LB cultures were used to inoculate M9 medium precultures with the indicated carbon sources for overnight cultivation.
General protocol information
Flux analysis method:
13C constrained MFA
Platform:
LC-MS
Methods description - Notes
Metabolic flux analysis
For steady state analyses, separate 13C-labeling experiments were performed with a mixture of 20% (wt/wt) [U-13C] labeled isotopologue (>99%; Cambridge Isotope Laboratories, Andover, MA) and 80% (wt/wt) of natural abundance carbon sources. Se
... parate 13C-labeling experiments were performed with 100% [1-13C]galactose, [1-13C]glucose, [1-13C]gluconate and [1-13C]fructose (>99%; Cambridge Isotope Laboratories, Andover, MA) and [1,3-13C]glycerol (>99%; CortecNet Voisins-Le-Bretonneux, France). Aliquots of fractionally 13C-labelled biomass were prepared from exponentially growing cultures and analyzed by gas chromatography mass spectrometry (GC-MS) [1].
Estimation of absolute fluxes was done by whole isotopologue balancing [2,3], using cumomer balances and cumomer to isotopologue mapping matrices [4] to calculate isotopologue partitioning of metabolites in a pre-defined stoichiometric network model for a given flux set. The flux set giving the best correspondence between measured and simulated 13C-label partitioning and physiology measurements of growth and extracellular fluxes was determined by non-linear optimization and selected as the final flux distribution. Standard deviations for metabolic fluxes were estimated through Monte Carlo simulations by re-estimating fluxes after adding Gaussian noise to the measured 13C-labeling data [5]. --------------------------------------------References---------------------------------------
[1] Zamboni, N., Fendt, S.-M., Rühl, M., and Sauer, U. (2009). Nat. Protoc. 4, 878–892. http://doi.org/b8ck9w [2] Kleijn, R.J., van Winden, W.A., van Gulik, W.M., and Heijnen, J.J. (2005). FEBS J. 272, 4970–4982. http://doi.org/bxpccx [3] Van Winden, W.A., van Dam, J.C., Ras, C., Kleijn, R.J., Vinke, J.L., van Gulik, W.M., and Heijnen, J.J. (2005). FEMS Yeast Res. 5, 559–568. http://doi.org/cgg3tw [4] Wiechert, W., Möllney, M., Isermann, N., Wurzel, M., and de Graaf, A.A. (1999). Biotechnol. Bioeng. 66, 69–85.
[5] Schmidt, K., Nielsen, J., and Villadsen, J. (1999). J. Biotechnol. 71, 175–189. http://doi.org/bjxbj5
LB cultures were used to inoculate M9 medium precultures with the indicated carbon sources for overnight cultivation.
General protocol information
Flux analysis method: 13C constrained MFA
Platform: LC-MS
Methods description - Notes
Metabolic flux analysis
For steady state analyses, separate 13C-labeling experiments were performed with a mixture of 20% (wt/wt) [U-13C] labeled isotopologue (>99%; Cambridge Isotope Laboratories, Andover, MA) and 80% (wt/wt) of natural abundance carbon sources. Separate 13C-labeling experiments were performed with 100% [1-13C]galactose, [1-13C]glucose, [1-13C]gluconate and [1-13C]fructose (>99%; Cambridge Isotope Laboratories, Andover, MA) and [1,3-13C]glycerol (>99%; CortecNet Voisins-Le-Bretonneux, France). Aliquots of fractionally 13C-labelled biomass were prepared from exponentially growing cultures and analyzed by gas chromatography mass spectrometry (GC-MS) [1].
Estimation of absolute fluxes was done by whole isotopologue balancing [2,3], using cumomer balances and cumomer to isotopologue mapping matrices [4] to calculate isotopologue partitioning of metabolites in a pre-defined stoichiometric network model for a given flux set. The flux set giving the best correspondence between measured and simulated 13C-label partitioning and physiology measurements of growth and extracellular fluxes was determined by non-linear optimization and selected as the final flux distribution. Standard deviations for metabolic fluxes were estimated through Monte Carlo simulations by re-estimating fluxes after adding Gaussian noise to the measured 13C-labeling data [5]. --------------------------------------------References--------------------------------------- [1] Zamboni, N., Fendt, S.-M., Rühl, M., and Sauer, U. (2009). Nat. Protoc. 4, 878–892. [2] Kleijn, R.J., van Winden, W.A., van Gulik, W.M., and Heijnen, J.J. (2005). FEBS J. 272, 4970–4982. [3] Van Winden, W.A., van Dam, J.C., Ras, C., Kleijn, R.J., Vinke, J.L., van Gulik, W.M., and Heijnen, J.J. (2005). FEMS Yeast Res. 5, 559–568. [4] Wiechert, W., Möllney, M., Isermann, N., Wurzel, M., and de Graaf, A.A. (1999). Biotechnol. Bioeng. 66, 69–85. [5] Schmidt, K., Nielsen, J., and Villadsen, J. (1999). J. Biotechnol. 71, 175–189.
KiMoSys (https://kimosys.org). Data EntryID 125 (Escherichia coli). [online], [Accessed 21 December 2024]. Available from: https://doi.org/10.34619/3x35-dd84
Back
Gerosa_2015.pdf
KIMODATAID125_v2.xlsx
Preview file
New Model