Escherichia coli has dispensable genome regions and eliminating them may improve cell use by reducing unnecessary metabolic pathways and complex regulatory networks. Although several strains with reduced genomes have already been constructed, there have been no reports of strains constructed with deletions assayed for influence on growth. To retain robust growth and fundamental metabolic pathways, the growth of each deletion strain and combination effects of deletions were checked using M9 minimal medium. Then a new strain, MGF-01, with a 1 Mb reduced genome was constructed by integrating deletions that did not affect growth. MGF-01 grew as well as the wild type in the exponential phase and continued growing after the wild type had entered the stationary phase. The final cell density of MGF-01 was 1.5 times higher than that of the wild-type strain. Using MGF-01 as a production host, a 2.4-fold increase in l-threonine production was achieved.