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

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.