showed that the protein had immuno-enicity. The virus neutralization test result showed that the micemmunized with the recombinant PEDV COE protein producedpecific serum antibodies with neutralizing activity, the neutraliza-ion titer reached 1:36. PEDV COE protein was correctly expressedn P. pastoris and the protein had a good biological activity.


Y-02

rotective effects of catalpol against hydrogen perox-de induced oxidative stress on preimplantation porcinembryos

eog-Bon Koo ∗, Yong-Hee Lee, Sung-Hun Min, Jin-Woo Kim, Jae-yun Ahn, Geon-Yeop Do, Sung-Kyu Chae

Daegu University, South Korea

Catalpol, an iridoid glucoside, isolated from the root ofehmannia glutinosa, possesses a broad range of biological andharmacological activity including anti-tumor, anti-inflammationnd anti-oxidant by acting as a free radical scavenger. Therefore,n the present study, the effects of catalpol on blastocyst develop-

ent and expression levels of reactive oxygen species (ROS) werenvestigated in preimplantation porcine embryos. After in vitro

aturation and fertilization, porcine embryos were cultured fordays in porcine zygote medium 3 (PZM-3) with catalpol (0,

00, 200 and 400 �M respectively). Blastocyst development wasot significantly improved in the catalpol treated groups whenompared the control group. However, subsequent evaluation of

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he intracelluar levels of ROS and numbers of apoptotic nuclein catalpol (100 �M) treated blastocysts revealed that ROS levelsf catapol-treated porcine blastocyst were decreased (P < 0.05) andhe numbers of apoptotic nuclei were reduced by catalpol treat-

ent in porcine embryos. Moreover, the blastocyst developmentnd total cell numbers of blastocysts were significantly increasedn the catalpol treated group relative to the untreated catalpolroup under H2O2 (200 �M) induced oxidative stress (P < 0.05).urthermore, the intracellular levels of ROS in catalpol-treatedroup were significantly decreased in the untreated catalpol groupnder H2O2 induced oxidative stress (P < 0.05). In conclusion, ouresults suggest that catapol improves the developmental compe-ence of porcine embryos via modulation of intracellular levels ofOS and the apoptotic index during the preimplantation stage.


Y-03

tudy on the establishment of the animal model infectedy influenza virus in Microtus brandti

efeng Wu ∗

Fujian Agriculture and Forestry University, China

In this study, the brandti voles were infected by influenza virus/WSN/33(H1N1) intranasally, in order to assess the susceptibilityf influenza virus on brandti voles. The brandti voles showed highusceptiblity to infection. The infection susceptibilty was furthervaluated to establish a possible susceptibility index for brandtioles model infected by influenza virus.

By means of the method of infecting brandti voles, influenzairus was separated and identified successfully. The result showedhat the virus was widely distributed in brandit vole tissues.hich also indicated that the brandti vole are highly sensitive

o influenza virus, and the major target organ was lung. The ani-al model of brandti voles infected by influenza virus has been

stablished successfully by means of some model indexes, suchs clinical symptoms, body weight, mortality, the index of theeparation of influenza virus and so on.


Y-04

Salmo salar diet based on a marine biosurfactant forhe profilaxis and treatment of Pisciricketsia salmonis

laudia Ibacache-Quiroga1,∗, M. Alejandro Dinamarca2, Juanjeda2, José Miguel Troncoso3

Centro Nacional Biotecnología/Micromarine Biotech, SpainUniversidad de Valparaíso, ChileEwos, Chile

Aquaculture is a fast growing economic activity due to an
ncrease of food demand worldwide, which main challenges areacterial infections that causes significant biomass and economicosses. Although classic antibiotic therapy was effective against

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acterial infections in aquaculture, nowadays these therapiesre unsuccessful due to an increase of antibiotic resistance.e recently reported that a specific marine biosurfactant (BS)

nterferes with quorum sensing (QS) cell-to-cell communicationystem, reducing virulence of bacterial fish pathogens. BS do notct modifying bacterial cellular processes, therefore no resistanceo BS is developed, making these molecules an alternative to these of antibiotics in aquaculture. The aim of this study was to incor-
orate the biosurfactant into fish food and to evaluate the effectf the supplemented diet on fish survival to Pisciricketsia salmonis.S was incorporated by emulsification into fish food. The effect ofhe supplemented diet was evaluated through a challenge assay,
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VETERINARY BIOTECHNOLOGY

n which Salmo salar was directly exposed to the fish pathogen. salmonis. Fish were fed with food supplemented with BS at anal concentration of 400 mg/kg of fish/10 days, during thirtyays. Fish diet with BS showed a survival rate 15% higher thanhe control diet (without BS). The immune makers IL-1� and IL-8valuated by qRT-PCR showed an increase in gills and intestineespect to the control diet. The food containing the BS and serumamples form treated fish were active for the inhibition of QS androwth of P. salmonis, demonstrating the presence of the active
rinciple.


