A convenient method for multiple insertions of desired genes into target loci on the <Emphasis Type="Italic">Escherichia coli</Emphasis> chromosome |
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Authors: | Email author" target="_blank">Daisuke?KomaEmail author Hayato?Yamanaka Kunihiko?Moriyoshi Takashi?Ohmoto Kiyofumi?Sakai |
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Institution: | (1) Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan |
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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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Keywords: | |
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