Genome mining and functional characterization of four type I sesquiterpene synthases from the Tiger Milk Mushroom Lignosus rhinocerus

The Tiger Milk mushrooms (Lignosus spp.) have long been used as traditional folk medicines throughout Southeast Asia. However, the chemical constituents of these species remain largely unexplored. In this study, four type I sesquiterpene synthases (LrhTS1-LrhTS4) from Lignosus rhinocerus TM02^® were functionally characterized through genome mining, in vitro enzymatic assays, and heterologous expression in an Escherichia coli host engineered to overexpress an exogenous mevalonate pathway. The enzyme products were analyzed by GC-MS and further purified and structurally elucidated by NMR spectroscopy. A total of 11 sesquiterpenes (1-11) were identified, among which two were unstable and converted to stable derivatives (2 to 2a and 7 to 7a). Notably, compounds 1-10 are reported from the genus Lignosus for the first time. LrhTS1 and LrhTS3 exhibited high catalytic specificity, whereas LrhTS2 and LrhTS4 displayed product promiscuity. This work expands the knowledge of terpene synthase from mushroom, and advances the understanding of the chemical basis underlying the bioactivity of the Lignosus rhinocerus TM02®.