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

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．