In this study, we found for the first time that filamentous fungi derived type I chimeric terpenoid synthases TvTS and MpMS can directly synthesize triterpenoid core skeleton using IPP, DMAPP or HexPP as substrates. Subsequently, the synthesis of triterpenoid skeleton was proved to be the natural property of this enzyme by in vitro reaction and homologous activation experiments. Then, the absolute configuration and cyclization mechanism of the products were analyzed by in vitro isotope feeding experiments. Finally, the mechanism of triterpenoid skeleton synthesis catalyzed by type I chimeric terpenoid synthase was analyzed through structural biology experiments and site-specific mutations of amino acids at key sites. The 3D structure of terpene synthases was predicted by AlphaFold2 in batch, and another two triterpene synthases were successfully predicted and screened based on the docking result. It was further proved that type I chimeric terpene synthase catalyzed the cyclization of HexPP into triterpene skeleton. It breaks the stereotype that the triterpene skeleton can only be synthesized with squalene as the starting unit.
