not specified

Glucose, acetate, ethanol and glycerol concentrations in the supernatant were determined with commercial enzymatic kits (Scil Diagnostics, Germany). Organic acids were quantified by high-pressure liquid chromatography (HPLC) using a Supelcogel C8 (4.6 by 250 mm) ion-exclusio ... n column. The column was eluted at 30°C with 2% sulfuric acid at a flow rate of 0.3 ml/min. The organic acids were detected using a PerkinElmer UV detector (Series 2000) at a wavelength of 210 nm. The physiological parameters maximum specific growth rate, biomass yield on glucose, and specific glucose consumption rate were calculated during the exponential growth phase. Analytical procedures and 13C-labeling experiments: All labeling experiments were carried out in batch cultures assuming pseudosteady-state conditions during the exponential growth phase [1, 2]. 13C-labeling of proteinogenic amino acids was achieved either by growth on 5 g/l glucose as a mixture of 80% (w/w) unlabeled and 20% (w/w) uniformly labelled [U-13C]glucose (13C > 99%; Martek Biosciences, Columbia, MD) or 100% [1-13C]glucose (> 99%; Omicron Biochemicals, South Bend, IN). Cells from overnight cultures were harvested by centrifugation and washed using sugar-free MM to remove residual unlabeled carbon sources. Cultures were routinely inoculated to an maximum OD600 of 0.03 and harvested by centrifugation at an OD600 ≤ 1. Residual medium was removed by washing the pellet with water. Cell protein was hydrolyzed for 24 h at 105°C in 6 M HCL and dried in a heating block at 85°C for 6 h. The free amino acids were derivatized at 85°C for 1 h using 15 μl dimethylformamide and 15 μl N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide [3]. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out as reported [2] using a series 8000 GC in combination with an MD800 mass spectrometer (Fisons Instruments, Beverly, MA). --------------------------------------------References---------------------------------------
[1] Fischer E, Sauer U. Eur J Biochem. 2003, 270: 880-891. http://doi.org/dg3q64
[2] Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wüthrich K, Bailey JE. J Bacteriol. 1999, 181: 6679-6688.
[3] Fischer E, Zamboni N, Sauer U. Anal Biochem. 2004, 325: 308-316. http://doi.org/c6kx35