(197b) Validation of Metabolic Network Model for E. Coli and Assumptions of 13C-Metabolic Flux Analysis and Using [U-13C]Glucose Tracer

Currently, the most powerful technique for measuring intracellular metabolic fluxes in living cells is based on the application of ¹³C-tracers and measurement of isotopic labeling by mass spectrometry and/or NMR. Two key assumptions of ¹³C-metabolic flux analysis (MFA) are that 1) enzymes doen’t discriminate between ¹²C and ¹³C-atoms, i.e. no kinetic isotope effect; and that 2) the metabolic network model that is used for fitting of isotopomer data is accurate and complete. In this study, we validated both assumptons experimentally for wild-type E. coli using mixtures of natural and [U-¹³C]glucose. E. coli were grown in parallel cultures on media containing 0%, 20%, 40%, 60%, 80% and 100% [U-¹³C]glucose (with balanced natural glucose). Extracellular measurements didn’t reveal any significant differences between the cultures. The growth rate, the biomass-specific glucose uptake rate and the specific product secretion rates were identical for all cultures. Thus, these results provide support for the assumption that presence of ¹³C-atoms in the substrate doesn’t affect growth of E. coli. To investigate the potental impact on intracellular metabolism, GC-MS measurements were collected for proteinogenic amino acids and the isotopomer data were fit to a previously established metabolic model of E. coli. The initial fits did not provide statisitically acceptable results. Further inspection of the isotopomer data  and the metabolic network model revealed that the network model was missing important reactions. After complementing the model with these additional, previously missed, reactions we obtained consistent flux results for all experiments. Thus, these results provide additional support for the hypothesis that the presence of ¹³C-atoms in intracellular metabolites doesn’t affect enzymatic reactions and that the outcome of metabolic flux analyses is independent of the amount of ¹³C-tracer used.