M9 sythetic medium: 48 mM Na2HPO4, 22 mM KH2PO4, 10 mM NaCl, 40 mM (NH4)zSO4 and 4 g/l of glucose. Components filter-sterilized separatory and then added (per liter of final volume): 1 ml 1 M MgSO4, 1 ml vitamin B1 (1 mgl stock), 1 ml 0.1 mM CaCl2, and 10 ml trace element solution containing (per liter): 0.55g CaCl2 1g FeCl3, 0.1 mg/l MnCl2.4H2O, 0.17 g ZnCl2, 0.043 g CuCl2.2H2O, 0.06 g CoCl2.2H2O, and 0.06 g Na2MoO4.2H2O).

13C-labeling experiments and sample preparation: The biomass sample was kept on ice for 2-3 minutes, and the sample was centrifuged at 6,000 rpm at 2°C for 15 minutes [1]. The cell pellets were washed three times with 20 mM Tris-HCl at pH 7.6 and suspended in 10 ml of 6 M HC ... l. The mixture was then hydrolyzed at 105°C for 15 hours in a sealed glass tube. During acid hydrolysis, tryptophan and cysteine were oxidized, and glutamine and asparagine were deaminated. The hydrolysate was evaporated to dryness. The dried material was dissolved in milli-Q water and filtered through a 0.22μm pore-size filter and evaporated to dryness. About 1.5 ml acetonitrile was added in the dried hydrolyte and incubated at room temperature overnight. After the color of liquid turned a color of pale yellow, it was filtrated through 0.22μm pore-size filter. The filtrate was then derivatized by N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBST-FA) (Aldrich, USA) at 110C for 30 minutes and was transferred to GC-MS sample tube for analysis [1]. 13C-labeling experiments were initiated after the culture reached steady state, which was inferred from the stable oxygen and carbon dioxide concentrations in the off-gas and stable OD in the effluent medium for at least twice as long as the residence time. The feed medium with 4 g/l of unlabeled glucose was then replaced by an identical medium containing 0.4 g of [U-13C] glucose, 0.4 g of [1-13 C] glucose, and 3.2 g of naturally labeled glucose per liter. Biomass samples for GC-MS analysis were taken after one residence time. Sample preparation, analytical procedures for GC-MS analysis, and flux computation are given elsewhere [2-4, 1]. Preparation of biomass hydrolysates and recording of the GC-MS spectra (PerklinElmer, Germany) were made as described previously [1,5,6].
--------------------------------------------References--------------------------------------
[1] Zhao J, Shimizu K. J Biotechnol. 2003, 101: 101-117. 17-23. http://doi.org/cdjhrn
[2] Peng L, Arauzo-Bravo MJ, Shimizu K. FEM Microbiology Letters. 2004, 235. http://doi.org/bn5mg6
[3] Siddiquee KAZ, Arauzo-Bravo MJ, Shimizu K. Appl Microbiol Biotechnol. 2004, 63: 407-417. http://doi.org/b9cm56 [4] Siddique KAZ, Arauzo-Bravo MJ, Shimizu K. FEMS Microbiology Letters. 2004, 235: 25-33. http://doi.org/dbcmv7 [5] Zhao J, Baba T, Mori H, Shimizu K. Metabolic Eng. 2004, 6: 164-174. http://doi.org/fxbfxd [6] Zhao J, Baba T, Mori H, Shimizu K. FEMS Microb Lett. 2003, 220: 295-301. http://doi.org/b3bb42