Discovery of unique CYP716C oxidase involved in pentacyclic triterpene biosynthesis from Camptotheca acuminata

Dozens of triterpenes have been isolated from Camptotheca acuminata, however, triterpene metabolism in this plant remains poorly understood. The common C28 carboxy located in the oleanane-type and ursane-type triterpenes indicates the existence of a functionally active triterpene, C28 oxidase, in this plant. Thorough mining and screening of the CYP716 genes were initiated using the multi-omics database for C. acuminata. Two CYP716A (CYP716A394 and CYP716A395) and three CYP716C (CYP716C80-CYP716C82) were identified based on conserved domain analyses and hierarchical cluster analyses. CYP716 microsomal proteins were prepared and their enzymatic activities were evaluated in vitro. The CYP716 classified into the CYP716C subfamily displays β-amyrin oxidation activity, and CYP716A displays α-amyrin and lupeol oxidation activity, based on gas chromatography-mass spectrometry analyses. The oxidation products were determined based on their mass and nuclear magnetic resonance spectrums. The optimum reaction conditions and kinetic parameters for CYP716C were determined, and functions were verified in Nicotiana benthaminana. Relative quantitative analyses revealed that these CYP716C genes were enriched in the leaves of C. acuminata plantlets after 60 d. These results indicate that CYP716C plays a dominant role in oleanane-type triterpene metabolism in the leaves of C. acuminata via a substrate-specific manner, and CYP716A is responsible for ursane- and lupane-type triterpene metabolism in fruit. This study provides valuable insights into the unique CYP716C-mediated oxidation step of pentacyclic triterpene biosynthesis in C. acuminata.