Application of Multiple Strategies To Debottleneck the Biosynthesis of Longifolene by Engineered Saccharomyces cerevisiae

Longifolene as an important sesquiterpene had enormous biological benefits. However, the low productivity of longifolene relying on chemical catalysis and plant extraction limited its wide application. Herein, the longifolene biosynthetic pathway was introduced into Saccharomyces cerevisiae, and multiple genetic strategies were applied to debottleneck the synthesis of longifolene, including the regulation of the rate-limiting enzymes, the elimination of the competitive pathways, the screening of the molecular chaperone to improve synthase activity, and the enhancement of the precursor supply. After combinationally applying these optimum strategies, the production of longifolene reached 27.30 mg/L in shake flasks and 1249 mg/L in fed-batch fermentation, respectively, which was the highest yield of longifolene reported thus far. It was demonstrated that the strategies applied in our work were effective in promoting the biosynthesis of longifolene, which not only laid a significant foundation for its industrial production but also provided a platform for the synthesis of other terpenoids.