The second call of ERA-IB offered funding possibilities to excellent innovative industrially relevant R&D and applied research projects. The participating countries were: Flanders (Belgium), Finland, France, Germany, Saxony (Germany), Poland, Portugal, Spain, Romania and The Netherlands. The organisations that participated in the second ERA-IB joint call for proposals are: IWT (Flanders-Belgium), MSES (Croatia), Tekes (Finland), ADEME (France), BMBF (Germany), SMUL (Germany - Freestate of Saxony), NCBiR (Poland), FCT (Portugal), MICINN (Spain), UEFISC (Romania), NWO, ACTS and NGI (The Netherlands). Out of the 46 applications 10 projects were funded within the second jointly coordinated, transnational call for project proposals in Industrial Biotechnology, namely: "Industrial biotechnology for Europe: an integrated approach" :
The total granted budget was 11.1 Million €.
The projects were focused on the following Industrial Biotechnology topics:
Novel Production Strategies for Biosurfactants
Project coordinator: Dr. Steffen Rupp - Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung e.V. - Germany
|Prof. Christoph Syldatk||Karslruhe Institute of Technology||Germany|
|Dr. Thomas Greiner-Stöffele||c-LEcta GmbH||Germany|
|Prof. Ludo Diels||Flemish Institute for Technological Reserach||Belgium|
|Dr. Eddy Laeremans||Tomans Engineering Noord BVBA||Belgium|
|Dirk Develter||Ecover Belgium NV||Belgium|
|Dr. Michael O'Donohue||LISBP||France|
Surfactants form an integral part of our everyday life with applications reaching far beyond our hygienic needs ranging from asphalt over food to fuel additives all the way to compounds with antibiotic activities. We aim at an increased replacement of petro-based surfactants by biosurfactants generated form renewable resources. Central topics are the identification of novel enzymes and microorganisms for new and more efficient biosurfactant production, understanding of cellular regulatory processes involved in biosurfactant production and consequent metabolic engineering for the improvement of the respective microorganisms also with respect to stress resistance during production, enzyme design combining rational and or evolutionary methods for enzymatic synthesis of surfactants and scale-up of bioprocesses including innovative down-stream processing using membrane technologies and biocatalyst recycling. These objectives will be achieved by connecting five technical work packages that address the entire biosurfactant value chain. The expected results include the identification of new, patentable microbial and enzymatically synthesized biosurfactants with significant economic exploitation potential for industrial applications. The construction of new production strains of biosurfactants with better productivity is envisaged as well as new enzyme products with advantageous properties towards the synthesis or modification of biosurfactants. In addition new technologies for fermentation and downstream processing of surfactants will be developed, including immobilized enzymes and in situ product removal from fermentations or biochemical conversions. By coupling of fermentation and separation technology we do not only expect to improve the down-stream process, but also envisage the improvement of surfactant production by avoiding product inhibition conditions. Exploitation is ensured by three companies. Ecover is a producer and supplier of detergents as well as biosurfactants. Ecover has both the marketing and distribution power to further develop the achieved R&D results into products and place them at the market. Enzymes and strains developed in the consortium will be commercialized by c-LEcta and engineering developments of in situ product recovery (ISPR) by Tormans, both SMEs.
