CENTERS OF EXCELLENCE WITH BASAL FINANCING

2nd YEAR PROGRESS REPORT GUIDELINES The report must be written following the format specified hereafter. It must cover only the annual period of execution (or the specific period indicated within). Once it is completed, it should be sent in printed to the following address: Programa Investigación Asociativa - CONICYT Moneda 1375, Piso 5, Santiago For further inquiries contact: ggonzalez@conicyt.cl. Phone: (56-2) 24354301 INDEX I. PRESENTATION ....................................................................................................................................................... 3

II. RESUMEN EJECUTIVO ....................................................................................................................................... 10

III. EXECUTIVE SUMMARY ....................................................................................................................................... 12

IV. OBJECTIVES AND RESULTS ACHIEVED .................................................................................................. 14

V. PROJECT MANAGEMENT ................................................................................................................................... 39

VI. LESSONS LEARNED ............................................................................................................................................ 41

VII. PERFORMANCE INDICATORS ........................................................................................................................ 42

VIII.COMMENTS ON THE INDICATORS ............................................................................................................. 42

IX. DOCUMENTATION REQUIRED ....................................................................................................................... 43

CENTERS OF EXCELLENCE WITH BASAL FINANCING

2nd YEAR PROGRESS REPORT

I. PRESENTATION

NAME OF THE CENTER CODE

Centre for Biotechnology and Bioengineering FB0001

DIRECTOR OF THE CENTER SIGNATURE

Juan Alfonso Asenjo De Leuze

EXECUTIVE / DEPUTY / CO-DIRECTOR

Gonzalo Navarro

MANAGER

Paz Zañartu

SPONSORING INSTITUTION

Universidad de Chile

ASSOCIATED INSTITUTION(S) Universidad de la Frontera Universidad de Los Lagos Universidad de Antofagasta Universidad de Santiago de Chile CENTER WEBSITE ADDRESS

www.cebib.cl

PERIOD INFORMED

May-2015 – Apr-2016

Research Lines

N° Title Objective Principal Researcher

Other Researchers (participating in the

line)

1.- Research line 1

Metabolomics and Metabolic Engineering (ME)

Metabolomic studies allow the investigation and optimization of the main metabolic fluxes in a cell. They also allow reconstruction of the cell metabolism from a sequenced genome as well as the design of totally novel pathways/cells “in silico” which can then be constructed experimentally using genetic engineering tools.

Juan A. Asenjo

Barbara Andrews Carolina Shene

2.- Research line 2

Protein Engineering (PE)

In our Protein Engineering studies we use site-directed and random mutagenesis based on PCR, molecular modeling and computational bioinformatics studies of structure function relationships. Our group is strong in the rational design of proteins and the study and analysis of information regarding structure-function relationships in protein molecules.

Barbara Andrews

Juan A. Asenjo Alvaro Olivera M.Elena Lienqueo

3.- Research line 3

Mathematical Modelling (MM)

Our aim is to develop mathematical models of separation processes in bioengineering systems of importance to industrial biotechnology. One of our goals is the understanding of iron transport phenomena associated with intestinal iron absorption through mathematical modeling and the development of mathematical models of tumor growth

María Elena Lienqueo (from July 2017 will be Carlos Conca)

Alvaro Olivera Carolina Shene M.Elena Lienqueo Juan A. Asenjo

4.- Research line 4

Bioinformatics (BI)

Computational biology problems have always been computation-intensive. Sequence databases storing trillion bases will soon emerge, and bioinformatic algorithms will have to cope with huge amounts of data. We plan to face this challenge with the combination of two tools: compressed data structures and distributed computing, most notably compressed suffix trees and arrays focused on exploiting repetitiveness of the data collections.

Gonzalo Navarro

Mauricio Marin Juan A. Asenjo Alvaro Olivera Carlos Conca

5.- Research line 5

Molecular Genetics, Extremophiles, and Ecophysiology (MG)

In Chile there are a series of environments that exhibit extreme and fluctuating conditions that represent an excellent source of new microorganisms and bioactive compounds. We will use multiple approaches to supply the pipeline of discovery of new drugs, including metagenomic analyses to describe the potential for bioactive compound production from these specific environments and the subsequent culturing of rare and useful microorganisms.

Alejandro Buschmann

Cristina Dorador Benito Gómez Juan A. Asenjo Bárbara Andrews M. Elena Lienqueo

Principal Researchers (i)

Other Researchers

Name Institution Email

Carlos Conca Rosende U. de Chile cconca@dim.uchile.cl

Patricio Cumsille Atala U. del Bío-Bío pcumsill@gmail.com

Oriana Salazar Aguirre U. de Chile orsalaza@ing.uchile.cl

Daniel Varela Zapata U. de Los Lagos dvarela@ulagos.cl

Ziomara Gerdtzen Hakim U. de Chile zgerdtze@ing.uchile.cl

Cristian Salgado Herrera U. de Chile jsalgado@ing.uchile.cl

Carolina Bonacic Castro U. de Santiago de Chile carolina.bonacic@usach.cl

Mario Inostroza Ponta U. de Santiago de Chile mario.inostroza@usach.cl

Nicolás Hidalgo Castillo U. de Chile nicolas.hidalgo@usach.cl

Erika Rosas Olivos U. de Santiago de Chile erika.rosas@usach.cl

Travis Gagie University of Helsinki Travis.gagie @cs.helsinki.fi

Miguel Ángel Martínez Prieto University of Valladolid migumar2@infor.uva.es

Diego Seco Naveiras U. de Chile dseco@udec.cl

Yakov Nekrich University of Kansas ynekrich@uwaterloo.ca

Susana Ladra Gonzalez U. de Chile sladra@udc.es

Alexandra Galetovic Carabantes U. de Antofagasta alexandra.galetovic@uantof.cl

Luis Cáceres Villanueva U. de Antofagasta luis.caceres@uantof.cl

Rodrigo Navia Diez U. de La Frontera rodrigo.navia@ufrontera.cl

Jorge Araya Rojas U. de Antofagasta jearoj@yahoo.com

Manuel Duarte Mermoud U. de Chile Manuel.duarte@die.uchile.cl

John Mackinnon Dahlgren U. del Desarrollo mackinnon.dr@gmail.com

Rodrigo Maureira Tenorio U. de Chile rmaureira@airson.cl

Vader Johnson Vera U. de Chile vaderonline@gmail.com

Sylvain Wielfrid Faugeron U. de Los Lagos sfaugeron@bio.puc.cl

Verónica Gil-Costa U. de Santiago de Chile ggvcosta@gmail.com

Senen Gonzalez U. de Santiago de Chile ulcango@gmail.com

Victor Sepulveda U. de Santiago de Chile palabragris@gmail.com

Roberto Uribe Paredes U. de Santiago de Chile roberto.uribeparedes@gmail.com

Ricardo Barrientos Rojel U. de Santiago de Chile ricardo.j.barrientos@gmail.com

Mauricio Silva Oyarzun U. de Santiago de Chile mauricio.oyarzun.silva@unap.cl

Oscar Rojas U. de Santiago de Chile oscar.rojas.d@usach.cl

Martha Hengst López U. de Antofagasta martha.hengst@uantof.cl

Pablo Moisset de Espanes U. de Chile pablo.moisset@gmail.com Changes in research personnel There have been no changes during the period.

Board of Directors The members of the Board of Directors of the Basal Centre CeBiB are the 5 senior Researchers of the CeBiB namely Juan A. Asenjo, Gonzalo Navarro, Alejandro Buschmann, Carlos Conca and Barbara Andrews and Felipe Alvarez(Vice-Dean of the Faculty of Physical and Mathematical Sciences and representative of the University) Fernando Lund (National Science Prize, Materials Science), Arturo Yudelevich, (Grupo Bios), Rodrigo Velasco (partner in Alessandri Abogados, patent lawyer) and Eugenio Correa (President of AsfalChile, Ph.D. in Philosophy and Co-Founder of the Instituto de Sistemas Complejos de Valparaiso, ISCV). During the second year the Board met on 29 October 2015. As stated in the minutes of this meeting all members were present, the performance of the first year of the Centre was evaluated and several interesting points were raised and suggestions made. In addition to praising CeBiB scientists for an excellent 2015 Workshop in July, that was focused at generating new collaborations amongst CeBiB researchers as well as consolidating existing ones, the external assessment of the first year progress report was considered to be very good. Since very good achievements were obtained after a relatively short period it was recommended the following:

- To identify a subject in which CeBiB has clear leadership. - To identify other centres comparable to CeBiB internationally and compare CeBiB with

these with well-defined metrics and considering level of funding. At this meeting the new Manager for Coordination and Product Evaluation, Ms. Isabel Guerra was introduced. Also the journalist and person responsible for outreach, with vast previous experience, Ms. Noemi Miranda was introduced. Finally it was also decided that, once we have moved to our new and “state-of-the-art” premises we should hold a Workshop with industrial potential partners and companies interested in CeBiB research activities in order to make an effort to raise more industrial funding.

II. RESUMEN EJECUTIVO El Centro representa una oportunidad única que ha comenzado a crear de forma muy activa un equipo interdisciplinario capaz de abordar e investigar preguntas clave en las ciencias de la biotecnología y bioingeniería modernas. El Centro tiene 3 características clave que son:1. El desarrollo de nuevas propuestas en ciencias biotecnológicas y de bioingeniería básicas utilizando herramientas de biotecnología de sistemas que deberían resultar en soluciones originales a problemas biotecnológicos más tradicionales. 2. Tener un impacto en problemas científicos tanto de importancia nacional así como internacional. 3. La aplicación del “know-how” desarrollado y existente en los distintos grupos del Centro en la solución de problemas aplicados de importancia tanto para Chile como internacional (Investigación Aplicada) tanto para las ciencias biotecnológicas básicas como para aplicaciones en la industria.

El objetivo del Centro es llevar a cabo investigación de frontera en biotecnología y bioingeniería y el desarrollar modelos de sistemas biológicos importantes usando herramientas de la bioingeniería moderna y un enfoque de biología de sistemas. Este enfoque está siendo aplicado de manera jerárquica desde la identificación de genes, la genómica (expresión de genes) y en muchos casos proteómica y metabolómica incluyendo función de las proteínas y regulación e ingeniería de proteínas utilizando herramientas matemáticas y computacionales modernas. Este enfoque holístico está siendo conducido por un equipo multidisciplinario de bioingenieros, biólogos (incluyendo moleculares, marinos y medioambientales), matemáticos y científicos de la computación lo que está comenzando a resultar en importantes desarrollos en la industria chilena.

La diversidad de las áreas de expertise de los científicos que participan en este Centro es una ventaja en un proyecto que ha comenzado a tener una opinión muy relevante en problemas tanto de importancia nacional como internacional. Chile es un país de solo 16 millones habitantes y tiene una comunidad científica pequeña. Por eso, un Centro formado por investigadores consolidados y potenciales en áreas relativamente diferentes de bioingeniería, matemáticas, bioinformática y ciencias biológicas, pero con un tema en común –biotecnología y bioingeniería- es importante para el desarrollo del país. Este es claramente el valor agregado “innovativo” del Centro. Los objetivos de investigación del Centro no serían posibles de obtener en ausencia del apoyo del Programa de Financiamiento Basal dado que la investigación llevada a cabo va mucho más allá que el campo de los laboratorios individuales.

El Centro está conformado por cinco componentes de investigación altamente integrados, cada uno de ellos liderado por un Investigador Titular Senior. Estos componentes son: Metabolómica e Ingeniería Metabólica (ME), Ingeniería de Proteínas (PE), Modelación Matemática (MM), Bioinformática (BI) y Genética Molecular, Extremófilos y Ecofisiologia (MG). Como se indica en la sección IV sobre Objetivos y Actividades, durante los dos primeros años ha existido una “fertilización cruzada” extensa entre los científicos participando en cada uno de ellos. Esto demuestra que como grupo estamos desarrollando proyectos mucho más comprensivos que aquellos de los componentes individuales. Estamos aumentando fuertemente estas interacciones en este Centro dado que hemos constatado que esta es una de nuestras principales fortalezas. La investigación aplicada llevada a cabo en el Centro consiste en la aplicación del “know-how” fundamental desarrollado en muchos de los grupos, tal como se hizo en forma más limitada en el pasado. Estamos aumentando fuertemente esta línea de trabajo en el Centro. Algunos ejemplos de estas investigaciones son:

• Desarrollo de enzimas con alta actividad a temperaturas extremas: basadas en aquellas encontradas en la Antártica (tres patentes) y las encontradas en microorganismos del desierto de Atacama.

