Synthetic medium: 48mM Na2HPO4, 22mM KH2PO4, 10mM NaCl, 45mM (NH4)2SO4, 4g/L glucose, 1mM MgSO4, 1mg/L thiamin.HCL, 5.6 mg/L CaCl2, 8 mg/L FeCl3, 1 mg/L MnCl2.4H2O, 1.7 mg/L ZnCl2, 0.43 mg/L CuCl2.2H2O, 0.6 mg/L CoCl2.2H2O and 0.6 mg/L Na2MoO4.2H2O
|Methods description - Notes
13C-labeling experiment - for metabolic flux analysis, 13C-labeling experiments were initiated after taking samples for transcriptome, proteome and metabolome analysis. For experiments with gene disruptants, the feed medium containing 4 g/l of natural glucose was replaced by
an identical medium containing 0.4 g/l of [1-13C] glucose, 0.4 g/l of uniformly labeled [U-13C] glucose and 3.2 g/l of natural glucose. For experiments examining the effect of changes in dilution rate on flux distributions, the composition of glucose in the feed medium was changed to 0.8 g/l of [1-13C] glucose, 0.8 g/l of [U-13C] glucose and 2.4 g/l of natural glucose. After two residence times, for gas chromatography-mass spectrometry (GC-MS) analysis, E. coli cells were harvested by centrifugation.
GC-MS analysis - the cells obtained from about 250 ml of culture were suspended in 4 ml of 6 M HCl and then hydrolyzed at 105 ºC for 16 h. After cooling, HCl was evaporated with a centrifugal evaporator (CVE-3100, Tokyo Rikakikai Co., Ltd., Japan). The dried hydrolysate was resuspended in water and then filtrated through a 0.22-μm pore size filter (Millipore Co., USA). The filtrate was dried again and redissolved in 1.5 ml of acetonitrile. For derivatization, the resulting 80 μl of biomass hydrolysate dissolved in acetonitrile was mixed with an equal volume of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide and then incubated at 110 ºC for 30 min. After cooling, the derivatized sample was used for the GC-MS analysis using a TurboMass Gold mass spectrometer (Perkin Elmer, USA). In the present study, two fragment ions, [M-57]+ and [M-159]+, of tert-butyldimethylsilylated (TBDMS-) amino acids (Ala, Gly, Val, Ile, Pro, Ser, Met, Phe, Asp, Glu and Tyr) were monitored. The analytical conditions for GC-MS were as described by Zhao et al .
Estimation of metabolic flux distribution - for metabolic flux analysis, we constructed a basic stoichiometric reaction model for the main metabolic pathways including glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, the glyoxylate shunt and the anaplerotic pathway. The biomass content reported by Li et al.  was used for calculations.
 J. Zhao, K. Shimizu, J. Biotechnol. 101, 101 (2003). http://doi.org/cdjhrn
 M. Li, P. Y. Ho, S. Yao, K. Shimizu, J. Biotechnol. 122, 254 (2006). http://doi.org/cr7qpd