Cofund on Biotechnologies

Biological conversion of CO2 to the platform chemical 3-hydroxypropanoic acid

Project coordinator: Peter Dürre - University of Ulm - Germany

Project leaders:

Summary

Current energy and chemical needs are met by the extraction and processing of the fossil fuels. Such resources are finite and their use causes environmental pollution and greenhouse gas (GHG) emissions. The challenge facing humankind is, therefore, to identify new, sustainable and cleaner processes for chemical and energy generation. Biological routes represent a promising option, but strategies to date rely on the use of microbes to convert through fermentation the easily accessible carbohydrates (sugar and starch) of plants (such as sugar cane or corn) into chemicals and fuels. This has led to concerns over competition with the use of these carbohydrates as food, and a
re-focussing of efforts on non-food, plant cell wall material (lignocellulose). However, lignocellulose is extremely resistant to being broken down into the sugar needed for fermentation. Overcoming this recalcitrance in a cost effective manner is proving extremely challenging.

There is, however, an exciting low-cost alternative, and that is to directly capture carbon, by harnessing the ability of certain bacteria to ‘eat’ single carbon GHG gases such as CO2. The gas is injected into the liquid medium of fermentation vessels where it is consumed by the bacteria and converted into the chemicals we need. Fortunately, such gases are an abundant resource, and may be derived from non-food sources such as waste gases from industry as well as ‘synthesis gas’ produced from the gasification (heating) of non-food biomass and domestic/ agricultural wastes. In this project, we will use this technology to make the platform chemical hydroxypropanoic acid.  It has a multitude of uses, including the manufacture of plastics, coatings, adhesives, floor polishes and paints. By using non-food, waste gas as a feedstock, competition with food and land resources is avoided while at the same time providing benefits to the environment and society through a reduction in GHG emissions.

Development and production of new insect chitosan and chitosan based functional coatings for yarns and textile fabrics

Project coordinator: Susanne Zibek - Fraunhofer Institute of Interfacial Engineering and Biotechnology - Germany

Project leaders:

Summary

Chitin is besides cellulose the second most important natural polymer in the world, found in marine crustaceans, like shrimps and crabs, insects and fungi. With the growth of the ecological awareness, the commercial interest for chitin and chitin-based products is increasing due to its biodegradability, biocompatibility, antimicrobial activity, and non-toxicity. Major end-user applications are found in wastewater treatment, animal feed, agriculture, cosmetics, and medicine. The total annual world production of purified chitin obtained from byproducts of the seafood industry is about 1600 tones, with Japan and USA being the main producers. The European market is increasing steadily. However, chitin and chitosan have to be imported to Europe for industrial application. A new European chitin source is insect chitin, a side-product within animal feed production. Insect larvae are used as protein source for animal feed whereas adult insects are used for reproduction. This leads to cocoons and insect bodies which can be used as chitin-rich resource. The aim of ChitoTex is the valorization of these insect chitin side-products and the application of insect chitin in functional coatings for textile surfaces, especially for technical purposes in mobile process development, home areas, packaging materials, building construction, civil engineering, geotextiles as well as clothing for working and protection. Through the interdisciplinary network and cooperation of microbiologists, chemists, and engineers an enzymatic-chemical approach will be developed for the allocation of chitin-based raw-materials with different properties (e.g. watersolubility) which can be further processed by the industrial textile partners to first functional coatings for textiles out of insect chitin.

Analysis and optimization of industrial microorganisms under dynamic process conditions

Project coordinator: Aljoscha Wahl - Delft University of Technology - the Netherlands

Project leaders:

Summary

The transition from the currently fossil feedstock based chemical industry to a sustainable, biobased feedstock will require efficient bioprocesses for the production of commodity chemicals. The efficiency of a bioprocess strongly depends on the performance of the microorganism used. Currently, only a few commodities like ethanol, 1,3-propandiol, 1,4-Butanediol (BDO), succinic acid can be produced from renewable feedstocks.

