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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...

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0308-1) contains supplementary material, which is available to authorized users.

BackgroundEnvironmental and intrinsic stress factors can result in the global alteration of yeast physiology, as evidenced by several transcriptional studies. Hypoxia has been shown to have a beneficial effect on the expression of recombinant proteins in Pichia pastoris growing on glucose. Furthermore, transcriptional profiling analyses revealed that oxygen availability was strongly affecting ergosterol biosynthesis, central carbon metabolism and stress responses, in particular the unfolded protein response. To contribute to the better understanding of the effect and interplay of oxygen availability and foreign protein secretion on central metabolism, a first quantitative metabolomic analysis of free amino acids pools in a recombinant P. pastoris strain growing under different oxygen availability conditions has been performed.ResultsThe values obtained indicate significant variations in the intracellular amino acid pools due to different oxygen availability conditions, showing an overall increase of their size under oxygen limitation. Notably, even while foreign protein productivities were relatively low (about 40–80 μg Fab/gDCW·h), recombinant protein production was found to have a limited but significant impact on the intracellular amino acid pools, which were generally decreased in the producing strain compared with the reference strain. However, observed changes in individual amino acids pools were not correlated with their corresponding relative abundance in the recombinant protein sequence, but to the overall cell protein amino acid compositional variations.ConclusionsOverall, the results obtained, combined with previous transcriptomic and proteomic analyses provide a systematic metabolic fingerprint of the oxygen availability impact on recombinant protein production in P. pastoris.

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