大肠杆菌PTS系统改造及重组菌生长性能测定  

Transformation of phosphotransferase system in Escherichia coli

作　　者：肖梦榕 张梁[1] 刘双平[1] 石贵阳[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室粮食发酵工艺与技术国家工程实验室,江苏无锡214122

出　　处：《生物工程学报》2014年第10期1561-1572,共12页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2012AA021201);教育部新世纪优秀人才支持计划(No.NCET-11-0665);江苏高校优势学科建设工程项目(No.PADP);江苏省2012年度普通高校研究生科研创新计划项目(No.CXLX12_0733);中央高校基本科研业务费专项资金(No.JUDCF12016)资助~~

摘　　要：利用Red重组系统对野生大肠杆菌Escherichia coli磷酸烯醇式丙酮酸-糖磷酸转移酶系统(Phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)进行修饰改造,敲除PTS系统中关键组分EⅡCBGlc的编码基因(ptsG),磷酸组氨酸搬运蛋白HPr的编码基因(ptsI),同时敲入来源于运动发酵单胞菌Zymomonas mobilis的葡萄糖易化体(Glucose facilitator)编码基因(glf),构建重组大肠杆菌,比较测定并系统评价了基因敲除和敲入对细胞的生长、葡萄糖代谢和乙酸积累的影响。敲除基因ptsG和ptsI造成大肠杆菌PTS系统部分功能缺失,细胞生长受到一定限制,敲入glf基因后,重组大肠杆菌能够利用Glf-Glk(葡萄糖易化体-葡萄糖激酶)途径,消耗ATP将葡萄糖进行磷酸化并转运进入细胞。通过该途径转运葡萄糖能够提高葡萄糖利用效率,降低副产物乙酸生成,同时能够使更多的碳代谢流进入后续相关合成途径,预期能够提高相关产物产量。We constructed several recombinant Escherichia coli strains to transform phosphoenolpyruvate： carbohydrate phosphotransferase system （PTS system） and compared the characteristics of growth and metabolism of the mutants. We knocked-out the key genes ptsI and ptsG in PTS system by using Red homologous recombination in E. coli and meanwhile we also knocked-in the glucose facilitator gene g/f from Zymomonas mobilis in the E. coli chromosome. Recombinant E. coli strains were constructed and the effects of cell growth, glucose consumption and acetic acid accumulation were also evaluated in all recombinant strains. The deletion of gene ptsG and ptsl inactivated some PTS system functions and inhibited the growth ability of the cell. Expressing the gene g/f can help recombinant E. coli strains re-absorb the glucose through Glf-Glk （glucose facilitator-glucokinase） pathway as it can use ATP to phosphorylate glucose and transport into cell. This pathway can improve the availability of glucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux in the metabolism pathway.

关 键 词：大肠杆菌 运动发酵单胞菌 PTS系统 glf Red同源重组 代谢工程 发酵 

分 类 号：Q93[生物学—微生物学]