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The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O 2 uptake and CO 2 evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO 2 evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.Mathematical models of in vivo enzyme kinetics in microorganisms are important for understanding metabolic control mechanisms operating on the level of the metabolome and can be used to assist the rational redesign of metabolic pathways to enhance desired functionalities of microbes (54). Kinetic parameters in this kind of model can be obtained by stimulusresponse experiments, in which cells grown in a (quasi-)steady state are perturbed by an external stimulus and the dynamic responses of intra-and extracellular metabolites are monitored. The time window of observation is usually within tens to a few hundred seconds after the application of the stimulus, and the responses are usually attributed to rapid (allosteric) enzyme-metabolite interactions (38). Kinetic parameters can be estimated from the measured responses, based on a set of (dynamic) material balances (7, 30, 49), as follows: dx/dt ϭ Sv (equation 1), where x is a vector of the metabolite concentrations, S is the stoichiometry matrix, and v is a vector of the reaction rates as a function of (yet) unknown kinetic parameters. The stimulus-response methodology is an ideal tool for obtaining kinetic information and has been applied to various microorganisms under different growth conditions (7,26,32,38,52).For Saccharomyces cerevisiae, a frequently applied perturbation is the addition of a concentrated glucose solution, i.e., a glucose pulse, to a glucose-limited chemostat culture, thereby inducing a short-term Crabtree effect. Theoba...

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An experimental platform has been developed for rapid sampling and quenching of chemostat cultivated Penicillium chrysogenum broth for metabolome analysis in highly dynamic experiments, aimed at the elucidation of the in vivo kinetic properties of metabolism. The sampling and quenching protocol available from Saccharomyces cerevisiae had to be modified for Penicillium chrysogenum mainly because of its filamentous character. Intracellular metabolites of glycolysis, TCA cycle, and adenine nucleotides were measured with isotope dilution mass spectrometry (IDMS) using a U-(13)C-labeled metabolite mix produced from yeast cells as internal standard. By addition of the U-(13)C internal standard mix prior to the metabolite extraction procedure, partial degradation of metabolites as well as non-linearity and drift of the LC-MS/MS could be successfully compensated for. It was found that there is a serious matrix effect on metabolite extraction between different organisms, which is however completely corrected for by the IDMS approach. Intracellular metabolites could be analyzed with standard deviations of around 5%. A comparison of the metabolite levels between Saccharomyces cerevisiae and Penicillium chrysogenum showed both significant similarities and large differences, which seem to be related to the presence of the penicillin pathway.