Novel Frontiers in the Production of Compoundsfor Biomedical Use,

Volume 1

Edited by

ANNIE VAN BROEKHOVENFRED SHAPIRO

lnnogenetics N. V, Ghent, Belgium

and

JOZEFANNECatholic University qfLeuven, Leuven, Belgium

KLUWER ACADEMIC PUBLISHERSDORDRECHT / BOSTON / LONDON

TABLE OF CONTENTS

EDITORS PREFACE v

PART 1 - GENOMICS: THE NEW APPROACH TO THEDISCOVERY OF NEW COMPOUNDS (3

The genomics approach: is it really the solution? 15Raul Goldschmidt and Karen Bush

Summary 15l.The Traditional Approach 152. The Biochemical Genetics Approach 163. The Genomics Approach 17

3.1 Target identification 173.2. Comparative genomics 17

4. Perspectives for an Integrated Approach 20References 21

The Contribution of Genomics to the Discovery of new Antibiotics 23David J. Holmes, John P. Throup, Nicola G. Wallis, Martin K. R. Burnham,Magdalena Zalacain, Sanjoy Biswas, Alison F. Chalker,Karen A. Ingraham, Andrea Marra, Alex Bryant, Gary Woodnutt, Patrick V.Warren, James R Brown, Martin Rosenberg

Abstract 231. Introduction 243. The Properties of an Antibacterial Target 24

3.1 Novelty 243.2 Spectrum/Selectivity 253.3 Expression during infection 253.4 Essential for cell viability 263.5 Amenable to high-throughput screening 26

4. Aminoacyl tRNA synthetases 275. Two Component Signal Transduction Systems 286. Discussion 29References 29

PART 2 - ANTIBIOTICS 33The antibiotic gallidermin - evolution of a production process 35

Markus Kempf,' Uwe Theobald and Hans-Peter Fiedlerl.The antibiotic gallidermin 352. The improvement of the production process 363. Stabilisation of product-formation 364. Stability test 375. Hs-value 37

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6. Economic improvement of the production process 416.1 Nutrient sources 426.2 Development of scale-up procedure 45

6.2.1 Development of a fed-batch process 456.2.2 Investigations of batch processes 496.2.3 Small scale fermentations 506.2.4 Variation of parameters in shake flask experiments 506.2.5 Pilot scale fermentations 516.2.6 An optimised scale-up procedure for the production of gallidermin . 52

References 54Resistance to P-lactams, a self-regenerating problem 57

Jozsef Aszodi* and Andre Bryskier1. Resistance to Beta-Lactams 572. Mode of Action of p-Lactams 573. Beta Lactamases 59

3.1. Class A P-Lactamases 603.2. Class C p-Lactamases 613.3. Class D p-Lactamases 613.4. Class B p-Lactamases 62

4. PBPs 624.1. Naturally Resistant Pathogens 634.2. Intrinsic Resistance Through Acquisition of Resistant PBPs 634.3. Mosaic Genes 654.4. Point Mutations 66

5. Penetration Barrier 666. Antibiotic Efflux 687. The future of P-Lactams 69

7.1. New Families 707.2. New Generations 707.3. Potentiators of P-Lactams 72

8. Conclusions 74References 75

Resistance to aminoglycoside antibiotics: Function meets structure 85Gerard D. Wright and Albert M. Berghuis

Abstract 851. Introduction 852. Aminoglycoside modifying enzymes 86

2.1. ANT 872.2. AAC 912.3. APH : 93

3. Conclusions 96Acknowledgements 96References 97

The genetics and biochemistry of resistance to glycopeptide antibiotics 99P. E. Reynolds

Summary 991. Action of Glycopeptides: Vancomycin and Teicoplanin 992. Potential Mechanisms of Glycopeptide Resistance 1003. Glycopeptide Resistance in Enterococci 101

3.1. The VanA phenotype 1013.1.1. Peptidoglycan Synthesis: aNew Pathway 1023.1.2. Peptidoglycan Synthesis: Control of Normal Host Pathway 102

