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), oxidosqualene cyclases HcOSC6, and acyltransferases (HcACT1, HcACT2) in Hemsleya chinensis. Meanwhile, isomultiflorenol synthases (HcOSC1, 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 Supplemental Table substrate for pharmaceutical cucurbitacin production and screening platform for candidate genes involved in cucurbitacin biosynthesis.

Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis

“…A total of six OSCs were annotated in this transcriptome, named HcOSC1-HcOSC6. As previously reported, HcOSC6 is the main enzyme involved in the synthesis of cucurbitacins in H. chinensis, catalyzing 2,3-oxidosqualene into cucurbitadienol, which serves as the starting substrate for CuIIa and other cucurbitacins (Chen et al, 2022). Additionally, previous study found that reported 4 novel OSCs from M. charanti, related to McCBS, McIMS, McBAS and McCAS (Takase et al, 2019).…”

“…Cucurbitacin F is commonly derived from cucurbitadienol. Previous studies have explored that HcOSC6 was clustered with characterized cucurbitadienol synthases from cucurbitaceae, and its expression pattern was regarded as responsible for the accumulation of cucurbitadienol, indicating HcOSC6 was a cucurbitadienol synthase involved in cucurbitadienol biosynthesis ( Chen et al., 2022 ).…”

“…Therefore, the first step in our study was to carry out computer modeling and molecular docking analysis to determine the biochemical role of HcOSC6. The full-length protein sequence of the HcOSC6 gene is from H. chinensis , which belongs to the Cucurbitaceae family ( Chen et al., 2022 ). Based on the protein sequence of H. chinensis , the first 3D structure of HcOSC6 in H. chinensis was predicted by training the model ( Figure 2A and Supplementary Figure 1A ).…”

“…Moreover, Hemsleya plants are used as traditional Chinese herbs, which contain cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb) involved in treating digestive and respiratory inflammations ( Figure 1 ). However, a few studies have investigated the biosynthesis of CuIIa and CuIIb ( Chen et al., 2022 ). Previous research has established that 2,3-oxidosqualene is cyclized to produce cucurbitadienol to further generate cucurbitacins ( Shibuya et al., 2004 ).…”

“…A previous study showed that substitutions, insertions, and deletions of bases can significantly affect OSC activity ( Chen et al., 2021 ). Furthermore, the highest contents of cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb) in tubers were 1.43 mg/g and 0.19 mg/g dw, respectively, and HcOSC6 was identified as the key enzyme for cucurbitacin production ( Chen et al., 2022 ). However, no reports have been found on how HcOSC6 affects the cucurbitacins accumulation.…”