Biosynthesis of bridged tricyclic sesquiterpenes in <i>Inula lineariifolia</i>

Presilphiperfolane-type sesquiterpenes represent a unique group of atypical sesquiterpenoids characterized by their distinctive tricyclic structure. They have significant potential as lead compounds for pharmaceutical and agrochemical development. Herein, we utilized a transcriptomic approach to identify a terpene synthase (TPS) gene responsible for the biosynthesis of rare presilphiperfolane-type sesquiterpenes in Inula lineariifolia, designated as IlTPS1. Through phylogenetic analysis, we have identified the evolutionary conservation of key motifs, including RR(x)8W, DDxxD, and NSE/DTE in IlTPS1, which are shared with other tricyclic sesquiterpene synthases in the TPS-a subfamily of Asteraceae plants. Subsequent biochemical characterization of recombinant IlTPS1 revealed it to be a multiproduct enzyme responsible for the synthesis of various tricyclic sesquiterpene alcohols from farnesyl diphosphate (FPP), resulting in production of seven distinct sesquiterpenes. Mass spectrometry and nuclear magnetic resonance (NMR) spectrometry identified presilphiperfolan-8β-ol and presilphiperfol-7-ene as predominant products. Furthermore, biological activity assays revealed that the products from IlTPS1 exhibited a potent antifungal activity against Nigrospora oryzae. Our study represents a significant advancement as it not only functionally identifies the first step enzyme in presilphiperfolane biosynthesis but also establishes the heterologous bioproduction of these unique sesquiterpenes.