• Compresión de datos genómicos para aplicaciones médicas y biotecnológicas. • Desarrollo de nuevos métodos para la síntesis de biocombustibles y productos químicos a

partir de biomasa renovable (p.ej. macroalgas café y verdes). • Microorganismos de ambientes extremos de distintos sitios del desierto de Atacama y marinos

como fuentes de nuevos antibióticos y anticancerígenos. • Matemáticas y algoritmos para la traducción de ondas sonoras a imágenes. • Desarrollo de vacunas (p.ej. contra el alcoholismo) y la identificación de proteínas

importantes en el cáncer. El Centro ha creado una Unidad de Comunicaciones a cargo de la difusión del centro, la que ha logrado a la fecha i) aumentar la presencia en medios de los investigadores CeBiB y está organizando un seminario para periodistas especializados en ciencia sobre avances en biotecnología, ii) participado en ferias científicas con charlas para estudiantes de enseñanza media y público general, ha organizado un programa de visitas a nuestros laboratorios de estudiantes de enseñanza básica y está a cargo del Ciclo 2016 de Conferencias de la Academia Chilena de Ciencias con el tema “Biotecnología, Bioingeniería y Bioinformática”, iii) organizado seminarios internacionales y nacionales con científicos de distintos campos abiertos a académicos universitarios e investigadores interesados en los temas, y iv) se encuentra organizando una serie de Charlas BioTech con la colaboración de importantes actores de la industria. Nuestros logros individuales y como grupo son muy sólidos dado que muchos de nosotros hemos colaborado en investigación y en la dirección de estudiantes de doctorado. En los últimos dos años hemos graduado una cantidad importante de estudiantes de doctorado y generado excelentes publicaciones científicas en revistas internacionales, muchas de ellas producto de la colaboración entre científicos del Centro. De la misma forma hemos sido muy activos en actividades de “outreach” y en interacciones con la industria y la sociedad en general. Tenemos una red de colaboradores internacionales y nacionales muy activa con centros de excelencia líderes como ha sido descrito en nuestra propuesta de investigación original y como se evidencia en algunas de nuestras recientes publicaciones científicas. El Centro incluye un número importante de científicos jóvenes y durante los últimos dos años hemos aumentado considerablemente el número de investigadores postdoctorales y colaboradores en nuestro Centro CeBiB. Ellos constituyen la semilla de una futura generación que va a asegurar el futuro de la ciencia, la tecnología e innovación en el país y va a fortalecer el rol de Chile en la escena científica mundial. En Julio 2015 se realizó un “workshop” de tres días con todos los miembros del Centro (científicos y estudiantes) en el Hotel Manquehue en Santiago. El objetivo principal de este “workshop” fue generar amplia discusión y propuestas de los diferentes grupos de investigación con el objeto de aumentar las colaboraciones interdisciplinarias. Un segundo “workshop” de tres días se llevó a cabo en Santa Cruz a comienzos de Diciembre 2015 nuevamente con la participación de todos los miembros del Centro. Tuvimos dos invitados plenarios del UK y de USA que, además de dar sus conferencias científicas, ambos dieron seminarios de sus experiencias en concebir y generar empresas “start-up” en sus ambientes académicos.

III. EXECUTIVE SUMMARY The Centre represents a unique opportunity that has started very actively to create an interdisciplinary team that is able to tackle and investigate key questions in modern biotechnological and bioengineering sciences. The Centre has 3 key features which are: 1. The development of novel approaches in basic biotechnological and bioengineering sciences using tools of systems biotechnology that should result in original solutions to more traditional biotechnological problems. 2. To have an impact on scientific problems of national as well as international importance. 3. The application of the know-how developed and existing in the different groups of the Centre to the solution of applied problems of importance in Chile and abroad (Applied Research), both to basic biotechnological science and to applications in industry. The goal of the Centre is to conduct frontier research in biotechnology and bioengineering and to develop models of important biological systems using tools of modern bioengineering and a systems biotechnology approach. This approach is being applied in a hierarchical fashion from gene identification, to genomics (gene expression), and in many cases proteomics and metabolomics, including protein function and regulation and protein engineering, using modern mathematical and computer tools. This holistic approach is being conducted by a multidisciplinary team of bioengineers, biologists (including molecular, marine and environmental), mathematicians and computer scientists that is starting to result in important developments of Chilean industry. The diversity of the areas of expertise of the scientists participating in this Centre is an advantage in a project which has started to have a strong say in the answer to problems of national as well as international importance. Chile is a country of only 16 million people and has a small scientific community. Hence a Centre formed by proven and potential investigators in relatively different areas of bioengineering, mathematical, bioinformatics and biological sciences but with a common theme – biotechnology and bioengineering - is important for Chile’s development. This is clearly the “innovative” added value of the Centre. The research goals would not be feasible in the absence of the Basal Funding Programme’s support for this Centre as the research carried out goes far beyond the scope of the individual laboratories. The Centre consists of 5 highly integrated research components, each led by a Senior Key associate Scientist. These components are: Metabolomics and Metabolic Engineering (ME), Protein Engineering (PE), Mathematical Modelling (MM), Bioinformatics (BI) and Molecular Genetics, Extremophiles and Ecophysiology (MG). As stated in section IV on Objectives and Activities, during the first two years there has been extensive cross-fertilization between the scientists participating in each of them. This demonstrates that as a group we are developing a strong capacity to interact and develop research projects which are more comprehensive than those of the individual components. We are strongly increasing this interaction in this Centre as we have realized that this is one of our major strengths. The Applied Research being carried out at the Centre consists of the application of the fundamental know-how developed in many of the groups, as has been done to a limited extent in the past. We are strongly increasing this line of work in the Centre. Some examples of this research are:

• The development of enzymes with high activity at extreme temperatures: based on those found in Antarctica (three patents) and those found in microbes in the Atacama Desert.

• Genome data compression for medical and biotechnology applications. • The development of novel methods for the synthesis of biofuels and chemicals from renewable

biomass (e.g. brown and green macroalgae). • Microorganisms from extreme environments, from different locations in the Atacama Desert

and in the sea as sources of novel antibiotics and anticancer agents. • Mathematics and algorithms for translating sound waves into images. • The development of vaccines (e.g. against alcoholism) and identification of proteins important

in cancer. The Centre has created a Communications Unit in charge of outreach that has i) increased the presence of CeBiB researchers in media and is developing a seminar for specialized journalists about cutting-edge biotechnology, ii) participated and imparted talks in scientific divulgation fairs for high school students and general audience, has organized regular visits to our labs from primary school students to foster scientific awareness and is in charge of the 2016 series of Conferences in the Chilean Academy of Sciences with the topic “Biotechnology, Bioengineering and Bioinformatics” iii) organized international and national seminars with scientists from different fields open to faculty professors and researchers interested, and iv) is in the process of organizing a series of BioTech Talks with collaboration of industry. Our achievements, individually and as a group, are very solid as many of us have collaborated in research and in the training of Ph.D. students. During the last two years we have graduated a substantial number of Ph.D. students and produced important scientific papers in international journals, many of them resulting from collaborations between the scientists participating the Centre. Similarly, we have been extremely active in outreach activities and in interactions with industry and society as a whole. We have a very active network of international and national collaborations with top centers of excellence as described in our original research proposal and as evidenced in some of our recent scientific publications. The Centre includes a large number of young scientists and during the last two years we have considerably increased the number of postdoctoral fellows and collaborators in the new CeBiB Centre. They are the kernel of a future generation who will ensure the future of science, technology and innovation in Chile and will make and strengthen Chile’s role on the worldwide scientific scene. In July 2015 a three day workshop was held with all members of the Centre (scientists and students) at the Hotel Manquehue in Santiago. The main aim of this workshop was to generate discussion and proposals of the different research groups in order to increase interdisciplinary collaborations. A second three day workshop was held in Santa Cruz in early December again with the participation of all members of the Centre. We had two invited plenary speakers from the UK and US and in addition to their scientific lectures they both gave lectures on their experience in conceiving and generating “start-up” companies in their academic environments.

IV. OBJECTIVES AND RESULTS ACHIEVED

1) Scientific Excellence

1. - Metabolomics and Metabolic Engineering (ME) a) Metabolic Flux Analysis of yeast producing bioethanol from Macroalgae The work of our postdoc Carolina Contador with the collaboration of several other members of the CeBiB is clearly reflected in the paper recently published in Metabolic Engineering Communications (Contador, C.A., Shene, C., Olivera, A., Yashikuni, Y., Buschmann, A., Andrews,, B.A., Asenjo, J.A. “Analyzing redox balance in a synthetic yeast platform to improve utilization of brown macroalgae as feedstock” (2015) Metab. Engin. Comm., 2, 76-84). When BAL constructed the yeast that should assimilate alginate and mannitol, the sugars present in macroalgae, it was experimentally observed that ethanol synthesis was seriously impaired. This publication investigates, using the tools of metabolic engineering, namely a Genome Scale Metabolic Model and detailed Metabolic Flux Analysis, which are the metabolic reasons for this limitation. An analysis of the redox balance was carried out. A context-specific model was derived from the available yeast genome-scale metabolic reconstructions. Flux balance analysis and dynamic simulations were used to determine the flux distributions. The model indicates that ethanol production is determined by the activity of DEHU (4-deoxy-L-erythro-5-hexoseulose uronate) reductase (DehR) and its preferences for NADH or NADPH which influences strongly the flow of cellular resources. Different scenarios were explored to determine the equilibrium between NAD(H) and NADP(H) that will lead to increased ethanol yields on mannitol and DEHU under anaerobic conditions. These results show that production of ethanol and other chemicals can be optimized if a redox balance is achieved. A possible methodology to achieve this balance is presented. This paper shows how metabolic engineering tools are essential to comprehend and overcome this limitation. b) Metabolic reconstruction of bacteria from Atacama Desert for production of antibiotics and anticancer agents Today there is a tremendous need for new antibiotics and novel cytotoxic compounds against cancer cells to develop efficient alternative treatment to chemotherapy. We have searched for highly active Streptomyces strains in the Atacama Desert in northern Chile. We have identified several new strains and found many novel antibiotics and anticancer agents (“Chaxamycins”, “Chaxalactins” and “Atacamycins”) from Streptomyces C34 and C38. We have totally sequenced the genome of Streptomyces leeuwenhoekii (Gomez-Escribano, J.P., Castro,J.F., Razmilic, V., Chandra G., Andrews, B.A., Asenjo, J.A., and Bibb, M.J. (2015), The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contings of the chromosome, circular plasmid pSLE1 and linear plasmid pSLE2. BMC Genomics, 16, 485). This has allowed to built a genome scale Metabolic Model. The genome scale model of the metabolism of Streptomyces leeuwenhoekii C34 has been developed from its genome sequence. The model includes 817 reactions including 147 for transport and secondary metabolite biosynthesis such as squalene, pentalenene and desferrioxamine. We have now included reactions for Chaxamycins, Chaxalactins and other polyketides such as the one synthesized by the halogenase cluster. A detailed Metabolic Flux Analysis is presently being carried out in order to optimize synthesis and production of Chaxalactins and Chaxamycins by recognizing appropriate pathways and useful knock-out sites to increase production of these secondary metabolites (Ph.D. thesis Valeria Razmilic). The gene cluster in S. leeuwenhoekii C34 responsible for the biosynthesis of the Chaxamycins and Chaxalactins has been identified and cloned into a much more efficient strain of Streptomyces, namely S. coelicolor A3(2) whose heterologous expression of gene clusters from other Streptomyces strains has recently been successfully tested (Ph.D. thesis of Jean Franco Castro, December, 2015). This was recently published (Castro, J.F., Razmilic, V., Gomez-Escribano, J.P.,

Andrews, B.A., Asenjo, J.A. and Bibb, M.J. (2015) Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii. Appl. Environm. Microbiol., 81, 1-12). J.F. Castro is now at the University of Newcastle in the UK working with Professor Mike Goodfellow on various aspects of Atacama desert microorganism isolation and metabolism. c) Metabolic Analysis of the production of Butanol from Macroalgae sugars As already described macroalgae are an abundant and potentially attractive carbohydrate substrate for fermentation processes given the low lignin content and the presence of abundant and complex sugars that include alginate and mannitol. E. coli and yeast have been and are being engineered in order to include a pathway that is able to degrade alginate and use it as a carbon source. On the other hand butanol is an attractive biofuel and also a potential building block for more complex molecules of interest and Saccharomyces cerevisiae shows a high tolerance to alcohol toxicity (Ph.D. thesis Jose Duguet). We have constructed an alginate degradation pathway in Saccharomyces cerevisiae considering an engineered secretion of endolytic and exolytic alginate lyases and a synthetic Enter-Dourodoff pathway for degradation, transport and integration of alginate as a carbon source. For butanol production, an analysis of a synthetic cluster of 6 genes (total 14 kb) is being carried out using pyruvate as a substrate. In order to improve butanol production and reduce its toxicity the cluster is being expressed in mitochondria as a strategy of compartmentalization. Both alginate degradation and butanol biosynthesis pathways are considered to be stable by genomic integration through in vivo homolog recombination. Our current results include the cloning of candidate enzymes for the cluster of genes that are being genomically integrated in the yeast. d) Reconstruction of the Metabolism of marine microbes (Salinispora tropica and other Salinispora) Bacteria within the order Actinomycetales are a well-known source of natural products such as antibiotics and anticancer agents, and the genus Salinispora is no exception. Salinispora tropica is a marine actinomycete that produces diverse secondary metabolites, including many that posess pharmaceutical properties such as Salinosporamide A (NPI-0052), a potent anticancer agent, and sporolides, candidates for antiviral compounds. We have already developed a genome-scale metabolic reconstruction for Salinispora tropica strain CNB-440 to be used in metabolic engineering of an improved strain. This was recently published in the Journal Antonie van Leeuwenhoek (Contador, C., Rodriguez, V., Andrews, B.A., Asenjo, J.A. (2015), Genome-scale reconstruction of Salinispora tropica CNB-440 metabolism to study strain-specific adaptation. Antonie van Leeuewenhoek, 108, 1075-1090.) The model was manually curated based on physiological and biochemical information for primary and secondary metabolism pathways. The reconstructed stoichiometric matrix consists of 1169 biochemical reactions, 204 transport reactions and 1317 metabolites. A total of 908 structural open reading frames (ORFs) were included in the reconstructed network. The number of gene functions included corresponds to 20% of all characterized ORFs in the S. tropica genome. The genome-scale metabolic model was used to study strain-specific capabilities by metabolic flux analysis using a constraint based-model approach. The genome-scale metabolic model was used to study strain-specific capabilities in defined minimal media. The iCC908 reconstruction was used to analyze growth capabilities in 41 different minimal growth-supporting environments. False predictions provided opportunities to gain new insights into the physiology of this species, and to gap fill the missing knowledge. The incorporation of modifications led to increased accuracy in predicting the outcome of growth/no growth experiments from 76 to 93 %. iCC908 can thus be used to define the metabolic capabilities of S. tropica and guide and enhance the production of specialised metabolites.

