Enzyme and Metabolic Engineering Enable High-Level δ-Cadinene Production in Serratia marcescens

Abstract δ-Cadinene is a bicyclic sesquiterpene with fruity and herbal aromas that is widely used in flavors, fragrances, and pharmaceuticals. In this study, Serratia marcescens HBQA7, which carries an integrated MVA pathway and tolerates terpenoid toxicity, was used as the chassis strain. Rational design and site-directed mutagenesis of δ-cadinene synthase from Gossypium arboreum yielded a high-yield variant, G531V. In addition, mutations at key residues S409A, Y411A, D452N, and G531E led to the formation of hedycaryol and germacrene D-4-ol as byproducts. By further constructing fusion proteins and balancing the metabolic flux, we increased the yield to 5.98 g/L after 48 h of shake-flask fermentation. In a 5 L bioreactor, 100 h of fed-batch fermentation yielded a final δ-cadinene titer of 55.7 g/L, the highest level of microbial δ-cadinene synthesis reported to date.