Metabolic and Enzyme Engineering for the Biotechnological Production of Partially Acetylated Chitosans
Project coordinator: Prof. Bruno Moerschbacher - Westfälische Wilhelms-Universität Münster - Germany
|Prof. Antoni Planas||Universitat Ramon Llull||Spain|
|Prof. Wim Soetaert||Bio Base Europe Pilot Plant vzw||Belgium|
|Dr. Katja Richter||Heppe Medical Chitosan GmbH||Germany|
|Prof. Wim Soetaert||Centre of Expertise - Ghent University||Belgium|
Just as people have to communicate to build a community and, eventually, a society, cells need to communicate to build a tissue and, eventually, an organism. Cells have evolved a sophisticated molecular language, and complex sugar molecules form key words of this language. One example are sugar structures on the cell surface that inform cells of their neighbors and that lead to the distinction between self and non-self as a basis of our immune system and successful defense against pathogens. Understanding this molecular language of sugars is a prerequisite for the molecular understanding of many diseases such as cancer which in fact results from failed cellular communication. Recent advances in glycosciences suggest that subtle patterns of substitution, such as the pattern of sulfation in heparin, are the bearers of crucial information, e.g. for blood coagulation. And evidence is accumulating that related sugar molecules such as chitosans with a specific pattern of acetylation may interfere with this cellular communication, allowing us to influence cellular behaviour in a targeted manner.
The ChitoBioEngineering project aims at establishing, through genetic, metabolic, and en-zyme engineering, biotechnological ways of producing fully defined, partially acetylated chito-san oligomers. Today’s commercially available chitosans are produced chemically from chitin isolated from shrimp shell wastes. They can be well defined concerning their degree of poly-merisation and degree of acetylation, but they are invariably characterised by a random pat-tern of acetylation (PA). We have recently hypothesised that the biological activities of chito-sans, such as their antimicrobial, plant strengthening, immuno-stimulatory or wound healing activities, should be greatly influenced by their PA. However, no methods are available today for the production of chitosans with defined non-random PA.
Microbial genes will be used to drive the biosynthesis of chitosan oligomers with defined ar-chitecture, i.e. with a specific, non-random PA. We will use novel chitin synthases and chitin deacetylases stemming from our extensive gene discovery projects for heterologous expres-sion in suitable micro-organisms to drive the production of a range of such oligomers which will be fully characterised using state-of-the-art analytical tools. Advanced genetic and en-zyme engineering will optimise the expression of the genes and fine-tune the properties of the enzymes, respectively, and metabolic engineering will maximise the yield of the well de¬fined chitosans. We will use our extensive experience as well as our wide-spread network within the chitosan scientific and industrial community to analyse the biological activities of these chitosans and to explore their potential applications in different market sectors, with a focus on cosmetics and pharmaceutical applications.
Genome mining for drug discovery: Activation of silent biosynthetic gene clusters
|Dr. Carmen Méndez||Universidad de Oviedo||Spain|
|Prof. Jolanta Zakrzewska-Czerwinska||University of Wroc³aw||Poland|
|Dr. E. Takano||University of Groningen||The Netherlands|
|Francisco Moris||EntreChem SL||Spain|
|Maria Holmbäck/Kristiina Ylihonko||Galilaeus Oy||Finland|
The GenoDrug proposal aims at the development of a new technology for drug discovery, i.e. the activation of previously silent biosynthetic gene clusters of microbial genomes. Microbial natural products have an outstanding track record as drugs and drug leads since more than sixty years. However, conventional screening programs increasingly result in the re-discovery of already known compounds. Sequencing of many microbial genomes, especially from actinomycetes, has now revealed that the genome of each strain contains gene clusters for the formation of 10-30 bioactive compounds ("secondary metabolites"). This implies that for any actinomycete strain most of its potential as producer of bioactive compounds is yet undiscovered. An economy which can harness this potential is likely to take an international lead in the development of new antibiotics, anticancer drugs and other pharmaceuticals in future.
The bottleneck in the economical exploitation of this strategy is the development of technologies which allow the utilization of DNA sequence data of secondary metabolic gene clusters to generate the encoded compounds, in quantities sufficient for characterization, pharmacological testing and preclinical drug development. The GenoDrug proposal will develop such technologies and demonstrate their applicability in drug discovery by the identification of several novel bioactive compounds.
Robust fermentation production of tacrolimus and related immunosuppressors: Molecular genetics and metabolic engineering to construct a by-product free superproducer.
