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

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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 (aroF fbr and tyrA fbr or aroF fbr and pheA fbr) 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.

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Acknowledgments

This work was partially supported by a Grant-in-Aid for Young Scientists (B) (19780082 and 21780105).

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Authors and Affiliations

  1. Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka, 536-8553, Japan

    Daisuke Koma, Hayato Yamanaka, Kunihiko Moriyoshi, Takashi Ohmoto & Kiyofumi Sakai

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  1. Daisuke Koma
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  2. Hayato Yamanaka
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  3. Kunihiko Moriyoshi
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  4. Takashi Ohmoto
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  5. Kiyofumi Sakai
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Correspondence to Daisuke Koma.

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Supplementary Table 1

Primers used in this study (DOC 113 kb)

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Koma, D., Yamanaka, H., Moriyoshi, K. et al. A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome. Appl Microbiol Biotechnol 93, 815–829 (2012). https://doi.org/10.1007/s00253-011-3735-z

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  • DOI: https://doi.org/10.1007/s00253-011-3735-z

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