A Unique CYP450 Enzyme Unlocks Antrodia Camphorata Lanostane Triterpenoid Diversity Through Oxidation and Skeletal Rearrangement.

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Citation abstract

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 Δ 14(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.