E. coli strain PPA305, which has a wild-type PTS system, and PPA316, which utilizes a proton-galactose symport system for glucose uptake, were used as host strains to harbor a phenylalanine overproduction plasmid pSY130-14 and to study the effects of using different glucose uptake systems on phenylalanine production. The non-PTS strain (PPA316/pSY130-14) produced much less phenylalanine, ranging from 0 to 67% of that produced by the PTS strain (PPA305/pSY130-14) depending on cultivation conditions used. The non-PTS strain PPA316/pSY130-14 had an intracellular PEP concentration only one-sixth that of the PTS strain, PPA305/pSY130-14. Additionally, PPA316/pSY130-14 had a substantially lower energy state in terms of the size of the pool of high-energy phosphate compounds and the magnitude of the pH difference across the cytoplasmic membrane. The non-PTS strain consumed oxygen at a higher rate, attained lower biomass concentration, and produced no acetate and phenylalanine during fermentation, suggesting more carbon was oxidized to CO2, most likely through the TCA cycle. Analysis of intracellular fluxes through the central carbon pathways was performed for each strain utilizing exponential phase data on extracellular components-and assuming quasi-steady state for intermediate metabolites. The non-PTS strain had a higher flux through pyruvate kinase (PYK) and TCA cycle which, in agreement with the observed higher oxygen uptake rate, suggests that more carbon was oxidized to CO2 through the TCA cycle. Further analysis using rate expression data for PYK and NMR data for the intracellular metabolites identified the regulatory properties of PYK as the probable cause for lower intracellular PEP levels in PPA316/pSY130-14.