Medium containing (per liter): 5.0 g of glucose, 1.0 g of NH4Cl, 2.7 g of (NH4)2SO4,6.8gofNa2HPO4,3.0gofKH2PO4, 0.6 g of NaCl, 0.2 g ofMgSO4 7H2O, 1.0 µg of thiamine HCl, 2.0 µL of polypropylene glycol 2000 as an antiform agent, and 10 mL of trace element solution [1].

MFA analysis: The carbon flux distribution in the bioreaction network was determined as a best fit to all extracellular flux measurements, the macromolecular biomass composition, and the relative intensities of the 13C-13C scalar coupling multiplets of the aforementioned 47 ... carbon positions of aminoacids and glycerol determined by 2D [13C,1H]-COSY. Theflux quantification was performed by a least-squares parameter fitting approach in the mathematical framework. Exchanges fluxes viareversible reactions were quantitatively considered in the flux calculations. Initially, the isotopomer balances of all metabolites in the bioreaction network are calculated from a random initial flux distribution. Relative 13C multiplet intensities are then simulated from this isotopomer distribution and compared to the experimental values. The quality of the fit was judged by the X2 (error) value. Multiple calculations were performed from different random starting points, and the best solution that was reproducibly attained was presented as the estimated result of flux distribution. A statistical error analysis of the estimated fluxes was included in the calculations. Moreover, the flux estimates were compared with the results of flux ratio analysis, since both methods employed are very different and thus flux ratio analysis can serve as an independent verification of the flux estimates. In addition, the calculation of the flux distribution in the pck deletion mutant was performed without any constraint on the flux through PEP carboxykinase.
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[1] Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wuthrich K, Bailey JE. 1999. J Bacteriol 181:6679–6688.