An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica

• An evolutionary engineering approach was developed for enhancing lipogenesis.
• Evolutionary engineering improved lipid production titer to 39.1 g/L in bioreactor.
• Achieved 0.243 g/g yield, 87% lipid content, 0.51 g/L/h productivity.
• Genome sequencing revealed the importance of GABA degradation pathway.

Lipogenic organisms provide an ideal platform for biodiesel and oleochemical production. Through our previous rational metabolic engineering efforts, lipogenesis titers in Yarrowia lipolytica were significantly enhanced. However, the resulting strain still suffered from decreased biomass generation rates. Here, we employ a rapid evolutionary metabolic engineering approach linked with a floating cell enrichment process to improve lipogenesis rates, titers, and yields. Through this iterative process, we were able to ultimately improve yields from our prior strain by 55% to achieve production titers of 39.1 g/L with upwards of 76% of the theoretical maximum yield of conversation. Isolated cells were saturated with up to 87% lipid content. An average specific productivity of 0.56 g/L/h was achieved with a maximum instantaneous specific productivity of 0.89 g/L/h during the lipid production phase in fermentation. Genomic sequencing of the evolved strains revealed a link between a decrease/loss of function mutation of succinate semialdehyde dehydrogenase, uga2, suggesting the importance of gamma-aminobutyric acid assimilation in lipogenesis. This linkage was validated through gene deletion experiments. This work presents an improved host strain that can serve as a platform for efficient oleochemical production.