For the success of current and new bio-processes, faster and more efficient development and design strategies for cell factories need to be developed. In this project, relevant partners study the properties of cells as factories under large-scale production conditions. Especially, effects that occur in large reactors are analyzed and the produced knowledge will be invaluable to improve the cellular properties in order to maximize fermentation performance under real-world, large-scale conditions.

The consortium focuses on a chemical that can be used as drop-in chemicals for fuels and the chemical industry, 1,4-butanediol (BDO) and isopropanol. The industrial partners are relevant players in the field and highly motivated to enable the change to a biobased economy. Genomatica has received the 2013 Kirkpatrick Chemical Engineering Achievement Award for its rapid commercialization of a direct fermentation process for 1,4-butanediol.

Next to the applied aspects, the project addresses very fundamental questions of metabolic regulation in bacteria. How does metabolism prevent overflow metabolism and substrate accelerated death by acidification of the intracellular environment upon sudden changes in the environment. Is there a training effect, especially are cells that have experienced many perturbations ‘fitter’ than ‘untrained’ cells? Which mechanisms are involved in the training phase and how does metabolism adapt to frequent perturbations.

Mastering the economics of adaptation  through constraint-based modeling in yeast

Project coordinator: Bas Teusink - VU University Amsterdam - the Netherlands

Project leaders:

Summary

Living cells evolved a remarkable ability to adapt to environmental conditions, or to withstand mutations. In biotechnology, this compromises success in metabolic engineering and causes instability of engineered strains. “Functional genomics” has allowed the cost-effective measurement of many of the components of the cell. However, we still mostly fail to understand how their interactions lead to cellular function and adaptation. It becomes clear, however, that physics and (bio)chemistry impose strong constraints on adaptation and evolution. Such constraints limit the total amount of protein that a cell can synthesize, and impact on how it should partition that limited resource over its processes to optimize fitness (“cellular economics”). From an industrial point, such knowledge is important to come with better metabolic engineering strategies that take into account the impact of novel genes and pathways on cellular economics, to develop processes with high yields that enable cost-effective bio-based chemicals and biofuels. In this proposal we will develop a modeling framework that will allow the integration of large data sets into comprehensive mechanistic models. These models are of genome-scale and will be able to compute the costs and benefits of implementing metabolic engineering strategies. The economic models will be used to provide proof-of-concept in two ways: (i) as tools for data integration and interpretation of adaptive responses; (ii) as predictive tool, through optimisation to predict more realistic theoretical yields and through exploration of metabolic engineering scenarios. q–‘s will be tested by a user case provided by our industrial partners, DSM and Roquette, involving succinate production, a versatile C4 diacid with a lot of potential applications, e.g. in polymers and resins (see www.reverdia.com). This project will thus provide the next generation of genome-scale metabolic models essential for metabolic engineering.

Novel approaches to develop filamentous micro-organisms for enzyme production

Project coordinator: Erik Vijgenboom - Institute of Biology Leiden University - the Netherlands

Project leaders:

Summary

In the coming decade the Biotech industry will invest strongly in new technology for the use of biomass in the production of chemical building blocks. This is the result of political and socio-economical pressure together with a steady decline in the availability of conventional petrochemical resources. The most critical aspect in the effort for the use of biomass in bio-based production is the need for enzymes capable of efficient and complete degradation of a wide variety of biomass. Current enzyme cocktails can only process a substantial part of the biomass but a significant percentage of biomass stays recalcitrant in current degradation approaches, leading to significant economic losses in feedstock-to-product conversion. The Filazyme consortium aims at contributing new enzymes and cell factories that can upgrade current enzyme cocktails towards complete degradation of biomass. The project is focused on new aspects of protein secretion in a commonly used filamentous fungal cell factory, ^spergillus and the development of a promising new filamentous bacterial cell factory, Streptomyces. Significant synergy is expected in reaching the final project goals by using these highly complementary cell factory platforms. Furthermore new enzymes will be identified, expressed and isolated to complement current enzyme cocktails. Fermentation protocols will be setup for the production of the enzymes at industrial relevant scale. The project entices participants representing the whole value chain from academia, to research institutes, to enzyme suppliers and end-users. The latter are innovative SME companies, representing the driving force in the new value chains within the bio-based economy. The project consist of a transnational consortium with participants from Northern and Southern European and upcoming near East markets, allowing penetration of the results of the research in areas with considerable and diverse growth potential.

