Expanding the Structural Diversity of Sesquarterpenes through Genome Mining of Noncanonical Class IB Terpene Synthases

Only a few sesquarterpenes (C₃₅ terpenes) have been found in nature, highlighting a chemical space requiring focused exploration of compounds. Noncanonical class IB terpene synthases (IB-TPSs) are the only enzymes that can cyclize C₃₅ prenyldiphosphate. Currently, ca. 6000 IB-TPS homologues from bacteria have been registered in the NCBI database. However, only two sesquarterpenes have been identified as IB-TPS products. In this study, we performed genome mining of 11 IB-TPSs from phylogenetically diverse bacterial species using an expression system in <i>Bacillus subtilis</i>. We revealed that 8 of the 11 homologues with ≥30% identity to <i>Bacillus subtilis</i> TPS (BsuTPS) synthesize the same product, tetraprenyl-β-curcumene, as BsuTPS. The absolute stereochemistry of the tetraprenyl-β-curcumene formed by BsuTPS and one of its homologues was determined to be (-)-7<i>R</i> using vibrational circular dichroism and specific optical rotation analyses. In contrast, 3 of the 11 IB-TPS homologues with <30% identity to BsuTPS produced 3 novel cyclic sesquarterpenes, 1 of which had an unprecedented 5/3-fused bicyclic sesquarterpene skeleton. These new sesquarterpenes could be synthesized through various carbocation quench mechanisms that differ from those of previously identified sesquarterpenes. In this study, we demonstrated that novel sesquarterpenes can be discovered with a high probability (3 out of 4) among IB-TPS homologues with <30% identity to BsuTPS, thereby expanding the structural diversity of sesquarterpenes.