Project coordinator: Prof. Juan-Francisco Martín - INBIOTEC Instituto de Biotecnología de León - Spain
|Prof. Wolfgang Wohlleben||Eberhard Karls Universität Tübingen||Germany|
|Dr. Marta Vas Mendes||IBMC Instituto de Biología Molecular e Celular||Portugal|
|Prof. Lutz Heide||Eberhard Karls Universität Tübingen||Germany|
|Eberhard Karls Universität Tübingen||Antibióticos S.A.||Spain|
Tacrolimus (FK506) and ascomycin (FK520) are closely related polyketide compounds with potent immunosuppressor activity that are widely used to avoid transplant rejection. They are produced by Streptomyces tsukubaensis and other Streptomyces species. These compounds bind the human FKBP protein causing a reaction cascade that gives rise to a reduction in the T-cell mediated human immune response thus avoiding transplant rejection. In addition, novel derivatives of these molecules (rapamycin analogues) have potential as antitumor compounds.
The biosynthesis of tacrolimus is controlled by complex networks involving phosphate and nitrogen limitation and strong aeration that causes oxidative stress. The aim of this project is to develop a robust fermentation process based on the use of genetically improved strains.
The project integrates the complementary efforts of five outstanding research groups from three different countries, very active in research at the international level in subjects that include molecular genetics of Streptomyces, physiology and metabolic regulation studies, oxidative stress and process optimization. The involvement of the medium-size biotechnology company, ANTIBIOTICOS S.A., guaranties the industrial usefulness of this study. Other European companies are also interested in this field.
The project is divided in six work packages that will be developed in close cooperation between the five partners. Close coordination will be supervised by Prof. Martin, who has a broad experience on coordination of EU projects.
Integral Engineering of Acetic Acid Tolerance in Yeast
Project coordinator: Dr. Ton van Maris - Delft University of Technology - The Netherlands
|Prof. Isabel Sá-Correia||Technical University of Lisbo||Portugal|
|Prof. Elke Nevoigt||Jacobs University Bremen||Germany|
|Prof. Jaoquín Ariño||Universitat Autònoma de Barcelona||Spain|
Carbon efficiency and food security dictate that a substantial replacement of current petrochemical production by industrial biotechnology should be based on crude plant biomass hydrolysates as feedstocks rather than on refined, food-grade carbohydrates. The presence of acetylated polymers in these crude hydrolysates implies that acetic acid tolerance of industrial microorganisms is and will remain a key issue in the implementation of sustainable, non-food feedstocks in industrial biotechnology.
The yeast Saccharomyces cerevisiae, one of the most important microorganisms in industrial production and in metabolic engineering, already has an innate degree of tolerance to weak organic acids and low pH. However, better understanding and improvement of the tolerance to acetic acid is essential for development, diversification and intensification of yeast-based bioprocesses in industrial biotechnology. Finding solutions to this problem is urgent, since the first full-scale factories for yeast-based production processes from lignocellulosic feedstocks (the first products will be biofuels) are anticipated within the next 5 years. Our highly complementary consortium will integrate classical genetic mapping, comparative genomics, genome-wide expression analysis, evolutionary engineering and global transcription machinery engineering with targeted genetic modification, with the aim to understand and rationally improve acetic tolerance in S. cerevisiae.
