Systematic Discovery of Bacterial Diterpene Synthases and Structure-Guided Functional Interconversion of ShHS and CbCS

Terpenoids constitute the largest and most structurally diverse family of natural products, and bacterial genomes harbor vast yet largely unexplored biosynthetic potential. Here, we performed large-scale genome mining combined with heterologous expression in yeast to systematically screen 313 bacterial type I terpene synthases, leading to the identification of 16 active diterpene synthases (DTSs) and the discovery of 10 previously unknown diterpenes, including 5 unprecedented carbon skeletons. The DTS ShHS from <i>Streptomyces hundungensis</i> produces a series of highly rearranged diterpenes featuring the complex hundungane scaffold, whereas CbCS, which shares identical early cyclization steps with ShHS, generates the structurally simpler sphaeroane skeleton. Isotope-labeling experiments in combination with density functional theory calculations reveal an intricate carbocation cascade and identify a key branching intermediate that governs skeletal divergence. The crystal structure of CbCS allowed for identification of active-site residues responsible for functional differentiation. Structure-guided mutagenesis enabled functional interconversion between complex and simple diterpene skeletons. These findings expand the known chemical space of bacterial diterpenes and demonstrate how subtle active-site features precisely control carbocation rearrangement trajectories in terpene biosynthesis.