Project: Generation of cell factories for production of poly-unsaturated fatty acids by in vivo recombination & metabolic engineering

Changing life style and increasing life span among the European population increases the number of well known life style diseases like cancer, cardiovascular and immune diseases, and obesity increases accordingly. The result is a significant reduction in quality of life for the people affected, along with increased health care costs for society with restoring side effects. Food intake has a vital role, and intake of nutraceutical ingredients and food enriched with premium food ingredients will allow consumers to take a proactive role in aiming at staying healthy. _x000D_The consortium is formed by two SMEs, Fluxome and Eviagenics, and will work on novel bioprocesses and the improved production of nutraceutical ingredients, particularly the omega-3 polyunsaturated fatty acid eicosapentaneoic acid (EPA). The project will support the market-introduction of a customer-tailored and biosustainable alternative to the traditional sources of omega-3 poly-unsaturated fatty acids (PUFAs) to the market, which is mostly fish-oil. EPA is produced by one of the consortium Ps, Fluxome, by fermentation of a well-known microorganism, baker's yeast, which has become genetically equipped to produce very-long chain PUFAs . The sustainable yeast-based bioproduction of this C20:5-omega-3 containing oil is new-to-market and novel-to-nature. EPA is an ambitious, high added-value product with excellent market perspectives. The realization of sugar-based, sustainable production of this compound is an important market driver. A novel EPA-oil bioprocess will strengthen European industrial biotechnology position, economically and scientifically. _x000D_EPA is essential for humans, and it is scientifically proven and broadly acknowledged that it has many beneficial effects on human health, including proper development of brain and visual functions and prevention of disease, such as cardiovascular disease and cancer. _x000D_Biosynthesis of very long chain PUFAs such as EPA occurs via a sequence of oxygen-dependent desaturations and elongations of monounsaturated fatty acids. Desaturases and elongases are two classes of enzymes critical for the enzymatic pathway leading to production of polyunsaturated fatty acids. Engineered variants of enzymes are often based on crystal structures of the polypeptides involved. The EPA producing enzymes are membrane-inserted and technically this crystallization option is limited at best and offers no means of high throughput methods to optimize the whole pathway with a realistic time line for an industrial product. Consequently, the project will take advantage of a recently developed technology platform using "in vivo" directed evolution, rather than using more rational protein design principles. As directed "in vivo" evolution mimics Darwinian evolution, the project has no need for detailed structure-function information and thereby significantly speeds up the process towards an optimized EPA-oil producing pathway. _x000D_In this project we want to combine Eviagenics' in vivo recombination technology with Fluxome's metabolic engineering in order to enhance the EPA-oil production in baker’s yeast. The application of the in vivo recombination technology allows the evolution of a complete metabolic pathway for the production of EPA. The idea of in vivo recombination for the evolution of whole metabolic pathways and metabolic engineering to design a cell to support the pathway is a particularly attractive concept, because most natural and novel compounds are produced by pathways rather than by one single enzyme. Metabolic pathway engineering usually requires the coordinated and parallel manipulation of all enzymes in the pathway. This means that a technology must have the capacity to recombine long DNA fragments. At the moment the Eviagenics' technology is the only technology capable to do this._x000D_Cells expressing complete metabolic pathways highly optimized through in vivo recombination joined with highly optimized cell metabolism through metabolic engineering will generally allow the replacement of chemical factories by environmentally and cost competitive cell factories . This will be exemplified in the present project by the design of an efficient cell factory for the production of the omega-3 fatty acid EPA._x000D_References: Gunnarsson N., Förster J and Nielsen JB (2010): Metabolically engineered Saccharomyces cells for the production of polyunsaturated fatty acids. Granted Patent. Publication number: US7736884 (B2) _x000D_Rayssiguier, C., THaler,D.S. and M. Radman, " The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants” . Nature (1989) 342: 396-401._x000D_Tavares S, Grotkjær T, Obsen T, Haslam RP, Napier JA, Gunnarsson N. (2011) Metabolic Engineering of Saccharomyces cerevisiae for Production of Eicosapentaenoic Acid, Using a Novel {Delta}5-Desaturase from Paramecium tetraurelia. Appl Environ Microbiol. 77, 1854-61._x000D__x000D__x000D__x000D__x000D__x000D__x000D_

Acronym PUFAREF (Reference Number: 6475)
Duration 01/09/2011 - 01/02/2014
Project Topic This project combines innovative in vivo recombination technology with innovative metabolic engineering of complete metabolic pathways to enhance the production of the omega-3 poly-unsaturated fatty acid EPA containing oil in baker’s yeast by fermentation.
Network Eurostars
Call Eurostars Cut-Off 6

Project partner

Number Name Role Country
2 FLUXOME Coordinator Denmark
2 EVIAGENICS Partner France