Severe Restriction of Glucose Import Enhances Recombinant Protein Production in Escherichia coli

Glucose uptake through the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) enables rapid growth of Escherichia coli but promotes overflow metabolism that limits recombinant protein production. To define how glucose transport capacity shapes cellular physiology and bioprocess performance, we compared wild‐type W3110 with two PTS‐deficient derivatives, VH33 (Δ ptsHIcrr , constitutive galP expression) and WHIC (Δ ptsHIcrr Δ mglABC ), in controlled stirred‐tank bioreactors containing 20 g L − 1 glucose. PTS inactivation reduced specific growth and glucose uptake rates by up to 63% and decreased acetate accumulation by more than 95%, while final biomass concentrations remained comparable. Transcriptomic analysis revealed metabolic rewiring in the PTS − strains, which displayed highly similar global transcriptional profiles. However, the upregulation of alternative carbon metabolism and fimbrial genes in VH33 indicates subtle regulatory differences that may result in differential allocation of cellular resources. When expressing GFP, VH33, and WHIC achieved 2.5‐ and 4.4‐fold higher titers than W3110, respectively, with WHIC exhibiting the highest biomass‐specific yield (37.5 mg g − 1 ). Flow cytometry demonstrated strain‐dependent population heterogeneity and enrichment of recombinant protein in filamentous cells. Across strains, GFP synthesis rates approached growth dilution rates, indicating sustained intracellular accumulation. These results demonstrate that severe restriction of glucose import improves recombinant protein production by reshaping metabolic and physiological states.