Characterization and Engineering of a Bisabolene Synthase Reveal an Unusual Hydride Shift and Key Residues Critical for Mono-, Bi-, and Tricyclic Sesquiterpenes Formation

Sesquiterpene synthases (STSs) catalyze carbocation cascade reactions with various hydrogen shifts and cyclization patterns that generate structurally diverse sesquiterpene skeletons. However, the molecular basis for hydrogen shifts and cyclizations, which determine STS product distributions, remains enigmatic. In this study, an elusive STS SydA was identified in the biosynthesis of sydonol, which synthesized a new bisabolene-type sesquiterpene <b>6</b> with a unique saturated terminal pendant isopentane. Extensive evidence from isotope labeling experiments, crystal structures of SydA and its variant, quantum chemical calculations, and mutagenesis experiments reveal a plausible mechanism for the formation of <b>6</b> involving an unusual 1,7-hydride shift, which may be a key branchpoint for monocyclic, bicyclic, and tricyclic products. Structure-based engineering resulted in SydA variants that promote different reaction pathways, leading to the production of bicyclic α-cuprenene and (+)-β-chamigrene and tricyclic 7-<i>epi</i>-β-cedrene and β-microbiotene. These findings not only reveal a new bisabolene and its biosynthesis but also provide insights into the molecular basis of the hydride shifts and cyclizations, which pave the way for engineering STSs to produce complex terpenoid products.