A Unique CYP450 Enzyme Unlocks <i>Antrodia Camphorata</i> Lanostane Triterpenoid Diversity Through Oxidation and Skeletal Rearrangement

Lanostane triterpenoids, key therapeutic components of medicinal mushrooms, such as rare Antrodia camphorata, face heterologous biosynthesis barriers due to the lack of enzymes for essential C21/C15 oxidation and skeletal rearrangement, limiting access to these rare therapeutics. In this study, we deciphered these missing steps through the characterization of a single CYP450 enzyme, AcCYP1. Notably, AcCYP1 not only catalyzes the indispensable C21 and C15 oxidations, but also represents the first CYP450 enzyme identified to directly rearrange a triterpenoid backbone. This rearrangement generates the uncommon lanostane skeleton characterized by a Δ¹⁴⁽¹⁵⁾ double bond and a C15 methyl group by disrupting canonical hydroxyl rebound and triggering cation-initiated rearrangement. Mechanistically, the catalytic performance of AcCYP1 is regulated by proximal active-pocket geometry and distal hydrophobicity. Mutating the key residue N520 markedly enhanced enzymatic activity, enabling controllable yeast-based production of lanostane triterpenoids with expanded structural diversity for more efficient than conventional artificial cultivation. Collectively, this work uncovers a non-canonical route for triterpenoid structural diversification beyond oxidosqualene cyclases, establishes a systematic strategy for deciphering biosynthetic pathways, and provides scalable, sustainable access to rare natural products.