Key deliverables from the project will include:
Multi enzyme systems involved in astin biosynthesis and their use in heterologous astin production
Project coordinator: Prof. Karl-Heinz van Pée - Technische Universität Dresden - Germany
|Prof. Jutta Ludwig-Müller||Technische Universität Dresden||Germany|
|Prof. Willem van Berkel||Wageningen University||The Netherlands|
|Dr. Jean-Yves Berthon||Greentech||France|
|Prof. Kaarina Sivonen||University of Helsinki||Finland|
|Prof. Wolfgang Wohlleben||Eberhard Karls Universität Tübingen||Germany|
|Prof. Phillippe Jacques (subcontractor)||University of Lille 1||France|
Astins are cyclic peptides isolated from the roots of the plant Aster tataricus and root extracts show antibacterial activity. Astin derivatives possess also a high anti-tumour potential. Since only very low amounts of astins can be isolated from plants and they are difficult to synthesise chemically without negative impacts on the environment. Therefore, this project aims at enhancing the production of astins using molecular genetic tools. Thus we will detect and clone the genes required for astin biosynthesis. To allow the detection of these genes, bioinformatic tools and sequence information from microbial nonribosomal peptide synthetases will be used to develop primers for peptide synthetases specific for the amino acids found in astins. Specific primers will also be constructed for the prolyl dehydrogenase and halogenase(s) catalysing the halogenation of prolyl residues. After detection, the genes will be cloned, sequenced and expressed in heterologous hosts such as bacteria or yeast. Alternatively, cell or organ cultures of aster can be used for homologous expression. After cloning and successful expression of the individual genes in heterologous hosts, the activity of the resulting nonribosomal peptide synthetases will be analysed using assays established in the groups working on this part of the project. Analogously, the gene(s) for the halogenase(s) and prolyl dehydrogenase will be expressed and analysed for activity. The genes of the individual enzymes will be combined in a “gene cluster” and will be introduced into heterologous host for over-expression. Over-expression of the astin biosynthetic “gene cluster” should result in enhanced production of astins. To allow the biotechnological production of astins, Streptomyces strains or alternatively plant cells will be used and a fermentation process will be developed by changing various fermentation parameters. With larger quantities of astins available, screening using DNA arrays can be performed to analyse the influence of astins on gene expression. Special attention will be given to oncogenes and “vital” genes. The study of the genes highlighted by this method and their implication in the biological functions can bring new perspectives for the development of new pharmacological or cosmetical applications of astins
Microreactor technology for continuous enzymatic reactions catalyzed by C-C-bond forming enzymes
Project coordinator: Prof. Ðurða Vasiæ-Raèki - University of Zagreb - Croatia
|Prof. Martina Pohl||Forschungszentrum Jülich GmbH||Germany|
|Prof. Pere Clapés||Instituto de Química Avanzada de Catalunya (IQAC) / Consejo Superior de Investigaciones Científicas (CSISC)||Spain|
|Mr Sergi Pumarola||Bioglane S.L.N.E.||Spain|
|Mr W. Bolt (subcontractor)||Micronit Microfluidics BV||The Netherlands|
The present project deals with the evaluation of microreactor technology for enzymatic carboligase reactions using thiamin diphosphate (ThDP)-dependent enzymes (TDEs)and D-fructose-6-phosphate aldolase from E.coli (FSA).The main goal for the TDEs is to develop micro-reactor technology as a screening tool to identify the appropriate enzyme for a desired carboligation reaction of two aldehydes yielding chiral 2-hydroxy ketones. Here, the prediction of the process relevant data concerning e.g. optimal substrate concentration, selectivity, enzyme stability and productivity with very small amounts of enzymes and chemicals will be tested using micro-reactor technology in comparison to conventional lab scale reactors. The main advantage of FSA is that it dispenses with the need for laborious preparation of a sensitive phosphorylated reagent, dihydroxyacetone phosphate (DHAP), essential for DHAP-dependent aldolase, and it is currently used for the large scale production of D-fagomine. Notwithstanding the obvious advantages, some emerging issues limit its broad synthetic applicability while others can be significantly improved making them attractive from industrial point of view. Among them, are the following:
A) Concerning the acceptor substrate selectivity, substrate inhibition, and the thermodynamic limitations of the catalyzed reactions.
B) Improving performance by cascade reactions with in situ aldehyde generation.
C) Improving molecular diversity by cascade two-aldol additions with in situ product formation. These issues can be effectively optimized in micro-reactors and/or in combination with protein engineering.