e) Metabolic reconstruction of the production of Hyaluronic acid from pentose (hemicellulose) and sugars in Macroalgae. With the aim of finding new ways of producing hyaluronic acid (HA), an Escherichia coli strain containing the sehasA gene, coding for Streptococcus equisimilis hyaluronan synthase, along with ugd encoding for E. coli UDP-glucose 6-dehygrogenase was constructed. Since xylose is a sugar which is abundant in forestry waste, an adaptive laboratory evolution (ALE) was carried out to improve the growth of E.coli on this sugar. The culture conditions of the HA-synthesizing E. coli were optimized and a flux balance analysis (FBA) was done to compare the evolved and not evolved strains. Conditions were optimized (Ph.D. Thesis of Daniela Vaisman, 2015 and Thesis of Natalie Feltes). Testing all these conditions together, an HA yield of 630 mg/(g DW of biomass) was obtained. This HA yield was 51% higher than the HA titer obtained with basal conditions. The FBA showed the metabolic improvement of the evolved strain compared to the not evolved one, which consists on average of 12% greater fluxes with xylose supplementation and an average of 29% greater fluxes with mannose supplementation for the PPP, glycolysis and TCA cycle. These results suggest that the ALE affected a glycolysis enzyme, since the changes were observed during xylose and mannose consumption and also allowed a greater hyaluronic acid and biomass production. In his Master’s degree thesis Jorge Meza has successfully developed a strain of E. coli which can produce hyaluronic acid (HA) using alginate and manitol as carbon sources. HA was obtained after 24 h. growth on minimal media supplemented with 2% alginate/manitol (2:1 ratio). f) Determination of pathways of production of antioxidants in seaweeds During this year Profs. Buschmann and Lienqueo have been working on the process of extraction of phlorotannins (antioxidants) from Macrocystis pyrifera. This work resulted in a publication in Algal Research (A Leyton, R Pezoa-Conte, A Barriga, A Buschmann, P Mäki-Arvela, J-P Mikkola; M E Lienqueo, Identification and efficient extraction method of phlorotannins from the brown seaweed Macrocystis pyrifera using an orthogonal experimental design (2016)). It is expected that this will lead to propose possible pathways for the production of these antioxidants since there is a Ph.D. thesis in progress on this subject (A. Leyton). 2. - Protein Engineering (PE) a) Testing of MOSST Algorithm in the prediction of mutations for improved enzymes We have continued our study of a cryophilic xylanase Xyl-L from Psychrobacter sp. 2-17 as a model enzyme for testing MOSST-SDM prediction. We have selected to change molecular properties that determine protein behaviour in ion exchange chromatography purification, while preserving enzymatic activity, cryophilicity and folding stability. As a test group, we have selected an orthologous group of thermophilic, mesophilic and cryophilic xylanases. We determined positions and conservations throughout the protein that putatively determine the target characteristics. We compared the information generated by this analysis with previously reported xylanase mutation data. We are also using mathematical models to correlate protein surface charge and charge distribution with retention time in ion exchange chromatography. These predictions and MOSST results will be integrated into an iterative algorithm that will contrast experimental measurements with predictions in order to guide the selection of cumulative mutations for xylanase Xyl-L to shift its elution profile. We are envisaging the use of similar algorithms in a general way for different target proteins. b) Construction of chimeric vectors for high-throughput site-directed mutagenesis. We developed a system for protein expression and autopurification in order to easily generate, purify and characterize protein variants in site-directed mutagenesis experiments. For this, we replaced the passenger domain of the antigen 43 (Ag43) of Escherichia coli K-12 with eGFP as a

model protein. With this system, eGFP was expressed associated to the external membrane of the expression host E. coli BL23(DE3), but it is time-dependently released to the medium. To demonstrate the mechanism and identify the hydrolysis site and active residues (if the mechanism is internal), we are using the Gibson assembly technique to clone eGFP in tandem with the Ag43 passenger domain and perform sequential truncations of the passenger domain. With this approach, we expect to identify the protease cleavage site, the active residues and the passenger domain sequence responsible for anchoring the cleaved protein to the Ag43 barrel domain. With these results, and taking into account protein structural models and sequence information, we will design mutations that could alter the cleavage reaction pH dependence, in order to modulate fusion protein cleavage and anchoring by pH control. In this way, we expect to develop systems for protein expression and autopurification in simple steps. c) Development of rationally optimized recombinant protein variants. Making peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome. A non common but interesting research area is related to peptides with intracellular targets. A high throughput recombinant expression and purification system for production of cell penetrating peptides in Escherichia coli has been designed and implemented. The system produces target peptides in an insoluble form by fusion to a hexahistidine tagged ketosteroid isomerase which is then separated by a highly efficient thrombin cleavage reaction procedure. The production process involves a low-temperature induction process and an efficient on-column intein-mediated cleavage, which allows an effective peptide recovery using a single chromatographic step providing a system suitable for recombinant production of peptides for scientific research, including biological assays. We have designed a second system for recombinant expression and purification of peptides which is being tested with several peptides, these results should be ready for publication by the latter half of 2016. We expect to increase peptide productivity by about 50%, respect to our previous work. Also, we have developed a new fusion tag for the target peptide which make this system suitable for a patent application. d) Development of a treatment for alcoholism. An attractive treatment against alcoholism is the reduction of ALDH2 levels through the administration of vectors that code for anti-Aldh2 antisense RNA molecules that inhibit ALDH2 gene expression. This inhibition has been achieved by the in vitro and in vivo administration of an adenoviral vector Ad5 and could be an attractive alternative to chemical treatments such as Disulfiram that have the same effect but high toxicity and low patient compliance. Adeno-associated viral vectors (AAV) have been used as a powerful tool for gene transfer in vivo studies in recent years. We have recently been evaluating the use of shRNA (small hairpin RNA) to improve the inhibition of the expression of the enzyme ALDH2. shRNA are more specific than the antisense gene that we have been using. We are testing 5 different shRNA for their ability to infect cells and inhibit enzyme expression. e) Determination and improvement of new enzymes for saccharification of seaweed Production of recombinant alginate lyases. We have cloned and expressed nine endo-acting alginate lyase genes from marine bacteria in E. coli. Accumulation of soluble protein has been optimized for three of these genes. The optimized parameters were: autoinduction/harvest time, expression in 4 different host strains, and testing 14 different protein-fusion partners. The best conditions have been translated to the remaining six strains resulting in robust soluble alginate lyase expression for four of them (seven soluble active enzymes total). We are currently comparing the activity of our recombinant products to the available commercial enzyme. On the other hand, we have also cloned and expressed ten exo-acting oligoalginate lyase genes from six marine bacteria, one plant-pathogen bacterium, and one

mollusk. We have tested the aforementioned optimized expression conditions, resulting in significant soluble enzyme activity for three of these genes. We are currently optimizing the assay conditions for these enzymes in combination with the recombinant alginate lyases. Production of recombinant cellulases and auxiliary enzymes Efficient hydrolysis or saccharification of lignocellulosic residues depends on the concerted action of cellulases and auxiliary proteins. We have isolated fungal proteins having auxiliary activity in order to design balanced and efficient mixtures of cellulase and auxiliary enzymes. Auxiliary proteins from rot fungus Gloeophyllum trabeum were identified by proteomic analyses of fungal proteins that showed cellulolytic enhancement activity on wheat straw. Among other hydrolytic and oxidative enzymes, a polysaccharide mono oxygenase (PMO) and a xylanase were identified. GtPMO1 and GtXYL1 were produced in the yeast Pichia pastoris and purified. Different combinations of these enzymes with Celluclast 1.5L were assessed in reactions with wheat straw as substrate. Results showed that individually GtPMO, GtXYL1 actually improve the production of reducing sugars by Celluclast by 30-50%, depending on the concentration of enzyme used. However, this enhancing effect is partially abolished by simultaneous combination of Celluclast with these two auxiliary proteins, suggesting that GtPMO can inhibit the GtXyl1 activity. Optimization of the combined use of these auxiliary enzymes is being conducted in order to take advantage of the individual benefits of each enzyme. f) Development of high-throughput exhaustive prediction tools for the effect of mutations One of the most difficult aspects of rational design in protein engineering to modify thermal stability of enzymes, is the conservation of catalytic activity. Taking this into account, in this period we have developed a new methodology to identify amino acid positions in an enzyme that can be modified to alter its thermal stability without significantly affecting its catalytic properties. This methodology consists of proposing mutations for a target protein and generating structural models of said mutants, subsequently performing docking experiments to calculate interaction energies between the mutant enzymes and their substrates, and finally calculating the normal modes of vibration of the protein, to calculate the molecule’s vibrational entropy. With this information, a statistical analysis is performed on the results to identify those amino acid positions that have a significant influence on the evaluated properties. Using a principal component analysis on the generated data for each new mutant protein structure model, it is possible to identify amino acids that cause entropy and/or docking energy changes. From these results, new mutations can be proposed based on regression models. This procedure can be repeated several times, and at each iteration a new set of amino acid mutations can be obtained. Mutations proposed at each iteration will be compared to identify those mutations that are conserved and statistically influential in several iterations, which are selected as the final set of proposed mutations of the methodology. This methodology is being tested on xylanase Xyl-L of Psychrobacter sp. 2-17. The proposed mutations will be performed and experimentally characterized with regards to thermal stability and catalytic activity, in order to validate the in silico predictions of the proposed methodology. g) Design and applications of ferritin-based bionanomaterials A new research line has been opened to use the protein ferritin to contain inorganic nanoparticles within. We propose to modify ferritin molecules using mutagenesis and protein fusion to explore its uses as carrier and support of nanoparticles on electrodic supports, to generate 2D or 3D nanoparticle networks, or to design hybrid nanomaterials able to selectively bind therapeutic targets. We have cloned, expressed, purified and characterized human L and H ferritin homomultimers. We have also genetically fused ferritin with specific peptides that could act as recognition anchors for biological or inorganic targets. Mutations are being performed to modify the external and internal surface of the protein to change its interaction properties, improve its recombinant production or alter the characteristics of nanoparticles within. We have been granted a U-Redes internal University of Chile fund to develop chemically and genetically modified ferritin

to generate hybrid functional materials that use the protein as a biocompatible matrix for carrying drugs or nanoparticles or to design new diagnostic and therapeutic devices. 3. - Mathematical Modelling (MM) One of the main aims of the Mathematical Modeling component is to establish mathematical models of cell functions and of separation processes in bioengineering systems of importance to industrial biotechnology. The main motivation is to use a system biology strategy, where a mathematical framework is established to treat the complexity of biological systems arising from the interplay of transport processes, metabolism, regulation and control processes. Main achievements during the second year according to the activities originally planned are summarized as follows: a) Mathematical modelling of Membrane Chromatography A promising separation technology amongst the adsorptive separation methods is membrane chromatography which is identical to a normal flow filtration membrane with the essential distinction that the pore surface is functionalized with ligands. This is the first approach to the use of a rate model to predict the behavior of the separation of proteins using membrane chromatography. In this process time and recovery liquid volume are reduced due to the absence of rate-limiting pore diffusion. A manuscript is being prepared to present the mathematical rate model for anion and hydrophobic membrane chromatography developed to simulate the elution of protein mixtures. A cost function that takes into account information from chromatograms, such as purity and yield, will help to predict the optimal operational conditions. Experimental data will be used to fit parameters and to validate the model predictions. b) Mathematical modelling of the elution of PEGylated proteins in affinity chromatography PEGylation is a strategy for improving the physico-chemical properties and bioavailability of therapeutic proteins in the organism. However, in a given reaction proteins with different degrees of PEGylation are produced; thus purification of the active isomer is a challenge. Modelling and simulation of the purification of PEGylated proteins could be used to define operational conditions for obtaining high yield and purity. We have developed a chromatographic method using a heparin resin and a linear salt gradient for elution of these proteins from a stationary phase; the results have shown a decreasing affinity regarding the degree of PEGylation. Simulation of elution curves of PEGylated proteins through the rate model will lead to the application of the current approach to lab-scale production. c) An inverse model for the determination of the Calcium channel distribution in the olfactory human system, and Stochastic Models of Calcium Dynamics in Microdomains of the Olfactory Cilia: Identification of detailed features of neuronal systems is a major issue in biological sciences. Mathematical inverse problems and methods have already proved to be efficient in this respect. We have developed an inverse model and derived a method to determine the spatial distribution of CNG ion channels along the length of an olfactory cilium (C. Conca, R. Lecaros, J. Ortega & L. Rosier Identifiability and stability of an inverse problem involving a Fredholm equation, Chin. Ann. Math. 2015). d) New mathematical models for biofilm growth We have published this year a paper on the mathematical modelling of biofilm growth (Cumsille, P., Asenjo, J.A. and Conca C., (2015) “Numerical solution of a novel biofilm growth model biofilm”, in Nonlinear Dynamics: Materials, Theory and Experiments, Springer Proceedings in Physics). In this work we simulate biofilm structures. The numerical method that we use in order

to carry out the computational simulations is new to the biological community, it sheds light on some central issues of biofilm growth: the pattern formation of heterogeneous structures, such as finger-like structures, in a substrate-transport-limited regime, and the formation of more compact structures, in a growth-limited-regime.