3.2. Similarity and diversity of the resistance operons of VanA-, VanB- andVanD-type enterococci 103

3.2.1. VanB-type resistance 1053.2.2. VanD-type resistance 105

3.3. VanC-type resistance 1083.3.1. VanE-type Resistance 109

4. Glycopeptide Resistance in Staphylococci 1095. The Future 110Acknowledgement 112References 112

p-Lactamases, an old but ever renascent problem 117Andre Matagne, Moreno Galleni, Nezha Laraki, Gianfranco Amicosante,Gianmaria Rossolini and Jean-Marie Frere

Abstract 1171. Introduction 1182. The target of penicillin and other P-lactams 1183. Resistance mechanisms 1194. p-Lactamases 1215. Carbapenems and carbapenem-hydrolysing P-lactamases 1236. Hydrolysis of third-generation cephalosporins : the TEM and SHV variantsl247. Inhibitor-resistant enzymes 1258. Overproduction by deregulation of the induction system 1269. Conclusion 127Acknowledgements 127References 128

Metabolic flux analysis in streptomyces coelicolor: 131Fereshteh Naeimpoor and Ferda Mavituna

Abstract 1311. Introduction 131

1.1 Streptomycetes ..." 1311.2 Streptomyces coelicolor 1331.3 Polyketides, peptide antibiotics and streptomycetes 1341.4 Metabolic engineering 135

2. Metabolic Flux Analysis in S. coelicolor 1352.1 Metabolite and Product Excretions 136

3. Results and Discussion 1363.1 Model description 1363.2 Effect of Different Nitrogen Sources on Biomass Yield 137

3.3 Metabolite excretion with different nitrogen sources 1384. Conclusion 140

Acknowledgements 142Nomenclature 143References 143

Metabolic engineering of the lysine pathway for P-lactam overproduction inPenicillium chrysogenum 147

Casqueiro, J., Banuelos, O., Gutierrez, S. and Martin, J.F1. Lysine biosynthesis: synthesis of a-aminoadipic acid a precursor of p-lactamantibiotics 1472. Relationships between lysine and penicillin biosynthesis 1493. Metabolic engineering of the lysine pathway in P. chrysogenum 151

3.1.- Metabolic engineering at the cc-aminoadipate reductase level:Channelling of lysine metabolic flux towards penicillin biosynthesis 1513.2.- Metabolic engineering at the homocitrate synthase level 152

4.- Future perspectives 156Acknowledgements 156References 156

Glycosylation of antibiotics and other agents from Actinomycetes 161Wolfgang Piepersberg

Summary 1611. Introduction : 1612. Sugars and Cyclitols as Building Blocks in Actinomycete SecondaryMetabolites: Pathways for Modified Sugars and Cyclitols 162

2.2. 6-Deoxyhexoses (6DOH) in Glycosylation of Antibiotics and OtherBioactive Secondary Metabolites 1622.3. Cyclitols 1642.4. Glycosyltransferases 164

3. Examples 1653.1 The Streptomycin Pathway 1653.2 Macrolide Sugars 1653.3 Lincosamine 166

4. Conclusion 166References 167

Enzymatic synthesis of amoxicillin 169A.C. Spiess and V. Kasche

1. Introduction 1692. Theory 170

2.1. Solid phase and dissolution 1702.2. Suspension to suspension conversion 1722.3. Limiting regimes 173

3. Materials and Methods 1743.1Enzymes and reagents 1743.2 Enzyme activity assay 1743.3 HPLC analysis 175

3.3 Solubility measurements 1753.4 Determination of kcatandKm 1753.5 Synthesis of amoxicillin in homogeneous reaction 1753.6 Synthesis of amoxicillin in heterogeneous reaction 1763.7 Suspension pictures 1763.8 Conjugation with fluorescent dyes 1763.9 CLSM (Confocal laser scanning microscopy) image acquisition 1763.10 Reactor cycle: 1773.11 Image processing: 1773.12 pH calibration and pH measurement using CLSM 1773.13 Bipolar membrane module. Construction and operation 1773.14 Module characterisation 177

4. Results and Discussion 1784.1. Solubility and rate of dissolution 1784.2. Kinetic parameters of amoxicillin synthesis and hydrolysis 1794.3. Evaluation of amoxicillin synthesis progress: pH, T, / - dependence.... 1804.4. Effect of concentration variation and pure substrate phase 1834.5. Comparison of soluble and immobilised enzyme 1844.6. Activity and selectivity of immobilised enzymes 1854.7. pH profiles in immobilised biocatalysts under reaction 1864.8. Mass transfer limit and yield prediction 1874.9. Proposal for integrated reaction separation process 187

