Sequence-guided engineering of a bacterial diterpene synthase induces further cyclization

Enzyme engineering serves as a powerful tool in biocatalysis, enabling the development of enzymes with improved stability, activity, and specificity for a range of academic, industrial, and pharmaceutical applications. However, a limited understanding of sequence–structure–function relationships in terpene synthases, the enzymes that form the complex polycyclic hydrocarbon skeletons of terpenoid natural products, presents a major challenge in predicting and engineering the products of terpene synthases. In this study, we investigated the product profiles of the first two bacterial variediene synthases, OdVS from Olivibacter domesticus and PsVS from Prauserella shujinwangii, and found that they share some products with the bacterial phomopsene synthase, PmS from Allokutzneria albata, despite low sequence similarity. Thirteen diterpenes were isolated and structurally elucidated including three previously unreported compounds. A series of variants of OdVS, PsVS, and PmS were constructed by targeting conserved residues around the active site and aided in the identification of key residues that help control the cyclization pathway. Ultimately, mutation of a single residue, PsVSY86L, was found to switch the major product of PsVS from the tricyclic variediene to the tetracyclic phomopsene.