e) Modelling, Simulation and Optimization of a Multiproduct Batch plant in biotechnology Gabriela Sandoval finished her Ph.D. thesis working on this subject and she has produced two solid papers. The first one was published in Computers and Chemical Engineering (G. Sandoval, D. Espinoza, N. Figueroa, J.A. Asenjo (2016) MILP reformulations for the design of biotechnological multi-product batch plants using continuous equipment sizes and discrete host selection, Computers and Chemical Engineering, 84, 1-11). In this article we present a new approach, relying on mixed-integer linear programming (MILP) formulations, for the design of multi-product batch plants with continuous sizes for processing units and host selection. The main advantage of the proposed approach is its scalability that allows us to solve, within reasonable precision requirements, realistic instances. Reproducibility of our results can be tested using our models and data available on-line at BPLIB1. The second paper (G. Sandoval, D. Espinoza, N. Figueroa, J.A. Asenjo (submitted) Optimization of a biotechnological multiproduct batch plant design for the manufacture of four different products: a real case scenario) has been submitted to Biotechnology and Bioengineering. Here, a mixed-integer linear programming (MILP) formulation developed in the first paper is used to optimize the equipment sizes of a hypothetical new biotechnological multi-product batch plant, based on information of real known processes for the production of 4 different biotechnological products. Size and time factors were computed and used as parameters to solve the aforementioned MILP reformulation. New constraints were included to permit the selection of some equipment –such as centrifuges and membrane filters- in a discrete set of sizes. For equipment that can be built according to customer needs -such as fermenters and stirred tanks- the original formulation was retained. Computational results show the ability of this methodology to deal with real data giving reliable solutions for a multi-product batch plant composed of 44 unit operations in a relatively small amount of time showing that in the case studied it is possible to save up to a 66% of the capital investment in equipment. f) Mathematical models for tumor growth coupled with tumor angiogenesis. New treatment strategies for cancer. Cancer is a large group of diseases that can affect any part of the body, characterized by abnormal cell proliferation, and an increasing migration rate that could derive in invasion and organ spreading, becoming the leading cause of death in the world. Hence, it is necessary to address new strategies for cancer understanding and treatment. In a recently published paper (P. Cumsille et al., Proposal of a hybrid approach for tumor progression and tumor-induced angiogenesis, Theoretical Biology and Medical Modeling, 2015), a mathematical model has been proposed to take into account all relevant processes in tumor progression, in particular the effect of systemic treatments and angiogenesis. Some experimental designs are suggested to obtain relevant biological or clinical data necessary to estimate relevant parameters of the model. We also define a hybrid approach as the feedback capacity between mathematical modeling and biological experimental design which is required for better understanding cancer, fitting parameters of a given model with a specific biological scenario, and for obtaining models with predictive capability.

g) Mathematical modeling of the dynamic storage of iron in ferritin. Mathematical modeling of iron homeostasis. In the study of transporters and iron absorption regulation, one of our goals is the understanding of iron transport phenomena associated with intestinal iron absorption through mathematical modeling. In a collaborative ongoing work with A. Olivera, Z. Gerdtzen and C. Salgado we have extended a recent model (C. Salgado et al. BCM 2010) to take into account the dynamics of iron transporters during the process of intestinal iron absorption. The next step of this research, which

is in progress, is to design a laboratory experiment that allows validation and calibration of the model. h) Mathematical modeling of a photobioreactor In the study of the effect of cultivation conditions of a photosynthetic microalga for the production of bioactive compounds such as highly unsaturated fatty acid eicosapentaeonic acid C20:5n3, and carotenoids) a mathematical model was derived (Shene et al. 2016, Algal Research). The model considers the mass transfer between the gas and liquid phases in the photobioreactor, the pH-dependent speciation of the dissolved organic carbon in the culture broth and the cells, the mass exchange between the cells and the culture medium, and the rates of the relevant biochemical reactions (i.e. carbonic anhydrase and RuBisCO mediated reactions; dark respiration). The parameters in the model were obtained from the literature. In view of the large number of parameters involved in the 60 model equations, and the fact that these were for different strains of Nannochloropsis and for diverse culture conditions, the model was used only to simulate the effects of pH and the CO2 level in the aeration gas on CO2 assimilation. i) Modeling, and simulation of bioprocess Mathematical models based on mass and energy balance equations can also be used to define the economic feasibility of a given process, i.e. all the steps needed for the conversion of a given raw material into a product and by products. The undergraduate thesis Modeling, simulation and economic feasibility of the process for the production of docosahexanoic acid by thraustochytrids (J. Peña, Universidad de La Frontera 2016) showed that although the project was technically feasible it was only economic feasible under certain conditions of the system (composition of the biomass, composition of the lipids, carbon source, biomass yield). The results obtained can be used to help in the screening of novel thraustochytrids with biotechnological potential by selecting those strains that met the conditions required for economic feasibility. Mathematical modeling and simulation were used to define conditions of a continuous sterilization process for sugar beet molasses in the production of bakers yeast (undergraduate project in industry, F. Fuentes 2016). 4. - Bioinformatics (BI) a) Compression and indexing of large repetitive collections. Gonzalo Navarro and his PhD student Alberto Ordóñez (U. Coruña, Spain) developed new faster compressed suffix trees for highly repetitive collections. These are 1000 times faster and only slightly larger than previous work. The suffix tree is the most powerful tool for sequence analysis in bioinformatics, and is used to perform all sorts of complex pattern matching operations on sequences. The result appeared in the ACM Journal of Experimental Algorithmics. Many sequence analysis processes extract a large lexicon from the sequence collection, for example all the k-mers or the nodes of the de Bruijn graph. Representing large lexicons efficiently is a challenge. Gonzalo Navarro, with Miguel Martínez-Prieto (U. Valladolid, Spain), Nieves Brisaboa (U. Coruña, Spain), Rodrigo Cánovas (U. Melbourne, Australia), and Francisco Claude, proposed novel lexicon representations and compared a wide variety of alternatives, obtaining recommendations on which to use depending on the type of collection. They published this result in the journal Information Systems. b) Complex pattern matching on Sequences. Gonzalo Navarro and S. Thankachan (Georgia Tech, USA) designed an index that finds in optimal time the pairs of consecutive occurrences of a pattern that are within some range of distances. This is a first step towards finding more complex patterns that are repeated within a short

distance in genomes. They published this result in the Combinatorial Pattern Matching Symp., and a journal version is accepted in Theoretical Computer Science. Gonzalo Navarro and his PhD student H. Ferrada improved the best existing structure for range minimum queries, a fundamental compressed data structure that is used for various complex pattern matching problems on strings and trees. The result was published in the Data Compression Conf. Gonzalo Navarro, his PhD student Roberto Konow, and Simon Gog (KIT, Germany) developed new fast indices for the problem of top-k document retrieval. This aims to return the sequences from a collection that score highest with respect to a query of interest. The problem, well solved on Natural Language text, is mostly open in the field of general sequences, such as DNA or Protein sequences. The new structure uses moderate space and answers queries within microseconds, outperforming the alternatives by orders of magnitude in space or time. The result was submitted to a journal. Gonzalo Navarro and his student Héctor Ferrada developed an alternative variant where a degree of approximation is allowed in the answer, showing that then it is possible to use much less space and obtain also very fast queries. The result was also submitted to a journal. c) Similarity search on collections of complex objects. An important problem when designing indices for similarity search is to understand the intrinsic dimensionality of the space, as it affects the performance of every index, whereas there are indices better suited to some range of dimensionalities. With C. Bustos, N. Reyes (U. San Luis, Argentina), and R. Paredes (U. Talca), Gonzalo Navarro studied the practical performance of various known intrinsic dimensionality estimators, obtaining recommendations on which perform better. They published the results in the conference Similarity Search and Applications and a journal version was invited to Information Systems. Gonzalo Navarro and his PhD student Nora Reyes (U. San Luis, Argentina) obtained new dynamic indices for metric data stored on secondary memory, which is essential when handling large datasets. The new indices improve upon the state of the art in number of distance evaluations or accesses to disk, at query or at update time, depending on the case. The article was accepted in the journal Information Systems. d) Gonzalo Navarro and Alvaro Olivera had started a collaboration for understanding the mathematical properties of the graphs defined by hydrogen bonds in proteins, with the aim of discovering which graph properties best predict actual interactions and dependencies inside proteins. To this effort they incorporated in 2015 an ex-PhD student of Gonzalo Navarro, Cecilia Hernández, who is an assistant professor at U. Concepción (Chile). Cecilia and her students also worked intensively on PPI (protein-protein interaction) networks, with the aim of using graph clustering techniques for the detection of protein complexes. The results have been sent to a journal. Mauricio Marin, with the parallel computing group at U. of Santiago, have developed strategies for improving query throughput in similarity searches for complex objects in metric spaces. In particular, new techniques combining caching and indexing strategies have been developed and published in two papers that appeared in the Lecture Notes in Computer Science Series of Springer. e) Compression Platforms and Services Gonzalo Navarro and Mauricio Marin are supervising the construction of a highly compressed distributed storage of genomic sequences. Currently there is an operational version of a compressed file system that works in a client-server mode where it is possible to keep compressed sequences on both sides (local user PC and server) with very efficient communication

between them. Experiments on real settings show impressive results. This has enabled us to talk with potential customers. In this line, we are currently working with a Chilean company that offers sequencing and analysis services to a large set of users abroad. The company is interested in improving the quality and diversity of their services by including (1) optimized data compression storages, (2) automatic detection of user communities, (3) high-performance parallel and distributed computing to speed up their process pipelines operating with many concurrent users, and (4) support for reproducible experimentation, documentation and user’s communication by means of formal specification languages. A platform like this would provide the company a competitive advantage in the market place. Thus in the near future we plan to use our file system as a base to automatically discover communities working on related sequences. We anticipate that this feature will enable us to produce a multi-user platform where collaborative work is encouraged by a recommendation system that discovers users with similar interests. There is also undergoing work on developing additional services built on top of the basic compression platform. Services such as: (1) phylogenetic analysis, (2) miRNA target prediction, (3) protein structure prediction and (4) virtual screening. f) Map-Reduce Scalability Bounds Mauricio Marin, with the parallel computing group at U. of Santiago, is devising tools to operate on top of the Map-Reduce compressed sequences file system. Each tool is a service that can be mounted as a plug-in on the file system. In terms of efficient implementation of these services using the Map-Reduce model of distributed computing, particularly those based on indexed sequences, a relevant topic to be investigated is the limits of Map-Reduce to support the realization of the plug-in services. In a recent work, we demonstrated formally lower bounds on the isoefficiency function for Map-Reduce applications, when these applications can be modeled as BSP jobs, which is the case for applications of bio-informatics. We also demonstrated how communication and synchronization costs can be dominant for Map-Reduce computations and discuss the conditions under which such scalability limits are valid. This work was published in J. Concurrency and Computation. g) Major Bioinformatics Applications Mauricio Marin and Carlos Conca are developing parallel and distributed numerical methods to solve differential equations modeling tumor growth in cancer. This is ongoing work where there is a first prototype operating. The next steps are to study the GPU parallelization of main operations involved in the computation of tumor evolution. h) Cluster of computers based realization of Bioinformatics Applications and Fundamental Algorithms for Bioinformatics. Mauricio Marin and the U. Santiago group are performing research on new techniques for (1) phylogenetic analysis based on multi-objective optimization (ongoing PhD thesis), (2) designing a high-throughput system for miRNA target prediction (ongoing MSc thesis), (3) design of robust metaheuristics for Protein Structure Prediction and protein-ligand docking and (4) analysis of high-throughput genetic data using multiobjective clustering techniques. This work is developed in collaboration with other researchers from CeBiB from problem formulation to evaluation of the solutions obtained. Results in these topics have been published in Computational Biology and Chemistry, J. Parallel Distributed Computing, and in the Genetic and Evolutionary Computation Conference 2015.

