Cofund on Biotechnologies

Bio-based production of chemical building blocks: Corynebacterium glutamicum as a platform for new and efficient bioprocesses

Project coordinator: Prof. Bernhard Eikmanns – University of Ulm – Germany

Project leaders:

Abstract

The project aims at developing Corynebacterium glutamicum as a designer bug, serving as a robust platform organism for new and efficient bioprocesses, such as the production of chemical building blocks from renewable resources. In an iterative way, we will construct and analyze C. glutamicum strains producing different dicarboxylic acids and amino acids. Additionally, the producer strains will be engineered to tolerate dicarboxylic acid stress, a relevant factor for process design and downstream processing of large-scale production processes. Using the producer strains, we will develop, scale-up and implement robust fermentation processes with integrated downstream processing solutions. The project has a high innovative potential for industrial applications and we regard the potential to improve the competitiveness of European companies as very high.

Enzyme Production in Optimized Streptomyces

Project coordinator:

• Dr. Erik Vijgenboom – Leiden University – The Netherlands
• Dr. Gilles van Wezel – Leiden University – The Netherlands

Project leaders:

Abstract

The exponentially growing demand for enzymes with the increasing human population, and the exponential growth of new economies sets new demands on modern biotechnology. These materials need to be provided in a sustainable manner, with less dependency on non-renewable fossil resources.

In industrial biotechnology the production of enzymes for sustainable and eco-efficient technologies in the textile-, paper-, consumer- and biofuel applications is a key objective. New enzyme production platforms must provide more choice and open up previously untapped enzyme sources. Due to their saprophytic nature streptomycetes contain a massive arsenal of industrial enzymes, including amylases, proteases, cellulases, xylanases and esterases. However, so far their use as industrial production platform is relatively limited, due to their slow mycelial growth, inefficient nutrient utilization and the lack of advanced expression systems. Other efficient enzyme production hosts (e.g. E. coli, B. subtilis) are currently used in the industry and via a plug-bug approach several of the Streptomyces enzymes can be expressed heterologously in such systems. However, the majority of the proteins requires host-specific machinery for folding, modification and/or secretion and can therefore not be produced in a bio-active form in other host systems. We will develop the industrially preferred enzyme producer Streptomyces lividans for optimal production of industrial enzymes, using xylanase as the model system. Integrating the latest know-how, the project will tackle the suboptimal parts of the existing production pathway, related to expression (transcription/translation), to secretion (machinery, signals), to the stability of the expressed protein, and to the organism itself (morphological engineering). To reach these objectives a consortium is set up consisting of academic institutions, located in four countries, with ample experience in industrial projects, and involving the relevant industry.

The outcome of this project is a new production platform consisting of a strain/vector combination capable of industrial level protein production and secretion. The Streptomyces production host has an optimal fermentation behavior, extended production growth phase, enhanced protein production and secretion capacity and strongly reduced protease activity. The developed expression vectors have the best available transcription, translation and secretion sequences.

Strains, expression systems and combinations of the two will be presented to the industry for exploitation after the project. Already existing formal and informal contacts of the partners with interested industrial parties will pave the way for this approach.

Novel enzyme tools for production of functional oleochemicals from unsaturated lipid

Project coordinator: Prof. Johanna Buchert – VTT Technical Research Centre - Finland

Project leaders:

Abstract

The objective of the project is to develop enzyme-aided technologies for the upgrading of low cost unsaturated fatty acids to functional oleochemicals to be used as coatings, adhesives, lubricants, plasticizers and natural plastics. A multidisciplinary approach is taken in the project by combining biotechnical expertise to chemical engineering and polymer technology. In addition, new advanced analytical methodologies will be implemented in the project for analyzing the characteristics of the chemicals produced.

The specific objectives of the project are to discover and produce novel enzymes being able to modify unsaturated fatty acids to reactive intermediates and to further convert these enzymatically obtained intermediates to oleochemicals. Finally the technical and economical feasibility of the developed oleochemical processes and products will be evaluated. The development of new, modern, environmentally friendly technologies to utilize abundant sources of lipids in novel product applications has very high business potential. The project will significantly enlarge the raw material base of the European oleochemical industry.

