Rational Engineering of a Membrane-Anchored Promiscuous Cytochrome P450 for the Efficient Biosynthesis of Valuable Ganoderic Acids

Lanostane-type triterpenoid ganoderic acids (GAs) possess interesting bioactivities, but their efficient biosynthesis remains challenging. CYP512W2, a promiscuous membrane-anchored cytochrome P450, was recently found to oxidize C7 (or C11) and C15 to form two important GAs, ganoderic acid Y (GA-Y) and ganodermic acid Jb (GA-Jb), but their catalytic routes were unclear. Herein, we report that this CYP exhibits a preference for C7 oxidation, followed by subsequent hydroxylation at C15 after C7 dehydration. Combining computation-aided design with experimental verification, we identified key residues I108, M114, M213, L294, and Y482 critical for CYP512W2 catalytic activity. Engineering these residues significantly increased the production titers of GA-Jb and/or GA-Y. Molecular dynamics analysis uncovered that the proportions of favorable C7- and C15-conformations were enhanced in I108A, and the active pocket and substrate entrance were enlarged in Y482F. Besides, the slightly improved C7-conformation and the reduced C15-conformation in M213R may be attributed to the regulation of substrate conformation mediated by electrostatic attraction. The successful identification of these residues in CYP512W2 provides insights into the design of microbial cell factories for efficient GAs biosynthesis.