5. Conclusion and prospects 189References 190

PART 3 - PRODUCTION OF THERAPEUTIC ANTIBODIES 193New Recombinant bi- and trispecific antibody derivatives 195

Nico Mertens, Reinilde Schoonjans, An Willems, Steve Schoonooghe,Jannick Leoen and Johan Grooten

Summary 1951 .Introduction 1962.Material and Methods 199

2.1 Cell lines 1992.2 Plasmids and gene assembly 1992.3 Production and purification of recombinant antibody fragments 2002.4 T-cell proliferation assay 200

3. Results and discussion 2003.1 Heterodimerization by CL-CH1 interaction in eukaryotic cells depends onextension with VI and VH domains 2004.2 Fab-scFv fusion molecule as a model system for intermediate sized BsAbproduction 2024.3 Fd:L mediated heterodimerization of two scFv molecules leads to efficientexpression of trispecific molecules 2034.4 Influence of linker length and composition on production andheterodimerization 204

4. Discussion 205

References 206Advantages of single-domain antigen-binding fragments derived from functionalcamel heavy-chain antibodies 209

Muyldermans Serge, Conrath Katja, Vu Khoa Bang, Serrao Teresa, BuschMagnus, Backmann Natasha, Silence Karen, Lauwereys Marc, DesmyterAline

1. Introduction 2092. Functional heavy-chain antibodies in sera of camelids 2103. Single antigen-binding domain of camel heavy-chain antibodies 2104. Cloning and selecting the camel variable domains of heavy chain antibodies

2115. Characteristics of the single domain antibody fragments 212

5.1. Expression yield 2125.2. Solubility 2125.3. Stability 2125.4. Specificity and affinity 2135.5. Enzyme inhibition 2135.6. Multivalent constructs 2145.7. Intrabodies 214

Acknowledgements 214References 214

PART 4 - HETEROLOGOUS PROTEIN PRODUCTION:NEW PRODUCTION STRATEGIES 217

Furin as a tool for the endoproteolytic maturation of susceptible recombinantbiopharmaceuticals 219

M. Himmelspach, B. Plaimauer, F. Dorner and U. Schlokat1. Introduction 2192. Sorting and processing of secretory proteins 220

2.1. The constitutive and regulated secretory pathways ; 2202.2. Endoproteolytic processing of precursor proteins 222

3. The pro-protein convertases 2223.1. Identification of eukaryotic pro-protein convertases 2223.2. Tissue distribution, sublocalisation and function 223

4. The endoprotease furin 2244.1. Structural organisation 2244.2. Subcellular localisation and trafficking 2264.3. Substrate specificity 227

5. Improved biotechnological processes by the use of furin 2315.1. Development of recombinant coagulation factors 2315.2. Von willebrand factor propeptide removal by full length furin 2325.3. Production of recombinant factor IX using a truncated soluble furinderivative 2365.4. Processing of recombinant factor x precursors using furin derivatives invitro 2365.5. Use of furin in transgenic animals ... 238

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6. Perspectives 239Acknowledgements 241References 241

Development of bioprocesses for the generation of and-inflammatory, anti-viraland anti-leukaemic agents 249

Mahmoud MahmoudianAbstract 2491. Introduction 2502. Process development for the generation of nucleoside 5'-carboxylic acids.. 2503. Abacavir (Ziagen™) 2554. Production of the anti-leukaemic agent 506U78 258Acknowledgements 264References 264

Apoptosis and bioprocess technology 267R.P. Singh and M. Al-Rubeai

1. Introduction 2672. Apoptosis: basic features of cell death 268

Apoptosis and the mitochondria 2693. Apoptosis and its control during industrial scale cell culture processes 2714. Conclusion 273References 273

Gram-positive Bacteria as host cells for Heterologous production ofbiopharmaceuticals 277

Lieve Van Mellaert and Jozef AnneAbstract 2771. Introduction 2782. The general secretion pathway 279

