自杀质粒同源重组技术敲除阴沟肠杆菌budC基因以提升乙偶姻的产率

Increase acetoin production of Enterobacter cloacae:budC gene knockout by suicide plasmid homologous recombination technology

为了研究阴沟肠杆菌(Enterobacter cloacae,E.cloacae)SDM的2,3-丁二醇脱氢酶(budC)基因对乙偶姻产生的影响,采用自杀质粒同源重组技术敲除该基因。根据E.cloacae SDM的budC基因序列设计引物,构建budC基因敲除质粒,然后将质粒转化进E.cloacae SDM中,根据表型筛选及PCR验证获得budC基因缺失菌株。进行发酵试验后,利用高效液相色谱检测乙偶姻产量。结果表明,目的基因budC敲除成功,与原始菌株相比,基因缺失菌株2,3-丁二醇的产量降低了76.34%,乙偶姻的产量提高了103.78%。budC基因的敲除阻断了乙偶姻进一步分解代谢产生2,3-丁二醇的途径,提高了乙偶姻的产量。该试验获得了E.cloacaeΔbudC突变株,为利用微生物法工业化生产乙偶姻奠定了基础。

In order to investigate the effect of 2,3⁃butanediol dehydrogenase(budC)gene of Enterobacter cloacae(E.cloacae)SDM on acetoin production,suicide plasmid homologous recombination technique was used to knock out this gene.Primers were designed according to E.Cloacae SDM budC,and the budC gene knockout plasmid was constructed.Then the plasmid was transformed into E.Cloacae SDM.The budC gene knocked⁃out strain was obtained through phenotypic screening and PCR validation.The yield of acetoin was tested using high performance liquid chromatography after fermentation experiment.The results indicated that the target gene budC was successfully knocked out.Compared with the original strain,the yield of 2,3⁃butanediol was decreased by 76.34%and the yield of acetoin was increased by 103.78%.The knockout of budC gene blocked the pathway of further catabolism of acetoin to produce 2,3⁃butanediol,and increased the yield of acetoin.E.cloacaeΔbudC mutant was obtained in this experiment,which laid a foundation for the industrial production of acetoin by microbial method.