(191aj) Rewiring Yarrowia Lipolytica Lipid Metabolism for the Production of Omega-3 Fatty Acid Using Alternative Substrates

Omega-3 long chain fatty acids are essential for human health and development. The stagnant production of omega-3 fatty acids from fish oils no longer meets the demands of the fast growing aquaculture and nutraceutical market. A sustainable and economic source of omega-3 fatty acids is urgently needed. Here we report a systematic approach to metabolic engineering of non-conventional yeast Y. lipolyticafor eicosapentaenoic acid (EPA) production from non-sugar substrates such as cheap plant oils and rendered animal fats. We first established an alternative EPA biosynthesis pathway into Y. lipolyticaby heterologous expression of a set of elongases and desaturases utilizing acyl-CoA instead of phospholipids, resulting in efficient production of EPA while avoiding accumulation of undesired intermediate fatty acids. We also enhanced the assimilation and synthesis pathways of triacylglycerides, and disrupted its degradation pathway to improve the efficiency of utilizing exogenous lipids as building block for EPA production. By redirecting carbon flux towards fatty acid elongation and desaturation, we further increased the amount of EPA that was incorporated into triacylglycerol. Finally, we have tested several strategies to increase acyl-CoA flux, including enhancing acyl editing and fatty acid transport. Along the way, novel genes in the Y. lipolytica genome were identified that enhanced acyl-CoA flux. Our results illustrate that the acyl-CoA pathway circumvents bottlenecks in the conventional phosphatidylcholine dependent desaturase pathways necessary for accumulation of triacylglycerides with high EPA content. This also suggests a new paradigm for the use of low-value alternative feedstocks for bioprocesses.