S147: Synergy of MFA & FBA for understanding metabolic adaption in ethanol producing E coli

Genome-based Flux Balance Analysis (FBA, constraints based flux analysis) has been used to predict the impact of perturbations on microbes. Steady state isotopic-labeling-based Metabolic Flux Analysis (MFA) allows such comparisons and insight into cellular regulation with the long term goal of improving ethanol production by E. coli. We used genome-derived model of E. coli MG1655 metabolism  for both FBA and 13C-MFA analyses of aerobic and anaerobic growth of these cells cultured on glucose in minimal medium. Substrate uptake and product secretion rates were quantified using NMR and enzymatic assays. For MFA the labeling of amino acids and a range of intracellular metabolic intermediates were measured using gas and liquid chromatographic mass spectrometry methods. The data with an isotopic network model were obtained and validated for carbon and energy flows through metabolism. The flux maps reveal that the maintenance ATP consumption is about two-fold higher under anaerobic (12.6 mmol ATP/g/h) than aerobic conditions (7.1 mmol ATP/g/h). While the FBA feasible range contains the fluxes established from MFA, the FBA predictions of “most-likely” internal fluxes yielded from sampling the feasible space were found to be at odds with experimentally derived MFA fluxes. The combination of FBA and MFA results also suggests that limited glucose supply fails to result in maximal growth efficiency under aerobic condition. The submaximal metabolic efficiency is due to the repressed oxidative phosphorylation. Both rational strain design and physiological studies employing flux analysis would benefit substantially from the use of both methods.