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east and filamentous fungi

Z-01

ew source of hyaluronidase – Fistulina hepatica

enka Bobková1,∗, Dzianis Smirnou1, Martin Krcmár1, Veronikaoravcová2, Martina Hermannová2, Vladimír Velebny3

Contipro Biotech s.r.o, Czech RepublicContipro Pharma s.r.o, Czech RepublicContipro Group s.r.o, Czech Republic

Hyaluronidases are a group of enzymes capable of hyaluroniccid (HA) depolymerization. These enzymes can be divided intoroups according to the mechanism of HA cleavage or sourcef origin. Bovine testicular hyaluronidase (hydrolase) and bacte-ial hyaluronidases (lyases) are the main ones used in cosmeticr medicinal applications nowadays. Fungi as a new source ofyaluronidases were not authentically described until recently [1].

This work was focused on novel hyaluronidase productiony the Basidiomycete Fistulina hepatica. The enzyme activity wasetected in culture medium. Characterization of the enzyme waserformed after chromatographic purification. Products of HAleavage were identified by HPLC and MS. The reaction pro-eeded in an eliminative manner and unsaturated hyaluronanligosaccharides were formed. This type of mechanism was soar described only for hyaluronan-lyases produced by bacteria.ptimal conditions for HA–oligosaccharide production were pHand 20 ◦C. The enzyme was stable for 8 days under these

onditions.

eference

].Bakke M, Kamei J, Obata A. Identification, characterization,and molecular cloning of a novel hyaluronidase, a member ofglycosyl hydrolase family 16, from Penicillium spp. FEBS Lett2011;585:115–20.


Z-02

articles prevent pellets: microparticle enhanced culti-ation MPEC increases 2-phenylethanol production inspergillus niger

aria M.W. Etschmann ∗, Ina Huth, Jens Schrader, Dirk Holt-ann

DECHEMA Research Institute, Germany

Adding microparticles to fungal shake flask cultivations pre-ents pellet formation and leads to finely dispersed and highlyroductive mycelia. This addresses a problem occurring in many

ndustrial processes using filamentous fungi in submerged cul-ures: formation of pellets. Fungi tend to grow in agglomerateshich can be as large as several centimeters in diameter. The cen-

er of the pellets is badly supplied with oxygen and nutrients and
an consist of essentially dead biomass. As only the biomass inhe outer shell is active, productivity of these processes can beow. Techniques to increase productivity are therefore in highemand.
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The production of the rose-like aroma compound 2-henylethanol (2-PE) by Aspergillus niger spp. is used as anxample to investigate the effect of microparticle enhanced culti-ation MPEC on the production of fungal secondary metabolites.he production of the higher alcohol 2-phenylethanol from L-henylalanine was up to now mainly investigated in yeasts.owever, Aspergillus niger DSM 821 is also capable of synthesizing-PE. It yields product concentrations of up to 700 mg/L, which iswice as much as previously investigated strains. Screening of 16ypes of particles of different chemical composition showed thathe application of MPEC increased the final concentration by a fur-her 30%. Experiments for downscaling the technique to 48 well

icrotiter plate scale is under way, results we be available by theime of the congress.


Z-03

he cellulase induction system in Trichoderma reeseitrains remains well conserved despite several genera-ions of random mutagenesis and screening

ante Poggi-Parodi1,∗, Aurelie Pirayre1, Thomas Portnoy1,ugues Mathis1, Thiziri Aouam1, Frédérique Bidard1, Stéphanee Crom2, Antoine Margeot1

IFP Energies Nouvelles, FranceUniversité Pierre et Marie Curie, Laboratory of Developmentaliology, CNRS UMR7622, Paris, France

Trichoderma reesei is the main industrial producer of enzymesegrading cellulosic and hemicellulosic biomass because of its highrotein secretion capacity. This outstanding level of cellulases pro-uction is the product of several rounds of random mutagenesisnd screening in the past.

