产印楝素前体tirucalla-7,24-dien-3β-ol的酿酒酵母细胞工厂构建  

Construction of yeast cell factories for production of azadirachtin precursor tirucalla-7,24-dien-3β-ol

作　　者：刘佳柔 苏新堯 刘金龙[1] 王彩霞[2] LIU Jia-rou;SU Xin-yao;LIU Jin-long;WANG Cai-xia(College of Biological Science and Engineering,Hebei University of Science and Technology,Shijiazhuang 050000,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]河北科技大学生物科学与工程学院,河北石家庄050000 [2]中国中医科学院中药研究所,北京100700

出　　处：《中国中药杂志》2021年第19期4959-4968,共10页China Journal of Chinese Materia Medica

基　　金：中央级公益性科研院所基本科研业务费专项(ZZ13-YQ-040);国家“重大新药创制”科技重大专项(2018ZX09201010);国家重点研发计划项目(2019YFC1906601,2019YFC1906603)。

摘　　要：印楝素是从印楝属植物印楝种子中提取的一种高度氧化的柠檬苦素类三萜化合物,是公认无害的植物源杀虫剂。在植物中含量很低,化学合成步骤繁琐,通过生物合成的方式来获取印楝素将对印楝素的商业用途提供很大的帮助,但生物合成途径未知限制了利用合成生物学进行外源合成。该研究在实验室优化过的甲羟戊酸途径(MVA)的代谢流的酿酒酵母菌株中,通过异源表达来自苦楝和印楝中的氧化鲨烯环化酶(OSCs)AiOSC1和MaOSC1基因,验证了上述2个OSC为苦楝素和印楝素合成的三萜骨架化合物。进一步将印楝氧化鲨烯环化酶基因(AiOSC1),连同拟南芥来源鲨烯合酶基因(AtAQS2),以及酿酒酵母来源截短的3-羟基-3-甲基戊二酰单酰辅酶A还原酶基因(PgtHMGR)共同整合到酿酒酵母染色体的delta位点上,构建了高产印楝素合成起始三萜骨架tirucalla-7,24-dien-3β-ol的酵母菌株。进一步将苦楝来源的细胞色素氧化酶基因(MaCYP71BQ5)导入上述高产tirucalla-7,24-dien-3β-ol酵母细胞中,成功在酵母细胞中检测到MaCYP71BQ5的产物。该研究不仅为tirucalla-7,24-dien-3β-ol生物合成奠定了基础,也为后期在酵母中研究印楝素生物合成途径中的细胞色素氧化酶(CYP450)提供了优势底盘菌株。Azadirachtin, as a botanical insecticide, is a highly oxidized limonoid triterpenoid existing in the seeds of Azadirachta indica. However, due to the low content in the seeds, the production of azadirachtin by seed extraction has low yield. Chemical synthesis of azadirachtin is characterized by complex process and low yield. Synthetic biology provides an alternative for the supply of azadirach-tin. In this study, two oxidosqualene cyclases AiOSC1 and MaOSC1 respectively derived from A. indica and Melia azedarach were identified in yeast. A yeast strain producing tirucalla-7,24-dien-3β-ol was constructed by integration of AiOSC1, Arabidopsis thaliana-derived squalene synthase gene(AtAQS2), and Saccharomyces cerevisiae-derived truncated 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene(PgtHMGR) into the delta site of yeast. Then, the function of MaCYP71BQ5 was successfully verified in yeast after this gene was introduced into the constructed yeast strain. This study not only laid a foundation for the biosynthesis of tirucalla-7,24-dien-3β-ol, but also provided a chassis cell for the functional identification of cytochrome oxidases(CYP450 s) in azadirachtin biosynthesis pathway.

关 键 词：氧化鲨烯环化酶 tirucalla-7 24-dien-3β-ol 酿酒酵母 

分 类 号：TQ453[化学工程—农药化工] Q78[生物学—分子生物学]