应用基因敲除快速构建大肠杆菌突变体改造脂肪酸代谢途径

Construction of Escherichia coli gene knock-out mutants for engineering of fatty acid metabolism

摘要:

摘要:【目的】基因敲除技术是研究基因功能的重要手段。我们试图建立一种快速、高效的大肠杆菌基因敲除方法。【方法】利用大肠杆菌(Escherichia coli)BW25113 单基因缺失体Keio 文库，将经典的Red 同源重组技术与P1噬菌体转导技术相结合，对E.coli MG1655脂肪酸代谢基因进行快速敲除。【结果】获得了大肠杆菌β-氧化途径的缺失菌株△fadD、△fadE和△fadD-△fadE;脂肪酸合成途径缺失菌株△fabH、△fabF和△fabH -△fabF。敲除fadD和fadE对生长情况没有影响;敲除fabH后，生长速度明显减慢;敲除fabF对生长几乎没有影响。FadD、FadE及双敲缺失体的脂肪酸含量18.2 mg/L、20.0mg/L和19.2 mg/L，略高于野生型17.5 mg/L;FabH、FabF及双敲缺失体的含量分别为12.6 mg/L、15.2 mg/L和11.2 mg/L，明显低于野生型。【结论】在单基因突变体文库基础上，利用P1 噬菌体转导、Red同源重组和抗性基因消除进行基因敲除，简化了构建大肠杆菌单基因和多重突变体的方法。

Abstract:

Abstract:［Objective］Gene knock out technique is very important for gene function study．We developed a simple and efficient method to knock out chromosomal genes in Escherichia coli．［Methods］Using the Escherichia coli Keio singlemutant library，we combined Red homology recombination with P1 phage transduction and developed a method to knock out the genes in Escherichia coli MG1655．［Results］We obtained β-oxidation mutants △fadD，△fadE and △fadD-△fadE and fatty acid synthesis mutants △fabH，△fabF and△fabH-△fabF. There were no obvious growth changes between △fadD or △fadE mutant strains and MG1655． However，△fabH and △fabH-△fabF mutant strains grew much slower than the wild type strain． The fatty acid contents in △fadD，△fadE and △fadD-△fadE were 18.2 mg/L，20.0 mg/L and 19.2 mg/L respectively，higher than 17.5 mg/L in wild type．The fatty acid contents in △fabH，△fabF and △fabH-△fabF were 12.6 mg/L，15. 2 mg/L and 11.2mg/L respectively，lower than that in wild type．［Conclusion］Using Keio mutant library，P1 phage transduction and resistant gene elimination，we have established a simple and efficient method for gene knock-out in Escherichia coli．