Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka, 536-8553, Japan.
Appl Microbiol Biotechnol. 2012 Jan;93(2):815-29. doi: 10.1007/s00253-011-3735-z. Epub 2011 Nov 30.
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.
我们开发了一种将多个目的基因插入大肠杆菌染色体靶位点的方法。该方法基于 Red 介导的重组、翻转酶和翻转酶识别靶位重组以及 P1 转导。使用该方法，可以将 6 个 lacZ 基因同时插入大肠杆菌染色体的不同位置。插入的 lacZ 基因可功能性表达，β-半乳糖苷酶活性与插入的 lacZ 基因数量成正比增加。该方法还用于代谢工程以产生芳香族化合物的高产菌。将莽草酸途径的重要基因（aroF(fbr)和 tyrA(fbr)或 aroF(fbr)和 pheA(fbr))引入染色体，以产生酪氨酸或苯丙氨酸高产菌。此外，将异源脱羧酶基因引入酪氨酸或苯丙氨酸高产菌的染色体，分别产生酪胺或苯乙胺高产菌。所得菌株选择性地过量生产目标芳香族化合物。因此，所开发的方法是大肠杆菌代谢工程生产有价值化合物的便捷工具。
