Sesquiterpene synthases from Inonotus obliquus: functional characterization and β-farnesene production.

The medicinal mushroom Inonotus obliquus is renowned for producing a wide array of pharmacologically active terpenoids, yet the enzymatic basis for this diversity remains partially elusive. Here, we functionally characterized 17 sesquiterpene synthase (IoSTSs) genes identified from the I. obliquus genome, and using Aspergillus oryzae as a heterologous host. Eight IoSTSs were confirmed to catalyse the formation of 15 sesquiterpene compounds, among which six enzymes showed consistent activities in vitro. Three key enzymes IoSTS3, IoSTS16, and IoSTS18 were identified as (+)-isoguaiene, (-)-daucene, and β-farnesene synthase, respectively, based on GC-MS and NMR analyses. By co-expressing the genes encoding 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl pyrophosphate synthase from A. oryzae in the AO-IoSTS18 transformant, further metabolic engineering enhanced the β-farnesene yield to 206.98 mg/kg of rice medium. This study broadens our understanding of the functional diversity of fungal sesquiterpene synthases. It also presents an effective and sustainable approach for the biotechnological production of medicinal and fuel-related terpenoids derived from I. obliquus.