中国中药杂志

2020, v.45(16) 3819-3825

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酿酒酵母细胞中β-香树脂醇代谢流调控的研究
Regulation of β-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells

晁二昆;钱广涛;孙梦楚;苏新堯;朱智慧;盛玮;王彩霞;薛建平;
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, Huaibei Normal University;Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences;

摘要(Abstract):

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 in β-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(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 the β-amyrin production. Compared with the control strain, the production of β-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the β-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 of β-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

关键词(KeyWords): CRISPR/CAS9;β-香树脂醇;酿酒酵母;合成生物学
CRISPR/CAS9;β-amyrin;Saccharomyces cerevisiae;synthetic biology

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 晁二昆;钱广涛;孙梦楚;苏新堯;朱智慧;盛玮;王彩霞;薛建平;
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, Huaibei Normal University;Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences;

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