In this study, using a well-studied lineage of improved strainsrom T. reesei, NG14 and RUT C30, we investigated the linksetween the mutations generated during the mutagenesis screens,hat allowed development of these strains, and the transcriptomeifferences in the first hours of the cellulases induction process.

Despite a high number of reported mutations and very differentroductivities, the transcriptome from both strains were surpris-

ngly similar, with few genes being over or under expressed inhe higher producer strain. Comparison of transcriptomes of the

other strain QM6a and RUT C30 before induction of cellulaseroduction revealed that most of observed changes between NG4 and RUT C30 were probably due to basal changes to the cellhysiology, and that the cellulase production system was essen-ially intact. Accordingly, very few mutated genes seem to belongo the cellulase induction system, suggesting that there is room formprovement regarding this system.

This study shows that systems biology methods can gathernformation about cellular systems that cannot be obtained byraditional techniques, and that modern strain developmentrograms should include both approaches to construct compet-

tive strains.


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Z-04

haracterization of the relevant genes and developmentf salt-tolerant yeast strains by transposon mutagenesis

yun-Soo Kim ∗, Hye-Min Kang

Department of Food Science and Industry, Jungwon University,outh Korea

Kimchi is a traditional, fermented Korean food prepared withifferent vegetables, spices, and ingredients and is an importantietary source of vitamins, minerals, and other nutrients. Theaste brine from manufacturing process of kimchi is releasednd eventually results in serious environmental pollution. Sac-haromyces cerevisiae strains tolerant to salt stress are importantor the production of single cell protein by using kimchi wasterine and removal of potential pollutants from the waste water.n this study, approximately 3000 transformants of the mTn3-

utagenized genome library were selected as leucine prototrophsnd replica-plated to both YPD agar and YPD agar containing 10%aCl. Three strains (Tn 1–3) tolerant to up to 10% NaCl were iso-

ated by screening a transposon-mediated mutant library. Threeransposon mutants showed higher ethanol production and grewaster than control strain when cultured in rich media contain-ng 5%, 7.5%, and 10% NaCl respectively. The determination ofransposon insertion sites and Northern blot analysis identifiedutative genes and revealed simultaneous down-regulations andisruptions of the genes indicating that salt tolerance can be con-erred. The genes identified in this study may provide a basis forhe application in developing industrial yeast strains.


Z-05

picoccum purpurascens (Didymellaceae, Ascomycota) asnon-model source of novel bioactive compounds:

iotechnological perspectives

ikhail Fokin1,∗, Bevan Weir2, Ting-Li Han3, Neethu Arun3,ydia Yamaguchi4, Massuo Jorge Kato4, Silas Villas-Boas3

University of Auckland, New ZealandLandcare Research, New ZealandCentre for Microbial Innovation, School of Biological Sciences,he University of Auckland, New ZealandChemistry Institute, University of São Paulo, Brazil

Filamentous fungi have been widely used as a source of valuableioactive compounds for medicine, agriculture and white biotech-ology. Recently, genomic studies have significantly improvedur knowledge of the functional background for known com-ounds and revealed a vast amount of novel secondary metabolitesot commonly expressed in the laboratory. Among fungi thesetudies have been focused mostly on a few model species. Epic-
ccum purpurascens is a widespread member of Dothideomyceteslass, which consists of over 20 thousands species. E. purpuras-ens has been isolated from different substrates such as plants,oil, and marine invertebrates. Several bioactive compounds were
mr

YEAST AND FILAMENTOUS FUNGI

solated from different E. purpurascens strains, including epicoc-aene, a broad-range fungicide, discovered and patented by ourroup. It is remarkable that the biology of this fungal speciesas not been much studied, and we have very little knowledgen its genetic and phenotypic diversity. Only recently the genuspicoccum was placed in the newly described family Didymel-aceae, and great intraspecific diversity of E. purpurascens has beeneported. We determined the genetic and metabolite profiles ofifferent E. purpurascens strains isolated in New Zealand, and weave found that metabolite profile was closely associated with thebility of each strain to produce biologically active compounds,hich did not match their similarities based on their genetic pro-le. We have now de novo sequenced and assembled the draftenome of our working E. purpurascens strain. Further annotationf the genome and specific mining for secondary metabolite genelusters will strongly facilitate biotechnological use of E. purpuras-ens.


