Enhancement of ß-Caryophyllene Biosynthesis in Saccharomyces cerevisiae via Synergistic Evolution of ß-Caryophyllene Synthase and Engineering the Chassis

ss-Caryophyllene is a plant-derived bicyclic sesquiterpene with multiple biological functions. ss-Caryophyllene production by engineered Saccharomyces cerevisiae represents a promising technological route. However, the low catalytic activity of ss-caryophyllene synthase (CPS) is one of the main restrictive factors for ss-caryophyllene production. Here, directed evolution of the Artemisia annua CPS was performed, and variants of CPS enhancing the ss-caryophyllene biosynthesis in S. cerevisiae were obtained, in which an E353D mutant enzyme presented large improvements in V-max and K-cat. The K-cat/K-m of the E353D mutant was 35.5% higher than that of wild-type CPS. Moreover, the E353D variant exhibited higher catalytic activity in much wider pH and temperature ranges. Thus, both the higher catalytic activity and the robustness of the E353D variant contribute to the 73.3% increase in ss-caryophyllene production. Furthermore, the S. cerevisiae chassis was engineered by overexpressing genes related to ss-alanine metabolism and MVA pathway to enhance the synthesis of the precursor, and ATP-binding cassette transporter gene variant STE6(T1025N) to improve the transmembrane transport of ss-caryophyllene. The combined engineering of CPS and chassis resulted in 70.45 mg/L of ss-caryophyllene after 48 h of cultivation in a test tube, which was 2.93-fold of that of the original strain. Finally, a ss caryophyllene yield of 594.05 mg/L was obtained by fed-batch fermentation, indicating the potential of ss-caryophyllene production by yeast.