Uwe Theobald

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305-316, 1997.

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae: II. Mathematical model

A mathematical model of glycolysis in Saccharomyces cerevisiae is presented. The model is based on rate equations for the individual reactions and aims to predict changes in the levels of intra- and extracellular metabolites after a glucose pulse, as described in part I of this study. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. The model structure and experimental observations are related to the aerobic growth of the yeast. The model is based on material balance equations of the key metabolites in the extracellular environment, the cytoplasm and the mitochondria, and includes mechanistically based, experimentally matched rate equations for the individual enzymes. The model includes removal of metabolites from glycolysis and TCC for biosynthesis, and also compartmentation and translocation of adenine nucleotides. The model was verified by in vivo diagnosis of intracellular enzymes, which includes the decomposition of the network of reactions to reduce the number of parameters to be estimated simultaneously. Additionally, sensitivity analysis guarantees that only those parameters are estimated that contribute to systems trajectory with reasonable sensitivity. The model predictions and experimental observations agree reasonably well for most of the metabolites, except for pyruvate and adenine nucleotides. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 592-608, 1997.

In vivo investigations of glucose transport in Saccharomyces cerevisiae.

In the present study, the glucose transport into the yeast Saccharomyces cerevisiae has been investigated. The approach suggested is based on a rapid sampling technique for studying the dynamic response of the yeast to rapid changes in extracellular glucose concentrations. For this purpose a concentrated glucose solution has been injected into a continuous culture at steady state growth conditions resulting in a shift of the extracellular glucose level. Samples have been taken every 5 s for determination of extracellular glucose and intracellular glucose‐6‐phosphate concentrations. Attempts to fit the experimental observations with simulations from existing models failed. The mechanism then proposed is based on a facilitated diffusion of glucose superimposed by an inhibition of glucose‐6‐phosphate. The use of the so‐called in vivo approach suggested in this article appears to be proper, because the investigations can be performed at defined physiological states of the microbial cultures. Furthermore, the experimental observations are not being corrupted by the preparation of the samples for the transport studies as it happens during radioactive measurements.

Fast purification and kinetic studies of the glycerol-3-phosphate dehydrogenase from the yeast Saccharomyces cerevisiae

The glycerol-3-phosphate dehydrogenase has been purified from Saccharomyces cerevisiae 140-fold to electrophoretic homogeneity by a simple procedure involving affinity and ion exchange chromatography. The purified enzyme was most active at pH 6.8 and 51 degrees C. Its molecular mass was determined to be 45000 +/- 2000 Da by SDS-polyacrylamide gel electrophoresis. At physiological pH values the thermodynamic equilibrium constant was determined to be 3.5 x 10(-3) (M-1). Product inhibition as well as competitive inhibition patterns were found which clearly indicate that the kinetic mechanism of the glycerol-3-phosphate dehydrogenase is random bi-bi with the formation of dead-end complexes. In vivo concentrations of selected metabolites and kinetic expression for G3P-DH were used to explain regulatory properties of this enzyme under conditions of short-term glucose effect in Saccharomyces cerevisiae.

Intracellular glucose 6-phosphate content in Streptomyces coelicolor upon environmental changes in a defined medium.

A new, chemically defined medium providing dispersed growth and high biomass formation and a method for quantitative extraction of intracellular metabolites was used to investigate the cellular response of Streptomyces coelicolor A3(2) during growth and upon changes in nutrient utilization. Fast changes of the glucose 6-phosphate content precisely signaled transitions in the flow of carbon sources. The results indicate that intracellular pool sizes may be used to detect early nutrient limitations in view of the onset of antibiotic production. Additionally the results disclose characteristics of the regulation of maltose and glutamic acid uptake and degradation in S. coelicolor A3(2).

Determination of in-vivo cytoplasmic orthophosphate concentration in yeast

To investigate in-vivo concentrations of cytoplasmic phosphate, especially during dynamic conditions, a method has been developed that enables reproducible determination of cytoplasmic phosphate from 5 μM up to 30 μM. The method involves fast sampling, spontaneous inactivation of cell metabolism and differential extraction procedure to gain porosity of the outer cell membrane exclusively. To determine very low cytoplasmic phosphate concentrations, an enzymatic assay was linked to a sensitive spectrophotometric cycling method to increase the detection limit.

Dynamics of orthophosphate in yeast cytoplasm

SummaryOrthophosphate concentration of bakers yeast Saccharomyces cerevisiae was investigated during dynamic conditions. As an example for those dynamics in cell metabolism the transition from glucose limitation to glucose excess (Crabtree-effect) was choosen. As a result of the metabolic switch from complete to partial oxidative metabolism, the cytoplasmic phosphate concentration increased suddenly from 8.4 mM to a maximum of 17.5 mM and transiently decreased to a minimum of 7.0 mM.

Use of HgCl2 to investigate dynamic phenomena in yeast cytoplasm

To investigate dynamic phenomena in yeast cytoplasm a method was developed that guarantees immediate inactivation of cell activity. Differential extraction procedure gains porosity of the outer cell membrane exclusively. Application of the method during transition from glucose limitation to glucose excess in Saccharomyces cerevisiae illustrates the dynamic behaviour of yeast metabolism and emphasized the necessity of intracellular compartmentation.

Modelling of Short Term Crabtree-Effect in Baker's Yeast

A mathematical model has been developed to simulate the dynamics of key reactions of the catabolism in growing cells of Saccharomyces cerevisiae. The structured model considers the reactions of glycolysis, tricarbon acid cycle, pentose phosphate shunt and respiratory chain. The mathematical description of the metabolism comprises of 22 dynamic mass balances which contain 21 reaction rates. The proposed model is able to predict the time-dependent states of Saccharomyces cerevisiae under in-vivo conditions during metabolic features of short-time dynamics of glycolysis satisfactorily.