(437f) Discovery of Novel Genes Regulating Acyl-CoA Availability in Yarrowia lipolytica

Yarrowia lipolyticais a nonpathogenic, oleaginous yeast that is currently used industrially for the production of eicosapentaenoic acid (EPA) and to produce triacylglycerides for biodiesel from simple sugars; however it also has the potential to use non-sugar substrates present in cheap plant oils and rendered animal fats. In the course of developing a novel 4-step acyl-CoA dependent pathway that converts exogenous fatty acuds into EPA, we characterized several previously uncharacterized genes important for fatty acid metabolism and lipid accumulation. Three new cytosolic acyl-CoA thioesterases, ACOT1-3 in Y. lipolytica, have the potential to improve EPA yields in a strain containing an acyl-CoA EPA pathway. We knocked out each gene individually and identified which candidates successfully improved EPA yields. Subsequent experiments used combinatorial knockouts to test any synergistic benefits through paired knockouts. The best knockout strain was paired with an overexpressed newly identified ABCA transporter, which we show improves exogenous fatty acid uptake and de novo fatty acid synthesis in the absence of exogenous fatty acids. Localization and substrate specificity tests have been performed on the uncharacterized ABCA transporter to help elucidate its function. By pairing ACOT knockouts with the overexpressed ABCA we seek to improve the uptake and flux of exogenous fatty acids through our engineered EPA pathway, while eliminating competing pathways that reduce the acyl-CoA substrate pool. This will improve the efficiency of the EPA biosynthesis pathway, improve the yields and cost effectiveness of producing EPA, and likely other oleochemical from cheap fats and oils.