Defined MOPS medium [1] supplemented with 0.1% glucose and 200 μg/ml erythromycin. The flasks were agitated at 150 rpm to ensure aerobic conditions.

Biomass samples for determining the fluxes were centrifuged (5000 × g, 10 min at 4 °C) and harvested in 150 μl of 6 m HCl and hydrolyzed at 105 °C for 24 h before drying at 85 °C. The hydrolyzate was dissolved in 50 μl of dimethyl formamide and derivatized using 30 μl of N-m ... ethyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. The mixture was incubated at 85 °C with shaking at 550 rpm for 60 min (Orbital Shaker CSA, Thermo Scientific) before GC-MS analysis, using previously described protocols [2,3]. From the mass isotope distribution patterns of the proteinogenic amino acids, relative pathway contributions (metabolic flux ratios) were computed that were used as constraints to infer metabolic activity [2,3]. An aliquot of 5 ml of the culture from mid-exponential phase was quenched in hot phenol (80 °C) and subsequently frozen at −20 °C for measuring the concentration of intracellular metabolites. The samples were processed as described previously [4] and analyzed using LC-MS/MS for quantitative determination of central carbon metabolites and cofactors. The values were normalized with biomass concentrations at the time of sampling. --------------------------------------------References---------------------------------------
[1] Neidhardt F. C., Bloch P. L., Smith D. F. (1974) J. Bacteriol. 119, 736–747.
[2] Fischer E., Sauer U. (2003) Eur. J. Biochem. 270, 880–891. http://doi.org/dg3q64
[3] Fischer E., Zamboni N., Sauer U. (2004) Anal. Biochem. 325, 308–316. http://doi.org/c6kx35
[4] Luo B., Groenke K., Takors R., Wandrey C., Oldiges M. (2007) J. Chromatogr. A. 1147, 153–164. http://doi.org/cxdj7w