A Branchpoint Cytochrome P450 CYP512A13 Interconverts Different Types of Ganoderma Triterpenoids

The enone is a widely occurring structural unit and functional motif in natural products. Its synthesis at specific positions on the lanostane skeleton represents a hallmark feature of type I ganoderic acids (GAs), a class of triterpenoids fromGanoderma lucidumrenowned for their potent pharmacological properties. However, the current knowledge on their biosynthesis is limited to the notion that type I GA serves as the direct precursor of type II GAs, another class of bioactive Ganoderma triterpenoids. Here, we report a groundbreaking discovery: a multifunctional cytochrome CYP512A13, which directly catalyzes the conversion of the carbon–carbon conjugated double bond on the lanostane skeleton, the hallmark feature of type II GA, into an enone. This transformation is facilitated by a water channel and the C15 hydroxyl group of the substrate, representing an exceptionally rare mechanism in enone biosynthesis. Beyond this remarkable activity, CYP512A13 demonstrates catalytic promiscuity, hydroxylating additional carbon positions on the same substrate to generate unreported type II GAs. By integrating computational modeling with experimental validation, we elucidated the catalytic mechanism of CYP512A13, revealing its dual capability to produce both enone-containing type I GAs and type II GAs. Leveraging these insights, we employed rational design to engineer CYP512A13, achieving selective production of these distinct GA classes. Our findings not only uncover a critical biosynthetic route for the interconversion of GA types but also significantly expand the synthetic biology toolkit, enabling the efficient biosynthesis of high-value Ganoderma triterpenoids.