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 and a non-steroidal anti-inflammatory drug that functions as the main ingredient of prescription Hemslecin capsules and tablets in China.Synthetic biology provides new strategies for production of such valuable cucurbitacins at a large scale;however,the biosynthetic pathway of cucurbitacin IIa has been unknown,and the heterologous production of cucurbitacins in galactose medium has been expensive and low yielding.In this study,we characterized the functions of genes encoding two squalene epoxidases(HcSE1-2),six oxidosqualene cyclases(HcOSC1-6),two CYP450s(HcCYP87D20 and HcCYP81Q59),and an acyltransferase(HcAT1)in cucurbitacin IIa biosynthesis by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana.We achieved high-level production of the key cucurbitacin precursor 11-carbonyl-20b-hydroxy-Cuol from glucose in yeast via modular engineering of the mevalonate pathway and optimization of P450 expression levels.The resulting yields of 46.41 mg/l 11-carbonyl-20b-hydroxy-Cuol and 126.47 mg/l total cucurbitacin triterpenoids in shake flasks are the highest yields yet reported from engineered microbes.Subsequently,production of 11-carbonyl-20b-hydroxy-Cuol by transient gene expression in tobacco resulted in yields of 1.28 mg/g dry weight in leaves.This work reveals the key genes involved in biosynthesis of prescription cucurbitacin IIa and demonstrates that engineered yeast cultivated with glucose can produce high yields of key triterpenoid intermediates.We describe a low-cost and highly efficient platform for rapid screening of candidate genes and high-yield production of pharmacological triterpenoids.

HMOD： An Omics Database for Herbal Medicine Plants

Dear Editor ,More than 50% of drugs are derived from chemical compounds that have been isolated from various plants （Fabricant and Farnsworth, 2001; Yarnell and Abascal, 2002）. With the development of sequencing technology and synthetic biology, we can obtain molecular information from the transcriptomic and genomic data of plants and then utilize bacteria to synthesize desired chemical compounds （Atanasov et al., 2015; Smanski et al., 2016）. Increasing numbers of researchers have started to publish omics data generated from herbal plants.