Z-06

he effect of carbon sources on upstream regulatoryegions controlling the expression of the Candida utilisaltase gene

án Krahulec ∗, Hanka Bonková, Veronika Lisková, Michaelasadská, Stanislav Stuchlík, Ján Turna

Faculty of Natural Sciences, Comenius University in Bratislava,lovakia

In the last few years it has been demonstrated that the industri-lly important yeast system Candida utilis represents a promisingxpression host, generating relatively high levels of recombi-ant proteins. Nevertheless, basic knowledge of its gene structurend regulation of gene expression, which is needed to allowore extensive use of this organism, is lacking. The current

tudy presents preliminary characterization of the carbon source-ependent expression of the C. utilis maltase gene in order toompare its regulation. Our results showed that C. utilis maltases able to hydrolyze cellobiose and soluble starch, and its affinityor cellobiose is even three times higher than for maltose. To fur-her investigate the function of maltase and the use of the maltaseromoter for the heterologous protein expression, we successfullypplied the Cre-loxP system to acquire a null mutant strain withisrupted maltase gene and promoter in the polyploid yeast C.tilis. The first step was to introduce a mutagenesis cassette har-oring a marker gene between two loxP sites into the target cells.fter identification of a partial mutant strain, the same mutagen-sis cassette was repeatedly used and the null mutant strain waselected on higher antibiotic concentration. The last step was tontroduce a Cre-expression plasmid and screen for marker rescue

Acknowledgements: This work is result of project imple-entation: “Production of biologically active agents based on

ecombinant proteins” (ITMS 26240220048) supported by the


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Z-07

haracterizing the sterol specificity of microbial andammalian acyltransferases

olly Stolterfoht1,∗, Barbara Petschacher1, Katharina Littringer1,artin Lehmann2, Hans-Peter Hohmann2, Harald Pichler3

ACIB GmbH, AustriaDSM Nutritional Products Ltd, AustriaInstitute of Molecular Biotechnology, TU Graz, Austria

Sterols are essential lipid components of eukaryotic membranesnfluencing membrane fluidity, membrane permeability as wells the activity of membrane-bound proteins. In case of excessiveterol formation, sterols are stored as sterol esters in lipid parti-les as energy reservoir or stock of membrane building blocks.he free sterol 3-OH group is acylated by the integral membraneroteins acyl coenzyme A: sterol O-acyltransferases (ACATs). Thistorage mechanism is conserved from yeasts to plants and mam-als, although the main membrane sterols differ between the

ingdoms of life. In yeasts, ergosterol is formed while mammalianembranes contain mainly cholesterol.For the two S. cerevisiae acyltransferases, differences in sterol

pecificity have been shown for Are2p preferring ergosterol andre1p esterifying also ergosterol precursors [1]. Here, we compare

n a cross-species study the sterol specificities of ACATs. Therefore,e expressed endogenous acyltransferases Are1 and Are2, and sev-

ral heterologous microbial and mammalian acyltransferases, inS. cerevisiae are1are2 knockout strain. ACAT substrate specifici-

ies were determined in vitro by activity assays using microsomalreparations, radioactively labeled oleoyl-CoA and ergosterol- andholesterol-like sterol substrates.

eference

].Zweytick D, Leitner E, Kohlwein SD, Yu Chunjiang, Rothblatt J,Daum G. Contribution of Are1p and Are2p to steryl ester synthesis in
the yeast Saccharomyces cerevisiae. Eur J Biochem 2000;267:1075–82.

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Z-08

election of preservation conditions for Debaryomycesansenii yeasts killer toxins

arbara Zarowska1, Lukasz Bobak2, Piotr Regiec3, Adam Figiel4,onika Grzegorczyk5, Marek Szołtysik2, Maria Wojtatowicz5,ymena Polomska5,∗

Wroclaw University of Environmental and Life Sciences, Wro-law, PolandThe Department of Animal Technology and Quality Manage-ent, Wroclaw University of Environmental and Life Sciences,roclaw, Poland

The Department of Food Storage and Technology, Wroclaw Uni-ersity of Environmental and Life Sciences, Wroclaw, PolandThe Institute of Agricultural Engineering, Wroclaw University ofnvironmental and Life Sciences, Wroclaw, PolandThe Department of Biotechnology and Food Microbiology, Wro-law University of Environmental and Life Sciences, Wroclaw,oland

Killer toxins of Debaryomyces hansenii yeast species are proteinsith an ecological regulation function: they are active againstther yeast species and filamentous fungi. The study investigatedhe possibility of protein concentration by ultrafiltration or evap-ration under vacuum, and then drying by means of spray dryingnd lyophilization.

