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

作　　者：Geng Chen Zhaokuan Guo Yanyu Shu Yan Zhao Lei Qiu Shaofeng Duan Yuan Lin Simei He Xiaobo Li Xiaolin Feng Guisheng Xiang Bo Nian Yina Wang Zhiyuan Li Chongkang Yang Yang Shi Yingchun Lu Guanze Liu Shengchao Yang Guanghui Zhang Bing Hao 

机构地区：[1]State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan,The Key Laboratory of Medicinal Plant Biology of Yunnan Province,National&Local Joint Engineering Research Center on Germplasms Innovation&Utilization of Chinese Medicinal Materials in Southwest China,Yunnan Agricultural University,Kunming 650201,China [2]Yunnan Characteristic Plant Extraction Laboratory,Kunming,Yunnan 650106,China

出　　处：《Plant Communications》2024年第6期154-172,共19页植物通讯（英文）

基　　金：supported by the Major Science and Technology Projects in Yunnan Province(2019ZF011-1);the Fundamental Research Project of Yunnan(202101AS070037);the Science and Technology Innovation team of Yunnan(202105AE160011);the Major Science and Technique Programs in Yunnan Province(202102AE090042);the Yunnan Characteristic Plant Extraction Laboratory(2022YKZY001);the First Projects of Science and Technology Plan in the Biomedical field in 2021(202102AA310048);the National Natural Science Foundation of China(grant nos.81960691 and 82160727).

摘　　要：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.

关 键 词：cucurbitacin IIa oxidosqualene cyclase OSC 11-carbonyl-20b-hydroxy-Cuol Saccharomyces cerevisiae Nicotiana benthamiana Hemsleya chinensis 

分 类 号：Q94[生物学—植物学]