Dynamic regulation combined with systematic metabolic engineering for high-level palmitoleic acid accumulation in oleaginous yeast

Palmitoleic acid (POA, C16:1Δ⁹) is widely recognized for its preventive and therapeutic effects in various chronic and cardiovascular diseases, but the current production practices based on plant extraction are both economically and ecologically unsustainable. Although Yarrowia lipolytica is capable of producing POA, it only accumulates to a small percentage of total fatty acids. The present study aimed to enhance the accumulation of POA by employing a two-layer engineering strategy, encompassing the modulation of the fatty acid profile and the promotion of the accumulation of POA-rich lipids. The fatty acid profile was subject to modulation through the engineering of the fatty acid metabolism by expressing heterologous specific fatty acid desaturases CeFat5 and implementing dynamic regulation based on a copper-responsive promoter. Then, the mechanism underlying this improvement of POA production capacity was elucidated. Finally, the POA-rich lipid accumulation ability was enhanced through engineering of the lipid metabolism by overexpressing the heterologous POA-specific triacylglycerol forming acyltransferase, introducing the artificial designed non-carboxylative malonyl-CoA production pathway, and preventing lipid degradation. The resulting optimized yeast strain achieved an impressive POA accumulation accounting for 50.62% of total fatty acids, marking a 37.7-fold improvement over the initial strain. Moreover, a record POA titer of 25.6 g/L was achieved in the bioreactor. Overall, this study introduces a framework for establishing efficient yeast platforms for the accumulation of valuable fatty acids.