5. - Molecular Genetics, Extremophiles and Ecophysiology (MG)

a) Isolation and culturing of extreme microorganisms. Extreme environments: This year we have generated new information regarding the microbial diversity of Salar de Huasco, which have revealed the presence of rare microbial groups. We have produced new metagenomes from microbial mats, increasing our knowledge regarding the functional role of microorganisms in high altitude wetlands. In collaboration with Dr. Steve Schmidt (Colorado University, USA) we participated in an expedition to the Llullaillaco volcano, one of the highest volcanos on Earth, collecting soil samples at 6000 masl. The objective was the isolation of extreme bacteria (probably endospore forming Firmicutes) with the potential to produce bioactive compounds. In response to a request from the Ministry of National Assets, we have investigated the limnology and microbiology of the hypersaline Laguna Cejar, in the Salar de Atacama, which exhibited a unique microbial diversity and chemical conditions. Model bacterium: We have selected one strain of our collection (Rhodobacter sp.) as a model for UV resistance experiments. Vilma Pérez (PhD student, Dra. Martha Hengst) undertook a 4 month research visit to Sterling University, UK, with Dr. Sabine Matallana obtaining the proteomics of the strain previously exposed to different radiation conditions in the laboratory and in situ at the Salar de Huasco. In addition, it has been possible to sequence the genome of this bacterium. Our results have revealed previously unknown pathways of UV protection. Lithium: We have established new methodologies for the isolation of halophilic Archaea and Bacteria from Salar de Atacama to test their potential to biosorb lithium. It has been possible to scale-up the cultures and currently we are working to test the tolerance to different saline conditions. Thanks to the collaboration with the lithium company (Rockwood Lithium), we have access to industrial samples. Bioleaching: In collaboration with Dr. Remonsellez (UCN), we are testing the bioleaching potential of bacterial isolates from thermal environments (Lirima wetland). Preliminary results show the capacity to use pyrite as an energy source. In the same area, we are sequencing the genome of Acidithiobacillus ferrivorans isolated from the Aromo river, Chilean Altiplano, which have the capacity to oxidise S and Fe at low temperatures. b) Seaweed cultivation and genetics During this second year of the proposal, we continue focusing our work on ecophysiology and genetics of macroalgae, particularly on our model seaweed: Macrocystis pyrifera. From the genetics point of view, after we determined the genetic population structure of the species along the Chilean coast, together with the fact that the species is exploited for alginate industry, conservation issues have alerted our team. We demonstrated and published in Algal Research (Barrento et al. 2016) how the time of maintenance of microscopic stages of kelp under controlled environmental conditions, sex and genetic origin determine the success of a germplasm for the conservation of the genetic variability of the species and to develop new strains for cultivation. In relation to kelp farming there are 2 additional manuscripts ready to be submitted to Aquaculture (related to Optimization of kelp production during the hatchery phase) and a second to Reviews in Aquaculture (on the sea-phase kelp production). In addition, we have been developing the experimental protocols to evaluate how gene expression changes under different culture conditions or different environmental triggers. During this period, we established a proper RNA extraction protocol, quantitative and qualitative analysis of RNA and RT-PCR analysis to set up our comparative CT (ΔΔCT) experiments.

c) Seaweed ecophysiology To continue our work on ecophysiology, and to embrace new questions, we developed new techniques at our laboratory, i.e. extraction of products of chemical interest (for example published by Leyton et al. 2016 Algal Research) and/or physiological-productive relevance; and establishment of photosynthesis experiment using Fluorescence (PAM) and Infrared Gas Analysis (IRGA) under outdoor conditions using adult individuals. A first congress presentation (International Seaweed Symposium) and manuscript will be prepared during 2016. In addition, an experiment was set up during 2015 to test growth performance and biochemical composition of 3 different genetic kelp populations under different nutrient conditions: 1) filtered seawater with no added nutrients and 2) effluent seawater from fish culture tanks. We analyzed the parameters such as: water nutrients (ammonia, nitrate and orthophosphate), light intensity, specific growth rate; final morphological characteristics including maximum length, width, individual weight, stipe length and width and blade angle; and final chemical composition including amino acids, manitol, alginate, glucose, phloroglucinol, DPPH, and fucoxanthin. The results indicate that both factors: nutrient source and population might affect the biochemical composition and the results will be ready for publication in 2016. d) Seaweed Products Finally, in a more applied context, during 2015 we finished pilot-scale experiment to test Kelp biomass transformation: production of biofuel (bioethanol) in a 70-L fermentor. The complete process involves an acid leaching and enzymatic depolimerization in 200L bioreactor followed by an enzymatic saccharification and final fermentation in 75L fermentor. The work is embodied in a finished manuscript ready to be submitted for publication during 2016. Additional work has been carried out by PhD student on kelp ecology studying aspects of kelp-bacteria interactions intending to understand the functional role that bacteria may exert on kelps. In addition, students are also studying the effect of human introduced nutrients into the environment enhancing algal blooms that can affect kelp development and the biomass availability in the future and the role of motile herbivores that affect the cultivation success. e) Screening of secondary metabolites from extreme bacteria (halophiles, thermophiles). One of the challenges has been the establishment of a battery of bioassays and the parallel work with analytical techniques (HPLC-MS). Nevertheless, we have been able to isolated halotolerant yeast from the Salar de Huasco determining their cytotoxic potential against Vibrio parahaemolyticus. Currently we are preparing a battery of bioassays in house to determine the anticancer potential of at least 50 crude extracts. Complex ecological interactions: another important source of bioactive compounds is via organisms that have particular biological interactions. In collaboration with Dr. Cristian Villagra (UMCE) we have been able to isolate fungi and bacteria that mediate the formation of galls and the colonization of insects in a native plant. Our results shows that several key components of this ecological interaction is mediated by the production of bioactive compounds with antimicrobial activity. f) Isolation of new microorganisms from Atacama. Microbial diversity in Atacama may now include three new members belonging to the Staphylococcus, Bacillus and Haloterrigena genera, isolated from Salar de Atacama, Andes highlands and Salar Grande, respectively. These microorganisms show properties that make them interesting candidates for biotechnological applications. Staphylococcus scuiri sp. LCHXa is a Gram positive bacterium able to grow at molar concentration of lithium. Studies are being conducted to learn how LCHXa tolerates Li, its adaptation mechanism

to high osmotic stress and if Li is absorbed or adsorbed in these cells. This type work may provide new applications for this microbe as a tool in lithium recovery, a metal with many industrial uses. Protocols for membranes and membrane protein isolation and proteomic studies are been designed to learn about the control on the expression of about 10 proteins that are differentially modulated in the presence/absence of lithium. Also, the draft genome of Staphylococcus sp. LCHXa is now available we will not publish it until we have the results from genomic and proteomic studies that are being conducted now. Nearly 3 million base pairs make the whole LCHXa genome, it is phylogenetically related to and probably a new strain of the Staphylococcus sciure group. Haloterrigena sp. SGH1 is an extreme halophilic Archaea that requires molar concentration of salt to grow; it is part of the microbial consortia colonizing halite rocks whose members until now were unculturable (Robinson et al., 2015. Microbial diversity and the presence of algae in halite endolithic communities are correlated to atmospheric moisture in the hyper-arid zone of the Atacama Desert. Environmental Microbiology Vol. 16(6) DOI: 10.1111/1462-2920.12364.). SGH1 is a carotenoid-rich Archaean and work is under way to identify them; preliminary evidence indicates that bacterioruberin, a C-50 carotenoid with important antioxidant properties, is one of the most abundant carotenoids made by SGH1. Genomic DNA from Haloterrigena sp. SGH1 has been recently sent for sequencing and this information will become available during May-June 2016. The draft genome of a third isolated microorganism was available in 2015 (Vilo et al. Draft genome sequence of a Bacillus bacterium from the Atacama Desert wetlands metagenome. Genome Announcements 3(4):e00955-15.doi:10.1128/genomeA.00955-15). This bacillus is part of the accompanying microflora of the edible cyanobacterium Nostoc sp. Llayta and its genome is ready for exploration. During this time, we have also confirmed the nutritious quality of the protein of Llayta and the content of essential amino acids may reach nearly 50% of the dry weight. The metagenome of Llayta is still under our scrutiny and we think new developments will be available soon. A new area of research on eukaryotes from Atacama has opened up with a recent collaboration with scientists from Brazil (Gonçalves et al., 2015. Fungi associated with rocks of the Atacama Desert: taxonomy, distribution, diversity, ecology and bioprospection for bioactive compounds. Environ. Microbiol. DOI: 10.1111/1462-2920.13005). This work support the evidence provided previously on the presence of fungi in the Atacama (Gómez-Silva, B. 2010. On the Limits Imposed to Life by the Hyperarid Atacama Desert in Northern Chile. In: Astrobiology: Emergence, Search and Detection of Life. Chapter 9, pp. 199-213. VA Basiuk (Ed.). American Scientific Publishers, Los Angeles, CA, USA.) Now we know a little bit more about whom and where they are. 6. - Collaborative Activities An has been described amongst the activities under the 5 main research lines of the Centre during the second year described above these include most collaborative activities as follows. - Under Metabolic Engineering (ME)

• Chemical analysis of crude extracts; separation methods (Dorador-Gómez / Asenjo – Andrews).

• Bioinformatics (Dorador – Gómez /Asenjo – Andrews).

• Determination of the pathway of production of antioxidants in seaweeds (Buschmann-

Lienqueo- Asenjo-Andrews-Olivera).

• Metabolic reconstruction of bacteria from Atacama desert for natural production (Andrews-Asenjo- Dorador-Gómez)

- Under Protein Engineering (PE)

• Determination and improvement of new enzyme for saccharification of seaweed (Asenjo-Andrews-Olivera- Lienqueo).

• Development of high-throughput exhaustive prediction tools for the effect of mutations

(Olivera-Asenjo- Andrews).

• Development of rationally optimized recombinant protein variants (Olivera-Andrews-Asenjo)

• Development of predictive databases of mutation effects in cancer and in protein design

(Olivera-Navarro-Asenjo-Andrews)

- Under Mathematical Modelling (MM)

• An inverse model for the determination of the Calcium channel distribution in the olfactory human system (Conca-Lecaros- Olivera).

• New mathematical models for biofilm growth (Asenjo-Conca- Cumsille-Olivera).

• Mathematical models for tumor growth coupled with tumor angiogenesis. New treatment

strategies for cancer (Asenjo-Conca-Cumsille-Olivera).

• Mathematical modeling of the dynamic storage of iron in ferritin (Conca-Olivera).

• Stochastic Models of Calcium Dynamics in Microdomains of the Olfactory Cilia (Andrews-Asenjo-

• Conca).

• Mathematical modeling of iron homeostasis (Conca-Olivera). - Under Bioinformatics (BI)

• Compression and indexing of large genome collections for bioinformatics applications (Navarro-Marin-Asenjo-Andrews).

• Cloud, GPU, and cluster based services for major bioinformatics applications (Navarro-

Marin-Asenjo-Andrews).

• Major Bioinformatics Applications adapted to Cloud services (Marin and Researchers from ME and MG groups).

• GPU based realization of Bioinformatics Applications (Marin-Navarro, and Researchers

from ME and MG groups).

• Cluster of computers based realization of Bioinformatics Applications (Marin-Navarro, and Researchers from ME and MG groups).

- Under Molecular Genetics, Extremophiles and Ecophysiology (MG)

• Genetic bases of ecophysiological plasticity and production responses of Seaweeds (Buschmann-Asenjo).

• Finally, Mathematical models for lignocellulosic biomass biorefinery (Shene, Lienqueo,

Asenjo, Andrews) is being carried out mainly by C. Shene and M.E. Lienqueo who have an undergraduate student developing his thesis on this subject.

2) Human Capital formation

The Centre’s main base is the Faculty of Physical and Mathematical Sciences of the Universtiy of Chile. A key policy of this Faculty is to attract both Chilean and foreign young academics who are at the frontier of science, technology and engineering to become full time faculty members. This Centre not only fully endorses this policy but will take full advantage of this in order to increase the number of young investigators in the next few years. This policy is also fully endorsed by the other Universities participating in this Centre (Los Lagos, La Frontera, Antofagasta, U. Santiago). Utilizing the resources made available by the basal funding of Conicyt we are planning to attract back to the country some of our students who have been carrying out their Ph.D. studies in the best Universities in the USA, in the U.K. and in Australia, amongst other top places, and whose profiles clearly match the focus as well as the key areas of work in Components I to V of the Centre. They include Dr. Pía Rodríguez, who carried out her Ph.D. at the University of Pennsylvania and published her work in Science (Rodriguez, P.L., Harada, T., Christian, D.A., Pantano, D.A.,Tsai, R.K., Discher, D.A. (2013) Minimal “Self” Peptides That Inhibit hagocytic Clearance and Enhance Delivery of Nanoparticles, Science, 339, 971-974) and is presently a postdoc at Stanford and Dr. Verónica Martínez, who has finished her Ph.D. working at the Australian Institute for Bioengineering and Nanotechnology (AIBN) at the University of Queensland in Australia and Camila Orellana carrying out her Ph.D. also at AIBN. Dr. Martinez is presently a postdoc at AINB and after that she will become a professor at the University of Chile or La Frontera and will become a young researcher at the CeBiB. Dr. Miguel Angel Campodonico who spent two years at the laboratory of Prof. Bernard Palsson at UCLA working at the “core” of Metabolic Engineering and Systems Biology and is now a postdoc at Novo Research Foundation in Denmark is also planning to return to the CeBiB as an academic and researcher within the next 3 years or so. Two Latin American students, Paul Acevedo (of Ecuador, University of Quito) and Duver Quinteros (of Colombia, University of Bogotá) defended doctoral theses in the field of mathematical modeling. Next July, the student Matías Godoy (Universidad de Chile) will defend his PhD thesis entitled: Detection of objects immersed in a fluid. In the University of Antofagasta, two master students defended their thesis in the field of biotechnology-based lithium extraction (Carolina Cubillos) and plastic degradation (Vinko Zadjelovic). Next semester, Bernardita Valenzuela will defend her PhD thesis about methanogenesis in El Tatio Geothermal Field. The University of Chile has signed a MOU (Memorandum of Understanding) with AIBN and we are planning to invite Profs. Lars Nielsen, Peter Gray and/or Anton Middleberg to spend substantial periods of time collaborating with us. The University of Chile has become a key partner of the University of Queensland in Latin America and Dr. Asenjo was invited to Queensland to deliver a key lecture at the end of July 2015. We expect this Centre to be an international pole of attraction in the field, competitive with the best internationally and as such we would like Prof. Alejandro Wolf, who very successfully carried out his Ph.D. at MIT, under Doug Lauffenburger, then was a postdoc at Harvard and has now a tenure track position of Assistant Professor at the University of Washington in Seattle, and

