Combining Metabolic Engineering and Lipid Droplet Storage Engineering for Improved -Bisabolene Production in Yarrowia Lipolytica

Bisaboleneis a bioactive sesquiterpene with a wide range of applicationsin food, cosmetics, medicine, and aviation fuels. Microbial productionoffers a green, efficient, and sustainable alternative. In this study,we focused on improving the titers of & alpha;-bisabolene in Yarrowia lipolytica by applying two strategies, (i) optimizingthe metabolic flux of & alpha;-bisabolene biosynthetic pathway and(ii) sequestering & alpha;-bisabolene in lipid droplet, thus alleviatingits inherent toxicity to host cells. We showed that overexpressionof DGA1 and OLE1 to increase lipidcontent and unsaturated fatty acid levels was essential for boostingthe & alpha;-bisabolene synthesis when supplemented with auxiliarycarbon sources. The final engineered strain Po1g & alpha;B10 produced1954.3 mg/L & alpha;-bisabolene from the waste cooking oil under shakeflask fermentation, which was 96-fold higher than the control strainPo1g & alpha;B0. At the time of writing, our study represents the highestreported & alpha;-bisabolene titer in the engineered Y. lipolytica cell factory. This work describes novel strategies to improve thebioproduction of & alpha;-bisabolene that potentially may be applicablefor other high-value terpene products.