Engineering oleaginous yeast Yarrowia lipolytica for enhanced limonene production from xylose and lignocellulosic hydrolysate

Limonene, a valuable cyclic monoterpene, has been broadly studied in recent decades due to its wide application in the food, cosmetics and pharmaceutical industries. Engineering of the yeast Yarrowia lipolytica for fermentation of renewable biomass lignocellulosic hydrolysate may reduce the cost and improve the economics of bioconversion for the production of limonene. The aim of this study was to engineer Y. lipolytica to produce limonene from xylose and low-cost lignocellulosic feedstock. The heterologous genes XR and XDH and native gene XK encoding xylose assimilation enzymes, along with the heterologous genes tNDPS1 and tLS encoding orthogonal limonene biosynthetic enzymes, were introduced into the Po1f strain to facilitate xylose fermentation to limonene. The initially developed strain produced 0.44 mg/L of limonene in 72 h with 20 g/L of xylose. Overexpression of genes from the mevalonate pathway, including HMG1 and ERG12, significantly increased limonene production from xylose to similar to 9.00 mg/L in 72 h. Furthermore, limonene production peaked at 20.57 mg/L with 50% hydrolysate after 72 h when detoxified lignocellulosic hydrolysate was used. This study is the first to report limonene production by yeast from lignocellulosic feedstock, and these results indicate the initial steps toward economical and sustainable production of isoprenoids from renewable biomass by engineered Y. lipolytica.