2.1. Early stage 2792.2. Middle stage 2812.3. Late stage 2812.4. Intrinsic features of secretory proteins 283

3. Improvement of secretion 2843.1. early stage secretion improvement 2843.2. middle stage secretion Improvement 2853.3. late stage secretion Improvement 285

3.3.1. SPase activity... 2863.3.2. Proteolytic breakdown 2863.3.3. Protein folding 287

4. Examples of Gram-positive bacteria as host cells for the production ofheterologous proteins 287

4.1. High-level production of biopharmaceutical compounds 288B. brevis 2894.2. Gram-positive bacteria as live vaccine delivery systems 291

4.2.1. Gram-positive bacteria used as antigen delivery systems 2914.2.2.Approaches for antigen presentation 293

4.2.3. Heterologous antigens presented by Gram-positive bacteria 294Clostridium tetani 295Clostridium tetani 2954.3. Other application areas 296

5. Conclusions and perspectives 296References 297

Multiple Pathways of Exoprotein Secretion in Gram-negative bacteria 301Anthony P Pugsley

1. Introduction 3012. The General Secretory Pathway 3033. The Type I or ABC secretion pathway 3064. The type III or Contact Secretion Pathway 3065. Progress and technical problems 3086. A specific example: the Klebsiella oxytoca pullulanase secreton 308References 309

Alterations of Metabolic Flux Distributions in Recombinant Escherichia coli inResponse to Heterologous Protein Production 313

Jan Weber and Ursula RinasSummary 3131. Introduction 3132.Methodology 315

2.1 Metabolic Flux Analysis 3152.2 Underdetermined Networks 3162.3 Why Linear Programming? 3172.4 Properties of the Metabolic Network 3182.5 Optimal amino acid drain for protein production 319

3. Applications 3203.1 Production of hfgf-2 by temperature shift 3203.2 Temperature-induced production of human insulin 3263.3 Effect of a stable and unstable recombinant protein on the host metabolism..S... 328

4. Summary and Concluding Remarks 329Appendix 332

Bioreaction Network of E. coli 332Phosphotransferase System '. 332Embden-Meyerhof-Parnas Pathway 332PEP Carboxykinase and PEP Carboxlyase 332By-products 332TCA Cycle 332Glydxylate shunt 332Transhydrogenase 332Oxidative Phosphorylation 333Pentose Phosphate Pathway 333Methylglyoxal Pathway 333Ammonium, Glutamate and Glutamine 333

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Amino Acids 333Protein 334Nucleotides 334RNA .;; 334DNA 334Lipids 334Lipopolysaccharide 334Peptidoglucane 334Glycogen 334One-carbon unit and Polyamine 334Biomass 335Miscellaneous 335Fibroblast Growth Factor hFGF-2 335Objective 335

References 335Dynamics of proteolysis and its influence on the accumulation of intracellularrecombinant protein 339

Rozkov A., Yang S. and Enfors S.-OSummary 3391 .Introduction 3392.Materials and Methods 341

2.1 Microorganism 3412 .2 Media and Cultivation: 3412.3 Product concentrations 3422.4 Determination of the proteolysis rate constant 342

3. Results and Discussion 3424. Conclusions 346References 346

PART 5 - ARTIFICIAL ORGANS AND XENOGRAFTING 349The impact of transgenesis and cloning on cell and organ xenotransplantation tohumans 351

Louis-Marie Houdebine , Bernard WeillSummary 3511. Why xenografting ? 3523. The mechanisms of xenograft rejection 3544. The preventive treatments of xenograft rejection 3555. Thebiosafety of xenografting 3586. The acceptability of xenografting 3597.Conclusion and perspectives 361References. 361

Reinforced Bioartificial Skin In The Form Of Collagen Sponge And Threads.... 365Eun Kyung Yang, Young Kwon Seo and Jung Keug Park

Abstract 3651. Introduction 3652. Materials and Methods 367

2.1 Collagen scaffolds 3672.1.1 Extraction of Type I Collagen Solution 3672.1.2 Fabrication of Macroporous Collagen Sponge 3672.1.3 Fabrication of Collagen Threads and Mesh 3682.1.4 Reinforcement by Cross-linking Treatments 3692.1.5 Reinforcement by Incorporating with Collagen Mesh 3702.1.6 Measurement of Mechanical Strength 3702.1.7 Morphological Analysis 370

