Temperature dependency and temperature compensation in a model of yeast glycolytic oscillations

Temperature sensitivities and conditions for temperature compensation have been investigated in a model for yeast glycolytic oscillations. The model can quantitatively simulate the experimental observation that the period length of glycolytic oscillations decreases with increasing temperature. Temperature compensation is studied by using control coefficients describing the effect of rate constants on oscillatory frequencies. Temperature compensation of the oscillatory period is observed when the positive contributions to the sum of products between control coefficients and activation energies balance the corresponding sum of the negative contributions. The calculations suggest that by changing the activation energies for one or several of the processes, i.e. by mutations, it could be possible to obtain temperature compensation in the yeast glycolytic oscillator.     

Sustained glycolytic oscillations in individual isolated yeast cells

Yeast glycolytic oscillations have been studied since the 1950s in cell-free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microfluidic chamber, we were able to observe sustained oscillations in individual isolated cells. Using a detailed kinetic model for the cellular reactions, we simulated the heterogeneity in the response of the individual cells, assuming small differences in a single internal parameter. This is the first time that sustained limit-cycle oscillations have been demonstrated in isolated yeast cells. DATABASE: The mathematical model described here has been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/gustavsson/index.html free of charge.     

On the mechanisms of glycolytic oscillations in yeast

This work concerns the cause of glycolytic oscillations in yeast. We analyse experimental data as well as models in two distinct cases: the relaxation-like oscillations seen in yeast extracts, and the sinusoidal Hopf oscillations seen in intact yeast cells. In the case of yeast extracts, we use flux-change plots and model analyses to establish that the oscillations are driven by on/off switching of phosphofructokinase. In the case of intact yeast cells, we find that the instability leading to the appearance of oscillations is caused by the stoichiometry of the ATP-ADP-AMP system and the allosteric regulation of phosphofructokinase, whereas frequency control is distributed over the reaction network. Notably, the NAD+/NADH ratio modulates the frequency of the oscillations without affecting the instability. This is important for understanding the mutual synchronization of oscillations in the individual yeast cells, as synchronization is believed to occur via acetaldehyde, which in turn affects the frequency of oscillations by changing this ratio.     

How yeast cells synchronize their glycolytic oscillations: a perturbation analytic treatment

Of all the lifeforms that obtain their energy from glycolysis, yeast cells are among the most basic. Under certain conditions the concentrations of the glycolytic intermediates in yeast cells can oscillate. Individual yeast cells in a suspension can synchronize their oscillations to get in phase with each other. Although the glycolytic oscillations originate in the upper part of the glycolytic chain, the signaling agent in this synchronization appears to be acetaldehyde, a membrane-permeating metabolite at the bottom of the anaerobic part of the glycolytic chain. Here we address the issue of how a metabolite remote from the pacemaking origin of the oscillation may nevertheless control the synchronization. We present a quantitative model for glycolytic oscillations and their synchronization in terms of chemical kinetics. We show that, in essence, the common acetaldehyde concentration can be modeled as a small perturbation on the "pacemaker" whose effect on the period of the oscillations of cells in the same suspension is indeed such that a synchronization develops.     

Model evaluation for glycolytic oscillations in yeast biotransformations of xenobiotics

Anaerobic glycolysis in yeast perturbed by the reduction of xenobiotic ketones is studied numerically in two models which possess the same topology but different levels of complexity. By comparing both models' predictions for concentrations and fluxes as well as steady or oscillatory temporal behavior we answer the question what phenomena require what kind of minimum model abstraction. While mean concentrations and fluxes are predicted in agreement by both models we observe different domains of oscillatory behavior in parameter space. Generic properties of the glycolytic response to ketones are discussed.     

Transduction of intracellular and intercellular dynamics in yeast glycolytic oscillations

Under certain well-defined conditions, a population of yeast cells exhibits glycolytic oscillations that synchronize through intercellular acetaldehyde. This implies that the dynamic phenomenon of the oscillation propagates within and between cells. We here develop a method to establish by which route dynamics propagate through a biological reaction network. Application of the method to yeast demonstrates how the oscillations and the synchronization signal can be transduced. That transduction is not so much through the backbone of glycolysis, as via the Gibbs energy and redox coenzyme couples (ATP/ADP, and NADH/NAD), and via both intra- and intercellular acetaldehyde.     