Implementing an Enzyme Engineering Technology Platform for the provision of tailor-made enzymes for biocatalytic synthesis

Project coordinator: Dr. Marc Struhalla - c-LEcta GmbH, Leipzig - Germany

Project leaders:

Abstract

According to a market study of Mc Kinsey nowadays approximately 5% of all chemical products corresponding to more then 50 billion € sales are already produced by biotechnological processes and they are growing with 10-20% per year. No matter whether isolated enzymes, whole-cell biotransformations or fermentation processes are used for the production of bio-chemicals, these entire innovative biotechnological processes are based on technologies which need to provide defined enzymatic activities. Nowadays mainly naturally occurring enzymes are used to setup new bio-based synthesis routes whereas artificially optimized enzymes obtained by enzyme engineering technologies are not playing a very important role. This is mainly due to the cost-intensive and timeconsuming efforts which are connected to the established technologies in this field. Within this joint project it is intended to build up an innovative technology platform based on new proprietary methods decreasing costs and increasing speed of enzyme engineering projects. This should allow making enzyme engineering technologies to become a much more important tool for the development of biocatalytic processes and to supply a powerful approach to sustainably improve the competitiveness of the European chemical industry. This proof-of-concept will be established on two enzyme classes of high industrial relevance capable of realizing direct chiral synthesis: Lyases and Transaminases. By broadening the substrate spectrum of these enzymes towards new molecules a number of new chemical products will become efficiently available. The joint project will focus on innovations along all parts of the enzymatic value chain. Enzyme screening and production processes, immobilization techniques and biocatalytic processes themselves will also be addressed. All activities are focused on increasing the efficiency, decreasing the development times and lowering the costs of the final process.

Integrated, multi-host approach for the improved microbial production of high quality therapeutic enzymes and proteins

Project coordinator: Prof. Antonio Villaverde – Universitat Autonoma de Barcelona - Spain

Project leaders:

Abstract

Many enzymes and other proteins of pharmacological interest and addressed to human therapy are presently produced by cultured mammalian cells through expensive procedures, resulting in high-priced products. The choice of mammalian cells instead of simpler microbial systems is often prompted by the occurrence of structural properties (disulfide bridge, proper conformation, glycosylation etc) linked to the requested biological activity, that cannot be fully reproduced in microbial cells.  However, the incorporation of new hosts for therapeutic protein production and the fast-moving advances in protein and metabolic engineering, strain and process design and physiological analysis of stress responses make the production of complex therapeutic proteins in microbial hosts a more feasible and economically competitive industrial biotechnology concept.

The objective of this proposal is to obtain functional therapeutic proteins by microbial production, and to create scientific data with wide applicability to diverse protein production problems in industrial biotechnology. The project will combine modular protein engineering approaches with metabolic and process engineering to obtain functional enzymes through microbial production processes, and will comparatively analyze the suitability of alternative microbial hosts, either conventional or novel, regarding the biological properties of a target therapeutic protein, the human alfa‑galactosidase A.

Targeting population heterogeneity at microscale for robust fermentation processes

Project coordinator: Dr. Anna Eliasson Lantz – Technical University of Denmark - Denmark

Project leaders:

Abstract

The overall objective of the proposed project is to establish a platform for more robust fermentation processes and production organism by understanding and controlling heterogeneity. It is essential to optimise fermentation parameters for achieving the most efficient production process. In most research projects on this topic, the microorganism population was considered homogeneous. However, research has shown that a typical population of microorganisms in a fermentation is heterogeneous. Due to continued technological developments in different fields, for example in genetics and molecular biology (reporter systems), flow cytometry and microfluidics (micro-bioreactors), we have now finally reached a phase where the investigation of the effect of cultivation parameters on the heterogeneity of a microorganism population has become possible, and this is precisely what will be done in this project. The central project hypothesis is that there exists an optimum level of heterogeneity leading to a robust fermentation process with sustained high productivity. To investigate this hypothesis, reporter systems for cell growth and productivity will be constructed for industrially relevant model organisms, which will allow to obtain a distribution of these properties for the population, e.g. by using flow cytometry. The effect of cultivation parameters on these properties will be investigated via. Both physiological adaptation to the signals it perceives in the culture, and genetic change allowing selection of optimally adapted forms to conditions in the culture will be researched. Experimental results at microscale will be extrapolated to labscale and pilotscale. This extrapolation will be supported by development of mathematical models, combining computational fluid dynamics with population balance models. In addition to methods for determining the level of heterogeneity both, other outcomes will be: knowledge to obtain more robust strains for biological production, which are more stress tolerant in a production setting; advanced micro-bioreactors and models able to simulate population behavior in large-scale fermenters.

