Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of 13C-labeled primary metabolites

Metabolic-flux analyses in microorganisms are increasingly based on C-13-labeling data. In this paper a new approach for the measurement of C-13-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. PKI13-7D 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. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.