Enhancing β-Elemene Production in Escherichia coli via Protein Scaffold-Mediated Co-Localization of Key Enzymes

β-Elemene, a sesquiterpene with anticancer activity, faces limited microbial production due to low yields and inefficient enzyme coordination. This study established a modular covalent enzyme cascade in Escherichia coli for high-efficiency β-elemene biosynthesis. Systematic screening identified the SnoopTag/SnoopCatcher-mediated covalent assembly of farnesyl diphosphate synthase and germacrene A synthase as the optimal configuration (strain FS07). Subsequent enzyme stoichiometry modulation or linker engineering did not surpass FS07’s performance, indicating a near-optimal design. Fermentation optimization elevated the β-elemene titer to 7.21 g/L in FS07. Fed-batch fermentation in a 1.3 L bioreactor subsequently increased the final titer to 31.21 g/L, representing a 4-fold improvement over scaffold-free controls and achieving the highest reported yield in E. coli to date. This work provided a robust enzymatic scaffolding strategy for high-level terpenoid production.