Escherichia coli expression systems are still preferred to other bacterial expression systems. However, by-product formation via glycolytic pathways inhibits protein production efficiency. In this paper, by-product-forming pathways were engineered to evaluate their effect on foreign protein production. Elimination of D-lactate dehydrogenase (encoded by IdhA) resulted in enhanced cell performance and 17.8% increase in recombinant beta-mannanase production. Single deletions of pflB (encoding pyruvate formate lyase), pps (encoding phoenolpyruvate synthase) or poxB (encoding pyruvate oxidase) also had an affirmative impact on recombinant protein production. Furthermore, simultaneous deletions of IdhA, pflB, pps and poxB increased cell mass by 29% and beta-mannanase production by 56% under shake-flasks cultivation. Meanwhile, overall acetate concentration showed a decrease of 33%. This quadruple mutant showed the best performance under bioreactor process, in which volume and specific activity of P-mannanase increased by 1.9 and 1.46 fold compared to the control strain respectively. The approach shown here indicated that rational engineering of glycolytic pathways can efficiently improve foreign protein production in E. coli. (C) 2014 Elsevier Ltd. All rights reserved.