Production of diesel fuels in Saccharomyces cerevisiae

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Verena Siewers, Shuobo Shi, Juan Octavio Valle Rodriguez, Bouke de Jong, Jens Nielsen

Chalmers University of Technology, Sweden

Efforts to replace petroleum derived products include the development of sustainable processes to produce diesel-like fuels. The current biodiesel (fatty acid alkyl ester) production is based mainly on plant oils and has a number of disadvantages, e.g. a low per hectare yield. We are therefore aiming at developing an efficient yeast cell factory for the synthesis of diesel fuels from biomass. The production of fatty acid ethyl esters (FAEEs) has previously been established in S. cerevisiae through introduction of a bacterial wax ester synthase. In this project, we optimized FAEE production in yeast through different strategies: (i) Improvement of wax ester synthase activity. Wax ester synthases from different sources were expressed in yeast to select the one with the highest activity, which was then integrated into the genome in multiple copies. We also developed a screening system to select for wax ester synthases with altered substrate specificity. (ii) Decrease of fatty acid consumption. Pathways competing with FAEE synthesis for the substrate, acyl-CoA, were eliminated. These included the formation of storage lipids and beta-oxidation. (iii) Increase of precursor supply. To increase the amount of acyl-CoA in the cytosol overexpression of a de-regulated version of acetyl-CoA carboxylase was combined with expression of a heterologous glyceraldehyde-3-phosphate dehydrogenase providing NADPH for fatty acid biosynthesis and overexpression of the endogenous acyl-CoA binding protein. In addition, the supply of acetyl-CoA was increased either by enhancing ethanol catabolism or through introduction of the heterologous phosphoketolase pathway. All these strategies proven beneficial when tested separately are now being combined in a single strain to create a platform strain for production of FAEEs and other fatty acid derived fuels and chemicals.