Production and Upgrading of 2,3-Butanediol from Biomass

Project coordinator: Dr. Ulf Pruesse – Johann Heinrich von Thunen Institute - Germany

Project leaders:

Abstract

The project objective is the development of an efficient fermentation process to produce the platform chemical 2,3-butanediol (2,3-BD) from various low-cost renewable feedstocks and its further upgrading.

2,3-butanediol is a valuable chemical useful as antifreeze agent and as raw materials for the production of pesticides, pharmaceuticals, plasticizers, fragrances, moistening agents etc. It can be further converted to 1,3-butadiene (1,3-BD), a multi million ton bulk chemical, mainly used for the production of synthetic rubber, several polyamides and other polymers as well as to methyl ethyl ketone useful as solvent or fuel additive.

2,3-BD can be produced from sugars or glycerol by different bacteria, e.g. K. oxytoca or K. pneumoniae. Although the biosynthesis of 2,3-BD is well understood, its fermentative production is not carried out on commercial scale, yet, due to low process economics. The project aspires to establish a commercially attractive fermentation process by overcoming the current drawbacks such as the low productivity and inefficient product isolation.

More specifically, different low-cost biomasses (optimized hydrolysates from wood, sugar beets or peels, raw glycerol) shall serve as substrates. New 2,3-BD producing microorganisms will be screened and optimized by mutagenesis and genetic/metabolic engineering with regard to productivity and tolerance for high substrate/product concentrations. Optimum fermentation conditions will be evaluated in lab-scale for different substrates and strains considering both free and immobilised cells. Promising processes will be further scaled-up. Novel processes for the isolation/purification of 2,3-BD will be developed as well as for the conversion of 2,3-BD to 1,3-BD.

In parallel to the whole project, process economics and life-cycle assessment will be carefully analyzed for the whole value chain and the main cost drivers will be identified in order to enable the development of the most cost-efficient and sustainable overall process.

Improvement of strength properties and reduction of emission of volatile organic compounds by enzymatic modification of lignin containing biopolymers and composites 

Project coordinator: Prof. Christian Wilhelm – Saxon Institute for Applied Biotechnology - Germany

Project leaders:

Abstract

The project concerns the development of biopolymers from lignin materials. Bio-composite material of engineering grade from residual lignin was developed a few years ago. The material combines the physical properties of solid wood and plastics in the manufacture process. However, the high emission rate of volatile organic compounds (VOC) and unfavourable odour characteristics prevent the material from being used for several value added products.

The main sources for these emissions are low molecular parts of lignin and hemicellulose. The emissions contain monoterpenes, sesquiterpenes, phenols, aliphatic alcohols and aldehydes. It became obvious that the odour characteristics of the material are very important for the application. The aim of the project is to develop enzyme complexes for the efficient degradation or polymerisation of lignin- and hemicellulose-based compounds, which are responsible for emissions of volatile organic compounds (VOC). For this purpose, enzymes with a specific spectrum of hemicellulases or lignin-oxidising activities will be developed and produced. Incubation procedures of lignin and lignocellulose fibres will be developed and optimised. Investigations on the mechanism of the enzymatic catalysed degradation and modification of the lignin and lignocellulose fibres will be carried out.

Injection moulding processes with modified materials and improved conditions will be run and optimised. Using enzymatic incubated lignin and short lignocellulosic fibres as raw materials, a fibre-reinforced biopolymer composite with reduced emission and improved physical properties will be developed from laboratory up to pilot scale. In addition to the development of specific enzyme complexes, the enzymatic modification mechanisms will be understood and used for the optimisation of the incubation process. New fibre-reinforced biopolymer with reduced VOC emission and improved physical properties will be used for the production of commercial composite products in cooperation with industrial partners.

The successful realisation of the project opens new value added applications for the by-product residual lignin and lignocellulose fibres. The scientific results will be used for patent application, published in scientific and technical journals, presented at scientific conferences and at international fairs and be taught at the participating universities. Several industrial partners from different industrial fields will be integrated in the project work.