Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of (13)C-labeled primary metabolites.
Metabolic-flux analyses in microorganisms are increasingly based on (13)C-labeling data. In this paper a new approach for the measurement of (13)C-label distributions is presented: rapid sampling and quenching of microorganisms from a cultivation, followed by extraction and detection by liquid chromatography-mass spectrometry of free intracellular metabolites. This approach allows the direct assessment of mass isotopomer distributions of primary metabolites. The method is applied to the glycolytic and pentose phosphate pathways of Saccharomyces cerevisiae strain CEN.PK113-7D grown in an aerobic, glucose-limited chemostat culture. Detailed investigations of the measured mass isotopomer distributions demonstrate the accuracy and information-richness of the obtained data. The mass fractions are fitted with a cumomer model to yield the metabolic fluxes. It is estimated that 24% of the consumed glucose is catabolized via the pentose phosphate pathway. Furthermore, it is found that turnover of storage carbohydrates occurs. Inclusion of this turnover in the model leads to a large confidence interval of the estimated split ratio.
van Winden
Wouter A
WA
Kluyver Laboratory for Biotechnology, Department of Biotechnology, Faculty of Applied Sciences, Bioprocess Technology Group, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. w.a.vanwinden@tnw.tudelft.nl
van Dam
Jan C
JC
Ras
Cor
C
Kleijn
Roelco J
RJ
Vinke
Jacobus L
JL
van Gulik
Walter M
WM
Heijnen
Joseph J
JJ
D002247:Carbon Isotopes / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D003470:Culture Media; D015966:Gene Expression Regulation, Fungal; D006019:Glycolysis; D013058:Mass Spectrometry / Q000379:methods; D010427:Pentose Phosphate Pathway; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*
D002247:Carbon Isotopes; D003470:Culture Media