Metabolic pathways reconstruction by frequency and amplitude response to forced glycolytic oscillations in yeast

The hypothesis that frequency and amplitude response can be used in a complicated metabolic pathway kinetics model for optimal parameter estimation, as speculated by its successful prior usage for a mechanical oscillator and a heterogeneous chemical system, is tested here. Given the complexity of the glycolysis model of yeast chosen, this question is limited to three kinetics parameters of the 87 in the in vitro model developed in the literature. The direct application of the approach, used with the uninformed selection of operating conditions for the oscillation of external glucose concentration, led to miring the data assimilation process in local minima. Application of linear systems theory, however, identified two natural resonant frequencies that, when excited by external forced oscillations of the same frequency, result in the expression of many harmonics in the Fourier spectra, that is, information-rich experiments. A single such information-rich experiment at one of the resonant frequencies was sufficient to break away from the local minima to find the optimum kinetics parameter estimates. The resonant frequencies themselves represent oscillation modes in glycolysis akin to those previously observed. Furthermore, operation of the bioreactor with large amplitude oscillations of glucose feed (25%) leads to enhanced ethanol average yield by 1.6% at the resonant frequency.     

Sensitivity of yeast glycolytic enzymes to chloroquine

Chloroquine at pH 8.0 and 10 mM concentration inhibits about 30% glucose consumption and ethanol formation in yeast cells. Out of the 11 glycolytic enzymes assayed, phosphoglycerate kinase and pyruvate decarboxylase have been found to be most sensitive to chloroquine. Next sensitive are hexokinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. Kinetic studies with the three kinases studied revealed competitive inhibition of chloroquine with ATP (hexokinase, phosphoglycerate kinase) or ADP (pyruvate kinase).     

Interaction of D-fructose and fructose 1-phosphate with yeast phosphofructokinase and its influence on glycolytic oscillations.

Fermentation of D-fructose- and D-glucose induced glycolytic oscillations of different period lengths in Saccharomyces carlsbergensis. Recent studies suggested, that D-fructose or one of its metabolites interacted with phosphofructokinase (ATP:D-fructo-6-phosphate 1-phosphofructokinase, EC 2.7.1.11), the core of the glycolytic 'oscillator'. In order to explore the kinetics of interaction, the influence of D-fructose and fructose 1-phosphate on purified yeast phosphofructokinase was studied. D-fructose concentrations up to 0.3 mM stimulated the enzyme, while a further increase led to competitive inhibition. The Hill coefficient for fructose 6-phosphate decreased from 2.8 to 1.0. Fructose 1-phosphate acted in a similar way, up to 1 mM activation and inhibition competitive to fructose 6-phosphate at higher concentration (2.0--3.5 mM) with the same effect on the Hill coefficient. The inhibition patterns obtained with D-fructose or fructose 1-phosphate suggest a sequential random reaction mechanism of yeast phosphofructokinase with fructose 6-phosphate and MgATP2-. The mode of interaction of phosphofructokinase with D-fructose and fructose 1-phosphate is discussed. The influence of both effectors resulted in altered enzyme kinetics, which may cause the different period lengths of glycolytic oscillations.     

From bistability to oscillations in a model for the isocitrate dehydrogenase reaction

Considered is a bienzymatic system consisting of isocitrate dehydrogenase (IDH, EC 1.1.1.42), which transforms NADP(+) into NADPH, and of diaphorase (DIA, EC 1.8.1.4), which catalyzes the reverse reaction. Experimental evidence as well as a theoretical model show the possibility of a coexistence between two stable steady states in this reaction system. The phenomenon originates from the regulatory properties of IDH. We extend the analysis of a theoretical model proposed for the IDH-DIA bienzymatic system and investigate the occurrence of different modes of bistability, with or without hysteresis, i.e. in the presence of two or only one limit point bounding the domain of multiple steady states. The analysis indicates that the two types of bistability may sometimes be observed sequentially as a given control parameter is progressively increased. We further obtain conditions in which sustained oscillations develop in the model. These results establish the isocitrate dehydrogenase reaction coupled to diaphorase as a suitable candidate for further experimental and theoretical studies of bistability and oscillations in biochemical systems.     