who presently has a research group working on Systems Biology applied to cancer, to be attracted in the next two years or so to become one of our research leaders. In the meantime he has the commitment to become a part-time faculty in the Centre spending time collaborating with our key researchers and co-supervising some Ph.D. students. We expect that the quality of the research being carried out and published in international journals as well as the solid international reputation of the participants and the resources being made available by the basal funding, will make the Centre a very attractive place to come and work for top level young and ambitious scientists from around the world. In the same line as discussed above and as mentioned in this proposal, many of our Ph.D. students spend periods of time, up to 1 ½ or even 2 years of their Ph.D. studies, in some of the best centres worldwide. These include the laboratories of E. Terry Papoutsakis presently at the University of Delaware, the laboratories of Prof. Sir Tom Blundell and Steve Oliver at the University of Cambridge, the laboratory of James Liao at the University of California in LA, the laboratory of Bernard Palsson at the University of California, San Diego, the laboratories of Mervyn Bibb at the John Innes Centre in Norwich, U.K., the laboratory of Marcel Jaspars at the University of Aberden and many others. Our Ph.D. students have spent important periods between 10 months and 2 years in all these centres mentioned above in the last three years. We expect to make visiting professor appointments to many of these collaborators as well as those that form part of our international network, so they will spend longer periods of time collaborating with our key researchers, co-supervising Ph.D. students as well as delivering short courses and participating in interdisciplinary workshops as they have done in the past to a more limited extent. Two undergraduate students (Jodie Esprabens of ENSIACET, Toulouse, France and Melanie Abrams of MIT, USA) spent six and two months, respectively, at CeBiB lab, to develop parts of their research unit in the Algal biorefinery area. Julian Naderi from Berlin Beuth Technical University is spending ca. 10 months at the Centre carrying out his final year research project and his Master’s Thesis working in the project of antibiotic synthesis optimization of Atacama desert Streptomyces.

3) Technology Transfer and Linkages with Other economic sectors and the Chilean society.

The Centre has created a technology transfer office. For this an experienced professional has been hired, Ms. M. Isabel Guerra with extensive technology transfer expertise. She was key in developing a spin-off in our Faculty called “Plasticopper” which is one of the first in the University. Another important achievement of our technology transfer office was the creation, in 2015, of the startup "Medical Innovation", aimed at the manufacturing and marketing of medical equipment, in particular the handheld portable ultrasound device TAOTE, in the process of patenting in Chile and in the USA with support from CeBiB. As shown in the Centre´s budget a substantial amount of resources has been set aside for this purpose. CeBiB has also supported the establishment of the pilot plant of the spin-off “Atacama Bioactives”, who together with the Universidad de Antofagasta created the product “Atacama Cream”, currently in the process of being patented. Some of the Centre’s researchers have international patents, for example 3 from cold active enzymes from Antartica. A small firm called STI, SoutheastTechInventures based in North Carolina U.S. has signed an option agreement in order to commercialize these patents as described below. This link has been strenghtend in the last few months. Similarly, other potential developments of the groups of the Centre such as a vaccine against alcoholism, new technologies developed for more efficient production of biofuels and chemicals and the development of Macroalgae carried out by Buschmann to make this an attractive substrate for commercial development are clear examples of what we expect in order to have an impact on the economic and social development of the country.

A solid example in this category is the development by the group of Carlos Conca and collaborators of a portable and cost effective ultrasound device which generates 2-D ultrasound images from inside a patient which is a totally new development worldwide. As a result of this project Dr. Conca and his collaborators have won several grants from Corfo (including “go-to-market”) which has allowed them to show the potential of this technology in several places in the USA (Stanford, New York, Chicago) and is resulting in the first “Spin-off” to come out of our Centre. Finally we should mention the fact that the support of Yahoo in the USA and Europe, to carry out frontier research by Chilean graduate students and Postdocs is an opportunity of having an international impact through research and development generated in Chile. The group of Professors Asenjo and Andrews has patented three commercially attractive enzymes with high activity at low temperatures which recently they have been actively attempting to commercialize through the Company STI (Southeast TechInventures) in North Carolina, USA. Finally the University of Chile signed the confidentiality agreement with STI after several months of negotiations between Professors Asenjo and Andrews and the University lawyers and authorities. This is a paradigmatic event since it is the first such agreement that the University signs to carry out the commercialization of these patents developed at the University. Given the fact that STI has shown a commercial interest in the three patents we expect the technology transfer will happen during the next few years. We have now hired Sergio Mercado, presently finishing his Ph.D. at the University of Cambridge in the UK, who was a key professional in the transfer of technology on cell culture and virus production between CeBiB and Butantan Institute in Brazil and later to the Biotechnology Centre in Montreal, to be the link and advisor between CeBiB and STI in North Carolina and also the Technology Transfer Office from the University of Delaware in the USA. He is presently developing a plan to bring all our 3 US patents on cold active enzymes closer to the market. Another case of technology transfer constitutes the research carried out by the groups of Marin and Navarro which is closely related and supported by the offices of Yahoo in the USA and Europe. The fact that Yahoo carries out frontier research both at the University of Santiago and at the University of Chile, through PhD students and Postdocs, is a great opportunity for our Centre, a clear example of technology transfer to the international computer industry. This is technology transfer already occurring and the resources of this transfer are clearly being channeled into the private/international funding of the Centre. The activities and support of Yahoo have been carried out during 2014 and 2015. Another case where technology transfer will take place is the seaweed ecology and aquaculture group of the i-mar Research Centre, which has made possible the creation of the BAL Biofuel Consortium and the installation of SpaceLab in Puerto Montt to promote innovation activities by providing chemical and biomass transformation infrastructure and equipment. The interaction of Professor Buschmann with BAL Biofuels who are clearly interested in commercializing different applications of the use of Brown Macroalgae that have several important economic potential applications such as some of its highly valued components and its transformation into biofuels and highly valued chemicals such as hyaluronic acid for application in medicine and cosmetics will be very fruitful. Knowledge transfer and expert consultancies related to aquaculture strategies towards the optimization of seaweed biomass production will be carried out. This will be done with consulting contracts between the i-mar Research Center and seaweed production companies and the latest example of this is the attendance of Dr. Buschmann to the Workshop on “Innovative Approaches to Ocean Cultivation and Processing of Macro Algae for the Production of Fuels, Chemicals, Feed, and Food” organized by the Departament of Energy in Washington DC (Advanced Research Projects Agency - Energy (ARPA-E)). Mauricio Marin and Gonzalo Navarro, with younger collaborators in the Center, are designing a product for the storage of large repetitive collections of genomic sequences, which will be offered to other research groups in the Center and abroad. The product is a storage service that takes

advantage of repetitiveness to significantly reduce the storage space. We are exploring Relative Lempel-Ziv as the underlying technology to exploit repetitiveness while supporting fast access to the data. The first prototypes of the product are being developed and tested with real biological data. CeBiB has established formal collaboration with the Agroindustry Institute at Universidad de La Frontera. This institute is at present working on a project for the creation of the Technological Centre for Innovation in Foods (CeTA, Corfo funds). In this Centre participate other national research institutes and it will be the platform for supporting the collaborative applied research in food technology not only with research groups from other institutions but also with industry.

4) Support to other research groups All main researchers in the Centre have and have had a large number of collaborations with other groups in the country. We have joint Ph.D. student supervision with them and carry out collaborative projects, thus supporting these research teams. Such groups include, in many cases, ex Ph.D. students. Clearly, thanks to the basal funding of Conicyt these collaborations are now better funded and thus very much enhanced. In addition to joint Ph.D. student supervision during the next year we are going to carry out workshops both with the participation of our international collaborators as well as our national ones as we have done in the past when we carried out two workshops. One on Microbes from the Atacama Desert and the other on Biofuels, Biorefinery and Protein Engineering. Both were extremely successful. In October we will have another workshop on Atacama desert microbiology and genetics with the participation of all our UK collaborators, Profs. Bull, Bibb, Goodfellow and Jaspars. Some examples of national collaborators are: Prof. Andrea Mahn, University of Santiago Prof. Juan Pablo Acevedo, University of Los Andes Prof. Loreto Parra, Catholic University Prof. Jeremy Barbay, Dept. of Computer Science, University of Chile Prof. Diego Arroyuelo, University Federico Santa Maria Prof Rodrigo Paredes, University of Talca Prof. Francisco Claude, University Diego Portales Prof. Diego Seco, University of Concepcion Prof. Patricio Cumsille, University of Bio-Bio Prof. Michael Seeger, UniversityFederico Santa Maria Prof. Bernabe Santelices, Catholic University Prof. Bernardo Gonzalez, University Adolfo Ibañez Prof. Monica Rubilar, Bioresource Nucleus, BIOREN, University of La Frontera Prof. Cesar Burgos, Agro-aquaculture Genomic Centre, University of La Frontera Prof. Julio Vasquez, Catholic University of the North Prof. Juan Correa, Catholic University Prof. Rene Carmona, University of Chile

5) Dissemination and Outreach activities to other sectors of Chilean

Society

At the end of the first year, CeBiB added to its staff a senior journalist specialized in scientific communication. In order to fulfill the goals defined in CeBiB’s proposal: (i) development of a diploma course in biotechnology and bioengineering for journalists (ii) communicating scientific advances in modern biotechnology to high school students via lectures given by the Centre´s scientists among other activities, (iii) delivering and sponsoring state-of-the-art lectures and conferences for the scientific community- a strategic communication plan was developed. This plan aims to: a) increase CeBiB’s presence in mass and social media b) increase contact of CeBiB’s scientists with general and selected public, particularly high-school students c) increase awareness of CeBiB as a reference centre in biotechnology and bioengineering in the scientific community. a) CeBiB’s presence in mass media and social media: Introduction: to create a connection between specialized journalists and CeBiB it is necessary first to create a strong presence in social and massive media. CeBiB must become a reference for biotechnology and bioengineering as a first step to achieve validation and therefore become the organizer of a diploma course for journalists in general. a.1) Digital Strategy: Creation of CeBiB channels on social media, starting with Facebook, followed by Twitter, Instagram, LinkedIn and Issuu, to reach different audiences. A first step was to aim massive audience and now, once achieved the goal for Facebook (+500 followers) and Twitter (+150), we will focus in 2016 on a selected audience thanks to the development of BioTech News, a magazine released online (Issuu) twice a year. In parallel, we will strengthen our digital contents aimed at younger public (high school students, young adults). For further details related to the digital component of the strategic communication plan, please, refer to appendix CeBiB Digital Strategy a.2) Mass Media Strategy: CeBiB first steps to strengthen its presence in mass media was to position its leading scientists as public speakers in topics of interest. We have increased our coverage by 100% in comparison with the first year with actions targeted to position our leading scientists as specialized sources on topics ranging from specific to general. We have also created alliances related to specific actions to link CeBiB with government institutions and strengthen relations with the communication channels of all the universities associated to CeBiB and the local media in their cities. There are challenges to achieve, such as creating a stronger bond between every associated university and CeBiB that allows presence from both institutions in media without “cannibalizing” each other. For a coverage report to the date, please, refer to CeBiB Coverage Report. a.3) Delivering Knowledge Strategy: Now that the most important scientific journalists have a relation with CeBiB, we have created a program called Biotechnology Up To Date, which consists of small seminars about state-of-the-art topics in biotechnology, bioengineering and bioinformatics. These seminars have been organized according to the journalist’s needs. For instance, for La Tercera newspaper, these seminars will take place at the newspaper offices. For public institutions, at CeBiB. This approach acknowledges the difficulties of the daily agenda of journalists and encourages bonds with mass media. The date will be defined by every interested media. a.4) CeBiB Microprograms: In alliance with Imago, a company specialized in science programs for TV, we have produced 5 TV programs to be broadcast in 2016-2017 on the 24 Horas Television Network. These programs refer to some of the different research lines at CeBiB and explain how these research lines address urgent needs. In parallel, we developed with Cabala Productions

