An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters.

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This article presents the dynamic responses of several intra- and extracellular components of an aerobic, glucose-limited chemostat culture of Saccharomyces cerevisiae to glucose and ethanol pulses within a time window of 75 sec. Even though the ethanol pulse cannot perturb the glycolytic pathway directly, a distinct response of the metabolites at the lower part of glycolysis was found. We suggest that this response is an indirect effect, caused by perturbation of the NAD/NADH ratio, which is a direct consequence of the conversion of ethanol into acetaldehyde. This effect of the NAD/NADH ratio on glycolysis might serve as an additional explanation for the observed decrease of 3PG, 2PG, and PEP during a glucose pulse. The responses measured during the ethanol pulse were used to evaluate the allosteric regulation of glycolysis. Our results confirm that FBP stimulates pyruvate kinase and suggest that this effect is pronounced. Furthermore, it appears that PEP does not play an important role in the allosteric regulation of phosphofructo kinase.