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 [6].

To rapidly quench the cell metabolism, 5 mL of culture suspension was cooled to 0°C in a −50°C methanol bath within 15–20 s. Cells were separated from the culture medium by centrifugation, and resuspended in cold 100% methanol immediately. Intracellular metabolites were extr ... acted by addition of cold chloroform at neutral pH based on the method of de Koning and van Dam [1]. This extraction method ensured minimal degradation of labile metabolites. After extraction, the aqueous phase was carefully collected, dried, and resuspended in MiliQ water. Shortly before the determination of metabolite concentrations, the cell extracts were filtered through a 0.2 m-poresize filter to remove possible small precipitates. The samples were stored for up to 5 days at −20°C for further analysis. The unstable metabolites (e.g., acetyl-CoA and oxaloacetate) were determined within 6 h after the extracts were obtained. Enzymatic determinations of the intracellular metabolites were performed on a microplate spectrofluorometer (SPECTRAmax GEMINI XS), following the changes in NAD(P)H fluorescence at the 355, 460-nm wavelength pair. The volume of the assay mixture was 200 L. The concentrations of fructose 1,6-bisphosphate, 3-phosphoglycerate, PEP, pyruvate, acetyl-CoA, isocitrate, L-malate, -ketoglutarate, oxaloacetate, L-aspartate, and adenine nucleotide (ATP and ADP) in the extracts were measured according to published protocols [2, 3, 4] with some modifications (such as the reduction of analytical volume). The value of the specific cell volume used for calculation of the intracellular concentrations was 1.77 L mgDW−1 [5]. All the intracellular concentrations were presented as the average of at least three measurements, with the corresponding standard deviation. ----------------------------------References--------------------------------------
[1] de Koning W, van Dam K. 1992. Anal Biochem 204:118–123.
[2] Bergmeyer HU. 1984. Methods of enzymatic analysis, 3rd edition, Vol. 6. Weinheim, Germany: VCH.
[3] Bergmeyer HU. 1985. Methods of enzymatic analysis, 3rd edition, Vol. 7. Weinheim, Germany: VCH.
[4] Williamson JR, Corkey BE. 1969. Methods in enzymology, Vol. 13. New York: Academic Press. p 434–513.
[5] Chassagnole C, Noisommit-Rizzi N, Schmid JW, Mauch K, Reuss M. 2002. Biotechnol Bioeng 79:53–73. http://doi.org/dj2nzc
[6] Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wuthrich K, Bailey JE. 1999. J Bacteriol 181:6679–6688.