A kinetic model of catabolic adaptation and protein reprofiling in Saccharomyces cerevisiae during temperature shifts.

In this article, we aim to find an explanation for the surprisingly thin line, with regard to temperature, between cell growth, growth arrest and ultimately loss of cell viability. To this end, we used an integrative approach including both experimental and modelling work. We measured the short- and long-term effects of increases in growth temperature from 28 °C to 37, 39, 41, 42 or 43 °C on the central metabolism of Saccharomyces cerevisiae. Based on the experimental data, we developed a kinetic mathematical model that describes the metabolic and energetic changes in growing bakers' yeast when exposed to a specific temperature upshift. The model includes the temperature dependence of core energy-conserving pathways, trehalose synthesis, protein synthesis and proteolysis. Because our model focuses on protein synthesis and degradation, the net result of which is important in determining the cell's capacity to grow, the model includes growth, i.e. glucose is consumed and biomass and adenosine nucleotide cofactors are produced. The model reproduces both the observed initial metabolic response and the subsequent relaxation into a new steady-state, compatible with the new ambient temperature. In addition, it shows that the energy consumption for proteome reprofiling may be a major determinant of heat-induced growth arrest and subsequent recovery or cell death.

A new regulatory principle for in vivo biochemistry: pleiotropic low affinity regulation by the adenine nucleotides--illustrated for the glycolytic enzymes of Saccharomyces cerevisiae.

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme's catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis-Menten constants, adding new perspective on these, 100 years after their introduction.

Measuring enzyme activities under standardized in vivo-like conditions for systems biology.

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme-kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme-kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113-7D grown in aerobic glucose-limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h(-1). The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo-like medium containing 300 mM potassium, 50 mM phosphate, 245 mM glutamate, 20 mM sodium, 2 mM free magnesium and 0.5 mM calcium, at a pH of 6.8. The V(max) values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme-specific, optimal conditions.

Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains.

The field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism.

Microbial systems biology: new frontiers open to predictive microbiology.

The field of Systems Biology is a rapidly evolving area of research. It follows on from the previous experimental and theoretical 'omics' revolution in biology. Now that we have through the use of these tools many 'indices' of biological systems available the next step is to actually start composing the systems that these indices specify. In this paper we will discuss the developments in the field of Systems Biology as they pertain to predictive food microbiology and give an example of state of the art current approaches. The data discussed in the case study deal with the resistance of the yeast Saccharomyces cerevisiae towards environmental temperature changes through adaptation of its metabolism, protein signalling and gene-expression. The results are integrated and its implications for the definition of new experiments discussed; the iteration between experiment driven model definition and model driven experimentation being characteristic for contemporary Systems Biology approaches. The stress condition discussed represents in no way a practical situation in food microbiology but what it teaches may well be applied in such cases. We will indicate how the latter may be achieved.

Activation of the protein kinase C1 pathway upon continuous heat stress in Saccharomyces cerevisiae is triggered by an intracellular increase in osmolarity due to trehalose accumulation.

This paper reports on physiological and molecular responses of Saccharomyces cerevisiae to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a tps1 mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such.

The metabolic response of Saccharomyces cerevisiae to continuous heat stress.

A study has been initiated to integrate molecular and physiological responses of Saccharomyces cerevisiae to heat stress conditions. We focus our research on a quantification of the energetics of the stress response. A series of continuous heat stresses was applied to exponentially growing cells of the strain X2180-1A at 28 degrees C, by increasing the growth temperature to 37, 39, 40, 41, 42, or 43 degrees C. Here, the results on cell growth and viability, as well as on anabolic and catabolic rates are presented. We observed a surprisingly 'thin line' for the cells between growing, surviving, and dying, with regard to growth temperature. The heat stress showed a dual effect on catabolism: immediately after the temperature increase a strong peak was seen, after which a new, steady level was reached. In addition, the yield on glucose decreased with increasing temperature. Our results indicate that life at elevated temperatures is energetically unfavourable and a non-lethal heat stress invokes a redistribution of catabolic and anabolic fluxes.