Modulation of the Glycerol Phosphate availability led to concomitant reduction in the citric acid excretion and increase in lipid content and yield in Yarrowia lipolytica

Abstract

In order to improve TriAcylGycerol (TAG) lipids accumulation in the yeast Yarrowia lipolytica on glucose, double over-expression of the major acyl-CoA:diacylglycerol acyltransferase encoding gene (ylDGA2) and of the glycerol-phosphate dehydrogenase encoding gene (ylGPD1) was carried out. The genes were over-expressed in a strain impaired for the mobilization of the accumulated lipids, through the deletion of the genes encoding acyl-coenzyme A oxidases (POX1-6 genes) and the deletion of the very efficient lipase attached to the lipid bodies, encoded by ylTGL4. This metabolic engineering strategy had the objective of pulling the C-flow into the TAG synthesis by increasing the availability of glycerol-3-phosphate and its binding to fatty acids for the TAG synthesis. This strain showed a strong improvement in production performances on glucose in terms of lipid content (increase from 18 to 55%), lipid yield (increase from 0,035 to 0.14gg ^-1) and by-product formation (decrease in citric acid yield from 0.68 to 0.4gg ^-1). For developing bioprocess for the production of triacylglycerol from renewable carbon sources as glucose it is of first importance to control the C/N ratio in order to avoid citric acid excretion during lipid accumulation. Our engineered strain showed a delay in the onset of citric acid excretion as suggested by the 15% modulation of the critical C/N ratio.

Keywords: Citric acid production; DGA2; Fed-batch; Lipid accumulation; Nitrogen limitation; Yarrowia lipolytica.