The project would bring new insights into green chemical syntheses reactions which are of vital interest to the field of high technologies, such as industrial biotechnology and approve a new concept for the production of enantiomerically pure diols and iminocyclitols. Technology transfer to end users, who are involved in the project, will be realized. Micro-reactors integrating oxidation, aldol reaction, enzyme separation and downstream processing are of utmost importance to develop a competitive process and the present project will provide technological solutions and will represent the first real example of industrial biotechnology development of an active process using aldolases.
Development of a process for the utilization both the carbohydrate and the lignin content from lignocellulosic materials of annual plants for the production of valuable products
Project coordinator: Prof. Christian Wilhelm - Saxon Institute for Applied Biotechnology at the Leipzig University - Germany
|Dr. Martina Bremer||Technische Universität Dresden||Germany|
|Prof. Martin Bertau||University of Mining and Technology Freiberg||Germany|
|Dr. Tarja Tamminen||VTT, Technical Research Centre||Finland|
|Prof. Mircea Ioan Popescu||Applied Biochemistry and Biotechnology Center||Romania|
|Dr. Carmen Boeriu (subcontractor)||Wageningen University||
The general aim of the project is the development of a process for the utilization of both the carbohydrate and the lignin content from lignocellulosic materials of annual plants, particularly wheat or maize straw. The investigations basically concern (i) the development of a pre-treatment process, which allows the separation of both the lignin and the carbohydrate content of lignocellulosic raw materials, (ii) the development of a fungal enzyme complex optimized for the saccharification of the carbohydrate content of lignocellulose in a simultaneous saccharification and fermentation (SSF) process, (iii) investigations on the SSF-process, using model microorganism-strains for the production of platform chemicals, like fermentation alcohols, and (iv) the modification of the separated lignin for the production of fibre-reinforced biopolymers as well as for the production of fine chemicals.
Results from subproject i, ii and iii will be invaluable as the projects furthermore aims for investigating the performance of the SSF-process and the developed enzyme-complex not only at lab but also up to pilot scale.
For the planned investigations concerning the production of fibre-reinforced biopolymers on the basis of wheat straw lignin we are going to include expertises from the ERA-IB-project EIB.08.025 in which the utilization of kraft lignin for the production of fibre-reinforced biopolymers is investigated. Lignin-modifying enzymes and invaluable knowledge concerning reduction of VOC emissions in kraft lignin have already emerged from the latter project thus providing a strong basis for this new project.
Finally the resulting processes will be evaluated economically in order to show whether it is commercially viable. As the main risk for the commercial utilization of the project results are enzyme costs and the effectiveness of the pre-treatment process, special attention will be paid to these objectives.
Pseudomonas 2.0: industrial biocatalysis using living cells
Project coordinator: Dr. Lars Blank / Prof. Andreas Schmid - TU Dortmund University - Germany
|Prof. Ralf Takors||University of Stuttgart||Germany|
|Dr. Susann Müller||Helmholtz Centre for Environmental Research ufZ Leipzig||Germany|
|Prof. Han de Winde||Delft University of Technology||The Netherlands|
|Prof. Bruno Zeliæ||University Zagreb||Croatia|
|Prof. Victor de Lorenzo||Centro Nacional de Biotecnologia Consejo Superior de Investigaciones Científicas||Spain|
|Dr. Marcel Wubbolts||DSM||The Netherlands|
|Dr. Andreas Karau||Evonik Rexim S.A.S.||France|
