Genome Mining for Sesterterpenes Using Bifunctional Terpene Synthases Reveals a Unified Intermediate of Di/Sesterterpenes

摘要

Genome mining is a promising method to discover novel secondary metabolites in the postgenomic era. We applied the Aspergillus oryzae heterologous expression system to functionally characterize cryptic bifunctional terpene synthase genes found in fungal genomes and identified the sesterfisherol synthase gene (NfSS) from Neosartorya fischeri. Sesterfisherol contains a characteristic 5-6-8-5 tetracydic ring system and is modified by cytochrome P450 mono- oxygenase (NfP450) to sesterfisheric acid. The cyclization mechanism was proposed on the basis of the analysis of in vivo and in vitro enzymatic reactions with isotopically labeled precursors. The mechanism involves C1 cation-olefin Ⅳ-olefin Ⅴ cydization followed by five hydride shifts, allowing us to propose a unified biogenesis for sesterterpenes branching from bicyclic (5-15), tricydic (5-12-5), and tetracydic (5-6-8-5) cation intermediates. Furthermore, the mechanism is distinct from that of a separate class of di/sesterterpenes including fusicoccins and ophiobolins. The difference between mechanisms is consistent with phylogenetic analysis of bifunctional terpene synthases, suggesting that the amino acid sequence reflects the initial cydization mode, which is most likely related to the initial conformation of a linear prenyl diphosphate.