CRISPR/Cas9技术敲除酿酒酵母gpd2基因对产2,3-丁二醇的影响  

Effects on 2,3-butanediol Production of Saccharomyces cerevisiae:gpd2 Gene Knockout by CRISPR/Cas9 Technology

作　　者：刘磊[1,2] 李娜 姜雪雍[1,2] 孙健 吕雨泽 葛菁萍[1,2] Liu Lei;Li Na;Jiang Xueyong;Sun Jian;Lv Yuze;Ge Jingping(Engineering Research Center of Agricultural Microbiology Technology,Ministry of Education,Heilongjiang University,Harbin 150500;Key Laboratory of Microbiology,College of Heilongjiang Province,School of Life Sciences,Heilongjiang University,Harbin 150080)

机构地区：[1]黑龙江大学农业微生物技术教育工程研究中心,哈尔滨150500 [2]黑龙江大学生命科学学院微生物省高校重点实验室,哈尔滨150080

出　　处：《中国农学通报》2020年第29期69-77,共9页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金“从2,3-丁二醇代谢角度构建工程微生物群体及其生态学机制研究”(31570492);黑龙江省麻类(工业)产业技术协同创新体系;黑龙江大学研究生创新科研项目(YJSCX2020-204HLJU)。

摘　　要：旨在利用CRISPR/Cas9基因编辑技术敲除酿酒酵母甘油-3-磷酸脱氢酶基因(gpd2),探究其对2,3-丁二醇产量的影响。根据酿酒酵母(Saccharomyces cerevisiae)W5甘油-3-磷酸脱氢酶基因(gpd2)设计供体片段及gRNA片段,将gRNA片段与可表达Cas9蛋白的敲除载体相连,之后将重组质粒及供体DNA片段转化到S.cerevisiae W5细胞中,根据表型筛选及PCR验证获得gpd2基因缺失菌株。结果表明目的基因gpd2敲除成功,基因缺失菌株与原始菌株经发酵实验相比,甘油产量下降22.01%,乙醇产量提高24.65%,2,3-丁二醇产量下降10.60%。gpd2基因的敲除并没有提高2,3-丁二醇的产量,原因可能是逐渐积累的NADH会优先被细胞内大量的乙醇脱氢酶所氧化,作用于乙醇的产生,而不是优先作用于2,3-丁二醇的合成。本实验构建了适用于酿酒酵母的基因敲除系统,该系统对进一步探究酿酒酵母其他代谢产物与2,3-丁二醇合成之间的关系具有实际的借鉴意义。This study aims at using CRISPR/Cas9 gene editing technology to knock out the glycerol-3-phosphate dehydrogenase gene(gpd2)in Saccharomyces cerevisiae,and investigating its effect on 2,3-butanediol production.Designing donor and guide RNA(gRNA)based on glycerol-3-phosphate dehydrogenase gene(gpd2)of S.cerevisiae W5,and linking the knockout vector could express Cas9 protein to the gRNA fragment,then the recombinant plasmid and donor DNA fragment were transformed into S.cerevisiae W5 cells.The gpd2 gene knocked-out strain was obtained through phenotypic screening and PCR validation,indicating that the gpd2 gene was successfully knocked out.Compared with the original strain,the gpd2 gene knocked-out strain increased ethanol production by 24.65%,and decreased glycerol production by 22.01%and 2,3-butanediol production by 10.60%.The production of 2,3-butanediol did not increase by knocking out the gpd2 gene,probably because that gradually accumulated NADH was preferentially oxidized by a large amount of alcohol dehydrogenase in the cell,and affected the production of ethanol rather than the synthesis of 2,3-butanediol.In this study,a suitable gene knockout system is constructed for S.cerevisiae,which has practical reference for further exploring the relationship between other metabolites of S.cerevisiae synthesis and 2,3-butanediol synthesis.

关 键 词：酿酒酵母 CRISPR/Cas9 2 3-丁二醇 载体构建 代谢工程 

分 类 号：S182[农业科学—农业基础科学]