AbstractAlthough glucose is an inexpensive substrate widely used as a carbon source in Escherichia coli recombinant fermentation technology, 10–30% of the carbon supply is wasted by excreting acetate. In addition to the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. Because glucose can enter the cell via several transport systems, isogenic strains defective in one or two of these transport systems were constructed. The effects of changes in the glucose uptake capacity on the in vivo flux distribution to a desired end product (β‐galactosidase) and to acetate were studied. The lack of one of the components (IICBGlc protein) of the glucose‐ phosphoenolpyruvate phosphotransferase system (Glc‐PTS) reduced the growth rate significantly. The maintenance of a low‐copy plasmid in this strain resulted in further arrest of the growth rate. However, β‐galactosidase production had no effect on growth rate. This strain directed more carbon into biomass and carbon dioxide, and less into acetate. β‐Galactosidase was produced in amounts not significantly different from the wild‐type strain from half the amount of glucose. An explanation for the experimental results is given, making use of published results on metabolic regulation. © 2005 Wiley Periodicals, Inc.