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

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.

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

“…Next, genetic traits were assembled by P1 transduction, and a kanamycin resistance marker was excised by FLP/FRT recombination to generate aromatic compound producers. The genetic trait of strain AR-G39 (acs::FRT-Km-FRT-T7p-ldhA) was incorporated into strains AR-G91 (tyrR::T7p-aroF fbr -pheA fbr ; Phe overproducer) and AR-G2 (tyrR::T7p-aroF fbr -tyrA fbr ; Tyr overproducer) (17), which were derived from E. coli strain BW25113(DE3), to generate PLA producer PAR-57 and 4HPLA producer PAR-3, respectively. Strain PAR-57 was further modified by assembling the genetic trait of strain AR-G85 (mtlA::FRT-Km-FRT-T7p-ldhA) to generate PLA producer PAR-58 harboring two copies of the chromosomal T7p-ldhA gene.…”

“…The timing to induce gene expression by the addition of isopropyl-␤-D-thiogalactopyranoside (IPTG) was varied depending on the strains because the growth of some constructed strains was depressed by IPTG addition and glucose consumption became poor. As a standard, the strains were cultivated at 37°C with shaking at 250 rpm/min (BR-23FH·MR; Taitec Co. Ltd.) in M9M medium until an optical density at 660 nm (OD 660 ) of 0.3 was attained (17). Strains PAR-47, PAR-51, PAR-53, PAR-63, PAR-64, PAR-65, PAR-75, PAR-77, PAR-79, PAR-83, PAR-89, PAR-90, PAR-91, PAR-92, and PAR-97, which were AR-G2 derivatives harboring T7p-ipdC at the mtlA locus of the chromosome, were cultivated in M9M medium at 37°C (250 rpm/min) until an OD 660 of 4.0 was attained.…”

“…The lactate dehydrogenase gene (ldhA) was amplified from genomic DNA from Cupriavidus necator JCM20644 (synonym Ralstonia eutropha) using the primer pair ReADH2-F-Nde (5=-CCAACCATATGCCTGCTCCCCAGATCCTCC-3= [the NdeI site is underlined]) and ReADH2-R-Xho (5=-CACTCGAGTTACAGCACTGGCG TCAGCAC-3= [the XhoI site is underlined]). The NdeI-XhoI-digested amplicon was introduced into the corresponding site of pET21a-FRT (17) to construct pARO133. The alcohol dehydrogenase gene (adhC) was amplified from genomic DNA from Lactobacillus brevis JCM1170 using the primer pair LbADH-F-Nde (5=-CCAACCATATGATGCAAATCAAAAC AGCTTTTTC-3=) and LbADH-R-Xho (5=-CACTCGAGTTAGAATGTG ATTACGGGC-3=).…”

“…Escherichia coli BW25113(DE3) was genetically modified by the previously developed method (17) to generate aromatic compound producers. The method consisted of Red-mediated recombination, FLP (flippase)/FRT (FLP recognition target) recombination, and P1 transduction.…”

“…We previously presented tyramine (TYM) and phenethylamine (PEA) production from glucose by expanding the shikimate pathway in E. coli (17). The TYM producer was constructed by overexpression of the tyramine decarboxylase gene from Lactobacillus brevis in a Tyr overproducer, whereas the PEA producer was constructed by overexpression of the aromatic amino acid decarboxylase gene from Pseudomonas putida in a Phe overproducer.…”