A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome
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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.
KeywordsEscherichia coli Chromosome Insertion T7 promoter Recombination P1 transduction Metabolic engineering
- Martinez-Morales F, Borges AC, Martinez A, Shanmugam KT, Ingram LO (1999) Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction. J Bacteriol 181:7143–7148Google Scholar
- Nelms J, Edwards RM, Warwick J, Fotheringham I (1992) Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase. Appl Environ Microbiol 58:2592–2598Google Scholar
- Parrott S, Jones S, Cooper RA (1987) 2-Phenylethylamine catabolism by Escherichia coli K12. J Gen Microbiol 133:347–351Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1998) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar