Biosynthetic pathway of prescription cucurbitacin IIa and high-level production of key triterpenoid intermediates in engineered yeast and tobacco

Cucurbitacin IIa is a triterpenoid isolated exclusively from Hemsleya plants, which is a non-steroidal anti-inflammatory drug that function as the main ingredient of prescription Hemslecin capsules and tablets in China. Synthetic biology provides new strategies for producing such valuable cucurbitacins in large-scale, however, the biosynthetic pathway of cucurbitacin IIa was still unknown and heterologous production of cucurbitacins in galactose medium was expensive and low-yield. In this study, the functions of two squalene epoxidases (HcSE1-2), six OSC genes (HcOSC1-6), two CYP450 (HcCYP87D20 and HcCYP81Q59) and acyltransferases (HcAT1) involved in cucurbitacin IIa biosynthesis were characterised by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana. The high-level production yeast the key cucurbitacin precursor, 11-carbonyl-20β-hydroxy-Cuol, were first time constructed from glucose via modular engineering of the mevalonate pathway and optimization of P450 expression levels, to produce 46.41 mg/L 11-carbonyl-20β-hydroxy-Cuol and 126.47mg/L total cucurbitacin triterpenoid in shake flasks which are the highest yield from known engineered microbes. Moreover, production of 11-carbonyl-20β-hydroxy-Cuol in transient expression of tobacco was employed to achieve 1.28 mg/g dry weight (dw) in leaves. This work uncovered the key genes involved in biosynthesis of prescription cucurbitacin IIa and constructed the engineered yeast cultivated with glucose to produce high-yield of key triterpenoid intermediates. We offered a low-cost and high-efficient platform for rapid screening candidate genes and high-yield production of pharmacological triterpenoids.