Discovery and Characterization of Two Novel Sesquiterpene Synthases Producing Pleostene in Oyster and Honey Mushrooms

Basidiomycetous fungi are prolific producers of terpenoids, yet many biosynthetic pathways remain unexplored. Here, we report the discovery of two novel terpene synthases: PoTS6 from the oyster mushroom (<i>Pleurotus ostreatus</i>) and AgTS2 from the bulbous honey fungus (<i>Armillaria gallica</i>). Both produce a unique terpene scaffold, pleostene (<b>1</b>), whose structure was elucidated by using NMR spectroscopy and GC-MS analysis. Despite sharing only 44% sequence identity, PoTS6 and AgTS2 exhibit highly similar three-dimensional conformations, including conserved active-site residues and their spatial arrangement within the substrate-binding pockets. Our finding highlights the importance of structural conservation over sequence similarity in product specificity. Phylogenetic analysis reveals two distinct clades of homologous enzymes, suggesting pleostene biosynthesis may be widespread among Agaricales fungi. Functional analysis demonstrates that the C-terminal region of PoTS6 (residues 331-407) is dispensable for catalytic activity. Genomic context analysis predicts that PoTS6 is not part of a gene cluster, whereas AgTS2 resides within a putative biosynthetic gene cluster containing 3 cytochrome P450 enzymes. These P450s may oxidize pleostene into bioactive derivatives such as clitocybulols and nebucanes, known for their cytotoxic and antimicrobial activities. These findings expand our understanding of fungal terpenoid biosynthesis and offer new opportunities for natural product biosynthesis.