Reprogramming product selectivity and activity in promiscuous terpene synthases via substrate conformation engineering

Terpenoids are structurally diverse natural products with broad applications, yet their biosynthesis is often constrained by the low catalytic efficiency and poor product selectivity of terpene synthases (TPSs). Simultaneously enhancing product specificity and catalytic performance remains a major challenge in TPS engineering. Here, we elucidate the regio- and stereoselectivity of a fungal germacrene A synthase, which catalyzes the 1,10-cyclization of farnesyl diphosphate to form a central sesquiterpene intermediate. The side-chain volume of a non-catalytic residue (T169) adjacent to the substrate pocket modulates substrate-binding conformations, thereby redirecting the carbocation cascade and reshaping the product profile. Mechanism-guided pocket engineering further yielded terpene synthase variants with altered product specificity. Notably, one variant increased (+)-germacrene A production in Escherichia coli by 24.3-fold, reaching 15.8 g L⁻¹ in a bioreactor—the highest titer reported in this host to the best of our knowledge. These results provide mechanistic insight into substrate-conformation-mediated reprogramming of TPS specificity, highlighting a promising strategy for the development of high-performance terpenoid biocatalysts. Simultaneously enhancing product specificity and catalytic performance remains a major challenge in terpene synthase engineering. Here, the authors elucidate the regio- and stereoselectivity of a fungal germacrene A synthase, which catalyses the 1,10-cyclization of farnesyl diphosphate to form a central sesquiterpene intermediate, and demonstrate that engineering substrate conformation enables simultaneous improvement of product specificity and catalytic performance.