Metabolic engineering of for overproduction of triacylglycerols.

Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase, , as well as the last two steps of TAG formation: phosphatidic phosphatase () and diacylglycerol acyltransferase (), ultimately leading to 129 mg∙gCDW of TAGs. Disruption of TAG lipase genes , , and sterol acyltransferase gene increased the TAG content to 218 mg∙gCDW. Further disruption of the beta-oxidation by deletion of , as well as glycerol-3-phosphate utilization through deletion of , did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter led to accumulation of 254 mg∙gCDW. The TAG levels achieved here are the highest titer reported in , reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.