Combining Metabolic Engineering and Lipid Droplet Storage Engineering for Improved α‑Bisabolene Production in <i>Yarrowia Lipolytica</i>

Bisabolene is a bioactive sesquiterpene with a wide range of applications in food, cosmetics, medicine, and aviation fuels. Microbial production offers a green, efficient, and sustainable alternative. In this study, we focused on improving the titers of α-bisabolene in Yarrowia lipolytica by applying two strategies, (i) optimizing the metabolic flux of α-bisabolene biosynthetic pathway and (ii) sequestering α-bisabolene in lipid droplet, thus alleviating its inherent toxicity to host cells. We showed that overexpression of DGA1 and OLE1 to increase lipid content and unsaturated fatty acid levels was essential for boosting the α-bisabolene synthesis when supplemented with auxiliary carbon sources. The final engineered strain Po1gαB10 produced 1954.3 mg/L α-bisabolene from the waste cooking oil under shake flask fermentation, which was 96-fold higher than the control strain Po1gαB0. At the time of writing, our study represents the highest reported α-bisabolene titer in the engineered Y. lipolytica cell factory. This work describes novel strategies to improve the bioproduction of α-bisabolene that potentially may be applicable for other high-value terpene products