Acetate, as a major by-product, was excreted by Escherichia coli when aerobic fermentation runs at high growth rates. In order to reduce the acetate secretion during the fermentation fundamentally, a list of genes related to acetate accumulation in E. coli was selected and knocked out. Physiological characterization of each mutant demonstrated that the growth and metabolites accumulation properties of these mutations exhibited significant change upon pathway engineering. The final engineered E. coli QZ1110 with ptsG, poxB, pta and iclR gene mutations was confirmed to accumulate 270% more biomass with 90% less acetate secretion than that of wild type E. coli in LB medium supplied with 1% glucose. Polyhydroxybutyrate biosynthesis experiment showed that the acetate reduction of the engineered strain in minimal medium also reduced 90% while the PHB accumulation increased almost 100% compare to wild type E. coli.