Expression of a yeast glycolytic gene is subject to dosage limitation

The Saccharomyces cerevisiae pyruvate kinase-encoding gene (PYK1) has been transformed back into yeast using a derivative of the multicopy vector, pJDB207. High levels of PYK1 expression in these transformants are limited by at least two separate mechanisms. Pyruvate kinase assays and polysome analyses demonstrate that the translation of the PYK1 mRNA is inhibited as its abundance increases. The abundance of the PYK1 mRNA per gene copy also decreases as the copy number of the PYK1 gene increases. This is the first report which demonstrates that a eukaryotic glycolytic gene is subject to dosage limitation at the translational level.     

From yeast genetics to biotechnology

Roots of classical yeast genetics go back to the early work of Lindegreen in the 1930s, who studied thallism, sporulation and inheritance of wine yeast strains belonging to S. cerevisiae. Consequent mutation and hybridization of heterothallic S. cerevisae strains resulted in the discovery of life cycle and mating type system, as well as construction of the genetic map. Elaboration of induced mutation and controlled hybridization of yeast strains opened up new possibilities for the genetic analysis of technologically important properties and for the production of improved industrial strains, but a big drawback was the widely different genetic properties of laboratory and industrial yeast strains. Genetic analysis and mapping of industrial strains were generally hindered because of homothallism, poor sporulation and/or low spore viability of brewing and wine yeast strains [1, 2]. In spite of this, there are a few examples of the application of sexual hybridization in the study of genetic control of important technological properties, e.g. sugar utilization, flocculation and flavor production in brewing yeast strains [3] or in the improvement of ethanol producing S. cerevisiae strains [4]. Rare mating and application of karyogamy deficient (kar-) mutants also proved useful in strain improvement [5]. Importance of yeasts in biotechnology is enormous. This includes food and beverage fermentation processes where a wide range of yeast species are playing role, but S. cerevisiae is undoubtedly the most important species among them. New biotechnology is aiming to improve these technologies, but besides this, a completely new area of yeast utilization has been emerged, especially in the pharmaceutical and medical areas. Without decreasing the importance of S. cerevisiae, numerous other yeast species, e.g. Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica have gained increasing potentialities in the modern fermentation biotechnology [6]. Developments in yeast genetics, biochemistry, physiology and process engineering provided bases of rapid development in modern biotechnology, but elaboration of the recombinant DNA technique is far the most important milestone in this field. Other molecular genetic techniques, as molecular genotyping of yeast strains proved also very beneficial in yeast fermentation technologies, because dynamics of both the natural and inoculated yeast biota could be followed by these versatile DNA-based techniques.     

A signal transduction pathway model prototype I: From agonist to cellular endpoint

The postgenomic era is providing a wealth of information about the genes involved in many cellular processes. However, the ability to apply this information to understanding cellular signal transduction is limited by the lack of tools that quantitatively describe cellular signaling processes. The objective of the current studies is to provide a framework for modeling cellular signaling processes beginning at a plasma membrane receptor and ending with a measurable endpoint in the signaling process. Agonist-induced Ca(2+) mobilization coupled to down stream phosphorylation events was modeled using knowledge of in vitro and in vivo process parameters. The simulation process includes several modules that describe cellular processes involving receptor activation phosphoinositide metabolism, Ca(2+)-release, and activation of a calmodulin-dependent protein kinase. A Virtual Cell-based simulation was formulated using available literature data and compared to new and existing experimental results. The model provides a new approach to facilitate hypothesis-driven investigation and experimental design based upon simulation results. These investigations may be directed at the timing of multiple phosphorylation/dephosphorylation events affecting key enzymatic activities in the signaling pathway.