Short-Term Metabolome Dynamics and Carbon, Electron, and ATP Balances in Chemostat-Grown Saccharomyces cerevisiae CEN.PK 113-7D following a Glucose Pulse

Abstract

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...

Measurement of fast dynamic intracellular pH in Saccharomyces cerevisiae using benzoic acid pulse

“…Glycolytic and TCA cycle intermediates were analyzed by ESI-LC-MS/MS method as described in van Dam et al (2002). ATP was analyzed by an ion pairing ESI-LC-MS/MS method as described in (Wu et al, 2006). Oxygen and carbon dioxide concentrations in the exhaust gas of the fermentation were measured on-line by combined oxygen (paramagnetic) and carbon dioxide (infrared) analyzer (Rosemount NGA, Rosemount Analytical, Solon, OH).…”

Impact of Thermodynamic Principles in Systems Biology

“…Many examples have been found which show this behaviour in Penicillium chrysogenum [3,4] and in Saccharomyces cerevisiae [26] such as for phospho glucose isomerase, enolase, phosphoglycerate mutase, phosphoglucomutase, fumarase.…”

Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: a chemostat study

“…The advantages of using chemostat cultures for physiological and metabolic studies have been widely recognized (Brauer et al, 2005;Hoskisson & Hobbs, 2005;Porro et al, 2003;Wu et al, 2006). Nevertheless, they have not yet found wide application in molecular and cell biology.…”