大肠杆菌ptsG和ptsM突变体的构建及特性研究

Knockout of the ptsG and ptsM Gene in Escherichia coli DH5α and Characterization of the Mutants

目的:敲除大肠杆菌DH5α中与葡萄糖磷酸化转运相关的ptsG、ptsM基因,考察缺陷株生长特性及其可能的应用。方法:PCR扩增靶基因,构建两翼带有靶基因序列并嵌合抗药基因标记的线性片段,利用Red同源重组技术敲除靶基因。结果:成功敲除了大肠杆菌DH5α的ptsG和ptsM基因;在含有葡萄糖的LB培养基中,DH5αΔptsG最高菌密度是亲本的2.8倍,添加吡咯喹啉醌或导入其生物合成基因后能够产酸;DH5αΔptsM最高菌密度是亲本的4/10,有明显的产酸现象。结论:DH5αΔptsG可用于大肠杆菌高密度发酵和吡咯喹啉醌生物合成基因缺陷株筛选。

Objective： To construct a ptsG deletion mutant and a ptsM deletion mutant of Escherichia coli DH5α by gene knockout and to investigate the growth properties and potential application of the mutants. Methods： Target genes were obtained by PCR, and a linear fragment was generated by insertion of antibiotic resistant genes into two wings of target genes. The mutants of ptsG and ptsM deletion of E.coli DH5α were constructed by Red recombination. Results： The ptsG and the ptsM gene were successfully knocked out from E.coli DH5α. In LB medium supplemented with glucose, the mutants of E.coliDH5α AptsG showed 2.4 fold biomass than the parent and the glucanic acid formation when pyrrolo- quinoline quinine （PQQ） was supplemented in the media or introduction of PQQ biosynthesis gene into the cell. The DH5α AptsM showed 0.4 fold biomass and acid formation in LB medium supplemented with glucose. Conclusion： The mu- tants of E.coli DH5α AptsG is available for high-density culture and the screening of strain deficient in PQQ biosynthesis gene.