M9 minimal medium supplemented with 2 g/liter of glucose in 500-ml Erlenmeyer flasks. M9 medium contained (per liter of deionized water) 0.8 g of NH4Cl, 0.5 g of NaCl, 7.5 g of Na2HPO4·2H2O, and 3.0 g of KH2PO4. The following components were sterilized separately and then added (per liter final volume of medium): 2 ml of 1 m MgSO4, 1 ml of 0.1 m CaCl2, 0.3 ml of 1 mm filter-sterilized thiamine HCl, and 10 ml of a trace element solution containing (per liter) 1 g of FeCl3·6H2O, 0.18 g of ZnSO4·7H2O, 0.12 g of CuCl2·2H2O, 0.12 g of MnSO4·H2O, and 0.18 g of CoCl2·6H2O. At the start of evolution, initial precultures of each mutant were grown overnight in LB medium before being transferred to minimal medium for adaptive evolution.

After 8 h of incubation at 37 °C and constant shaking, LB precultures were used to inoculate M9 medium precultures that were grown overnight for inoculation of cultures for physiological or 13C-labeling experiments. For 13C-labeling experiments, glucose was added either enti ... rely as the 1-13C-labeled isotope isomer (>99%; Euriso-top, GIF-sur-Yvette, France) or as a mixture of 20% (w/w) U-13C (>98%; Isotech, Miamisburg, OH) and 80% (w/w) natural glucose. Cell growth was monitored by following the A600. Glucose and acetate concentrations were determined enzymatically using commercial kits (Beckman-Coulter (Zurich, Switzerland) or Dispolab (Dielsdorf, Switzerland)). Other organic acids in culture supernatants were detected by high pressure liquid chromatography analysis (PerkinElmer Life Sciences) at a wavelength of 210 nm, using a Supelcogel C8 column (4.6 × 250 mm) at 30 °C and a mobile phase of 2% (v/v) sulfuric acid at a flow rate of 0.3 ml/min.
The following physiological parameters were determined during the exponential growth phase as described previously [1]: maximum growth rate, biomass yield on glucose, specific glucose consumption rate, and specific byproduct production rates, using a predetermined correlation factor of 0.44 g of cellular dry weight per liter and A600 unit.
Metabolic Flux Ratio (METAFoR) Analysis by Gas Chromatography-Mass Spectrometry—Samples for gas chromatography-mass spectrometry analysis were prepared as described previously [2]. 13C-Constrained Net Flux Analysis—Intracellular net fluxes were estimated with a stoichiometric model that contained all major pathways of central carbon metabolism [3]. See more information in the original article.
-----------------------------------References---------------------------------
[1] Sauer, U., Lasko, D. R., Fiaux, J., Hochuli, M., Glaser, R., Szyperski, T., Wüthrich, K., and Bailey, J. E. (1999) J. Bacteriol. 181, 6679-6688.
[2] Fischer, E., and Sauer, U. (2003) Eur. J. Biochem. 270, 880-891. http://doi.org/dg3q64
[3] Fischer, E., Zamboni, N., and Sauer, U. (2004) Anal. Biochem. 325, 308-316. http://doi.org/c6kx35