磷酸水解酶对大肠杆菌工程菌合成番茄红素的影响  

The Impact of Phosphatases on Lycopene Biosynthesis in E.Coli

作　　者：刘洋洋 王天民 郭佳慧 张翀[2,3] 薛正莲 邢新会[2,3] LIU Yangyang;WANG Tianmin;GUO Jiahui;ZHANG Chong;XUE Zhenglian;XING Xinhui(College of Biological and Chemical Engineering,Anhui Polytechnic University,Wuhu 241000,China;MOE Key Lab of Industrial Biocatalysis,Institute of Biochemical Engineering,Department of Chemical Engineering,Tsinghua University,Beijing 100084,China;Center for Synthetic ＆ System Biology,Tsinghua University,Beijing 100084,China)

机构地区：[1]安徽工程大学生物与化学工程学院,安徽芜湖241000 [2]清华大学化学工程系生物化工研究所,北京100084 [3]清华大学合成与系统生物学中心,北京100084

出　　处：《安徽工程大学学报》2018年第4期1-5,42,共6页Journal of Anhui Polytechnic University

基　　金：国家自然科学基金资助项目(NSFC21627812)

摘　　要：实验以大肠杆菌DH1为宿主菌,在已有生产番茄红素大肠杆菌工程初始菌株lyc001的基础上,增加合成番茄红素途径中的关键基因crtEIB拷贝数;表征结果表明,相较于初始菌lyc001,番茄红素产量提高了34%,并将此菌命名为lyc011.利用CRISPR干扰(CRISPR interference,CRISPRi)原理以及Golden-Gate组装方法成功构建了可以靶向得到的57个磷酸水解酶基因的双single guide RNA (sgRNA)的质粒.将dCas9质粒和sgRNA表达质粒导入lyc011菌株中,通过下调这57个磷酸水解酶基因表达量,最终通过发酵表征成功筛选到了15个对番茄红素产量提高具有正向调节结果的磷酸水解酶基因,分别为:yjjG,aphA,nagD,yqaB,yidA,pphA,bacA,glpQ,pgpB,spoT,cpdB,nudJ,dgt,phoQ.Escherichia coli DH1 has been chosen as host strain. Starting from the previously constructed lycopene producing kickoff strain named lyc001, one additional copy of crtEIB cluster was integrated, the key genes in the lycopene biosynthesis pathway at the smf site. This modification led to 34% in crease in lycopene yield compared with the parent strain lyc001.This engineered strain named as lyc011 was used as the host for the following phosphatase screen.CRISPR interference （CRISPRi） and Golden Gate assembly were employed to construct 57 plasmids containing double single guide RNAs （sgRNA） targeting genes. The pdCas9 plasmid and sgRNA expression plasmid were introduced into the lyc011 strain and the lycopene yield was assessed for each construct. Finally, 15 genes encoding phosphatases （ yjjG ,aphA , nagD , yqaB , yidA , pphA , bacA , yahA , gZpQ, pgpB , spoT, cpdB , nudJ , dgt , phoQ ） were identified with positive effects on lycopene production.

关 键 词：大肠杆菌DH1 番茄红素 磷酸水解酶基因 CRISPRi GOLDEN GATE 

分 类 号：TQ28[化学工程—有机化工]