(another company specialized in science documentaries) a serie of eleven micro-documentaries, dedicated to explain the work of our principal and associated researchers. These programs will be delivered to schools, social network and CeBiB channels. b) CeBiB’s contact with general and selected public, particularly high-school students The digital strategy in social media speaks also to younger audiences, especially high-school students who consume information posted on social networks, such as Facebook. However, there are also different channels to have direct contact with them, some of them linked to activities organized by the Chilean Secretary of Education, other to CeBiB associated universities. To tackle this point, we developed a strategy directed –in an early phase- to attract interest from high school students and generate interest among our scientists to become specialists in divulgation. Now that CeBiB is better known among school teachers and students, we are developing a program of seminars in CeBiB which includes a lecture, followed by questions and a visit to our laboratories guided by our researchers to demonstrate the work they do. To see in detail the results in Students Outreach, please refer to file CeBiB Outreach Participation b.1) Campaign to increase awareness of our role as scientific divulgators: CeBiB has created an internal communication campaign for young scientists, recognizing their support as Educational Contents Coordinators and launching an internal science-photo contest, the results of which will be communicated massively and via social media, among other activities. The role of our Educational Contents Coordinators have been highlighted in social media, to foster the link between younger public and young scientists. b.2) Active participation in activities with high-school students: The program Explora has the most extended network of schools, universities and research centres connected with the goal of divulgation of science and technology to students and the general public. CeBiB had meetings with Explora Antofagasta, located at the University of Antofagasta, and Explora Puerto Montt, located at the University of Los Lagos. These meetings resulted in cooperation agreements with CeBiB and during 2015-2016 our researchers participated in more than 20 conferences to high-school students, reaching more than a thousand teens from Antofagasta to Puerto Montt. Special mention to the Science Week organized by Explora Santiago: CeBiB participated in this five day event with a stand consisting of an exhibition and 20 minutes interdisciplinary seminar (repeated through the day with recesses of 10 minutes) of Chilean biotechnology potential from north to south, with scientists from the universities of Antofagasta, Chile and Los Lagos. This stand received more than 1.500 visits. We also participated in a conference organized by the Universidad Mayor, with an average assistance of 400 high school students. CeBiB will be part of the Science Expo organized bi-yearly by the Faculty of Physical and Mathematical Sciences, an activity that receives on average more than 1.500 high school students. b.3) Program “Knowing Your Science”: CeBiB has created a program called “Knowing Science” which consist of a class given in CeBiB and practical activities. CeBiB received five groups in 2015 of primary students to test the methodology but also to reach primary students, their parents and teachers. For 2016 we have defined the audience of the program to reach high school students, via their schools and science clubs. We have already organized visits for June and July from Deutsche Schule of Santiago and Sek Institute. b.4) In alliance with the Faculty of Physical and Mathematical Sciences we have organized a seminar for high school science teachers with three topics: The Unknown Macroalgae World, The Potential of Extremophiles from the Atacama Desert and Biotechnological Applications Based On Chilean Resources, to be held in June. b.5) In alliance with the Chilean Academy of Sciences CeBiB will offer a serie of seminars for the general public and high school students under the general topic “The year of Biotechnology, Bioengineering and Bioinformatics” in which will participate at least nine of our principal

researchers. These seminars will take place at the Chilean Academy of Sciences from June to August 2016. b.6) CeBiB organized the participation of iGEM UChile OpenBio in the iGEM Competition in Boston (USA), the most important contest on Synthetic Biology worldwide in which more than 3.000 students participate. This was the first time the University of Chile had a team representing the science done in this University. This team visited schools and hosted talks about Synthetic Biology supported by CeBiB researchers. The aim of this action was to attract high school students to the topic and was widely covered by media and government institutions. For further details please refer to CeBiB and iGEM UChile OpenBio. c) CeBiB as a reference centre in biotechnology and bioengineering in the scientific community CeBiB has developed international seminars with researchers from different universities/countries delivering specialized lectures and conferences to the scientific community. Also, every researcher of the Centre presents at least once a year their work and state of development in open seminars. These and other contents will appear also in BioTech News, the magazine developed by CeBiB which aims to reach general audiences with scientific culture, but mostly the scientific community. For CeBiB researcher participation in congresses, please, refer to Annual Productivity Register. In parallel, the Technology Transfer and Communication Departaments have developed a common strategy to increase awareness of CeBiB as a reference centre in biotechnology and bioengineering. This strategy has different audiences: government, industry, associations related to science and industry, other research centers, universities and the scientific community. According to this strategy, we developed a serie of profiles of CeBiB Technologies of interest to industry that have been presented in seminars, congresses and via email. We are also organizing new thematic seminars in alliance with the Pfizer Center of Excellence in Precision Medicine and ChileBio, with speakers from different fields giving lectures on state-of-the-art topics ranging from biomedicine and cancer, to transgenic seeds. Special attention to CeBiB support to the Taote initiative, the medical device for mobile ultrasound examination, with a strong media campaign that obtained important coverage and participation in contests and programs of other institutions and universities nationally and internationally. We are also creating an alliance with the Chilean-American Chamber of Commerce (AmCham Chile) to participate in their Biotechnology Programme, congresses and visits to related industries in the United States and in Chile. To increase awareness in public leaders, CeBiB Director, Juan A. Asenjo, participated in important events, which were communicated massively by CeBiB. In those events, Juan A. Asenjo referred to the interdisciplinary research made in CeBiB and the necessity to have a Science and Technology Ministry. Dr. Asenjo continues to be quoted as an expert in public policies to foster science and technology development.

Specific questions to answer in this section

1) Technology Transfer external advisories and firms commitment The Centre has consulted and received advice from Bernardita Araya and other business experts related to the application for funds from CORFO to develop and translate applications of potential commercial interest into “start-up” companies in Chile. Also a close associate of Pablo Valenzuela’s Bios-Group, namely Dr. Arturo Yudelevich is now a member of the Board of Directors of CeBiB providing advice on technology transfer issues. Regarding licensing our three US patents on cold active enzymes from Antarctica, we are actively increasing our interaction with Southeast Techinventures, STI, in North Carolina. We have now hired Sergio Mercado, presently finishing his Ph.D. at the University of Cambridge in the UK, who was a key professional in the transfer of technology on cell culture and virus production between CeBiB and Butantan Institute in Brazil and later to the Biotechnology Centre in Montreal, to be the link and advisor between CeBiB and STI in North Carolina and also with the Technology Transfer Office from the University of Delaware in the USA. He is presently developing a plan to bring all our 3 US patents on cold active enzymes closer to the market. Our relationship with Bioarchitecture Laboratories, BAL Chile, for the utilization of macroalgae for biofuels and chemicals (such as butanol and hyaluronic acid) continues to grow as is explained in section IV on Objectives and Results achieved. Also the involvement of CeBiB with the international computer company Yahoo! Inc. was very active during 2014 and 2015 and they provided substantial funding for scientists and Ph.D. students mainly working in the group of Mauricio Marin. The funding was provided by Yahoo! Europe that was managed by our colleague Ricardo Baeza, also a part-time academic at our Faculty at the University of Chile. Unfortunately Yahoo decided to close their office in Europe hence we are looking for other sources of external funding for the Bioinformatics groups as described below in this section. The project related to the obtention of marine sediment samples to find novel microbial strains in the Atacama trench, which was an important subproject of the PharmaSea international consortium, has been postponed since the cost of this deep sea expedition was higher than initially estimated in the project. Finally, we are actively exploring the support of the copper mining companies to CeBiB. Although the mining industry is facing strong financial limitations, the company Ecometales has shown a key interest in having a collaboration with our Centre and we are having a meeting with the general manager, Ivan Valenzuela, and several experts of Ecometales on May 13, 2016. In addition to the efforts described above to generate links with industry in order to generate external funds and bring some of the potential products closer to market, we have made several new efforts with Genoma Mayor, Pfizer, Nactive, Veterquimica and Maqui New Life as described below. a) Genoma Mayor is a spin off of the Universidad Mayor, associated with the Center for Genomics and Bioinformatics at the Faculty of Sciences of this University. The company offers a Sequencing and Bioinformatics service platform. In addition, Genoma Mayor works in projects of Personalized Medicine, that use information about a person’s genes, proteins, and environment to prevent, diagnose, and treat disease. Due to the reduced costs of sequencing and increased process efficiency, many projects based on genomics are producing large amounts of data that need to be stored, distributed and analyzed, creating a major challenge for researchers in the areas of biological and computer science. In this context the research line Bioinformatics at CeBiB, through its principal investigators Gonzalo

Navarro and Mauricio Marin, have developed a technology that allows the management of genomic data in a compressed form. These researchers and Genome Mayor are starting a collaboration to address current storage problems in genomic information and study others such as optimization of the pipeline process of the company, with the aim of improving and expanding the current services provided to strengthen its national and international competitive position. A first step in this relationship will be to apply the computer science doctoral thesis of Manuel Villalobos to the National competition doctoral thesis in the productive sector of CONICYT, sponsored by Genoma Mayor. b) Pfizer is a global biopharmaceutical company based on applied science and the company global resources to bring therapies to people that extend and significantly improve their lives through the discovery, development and production of products for health care. Currently the CeBiB is working to sign a working agreement with Pfizer Chile to explore projects of research and development related to the area of biotechnology, bioengineering and medicine in order to complement capabilities and increase the value of technologies by conducting scientific collaborations. c) Nactive is a company dedicated to the development, production and marketing of active ingredients based on endemic natural resources from Chile, for cosmetic and nutraceutical industries. It has a robust international distribution network and currently is searching for new compounds for their product portfolio for the healing of exposed dermis caused by chronic diseases and post-surgery to prevent keloid formation. Nactive has shown interest in using a patented technology “Protein and Nucleic Acid Sequence Encoding a Krill-Derived Cold Adapted Trypsin-Like Activity Enzyme”. In addition, the company has participated in the application of the project " Obtaining high molecular weight Hyaluronic Acid with a recombinant E. coli strain using sustainable carbon sources" presented by CeBiB to the III IDeA Contest in stage II of the IDeA Program of Fondef, CONICYT. d) Veterquímica is a national company with more than 45 years of presence in Latin America, with four business areas: Animal Health, Animal Nutrition, Hygiene and Biosafety, and Laboratory Services. It has plants and laboratories of pharmaceutical high-tech production covering approximately 4,000 m2 of installed physical plant, including: laboratories and isolated cell culture plants, pharmaceutical production, production of biological and aquariums at GMP level. Currently the company produces viral vaccines for pigs in PK15 cells growing in adherence, but Veterquímica is interested in modifying its current process. The experience of the researcher Ziomara Gerdtzen in animal cell culture and of networks and metabolic phenomena involved in the processes of cell growth and product generation, has led to a partnership on the adaptation of PK- 15 cells to suspension growth, determination of parameters and nutritional requirements of the cells in culture medium for design and optimization of culture conditions, with the aim of improving biomass production and product generation, and finally to obtain a scalable process design. e) Maqui New Life was born as a spin-off of CTI HEALTH in Chile (Consortium for Technology and Research for Health), and now opened its operations in Switzerland. The company manufactures and commercializes Delphinol®, a standardized maqui extract that grows exclusively in southern Chile and take advantage of its rich content of specific flavonoids known as anthocyanins species, especially a subcategory of these known as delfinidinas. Among its many applications Delphinol® is a new dietary option for naturally controlling the sugar blood level after meals. In collaborative work with the company through the principal researcher Alvaro Olivera, in the context of studies of glycemic metabolism and effects of Delphinol ®, they are developing a mathematical model of the possible action mechanisms of Delphinol ® over the glycemic and insulinemic metabolism in patients who have undergone Oral glucose tolerance tests with insulin measurements (OGTT-I).

2) Commitment of knowledge dissemination or outreach

Regarding the commitment of knowledge dissemination or outreach (diploma course about scientific advances in modern biotechnology to journalists and lectures about scientific advances in modern biotechnology to high school students): a) How have these objectives been tackled? a1) Diploma Course about scientific advances in modern biotechnology to journalists: To address this commitment, CeBiB created a strategy to, first, increase awareness about CeBiB as a leader in interdisciplinary research in biotechnology, bioengineering and bioinformatics. To reach journalists, CeBiB first increased its presence in media. Second, once this goal has been achieved and CeBiB researchers are well known in media, and as stated in point 5.a.3, we are organizing the Biotechnology Up To Date Seminars, to be held in the media offices. This means that CeBiB will deliver in 2016 these seminars, for instance, in La Tercera and El Mercurio newspapers, to strengthen the bond between media and CeBiB by acknowledging the difficulties and restrictions of their agendas. These seminars will conclude with a qualitative survey to receive feedback and create, as a third phase, the Diploma Course. In parallel, CeBiB is working with the National Association of Science Journalists, the Faculty of Physical and Mathematical Sciences and the Vice Presidency of Outreach and Communication to create the academic plan for the Diploma Course.

a2) Lectures about scientific advances in modern biotechnology to high school students: As stated in point 5.b.3 and 5.b.4, CeBiB has increased importantly its participation in outreach activities organized, for instance, by Explora Conicyt in Santiago, Antofagasta, Temuco and Puerto Montt, reaching only in the last year around 3.500 high school students. We are now preparing visits to CeBiB from schools in Santiago: their students will be part of a seminar and visit to our labs. In Antofagasta, Temuco and Puerto Montt we will continue working with Explora Conicyt: the quantity of schools are smaller in comparison with Santiago, their activities more restricted and, therefore, we prefer to support Explora instead of diverting audience.

b) Which have been the main mechanism used for reaching this objective? The mechanisms to tackle these two points are: increasing CeBiB public awareness, increase the position of CeBiB as a leader in its fields, increase CeBiB participation in public activities targeted to these audiences, create particular contacts with, for instance, media and schools, target media and schools with specific proposals, attract them to CeBiB and then deliver lectures and talks. The key point to succeed in this was for CeBiB to be recognized as a scientific center of excellence in its field.

