To obtain strain YP211 with a high tendency for accumulating pyruvate, central metabolic pathways were modified in Escherichia coli MG1655. Specifically, seven genes (ldhA, pflB, pta-ackA, poxB, ppc, frdBC) were knocked out sequentially and full pyruvate dehydrogenase was retained. In batch fermentation with M9 medium, pyruvate yield and production rate reached 0.63 g/g glucose and 1.89 g/(1 h), respectively. Meanwhile, the production of acetate, succinate, and other carboxylates was effectively controlled. To understand the physiological observations, we further completed genome-wide transcription analysis of wild-type and YP211. As the acetic acid pathways were blocked, the pathways of convertion of pyruvate to phosphoenol pyruvate and acetyl CoA were enhanced. The transcription of pck, as an alternative gene for ppc, was increased by 2.6 times. So even if gene ppc was inactivated, the tricarboxylic acid pathway was still enhanced in YP211. In order to balance intracellular NADH/NAD(+), oxidative phosphorylation and flagellar assembly system were also up-regulated significantly.