Highly efficient production of β-caryophyllene in Saccharomyces cerevisiae using a novel synthase from Artemisia argyi

β-Caryophyllene, a bicyclic sesquiterpene shows remarkable application potential in agriculture for plant disease prevention, pest control and green pesticide development. However, conventional production methods—relying on plant extraction or chemical synthesis—exhibit inherent limitations that hinder both the achievable yield and the commercial scalability of β -caryophyllene manufacturing. In this study, we mined and identified a highly efficient β -caryophyllene synthase, designated ArarCARS. Through molecular docking and ancestral sequence reconstruction, we screened mutants at key residues and revealed that the F117Y variant significantly enhanced production. Molecular dynamics simulations demonstrated that this mutation improved product formation through enhanced enzyme stability. Based on these results, we engineered a high-titer β -caryophyllene-producing Saccharomyces cerevisiae strain via a series of metabolic engineering strategy. Fed-batch fermentation at low temperatures achieved a final β -caryophyllene titer of 35.4 g/L, representing the highest level documented so far. This work not only provides an effective strategy for β -caryophyllene biosynthesis but also offers engineering insights for the efficient microbial production of other valuable terpenoids.