Escherichia coli has been the most widely used host for the production of many industrially important compounds. But it has some drawbacks such as ill-defined cellular processes, use of too much energy, and production of many unnecessary compounds. In order to direct more metabolic energy into the production of useful materials and to decrease the production of unnecessary compounds, development of a new E. coli strain, which contains only the functional genes, was explored by deletion of large genomic segments using Cre/loxP site-specific recombination system. As a preliminary experiment we selected a target region between argF and hemB, which is 100kb apart, because this region contains a lac operon for the easy detection of the deletion. Two loxP recognition sequences for a site-specific recombinase Cre, were inserted into the E. coli chromosome at the pre-determined sites by homologous recombination and P1 transduction. The Cre, which is transiently expressed upon induction, deleted the targeted genomic segment flanked by two loxP sites. The new E. coli strain with the engineered chromosome showed normal growth pattern on rich-media. The feasibility of minimizing E. coli genome for a metabolic engineering was successfully demonstrated here by deleting the 100kb argF-hemB region of the E. coli MG1655 genome.