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 Streptomyces hundungensis 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.
Hu et al. (Mon,) studied this question.