Engineering precursor pools for increasing production of odd-chain fatty acids inYarrowia lipolytica

Abstract

Microbial production of lipids is one of the promising alternatives to fossil fuels with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five- carbon chain (β-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica. We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve β-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding β-ketothiolase in the producing strain, and the OCFA production was increased by 33 % compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications.

Metabolic Engineering for Unusual Lipid Production in Yarrowia lipolytica

“…Y. lipolytica was further engineered (mfe1∆ tgl4∆ pTEF-GPD1 pTEF-DGA2) to accumulate large amounts of lipids, increasing both OCFA levels and total lipid content; the final strain produced 0.75 g/L of OCFAs, mainly C17:1-FA, thanks to an optimized fed-batch co-feeding strategy [66]. This strain was further modified to boost propionyl-CoA pools by screening for propionate-activating enzymes [67]. The highest levels of OCFA production (3.8 times higher than those in the control strain) were obtained by expressing propionyl-CoA transferase (PCT) from Ralstonia eutropha.…”