Ultrafiltration on a membrane with 18 kDa cut-off point hasroved to be an effective method for killer protein concentrationf the tested strains. The use of membranes with a higher cut-offesulted in the activity decrease of the killer protein in the obtainedoncentrates, and at the same time, relatively high activity in fil-rates. Comparably good results, as in the case of the ultrafiltrationith membrane of 18 kDa cut-off, were obtained using the vacuum

vaporator. Spray drying resulted in the loss of activity of lethalreparations. The smallest losses of 15% were observed in prepa-ations dried at 100 ◦C of inlet air. The best method to stabilizehe killer toxin was freeze-drying. Immediately after lyophilizationnd after three months of storage killer toxins exhibited activityimilar to that before preservation.

Acknowledgements: This work was supported by researchrant POIG.01.03.01-02-080/12, co-financed by the Europeannion from the European Regional Development Found.


Z-09

valuation of Geotrichum candidum deacidificationotential for yoghurt keeping quality

mran Muhammad, Saima Riaz, Nawaz Farah ∗

Department of Microbiology, Quaid-i-Azam University,slamabad, Pakistan

Lactic acid bacteria have received a lot of research attention dueo their diverse technological and metabolic characteristics in fer-

ented dairy products. The present study was designed to studyhe impact of microbial population on maintaining the quality of

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ermented milk. Post fermentation acidification is a serious con-ern in dairy industry owing to its potential detrimental effectsn yogurt safety and quality. In order to rectify post acidificationroblem in yogurt, the deacidifying ability of G. candidum was uti-

ized. G. candidum and starter culture when used in combinationere found to have positive influence on the stability and qual-

ty of the yogurt. Growth parameters such as pH, acidity, syneresis,otal solid content, sensory evaluation and viscosity were analysed.esults have shown that G. candidum remarkably maintained pH to.75 from 48 until 196 h as compared to control yogurt. Similarlyolid contents were higher and reduced syneresis was recorded forogurt with G. candidum.

In the light of above findings it could be inferred that this mixulture starter could be a better option for industry for controllinghe post fermentation acidification problem in fermented dairyroducts thus contributing towards safety and stability of finalroduct.


Z-10

enes encoding DNA polymerases on linear dsDNA plas-ids of Debaryomyces hansenii yeasts share very high

omology

ymena Połomska1,∗, Cecile Neuveglise2, Zbigniew Lazar3, Bar-ara Zarowska4

The Department of Biotechnology and Food Microbiology, Wro-law University of Environmental and Life Sciences, FranceINRA, MICALIS, AgroParisTech, Thiverval-Grignon, FranceWrocław University of Environmental and Life Sciences; INRA,ICALIS, AgroParisTech, Thiverval-Grignon, France

Department of Biotechnology and Food Microbiology, Wrocław
niversity of Environmental and Life Sciences, Wrocław, Poland
Debaryomyces hansenii yeasts can keep natural plasmids in theirytoplasm occurring in systems with two or three linear dsDNA

YEAST AND FILAMENTOUS FUNGI

olecules of different size. Those plasmids replicate independentlyrom cell nucleus due to the encoding of their own DNA poly-

erases together with the terminal protein being a very importantlement of replication initiating complex.

In this work seven new D. hansenii plasmid systems (four dou-le and three triple) were sequenced by NGS and the sequencesf genes encoding DNA polymerases were compared to each othernd to previously known plasmid sequences from D. hansenii andther yeast species. All genes of D. hansenii DNA polymeraseshared very high homology with corresponding plasmids (themallest or the largest plasmids from the systems) with identityn the level of 95–96%. Also high homology, about 72% of iden-ity, was observed with genes of plasmids from other yeast speciesike Kluyveromyces lactis, Babjeviella inositovora, MIllerozyma acaciae,chwanniomyces etchellsii. Within one plasmid system only resid-al similarity in analyzed genes was observed; a few percent of

dentity.Acknowledgements: This work was financially supported by

olish Ministry of Science and Higher Education. Project N N 312


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