A Synthetic Biology approach for bacterial bioconversion of lignin into renewable chemicals

Project coordinator: Timothy Bugg - University of Warwick - United Kingdom

Project leaders:

Summary

Lignin is a polymer found as 15-30% of plant biomass, and is produced industrially as a low-value by-product of pulp/paper manufacture and biofuel production. It contains phenolic monomer units, hence it represents a possible renewable source of aromatic chemicals that are needed industrially to make plastics, pharmaceuticals and agrochemicals, currently derived from crude oil. As part of the effort to develop a sustainable European bio-based chemical industry, we wish to convert lignin into renewable chemicals, however, due to its highly inert structure, no commercial process has yet been developed for conversion of lignin to renewable chemicals. Recently Prof. Bugg’s group at Warwick have discovered two novel bacterial lignin-degrading enzymes, and have shown that the lignin breakdown pathways in Rhodococcus jostii RHA1 can be modified genetically in order to generate vanillin, a chemical used in the food/flavour industry. However, at present the metabolic pathways and regulatory systems for lignin breakdown in bacteria are not well understood.

Therefore, in this project we will study Pseudomonas putida KT2440, a lignin-degrading bacterium whose genome has been sequenced and whose aromatic metabolic network is the best known in bacteria, to elucidate the metabolic and regulatory networks for lignin degradation. We will then use this knowledge to reprogramme this bacterium, using high activity lignin-degrading enzymes, to produce two target chemicals that are of interest for the production of renewable bioplastics. With industrial partner Biome Bioplastics, we will then develop a fermentation-based process for the production of renewable aromatic chemicals from lignin.

A metagenomic collection of novel and highly efficient biocatalysts for industrial biotechnology

Project coordinator: Wolfgang Streit - University Hamburg - Germany

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Summary

Chemical and bio-industries have a steadily growing demand for enzyme biocatalysts, which can catalyze a huge variety of different chemical reactions with high activity, substrate specificity and enantioselectivity. However, enzymes have been evolved by nature to work in living cells and under mild reaction conditions; consequently, most enzymes cannot be applied in industrial processes directly. Although it is known that few enzymes exist, which are well suited for biotechnological applications, the molecular basis is unknown. The MetaCat project will deliver innovative tools and knowledge for the identification of such robust “all-round frequent hit” enzymes (AFHs). Metagenomic resources will be exploited by novel function- and sequence-based screenings to identify nitrilases, transaminases, ketoreductases, glycosyl hydrolases and lipases/esterases. A combination of metagenomics and metacatalysis will be used together with structure-based and high-throughput technologies, generic model substrates mimicking challenging chemical synthesis steps, next generation sequencing technologies as well as in silico data mining. New genetic tools using synthetic biology approaches will be developed to construct a cell-free function-based screening platform for faster and improved screening. An innovative single-cell laser trapping technology will be established to give access to a new previously unknown enzyme diversity. The identified enzymes will form a marketable versatile biocatalyst collection together with a comprehensive database and can be used as starting points for modeling and in vitro evolution experiments. They will be applied to improve chemical production processes regarding e.g. timelines, purity of the products, environmental sustainability and will lead to value-added products. Thus, MetaCat will contribute to shorten timelines for development of biotechnological processes and thus to make industrial biocatalysis more attractive and profitable.

Next Generation Immunosuppressants: Brasilicardin synthesized by Nocardia spp.