2.2 Bioartificial skin 3712.2.1 Primary Cell Culture 3712.2.2 Culture of Bioartificial Skin 3712.2.3 Morphology Examination 372

3. Results and Discussion 3723.1 Collagen scaffolds 372

3.1.1 Preparation of Collagen Threads 3723.1.2 Ultimate Tensile Strength of Collagen Sponges 374

3.2 bioartificial skin 3774. Conclusion and Future work 379Acknowledgement 379References 380

PART 6-ANTITUMOUR COMPOUNDS 381Towards the generation of novel antitumour agents from actinomycetes bycombinatorial biosynthesis 383

Jose A. Salas, Gloria Blanco, Alfredo F. Brana, Ernestina Fernandez,Ma Jose Fernandez, Jose Garcia Bernardo, Ana Gonzalez, Felipe Lombo,Laura Prado, Luis M. Quiros, Cesar Sanchez and Carmen Mendez

1. Introduction 3832. Anticancer biosynthetic gene clusters 3853. The aureolic acid group 386

3.1. Genes involved in the biosynthesis of the polyketide moiety 3873.2. Genes encoding enzymes modifying the polyketide skeleton 3883.3. Genes encoding an activated methyl cycle 3883.4. Genes encoding sugar biosynthetic enzymes 3883.5. Genes encoding glycosyltransferases 3893.6. Genes responsible for resistance and secretion 389

4. Generation of novel compounds 3904.1. Insertional inactivation 3904.2. Tailoring modification 3924.3. Combinatorial biosynthesis 394

5. Concluding remarks 397Acknowledgements 397References 397

Cell immobilisation of Taxus media 401Chi Wai Tang and Ferda Mavituna

Summary 401

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1. Introduction 4012. Materials and Methods 402

2.1 Plant Material and Callus induction 4022.2 Callus growth measurement 4032.3 Suspension Culture and Cell Immobilisation: 4032.4 Bioreactor: 403

3. Results and Discussion 4033.1 Effect of media on callus initiation from explants: 4033.2 Suspension culture: 4063.3 Immobilisation: 4063.4 Bioreactors: 406

4. Conclusion 406References 406

PART 7 - PRE AND PROBIOTICS 409The role of prebiotics inhuman gut microbiology 411

Catherine E. Rycroft, Robert A. Rastall and Glenn R. GibsonAbstract 4111. The Human Large Intestine 4122. Beneficial and Pathogenic Bacteria 4133. The Prebiotic Concept 4134. Methods for Evaluating Prebiotics 413

4.1. In vitro methods 4144.2. In vivo methods 4144.3. Use of molecular methods 414

5. Bifidogenic Factors 4156. Oligosaccharides as Prebiotics 415

6.1 Lactulose 4166.2. Inulin and fructo-oligosaccharides 4166.3. Galacto-oligosaccharides 4186.4. Soybean oligosaccharides 4206.5. Lactosucrose 4216.6. Isomalto-oligosaccharides 4226.7. Gluco-oligosaccharides 4236.8. Xylo-oligosaccharides 425

7. Conclusions 425References 425

The influence of intestinal microflora on mucosal and systemic immune responses429

Stephanie Blum, Dirk Haller, Susana Alvarez, Pablo Perez and Eduardo J.Schiffrin

Summary 429Abbreviations 4301. Introduction 4302.The innate immune system 4303.Adaptive immunity atmucosal sites 431

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3.1 The mucosal secretory immune system 4313.2 Stimulation of IgA production by the probiotic microorganism L.johnsoniiLai 432

4. The epithelial compartment 4334.1 Intraepithelial lymphocytes 4334.3 . Intestinal epithelial cells as active partners in mucosal immune defenses

4345. Modulation of the mucosal immune response by commensal bacteria 434

5.1 Regulation of the immune phenotype of intestinal epithelial cells in vitro434

5.2 Interaction of non-pathogenic bacteria with mixed mucosal cellpopulations: 436Human CaCo-2/leukocyte co-cultures in vitro 436

6. Modulation of the systemic immune response 4386.1 Interaction of non-pathogenic bacteria and blood leukocytes 438

7. Regulation of the mucosal immune response by luminal factors. Newperspectives 4428. Conclusion 443References 443

INDEX 447

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