M9 minimal medium (per liter): 8.5 g of Na2HPO4.2H20, 3 g of KH2PO4, 1 g of NH4Cl, 0.5 g of NaCl. The following components were sterilized separately and then added (per liter of final medium): 1 ml of 0.1 m CaCl2.2H2O, 1 ml of 1 m MgSO4.7H2O, 1 ml of 50 mm FeCl3.6H2O, and 10 ml of trace salt solution. The trace salts solution contained (per liter): 170 mg of ZnCl2, 100 mg of MnCl2.4H2O, 60.0 mg of CoCl2.6H2O, 60.0 mg of Na2MoO4.2H2O, and 43.0 mg CuCl2.2H2O. When preparing the medium, the base salts were added first followed by CalCl2, MgSO4, FeCl3, and finally the trace elements.

Physiological Parameters: Extracellular substrate and byproduct concentrations were measured by HPLC analysis using an Agilent 1100 series HPLC stack in combination with an Aminex HPX-87H polymer column (Bio-Rad). Sugars were detected with a refractive index detector, and or ... ganic acids were detected with a UV-visible detector. Specific rates were calculated by regression analysis for at least five time points during the exponential growth phase as described previously [1]. Cell growth was monitored photometrically at 600 nm, and cell dry weight was inferred from a predetermined conversion factor of 0.48 g of cells/A600 [2]. All physiological parameters were determined during the exponential growth phase, typically ranging from 0.10 to 1.5 A600. Metabolic Flux Analysis: Metabolic fluxes were derived using whole isotopologue analysis [3,4]. In short, the procedure uses the cumomer balances and cumomer to isotopologue mapping matrices introduced by Wiechert et al. [5] to calculate the isotopologue distributions of metabolites in a pre-defined stoichiometric network model for a given flux-set. The flux-set that gives the best correspondence between the measured and simulated 13C-label distribution is determined by non-linear optimization and denoted as the optimal flux-fit. All calculations were performed in Matlab 7.6.0.
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[1] Sauer U., Lasko D. R., Fiaux J., Hochuli M., Glaser R., Szyperski T., Wüthrich K., Bailey J. E. (1999) J. Bacteriol. 181, 6679–6688. [2] Tännler S., Decasper S., Sauer U. (2008) Microb. Cell Fact. 7, 19. http://doi.org/cjbk3x [3] van Winden W. A., van Dam J. C., Ras C., Kleijn R. J., Vinke J. L., van Gulik W. M., Heijnen J. J. (2005) FEMS Yeast Res. 5, 559–568. http://doi.org/cgg3tw [4] Kleijn R. J., van Winden W. A., van Gulik W. M., Heijnen J. J. (2005) FEBS J. 272, 4970–4982. http://doi.org/bxpccx [5] Wiechert W, Möllney M, Isermann N, Wurzel M, de Graaf AA (1999). Biotechnol Bioeng 66: 69–85.