Project coordinator: Harald Gross - Pharmaceutical Institute of the University of Tuebingen - Germany

Project leaders:

Summary

NeBrasCa stands for Next generation immunosuppressive drugs: Brasilicardin from kocardia. Brasilicardin is a terpenoid secondary metabolite which is known to have an immunosuppressive activity and is produced by the soil bacterium Nocardia terpenica. The potency of this natural product is higher than today’s standard drugs tacrolimus and cyclosporin, and brasilicardin has the advantage of being less toxic, i.e. shows less side effects. It targets the amino acid transport system L and exhibits therefore a completely different mode of action than the established drugs. Taken together, brasilicardin based compounds represent in future a valueable drug option that can expand and complement todays immunosuppressant therapy. The NeBrasCa project aims at the biotechnological development of a brasilicardin analogs as new immunosuppressant drugs. 

Currently, the development is halted due to supply issues. The producer strain shows only a low production titer and is furthermore categorized as biosafety level 2 organism. Both facts make the production of the original brasilicardin by classic fermentation expensive and elaborate. In addition, despite considerable efforts, it is neither practical nor reasonable to generate the molecule by total synthesis.

NeBrasCa wants to overcome this bottleneck with a biotechnological approach in order to produce an optimized brasilicardin congener, which can be developed to the point of regulatory preclinical trials.

Optimized laccase systems for high-value bio-plastics production from biomass

Project coordinator: Alexander Wentzel - Stiftelsen SINTEF - Norway

Project leaders:

Summary

These days, the bio-based plastics market is in rapid growth, and bio-based plastic products will more and more replace petroleum-based plastic products, leading to reduced CO2 emissions and therewith a positive environmental impact. The use of biocatalysts (enzymes) in the bio-plastics production value chain has the potential to add largely to this positive impact as it may substitute toxic chemical catalysts in processes using to produce polymers from bio-based feedstock. Lignin is the largest available source of aromatic building blocks for bio-based aromatic derivatives, and its abundance, e.g. from the pulp and paper industry, makes it a highly interesting potential source for novel aromatic chemicals and polymer precursors that may be polymerized by chemical and biocatalysts.

The OXYPOL project addresses the bio-based plastics value chain using biocatalysis for the degradation of lignin into aromatic building blocks and the subsequent controlled polymerization of these aromatic precursors into bio-based polymers. Highly efficient laccase mediator systems (LMS) based on abundant potential low-cost mediator compounds directly derivable from selected lignin preparations will be developed, applying enzyme engineering strategies. In combination with improving enzyme-recycling approaches for LMS involving immobilization on polymer particles and whole-cell biocatalysis, we aim at breaking crucial bottlenecks in the application of LMS based biocatalysis towards large scale industrial applications in bio-plastics production.

OXYPOL is a pan-European endeavour integrating complementaryscientific excellence and market driven R&D competence of the totally 8 academic, institutional and commercial project partners, aiming at making an impact on the biocatalysis-based bio-plastics production, with positive implications for the European bio-economy.

The exploitation of Xanthophyllomyces dendrorhous as a sustainable platform for the PROduction of high-value CARotenoids

Project coordinator: Paul Fraser - Royal Holloway University of Londen - United Kingdom

Project leaders:

Summary

Most of the colours in the natural world are due to the presence of compounds known as carotenoids. These natural pigments have been utilised by human civilisations for millennia, and exploited by industry commercially as colorants, and health and dietary supplements. Two particular carotenoids namely zeaxanthin and phytoene, are in high demand and it is estimated that the global market for zeaxanthin alone will reach$334 million by 2018. This interest in zeaxanthin is associated with its use as a treatment and preventative measure against Age Related Macular Degeneration (AMD), a chronic eye disorder that accounts for over half of all cases of blindness in Western societies. Phytoene is a colourless carotene used as a bioactive ingredient in the rapidly expanding cosmetic industry. Its beneficial activities include protection against UV and oxidative damage, which lead to pre-mature ageing and other phenomena.

Presently chemical synthesis is the production method used to produce these pigments/compounds. However, with the advent of new technologies microbial production is a competitive sustainable alternative to plant and chemical based production. This approach also does not compete with land resources for food production or impact on our dwindling sources of fossil fuel. In the PROCAR project state of the art technologies will be used to develop the red yeast Xanthophyllomyces dendrorhous as a renewable source of natural zeaxanthin and phytoene to meet the demands of the European consumer.