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

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.