Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities. In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumulation in Escherichia coli is presented. The alsS gene from Bacillus subtilis encoding the enzyme acetolactate synthase was introduced into E. coli cells using a multicopy plasmid. This newly introduced heterologous enzyme modifies the glycolytic fluxes by redirecting excess pyruvate away from acetate to acetolactate. Acetolactate is then converted to a nonacidic and less harmful byproduct acetoin, which appears in the broth. Furthermore, comparative fermentation studies show that the reduction in acetate accumulation leads to a significant improvement of recombinant protein production. The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the specific protein activity (to a level of 30% of total cellular protein) and 50% in terms of the volumetric activity in a batch fermenter. In fed-batch cultivation, the engineered strain achieved a volumetric recombinant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-galactosidase), which represented a 220% enhancement over the control strain. In the meantime, acetate excretion was maintained below 20 mM compared with 80 mM for the control, and the final cell density was improved by 35%.