酿酒酵母细胞中β-香树脂醇代谢流调控的研究  

Regulation ofβ-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells

作　　者：晁二昆 钱广涛 孙梦楚 苏新堯 朱智慧 盛玮[1] 王彩霞[2] 薛建平[1] CHAO Er-kun;QIAN Guang-tao;SUN Meng-chu;SU Xin-yao;ZHU Zhi-hui;SHENG Wei;WANG Cai-xia;XUE Jian-ping(College of Life Sciences y Haaibei Normal University,Huaibei 235000,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]淮北师范大学生命科学学院,安徽淮北235000 [2]中国中医科学院中药研究所,北京100700

出　　处：《中国中药杂志》2020年第16期3819-3825,共7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(2018YFC1706500);国家自然科学基金项目(31501368,81573518);安徽省教育厅高校科研平台创新团队项目(KJ2015TD001)。

摘　　要：该研究通过使用CRISPR/CAS9基因编辑技术,成功在实验室前期保存的产β-香树脂醇酿酒酵母细胞中敲除编码胞质苹果酸合成酶(citrate synthase,CIT2)和过氧化物酶体柠檬酸合酶(malate synthase,MLS1)的CIT2和MLS1基因,并将编码磷酸葡糖异构酶(phosphoglucose isomerase,PGI1)的PGI1基因启动子替换成编码酵母线粒体蛋白细胞色素c氧化酶亚基Ⅶa(cytochrome c oxidase subunitⅦa)的核基因Cox9的启动子,从而弱化PGI1基因的表达,调节乙酰辅酶A和胞内NADPH供给,进而调控β-香树脂醇的产量。发酵实验结果表明,删除CIT2基因对β-香树脂醇的产量没有影响;删除MLS1基因后,β-香树脂醇的产量是对照菌株的1.85倍,达到了3.3 mg·L^-1。弱化PGI1基因表达后,β-香树脂醇的产量是对照菌株的3.75倍,达6.7 mg·L^-1。该研究通过使用CRISPR/CAS9基因敲除技术成功的敲除CIT2和MLS1基因并弱化PGI1基因,提高了β-香树脂醇的产量,也为后期研究β-香树脂醇的酵母异源合成提供一定的借鉴意义。In this study,citrate synthase gene(CIT2),and malate synthase gene(MLS1)were successfully knocked out inβ-amyrin-producing yeast cells by using CRISPR/CAS9.The promoter of phosphoglucose isomerase gene(PGI1)was replaced by that of cytochrome c oxidase subunitⅦa(Cox9)to weaken its expression,aiming to channel more carbon flux into the NADPH-producing pathway.The fermentation results showed that CIT2 deletion had no effect on theβ-amyrin production.Compared with the control strain,the production ofβ-amyrin was increased by 1.85 times after deleting MLS1,reaching into 3.3 mg·L^-1.By replacing the promoter of PGI1,theβ-amyrin yield was 3.75 times higher than that of the control strain,reaching up to 6.7 mg·L^-1.This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9,which directly influenced the production ofβ-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

关 键 词：CRISPR/CAS9 β-香树脂醇 酿酒酵母 合成生物学 

分 类 号：Q78[生物学—分子生物学] R284[医药卫生—中药学]