Applied Microbiology and Biotechnology

, Volume 93, Issue 2, pp 815–829

A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome

  • Daisuke Koma
  • Hayato Yamanaka
  • Kunihiko Moriyoshi
  • Takashi Ohmoto
  • Kiyofumi Sakai
Methods and protocols

Abstract

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroFfbr and tyrAfbr or aroFfbr and pheAfbr) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.

Keywords

Escherichia coli Chromosome Insertion T7 promoter Recombination P1 transduction Metabolic engineering 

Supplementary material

253_2011_3735_MOESM1_ESM.doc (113 kb)
Supplementary Table 1Primers used in this study (DOC 113 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Daisuke Koma
    • 1
  • Hayato Yamanaka
    • 1
  • Kunihiko Moriyoshi
    • 1
  • Takashi Ohmoto
    • 1
  • Kiyofumi Sakai
    • 1
  1. 1.Osaka Municipal Technical Research InstituteOsakaJapan

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