The carbon dioxide (CO2) produced during cell metabolism may accumulate to high concentration under high cell density cultures (HCDC), particularly in industrial scale fermenters with high hydrostatic pressures. CO2 partial pressures (pCO2) of more than 150-200 mbar may have inhibitory effects on growth and metabolism of most industrially important microorganisms. We have previously determined that maximum pCO2 in recombinant BL21 E. coli cultures, at biomass concentrations of 60 g/L (dry weight), can be as high as 110 mbar. Considering a simulated industrial culture operated under similar conditions, maximum pCO2 values can be in excess of 200 mbar. In this work we present a comparative study of the effect of pCO2 on the performance of batch cultures of E. coli strain W3110 (ATCC 27325), transformed with pV21 plasmid. Such plasmid contains a spectinomycin resistance gene and the green fluorescence protein (GFP) gene (gfp), placed under control of the lacZ gene promoter. pCO2 was continuously measured using an in situ (Cole Parmer) probe and controlled by a proportional algorithm, at constant values of 20, 70, 150, and 300 mbar, by manipulating through mass flow controllers the inlet gas composition (air, N2, and CO2). Dissolved oxygen tension, pH, and temperature were controlled at constant values in all cultures. All conditions were evaluated in duplicate and compared with a reference culture without CO2 control. Compared to the reference culture, the specific growth rate decreased by 19, 33, and 42 % for cultures maintained at constant pCO2 of 70, 150 and 300 mbar, respectively. In contrast, biomass yield was unaffected for cultures at 20 and 70 mbar, and only a negative effect was detected for pCO2 above 70 mbar. Acetate accumulation increased up to 2-fold with increasing pCO2. Maximum GFP concentration, compared to the reference culture, increased 10, 24, and 19% in cultures at 20, 70, and 150 mbar, respectively. However, a larger delay in GFP production was observed as pCO2 increased. In addition to determining the effect of pCO2 on the main macroscopic kinetic and stochiometric parameters, a comparative transcription analysis was performed. Fifteen genes were analyzed, including genes involved in central carbon metabolism (aceA, aceF, icdA, lpdA, sucA, sucB), acetate production pathway (ackA, poxB), anaplerotic metabolism (pckA, ppc), acid resistance system (adiA,, inaA, gadA, gadC), and the heterologous gene (gfp). The relative transcription expression values for anaplerotic reactions, the heterologous gene, and acetate production pathway genes did not change as pCO2 increased. Accordingly, these results reveal that the observed effect of high pCO2, at least partially, occurs at the level of enzymatic activity. Interestingly, the relative transcription expression for acid resistance genes (gadA, gadC) increased 2.5 and 3 times for 70 and 150 mbar, respectively. Furthermore, sucA and sucB genes, that code for α-oxoglutarate dehydrogenase complex, as well as icdA gen, that codes for isocitrate dehydrogenase, were down-regulated for all conditions. This represents a negative effect of CO2 on decarboxylation reactions of TCA. Altogether, the results of this work are important for developing improved culture operation strategies and metabolic-engineering approeaches for improving CO2 tolerance.