The culture of E. coli for the commercial production of recombinant proteins has increased significantly in recent years. The production of acetate as a byproduct has been a significant problem because it retards cell growth, inhibits protein formation, and diverts carbon from biomass and protein product. Our approach to reducing acetate accumulation was to disable the phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) by deleting the ptsHI operon in the wild-type E. coli strain GJT001. The mutant strain (TC110) displayed a severely reduced growth rate and glucose uptake rate in glucose-supplemented M9 minimal medium, which confirmed the mutation. TC110 apparently metabolized glucose by a non-PTS mechanism that we are currently investigating, followed by phosphorylation by glucokinase. In complex medium such as 2xLB broth with 2% glucose, TC110 was able to grow quickly and still retained the phenotype of reduced acetate accumulation (9.1 ± 6.6 mM in TC110 vs. 90.4 ± 1.6 mM in GJT001). The reduced acetate accumulation resulted in a significant improvement in final OD (23.5 ± 0.7 in TC110 vs. 8.0 ± 0.1 in GJT001). We tested the strains for the production of model recombinant proteins such as green fluorescent protein (GFP) and β-galactosidase. TC110 had a 385-fold improvement in final volumetric productivity of GFP over GJT001 in shake flask cultures (2xLB broth with 2% glucose). The distribution of GFP fluorescence in the cell population, as determined by flow cytometry, was much broader in GJT001 (coefficient of variation = 466% ± 35%) than in TC110 (coefficient of variation = 55% ± 1%). We also tested the strains in corn steep liquor medium with 2% glucose and saw a 28-fold improvement in final volumetric production of GFP. TC110 had a 7.5-fold improvement in final volumetric productivity of β-galactosidase over GJT001 in 2xLB broth with 2% glucose medium. When tested in a batch bioreactor cultures with 2xLB broth with 2% glucose medium, the volumetric production of GFP by TC110 was 25-fold higher than that of GJT001. In summary, the ptsHI mutant of GJT001 resulted in reduced acetate accumulation, which led to significant improvements in recombinant protein production in batch bioreactors.