Pathway elucidation of pharmaceutical cucurbitacin IIa in Hemsleya chinensis and high-level production of precursor cucurbitadienol in engineered Saccharomyces cerevisiae and Nicotiana benthamiana

Abstract Cucurbitacin IIa is a triterpene isolated exclusively from Hemsleya plants, which is non-steroidal anti-inflammatory drug that function as the main ingredient of Hemslecin capsules and Supplemental Tablets in China. In this study, the biosynthetic pathway of cucurbitacin IIa was elucidated by characterization of squalene epoxidases (HcSE1, HcSE2), cucurbitenol synthases (HcOSC6) and acyltransferases (HcAT1) in Hemsleya chinensis . Meanwhile, cycloartenol synthases (HcOSC1), isomultiflorenol synthases (HcOSC5) and β-amyrin synthase (HcOSC2-4) involved in sterol and triterpenes biosynthesis were functionally illustrated. The high-level production of yeast the key cucurbitacin precursor, cucurbitadienol, was constructed to produce 296.37 mg/L cucurbitadienol and 722.99 mg/L total triterpenoid which is the highest yield cucurbitadienol from known engineered microbes. Moreover, production of cucurbitenol in transient expression of tobacco was employed to achieve 94.8 mg/g dry weight (dw) cucurbitenol from leaves. In this study, the key genes involved in cucurbitacin IIa biosynthesis were identified to facilitate its medical applications via biosynthetic strategy. Meanwhile, the high-level production of cucurbitadienol chassis yeast and tobacco transient expression offered a robust and sSupplemental Table substrate for pharmaceutical cucurbitacin production and screening platform for candidate genes involved in cucurbitacin biosynthesis.