3) Commitment of levering up resources from non-CONICYT sources

Our Centre, CeBiB, has been quite successful in obtaining external resources from non-Conicyt sources, particularly from international projects. During 2014 and 2015 we obtained important funding from Yahoo! Europe. We also obtained resources from the EU project PharmaSea and from a UK funded project from the Newton fund. Starting in January 2016 the group of Gonzalo Navarro and several other laboratories were awarded a grant from the European Union to carry out the project: “Bioinformatics and Information Retrival, Data Structures, Analysis and Design” (BIRDS) for a total funding of 648,000 Euros for 48 months. The contribution to Navarro’s group and hence the Centre corresponds to ca. 90,000 Euros. Furthermore we organized an international conference “Affinity 2015” which provided quite a substantial funding. The company Recalcine had a strong commitment to provide external funding to continue with the project on the generation and development of a vaccine against alcoholism. This project, initially funded by FONDEF and Recalcine was very successful since we developed the cell culture and virus production and purification technology and the pilot plant protocols which allowed manufacture of the vaccine in Montreal, Canada and extensive pre-clinical trials to be done in Mumbai, India. However, although Recalcine was prepared to provide substantial funding in a project presented to CORFO, the project evaluators did not understand the scope and relevance of the project and hence it was not approved. Subsequently Recalcine was bought by Abbot Laboratories and they defined different priorities. With Asfalchile, given the fact that Eugenio Correa (President) is a member of the Board of Directors of CeBiB, we are in conversations with him and expect to have a contribution from Asfalchile during 2016-2017. Juan Rayo Ingenieria is an important consulting company in the copper mining sector as we have had projects funded by them on mathematical modeling in the past. We had a meeting with Juan Rayo and Nelida Heresi on 18 August, 2015 and explored possibilities of collaboration but, since the mining industry is undergoing strong financial restrictions, this has not been consolidated into a real commitment thus far. With Bio Sigma we had a meeting with the General Manager, Pilar Parada, and the relevant company scientists on 24 August, 2015 and explored possibilities of collaboration. With Bios Chile we expect to increase potential collaborations given the fact that they made contributions to our three projects on cold active enzymes and that Arturo Yudelevich is a member of our Board of Directors.

• PROJECT MANAGEMENT

Our Centre has developed a very solid management strategy both at the Faculty of Physical and Mathematical Sciences (FCFM) at the University of Chile and at the regional universities involved in the Centre, the University of Antofagasta, the U. of La Frontera, the U. of Los Lagos and the U. of Santiago. The organizational chart is shown below and the decision making council is the Board of 10 Principal Researchers from all institutions that meet 6 or 7 times a year at the Centre’s Council Room at the FCFM. All administrative decisions are taken by this Board. We have an Executive Director (Operational Manager) who is responsible so that all the decisions of the Board are implemented; she is also the link with the financial office at the FCFM and in charge of the transfer of resources to the regional universities. During the period several activities have been carried out by the Operations Management in order to promote and nurture the relationship with the partner institutions and staff belonging to the Centre. Setting up the co- execution agreements between the sponsoring and associated institutions, which allowed the transfer of resources was a major achievement. The Executive Director met authorities and administrative personnel of associated universities first to coordinate the implementation of the work plan of the Centre and secondly to standardize administrative and financial procedures related to budget execution. The support staff maintains fluid communication with researchers and administrative personnel, together with the permanent monitoring of our accounting office who provides ongoing support for the correct accountability. She is also largely the link with the Office in Conicyt that is in charge of the Basal Centres such as CeBiB. We also have Coordination and Product Evaluation Manager. She is in charge and following all the collaboration projects that were agreed at the outset of our Centre and those new ones being proposed at our Workshops. The aim of the Workshop in July 2015 at the Manquehue Hotel was mainly the generation of new interactions and collaborations. She has also had many contacts with industry, with the Innovation offices at the different Universities and made a visit to Stanford and also the East Coast in the US in order to explore the possibilities of raising funds to promote new “Start-up’s”. She is the link with Conicyt on these matters. The role of the Board of Senior Researchers is to evaluate academic outcome, evaluate possible postdoc candidates and propose promotions within the Centre. The role of the Board of Directors has been described at the beginning of this report (I.) and is mainly related with general policies of the Centre, its relationship with industry and society in general including national and international impact. The general policies developed by the Centre as well as the frequent meetings of the Board (10 Principal Researchers) and the two yearly workshops held has guaranteed the cohesion, sense of belonging and proper management of the Centre. In addition to fostering interdisciplinary work and reduce the geographic dispersion, researchers from different groups held meetings to focus collaborative work. The principal investigators Navarro and Marin toured our various partner universities, conducting a survey of needs and opportunities for working with bioinformatics and mathematical modeling. Similarly, a meeting was conducted at the Universidad de la Frontera to define different projects in the research on macroalgae, in which researchers A. Buschmann, C. Shene and ME Lienqueo participated, along with other associates and our technology transfer manager. Also, the principal investigators, A. Olivera and A. Buschmann met at the Imar Center at the Universidad de Los Lagos, to work on various collaborative themes. Also noteworthy, in the effort to achieve and maintain cohesion and sense of belonging to the Center, was the realization of a winter course on Seaweed in the Future

of Chilean Aquaculture, during August 2015 at the Imar Center at the Los Lagos University, attended by all members of CeBiB interested in the topic.

Current Organizational Chart of the Center

V. LESSONS LEARNED Our Centre CeBiB has brand-new facilities occupying the whole 7 th floor of the western block of the new Beauchef 851 building. This requires an important level of infrastructure investment which is considered in the budget of our Centre. The difficulties mentioned in our first year report were finally sorted out by our Executive Director and the finance office (as well as the architects) of the Faculty (FCFM) of the University and the move of all our CeBiB facilities to Beauchef 851 was carried out on the second half of January 2016. This includes our excellent offices and new Laboratories. This resulted in a very impressive inauguration ceremony on Thursday 28 April with the presence of the Dean, Patricio Aceituno, who gave a very nice speech regarding the importance of CeBiB to the Faculty and the University and also the participation of the regional Universities as part of it, which are all public universities. Also, in addition to a presentation given by the Director of CeBiB who explained the strengths, importance and scope of CeBiB, emphasizing the role of the regional universities which constitutes a nationwide effort, there were two interventions by members of the Board of Directors: Mr. Rodrigo Velasco and Dr. Arturo Yudelevich. They stressed the importance of generating new patents and also the transfer of basic and applied science and “know-how” developed at the Centre to industry and the generation of “start-up” companies. This inauguration was attended by well known Chilean scientists, the new President of the Chilean Academy of Sciences, National Science Prize holders, Members of Government, the professionals of the Basal Centre programme of Conicyt, the previous Dean, Francisco Brieva, largely responsible for the idea of putting up this excellent building and the member of parliament responsible for the Committee of Science and Technology, Senator Guido Girardi. During this second year we have also managed to tackle the question of hiring Ph.D. Students and postdocs, both Chilean and from overseas. This has also been true for the hiring of researchers in the associated institutions, mainly thanks to the strong efforts of our management team. Regarding operational expenses, together with the finance office of the Faculty (FCFM) we have also managed to find the ways to smoothen at least partially the process of purchasing of consumables using the system “Chilecompra”, so that this bureaucratic system does not seriously affect the functioning of our Centre. This can be partially bypassed also, for very specific products, services, equipment, maintenance and others via a direct purchase (trato directo) which requires a resolution and a decree which can be slow and has to be done well in advance. As has already been stated in the Project Management section we were finally able to transfer the funds destined to the researchers at other institutions as the agreements between the University of Chile and the associated Universities were finally approved and signed. This was largely done due to the important efforts of our project management team that had long discussions with the regional universities which created in some cases specific “project offices” in order to consolidate the transfer of funds and ensure the correct functioning of the Centre’s laboratories at these universities.

VI. PERFORMANCE INDICATORS This section has been modified since this information is already provided in Appendix 1. VII. COMMENTS ON THE INDICATORS All 4 indicators of Scientific Excellence are very good, particularly the impact level of the journals where the Centre’s researchers have published their articles. The Technological Transfer indicators are also very good. Although there were no new patent applications presented, as has been described in several sections of this report, the Centre has taken important measures and is making a strong effort in order to consolidate its technological transfer to industry. We were also able to insert 3 PhD students in the industrial sector, which makes it 7 in the first two years, which is quite unusual for Chilean Centres. Also the number of projects developed and carried out by enterprises and/or internationally cofinanced (10) is a very good number. We are also proud of the results of Training of Human Resources, 5 PhD candidates graduated in this period, which makes it 15 in our first 2 years, an important number. Also, thanks to the Basal Centre funding we have an important number of postdoctoral fellows (14). The number of visiting researchers has substantially increased which is also partly thanks to the basal funding. The Centre has also increased the number of ISI publications co-authored with international researchers from other institutions. The figures for International Networks and interaction with them are also very positive. Several researchers of other national research groups have used our facilities, such as Loreto Parra, Assistant Professor at the Catholic University, whose students are using our laboratories. There are several other examples in the different universities participating in this Centre. Finally, the number of outreach activities has increased substantially during our second year particularly since the communication of our research output and scope to students and to society as a whole has become a very important issue for our Centre and that is why we have established a strong outreach office.

VIII. DOCUMENTATION REQUIRED This documentation has to match the information provided in the tables of output.

a) Publications Pdf files of the publications resulting from the informed period or the website where to access it.

b) Organization of Scientific Events

Include a digital copy of the abstracts or full manuscript of the presentations at congresses, courses, conferences, workshops or symposia organized by the Center or the website where to access this information.

c) Participation in Scientific Events

Include a digital copy of the abstracts that reflect the attendance of members of the Center to congresses, courses, conferences, workshops and symposia and attach a digital copy of the front page of the scientific event.

d) Theses If the theses are already finished, please attach a digital copy of the abstract and the subject index.

ANNEXE PUBLICATIONS LIST

1. Castro, J.F., Razmilic, V., Gomez-Escribano, J.P., Andrews, B.A., Asenjo, J.A. and Bibb,

M.J. (2015) Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii. Appl. Environm. Microbiol., 81, 1-12

2. Contador, C. A., Shene, C., Olivera, A., Yoshikuni, Y., Buschmann, A., Andrews, B.A, and Asenjo, J.A. (2015), Analyzing redox balance in a synthetic yeast platform to improve utilization of brown macroalgae as feedstock. Metab. Engin. Comm., 2, 76-84.

3. Gomez-Escribano, J.P., Castro,J.F., Razmilic, V., Chandra G., Andrews, B.A., Asenjo, J.A., and Bibb, M.J. (2015), The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contings of the chromosome, circular plasmid pSLE1 and linear plasmid pSLE2. BMC Genomics, 16, 485.

4. Contador, C., Rodriguez, V., Andrews, B.A., Asenjo, J.A. (2015), Genome-scale reconstruction of Salinispora tropica CNB-440 metabolism to study strain-specific adaptation. Antonie van Leeuewenhoek, 108, 1075-1090.

5. Elsayed, S., Trusch, F., Deng, H., Raab, A., Prokes, I., Busarakam, K., Asenjo, J.A., Andrews, B.A., van West, P., Bull, A., Goodfellow, M., Yu, Y., Ebel, R., Jaspars, M. and Rateb, M. (2015), Chaxapeptin, a lasso peptide from the extremotolerant Streptomyces leeuwenhoekii strain C58 from the hyper-arid Atacama Desert. J. Organic Chemistry. J.Org.Chem., 80, 10252-10260.

6. Sandoval, G., Espinoza, D., Figueroa, N. and Asenjo, J.A. (2016), MILP reformulations for the design of biotechnological multi-product batch plants using continuous equipment sizes and discrete host selection. Comput. Chem. Eng., 84, 1-11.

7. Conca, C., Lecaros, R., Ortega, J., Rosier, L. (2015), Identifiability and Stability of an

Inverse Problem Involving a Fredholm Equation. Chinese Annals of Mathematics, 36B, 737-762

8. Acevedo, P., Amrouche, Ch., Conca, C. (2015), Boussinesq system with non-

homogeneous boundary conditions. Appl. Math. Lett., 53, 39-44. 9. Caubet, F., Conca, C., Godoy, M. (2016), On the detection of several obstacles in Stokes

flow: Topological sensivity and combination with shape derivative. Inverse Problems & Imaging. (in press)

10. Cumsille, P., Coronel, A., Conca, C., Quiñinao, C., Escudero, C. (2015), Proposal of a

hybrid approach for tumor progression and tumor-induced angiogenesis. Theoretical Biology and Medical Modelling, 12, 1-22.

11. Conca, C., Dambrine, M., Quinteros, D., Mahadevan, R. (2016), Minimisation of the ground

state of the mixture of two conducting materials in a small contrast regime. Math. Methods Appl. Sci., doi: 10.1002/mma.3797. (in press)

12. Conca C., Donato P., José, E.C. Mishra I.(2016), Asymptotic analysis of optimal controls of

a semilinear problem in a perforated domain. J. Ramanujan Mathematical Society (in press).

13. Navarro, G., Ordoñez, A. (2016). Faster Compressed Suffix Trees for Repetitive Collections. ACM Journal of Experimental Algorithmics, 21, Art 1.8 (in press)

14. Martínez-Prieto, M.A., Brisaboa, N., Cánovas, R., Claude F., Navarro, G. (2016). Practical

compressed string dictionaries. Information Systems, 56,73-108. 15. Navarro, G., Thankachan, S. (2016). Reporting Consecutive Substring Occurrences Under

Bounded Gap Constraints. Theoretical Computer Science, doi:10.1016/j.tcs.2016.02.005 (in press)

16. Navarro, G., Reyes, N. (2016) New Dynamic Metric Indices for Secondary Memory.

Information Systems. doi:10.1016/j.is.2016.03.009 (in press) 17. Tomova, A., Ivanova, L., Buschmann, A.H., Rioseco, M., Kalsi, R.K, Godfrey, H.P.,

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OTHERS

i Please remember that changes in the staff of Principal Researchers, must be evaluated previously by reviewers and authorized by the Associative Research Program.