Engineering Saccharomyces cerevisiae for the high-level production of free fatty acids.

Advances in biotechnology have enabled potential solutions to the oil-dependency of the chemical industry through renewable feedstocks for the production of current compounds, the discovery and production of new compounds with novel properties, and in some cases, the synthesis of chemicals at a lower cost than with traditional processes. The yeast Saccharomyces cerevisiae has been widely used in industry to produce ethanol, proteins, nutritional supplements and pharmaceuticals. A recent major focus has been the use of microorganisms to produce biofuels, commodity chemicals and specialty chemicals. Long-chain fatty acids (LCFA), medium-chain fatty acids (MCFA), and polyketides are good targets for the biochemical industry as they supply platform chemicals that can be later derived to a library of final products by chemical catalysis. The focus of our work has been the metabolic engineering of S. cerevisiae for the synthesis of free fatty acids of defined length. We constructed a yeast strain devoid of its native fatty acid metabolism and expressing the heterologous type II fatty acid synthase (FAS) system from Escherichia coli and a C14-specific thioesterase. This FAS system increased total fatty acids and shifted the fatty acid profile, increasing the percentage of C14 fatty acids from less than 1% to 33%. Yeast β-oxidation was engineered for MCFA production by a series of pathway interventions. Disruption of two genes, the essential gene ECI1 for mono-unsaturated fatty acid (mUFA) degradation and FAA2 for the peroxisomal MCFA activator, led to an increase in C12:1 production from 50 mg/L to 1 g/L in oleic acid medium. CRISPR/Cas9 was used to swap the native FAS1 and FAS2 promoters to the strong constitutive TEF1 promoter to increase expression of the yeast FAS. This resulted in a 3.8-fold increase in the production of total MCFA from glucose media relative to the faa2∆ eci1∆ strain. LCFA production and secretion was increased by deleting three genes for enzymes involved in fatty acid activation and three genes for enzymes in the fatty acid degradation pathway. Most importantly, a novel approach overexpressing genes for a lipid particle-forming enzyme (DGA1) and a triacylglycerol lipase (TGL3) further increased the production of secreted LCFA in shake flack culture to 2.2 g/L, the highest by S. cerevisiae to date. Finally, we performed preliminary work to increase acetyl-CoA pools for the synthesis of the closely related polyketides by downregulating competing pathways, such as fatty acid synthesis and the glyoxylate shunt. We evaluated downregulation of FAS1 and FAS2 using the CRISPR interference (CRISPRi) system, and also a more traditional promoter engineering strategy