Functional Characterizationof a Thujopsene-Type SesquiterpeneSynthase from Nemania Diffusa

Thujopsene, a 3/6/6 tricyclic sesquiterpene, is widely distributed in the essential oils of various plants with diverse biological activities, such as antibacterial, antifungal, and antitermite properties. Here, we identified a sesquiterpene synthase (NdSTS) from the fungus Nemania diffusa that catalyzes the formation of thujopsene and thujopsan-2β-ol, as demonstrated by both in vivo and in vitro assays. This study represents the first report of a thujopsene-type sesquiterpene synthase originating from a fungal source. In addition, NdSTS displays broad substrate promiscuity and is able to utilize GPP as a substrate to produce the acyclic monoterpene alcohol geraniol in vitro. Furthermore, protein modeling combined with site-directed mutagenesis identified seven residues that are essential for the catalytic activity of NdSTS. Overall, this work expands our understanding of the catalytic diversity of fungal terpene synthases and provides novel genetic resources for thujopsene biosynthesis.