Metabolic Engineering of Saccharomyces Cerevisiae for the Synthesis of Polyketides and Fatty Acids

and Fatty Acids

Nancy A. Da Silva, Christopher Leber, Javier Cardenas, Jin Wook Choi, Ruben Fernandez-Moya

Department of Chemical Engineering and Materials Science, University of California, Irvine

Polyketides and fatty acids are of critical importance as biorenewable chemical precursors, biofuels, and pharmaceuticals. Both are synthesized via complex polyketide or fatty acid synthases, with many using acetyl-CoA and malonyl-CoA as starter and extender units. We have engineered the yeast Saccharomyces cerevisiae for the production of these valuable compounds and to allow the synthesis of novel variants. We have combined enzyme engineering (of the pathway and synthase enzymes), extensive metabolic pathway engineering for increased cofactor and precursor pools, and cultivation strategies to substantially increase titers and yields of a variety of products, including 6-methylsalicylic acid (6-MSA), dihydromonocolin L (DML; precursor to lovastatin), fatty acids (FAs) of varying lengths, and triacetic acid lactone (TAL). Fatty acids and triacetic acid lactone are attractive biomolecules due to their straightforward conversion into a broad range of biofuel and biochemical products. TAL can be converted via chemical catalysis to sorbic acid, 1,3-pentadiene, and a variety of other end products. S. cerevisiae was engineered for the high-level production of TAL by overexpression of the Gerbera hybrida 2-pyrone synthase (2-PS), engineering of the yeast metabolic pathways, and implementation of various cultivation strategies. These interventions increased both yield and titer 40 to 150-fold (>5 g/L TAL), levels far higher than previously reported. Fatty acids are also of interest as both biofuel and chemical precursors. We have introduced heterologous fatty acid synthases into S. cerevisiae to allow the synthesis of short (C6-C8) and medium (C10-C12) chain free fatty acids, and have done extensive pathway engineering to increase the levels and secretion of these and long-chain free fatty acids (C16-C18) to the culture medium. As an example, S. cerevisiae was engineered to overproduce free fatty acids by increasing carbon flux from glucose into the fatty acid and neutral lipid forming pathways, and by preventing the degradation and reactivation of these fatty acids. A unique combination of six gene knockouts and overexpression of two genes resulted in extracellular FFAs at a titer of 2.2 g/L, 4.2-fold greater than the highest level of extracellular free fatty acids reported for S. cerevisiae. In the presentation, we will discuss the critical pathways engineered, examine the synergy between successful strategies for the various polyketide products, and quantify the improvements obtained.