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.

The genus Agrocybe, situated within the Strophariaceae family, class Agaricomycetes, and phylum Basidiomycota, encompasses a myriad of species exhibiting significant biological activities. This review presents an integrative overview of the secondary metabolites derived from Agrocybe species, elucidating their respective biological activities and potential pharmacological applications. The metabolites under scrutiny encompass a diverse array of biological macromolecules, specifically polysaccharides and lectins, as well as a diverse group of 80 documented small molecular chemical constituents, classified into sterols, sesquiterpenes, volatile compounds, polyenes, and other compounds, their manifesting anti-inflammatory, anticancer, antioxidant, hepatoprotective, antimicrobial, and antidiabetic activities, these metabolites, in which polysaccharides exhibit abundant activities, underscore the potential of the Agrocybe genus as a valuable source of biologically active natural products. The present review emphasises the need for escalated research into Agrocybe, including investigations into the biosynthetic pathways of these metabolites, which could foster the development of novel pharmaceutical therapies to address various health challenges.