Oxidosqualene cyclases and cytochrome P450s involved in the biosynthesis of diverse triterpenes in Centipeda minima

Abstract

Centipeda minima (L.) A. Braun et Aschers, a widely used traditional herbal medicine, is abundant in volatile oils and terpenes with notable antitumor, anti-inflammatory, and antiallergic activities. However, the key enzyme genes involved in the triterpene biosynthesis pathway of C. minima remain unexplored. In this study, six 2,3-oxidosqualene cyclases (CmOSC1–CmOSC6) and three cytochrome P450 enzymes (CmCYP716A1–CmCYP716A3) were discovered through transcriptome sequencing. Upon them, five OSC genes and two CYP450 genes were functionally characterized. CmOSC1 was identified as a cycloartenol synthase, CmOSC2 and CmOSC3 as multifunctional synthases, while CmOSC5 and CmOSC6 as lupeol synthases. CmCYP716A2 catalyzed one- to three-step oxidations at the C-28 position of oleanane-, ursane-, and lupane-type triterpenoids, producing compounds such as ursolic acid, oleanolic acid, betulin, and other intermediates. CmCYP716A3 catalyzes a single-step oxidation at the C-16 position of β-amyrin, α-amyrin, and lupeol, and a three-step oxidation at the C-28 position of β-amyrin, yielding oleanolic acid. A yeast heterologous expression system was employed to synthesize triterpenoid compounds with potent anti-inflammatory and antitumor activities, laying a foundation for future research on the biosynthesis of triterpenoids in C. minima.