Genome mining reveals a new cyclopentane-forming sesterterpene synthase with unprecedented stereo-control

Abstract

Fungal bifunctional terpene synthases (BFTSs) catalyze the formation of diverse ring systems in diterpene/sesterterpene structures. Through genome mining of fungal BFTSs, we discovered a novel sesterterpenoid gene cluster pst, consisting of a BFTS gene pstA and a P450 monooxygenase gene pstB. Heterologous expression of both genes led to the identification of three new compounds, penisentene (1), penisentenol (2) and penisentone (3). Their planar structures were elucidated by GC-MS, HRESIMS, 1D and 2D NMR analysis. Their stereochemistry was determined by electronic circular dichroism (ECD), [Rh₂(OCOCF₃)₄]-induced ECD studies and chemical transformations. Noticeably, 1–3 possess the unique 5/15 cis-fused ring system, while all other ring systems produced by known BTFSs are trans in an equivalent 5/n fused position. In addition, the heterologous expression of the enzyme in Escherichia coli led to the production of 1. This unprecedented mode of ring fusion suggests that PstA controls the stereochemical outcome of the initial cyclization with a novel mechanism, which was discussed by an in vivo isotopic [1-¹³C,²H₃] acetate labeling experiment and density functional theory (DFT) calculations.