The potential of non-pathogenic Pseudomonas as a platform host for Industrial Biotechnology has been discussed for decades in Europe, mainly inspired by its metabolic versatility, ease of genetic programming and high solvent tolerance. These properties enable growth in the presence of a second phase of toxic solvents, such as styrene or octanol, or high concentrations of inherently toxic compounds originating from cheap renewable feedstocks (e.g., biomass hydrolysates), like furaldehydes. Furthermore, Pseudomonas displays an extensive enzymatic inventory (e.g., hydrocarbon degradation pathways), and the potential to regenerate redox cofactors at a high rate (Blank et al., FEBS J. 2008 275/20). On this background, it comes as a surprise that Pseudomonas strains are still a minor player as genomic and metabolic chassis for the Bio-Industry, where most key processes are still dominated by Bacillus, Corynebacterium glutamicum, and Escherichia coli. We argue that by tackling and overcoming the few molecular bottlenecks that still make non-pathogenic Pseudomonas less efficacious than bacterial alternatives, we can contribute to place European Bio- Industry into a prime position within the global Biotechnology scenario. Novel biocatalytic processes, must successfully overcome economic barriers before realization. This necessitates high solvent tolerance, a high rate of redox cofactor regeneration, carbon efficiency, and biocatalytic stability. Preferably, these parameters determining whole-cell biocatalyst performance are optimized simultaneously. Explicitly, this performance has to be transferable to industrial environments, including large scale fermenters, which will be a main focus of Pseudomonas 2.0. The transfer of excellent academic research findings into industrially useful technology will be achieved by truly cooperative work between the 6 academic partners and Rexim-Evonik and DSM, 2 of the major European chemical companies. The outcomes of the project Pseudomonas 2.0 will propel the development of Industrial Biotechnology in Europe, supporting a bio-refinery approach in the chemical industry on the basis of the European Lead Market Initiative.
Combining efforts in enzyme and process engineering to improve access to multifunctional chiral intermediates
Project coordinator: Prof. Martin Bertau - Freiberg University of Mining and Technology - Germany
|Dr. Antje Eichler||Junior Research Group Industrial Biotechnology||Germany|
|Prof. Wladimir Reschetilowski||Technische Universität Dresden||Germany|
|Dr. Thomas Greiner-Stöffele||c-LEcta GmbH||Germany|
|Prof. Adrzej Kolinski||University of Warsaw||Poland|
|Prof. Volker Hesse||Eindhoven University of Technology||The Netherlands|
|Wessel Hengeveld (industrial observer)||Flowid B.V.||The Netherlands|
|Dr. Wille Gregor (industrial observer)||Sigma-Aldrich Production GmbH||Gregor|
Most of the top-selling drugs today, like the cholesterol lowering “Atorvastatine”, are chiral, i.e. they contain one or more stereogenic centres. To produce these chiral drugs, small chiral building blocks are required as a starting material. Hence provision and thus synthesis of the latter in an efficient, economical and preferentially ecological way is of increasing importance in order to ensure the supply of state-of-the-art drugs at a reasonable price.
This project addresses the latter issue by focusing on the synthesis of a certain group of chiral building blocks named a-amino-alcohols. These substances are known to be valuable in the synthesis of various a-sympathomimetics, anti-parkinsons-disease-drugs and even antibiotics.
Enantiomerically enriched a-amino-alcohols are theoretically accessible in an ecological and economical way by using enzymes. However, most of the amino-alcohol producing enzymes known today only accept a limited number of substrates and show unsatisfying stereoselectivity. Thus the first aim of this project is to identify new enzymes which are subsequently optimised and adjusted to the process needs using mutagenesis guided by in silico enzyme modelling.
A further challenge which hampers application of enzymes in biocatalytic synthesis of chiral amino-alcohols lies in the reversibility of the reaction. As a consequence a high yield is only achievable if measures are taken to shift the equilibrium of the reaction towards the side of the products. The second aim of the project will attend to that by investigating consecutive reactions coupled to the primary one in order to make the overall reaction practically irreversible. This will also be facilitated by innovations in process chemistry. Thereby the project is aimed at running the process in micro-structured reactors allowing operation in novel process windows, in which the reaction is intensified and run continuously.
With the established project consortium bringing together experts from various fields such as biochemistry, bioinformatics, industrial chemistry and micro-reactor engineering, the project is expected to yield new enzymes and a new process which together will improve access to enantiopure a-amino-alcohols. As enzymes and the process are expected to be commercially available, industry is enabled to benefit from the results of the project.