Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

The mechanism of changes in respiratory activity during callus formation in carrot root slices cultured in vitro

The rates of both O₂ uptake in air and CO₂ output in N₂ perprotein nitrogen and per cell increased markedly during thelag phase of callus formation, and decreased rapidly then graduallyduring the exponential and subsequent phases in carrot-rootphloem slices cultured in vitro. Rates of ¹⁴CO₂ output fromlabeled citrate and succinate supplied to the slices changedsimilarly. Respiration of the cultured slices became more sensitiveto fluoride and cyanide during the lag phase and less sensitiveduring the exponential phase. These results suggest that activitiesof both the glycolytic pathway and the TCA cycle rise duringthe lag phase and decline during the exponential one. The activities per protein nitrogen of the glycolytic enzymes,hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolaseand pyruvate kinase remarkably increased during the lag phaseafter a little decline in the first day of culture, and decreasedas the callus developed. A similar pattern of change was observedin the number and the respiratory activity of mitochondria percell. It was concluded, therefore, that changes in respiratoryactivity during callus formation may depend mainly on changesin capacity of the respiratory machinery, although the increasein respiratory activity at the beginning of culture may be dueto some other mechanism, such as an increase in the turnoverrate of ATP. (Received May 11, 1972; )     

Dynamics of Glycolytic Regulation during Adaptation of Saccharomyces cerevisiae to Fermentative Metabolism

The ability of baker's yeast (Saccharomyces cerevisiae) to rapidly increase its glycolytic flux upon a switch from respiratory to fermentative sugar metabolism is an important characteristic for many of its multiple industrial applications. An increased glycolytic flux can be achieved by an increase in the glycolytic enzyme capacities (V_max) and/or by changes in the concentrations of low-molecular-weight substrates, products, and effectors. The goal of the present study was to understand the time-dependent, multilevel regulation of glycolytic enzymes during a switch from fully respiratory conditions to fully fermentative conditions. The switch from glucose-limited aerobic chemostat growth to full anaerobiosis and glucose excess resulted in rapid acceleration of fermentative metabolism. Although the capacities (V_max) of the glycolytic enzymes did not change until 45 min after the switch, the intracellular levels of several substrates, products, and effectors involved in the regulation of glycolysis did change substantially during the initial 45 min (e.g., there was a buildup of the phosphofructokinase activator fructose-2,6-bisphosphate). This study revealed two distinct phases in the upregulation of glycolysis upon a switch to fermentative conditions: (i) an initial phase, in which regulation occurs completely through changes in metabolite levels; and (ii) a second phase, in which regulation is achieved through a combination of changes in V_max and metabolite concentrations. This multilevel regulation study qualitatively explains the increase in flux through the glycolytic enzymes upon a switch of S. cerevisiae to fermentative conditions and provides a better understanding of the roles of different regulatory mechanisms that influence the dynamics of yeast glycolysis.     

Studies of protein-protein interaction using countercurrent distribution in aqueous two-phase systems: partition behavior of five glycolytic enzymes from crude baker's yeast extract

The partition behavior of five glycolytic enzymes, in extracts from baker's yeast (Saccharomyces cerevisiae), between two aqueous phases has been studied by countercurrent distribution. All enzymes showed distribution patterns which indicated homogeneity and a similar partition behavior. In purified form, three of the enzymes (glyceraldehyde-phosphate dehydrogenase, 3-phosphoglycerate kinase, and enolase) showed the same partition behavior as in the extracts. Pure 6-phosphofructokinase, on the other hand, changed its partition distinctively relative to what was found in the extracts. These results indicate interactions between this enzyme and macromolecular compounds in the extracts and support a model suggested by Kurganov et al. (1985, J. Theor. Biol. 116, 509-526) describing a "glycolytic particle."     

Inactivation of the ppn1 gene exerts different effects on the metabolism of inorganic polyphosphates in the cytosol and the vacuoles of the yeast Saccharomyces cerevisiae

Inactivation of the PPN1 gene, encoding one of the enzymes involved in polyphosphate metabolism in the yeast Saccharomyces cerevisiae, was found to decrease exopolyphosphatase activity in the cytosol and vacuoles. This effect was more pronounced in the stationary growth phase than in the phase of active growth. The gene inactivation resulted in elimination of a approximately 440-kDa exopolyphosphatase in the vacuoles but did not influence a previously unknown vacuolar exopolyphosphatase with a molecular mass of >1000 kDa, which differed from the former enzyme in the requirement for bivalent cations and sensitivity to heparin. Inactivation of the PPN1 gene did not influence the level of polyphosphates in the cytosol but increased it more than twofold in the vacuoles. In this case, the polyphosphate chain length in the cytosol increased from 10-15 to 130 phosphate residues both in the stationary and active growth phases. In the vacuoles, the polyphosphate length increased only in the stationary growth phase. A conclusion can be made that the PPN1 gene product has different effects on polyphosphate metabolism in the cytosol and the vacuoles.     

Diauxic Growth of Azotobacter vinelandii on Galactose and Glucose: Regulation of Glucose Transport by Another Hexose

The growth curve of Azotobacter vinelandii was biphasic when the organism was grown in a medium containing a mixture of galactose and glucose. Galactose was the primary carbon source; glucose was also consumed, but the rate at which it was consumed was lower than the rate at which galactose was consumed during the first phase of growth. Metabolic pathways for both sugars were induced. Cell cultures exhibited a second lag period as galactose was depleted. The length of this lag phase varied from 2 to 10 h depending on the pregrowth history of the cells. The second log growth phase occurred at the expense of the remaining glucose in the medium and was accompanied by induction of the high-maximum rate of metabolism glucose-induced glucose permease and increases in the levels of glucose metabolic enzymes. The second lag phase of diauxie may have been due to the time required for induction of the glucose-induced glucose permease.     

Increased glucose metabolism and ATP level in brain tissue of Huntington's disease transgenic mice

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by multifarious dysfunctional alterations including mitochondrial impairment. In the present study, the formation of inclusions caused by the mutation of huntingtin protein and its relationship with changes in energy metabolism and with pathological alterations were investigated both in transgenic and 3-nitropropionic acid-treated mouse models for HD. The HD and normal mice were characterized clinically; the affected brain regions were identified by immunohistochemistry and used for biochemical analysis of the ATP-producing systems in the cytosolic and the mitochondrial compartments. In both HD models, the activities of some glycolytic enzymes were somewhat higher. By contrast, the activity of glyceraldehyde-3-phosphate dehydrogenase was much lower in the affected region of the brain compared to that of the control. Paradoxically, at the system level, glucose conversion into lactate was enhanced in cytosolic extracts from the HD brain tissue, and the level of ATP was higher in the tissue itself. The paradox could be resolved by taking all the observed changes in glycolytic enzymes into account, ensuing an experiment-based detailed mathematical model of the glycolytic pathway. The mathematical modelling using the experimentally determined kinetic parameters of the individual enzymes and the well-established rate equations predicted the measured flux and concentrations in the case of the control. The same mathematical model with the experimentally determined altered V(max) values of the enzymes did account for an increase of glycolytic flux in the HD sample, although the extent of the increase was not predicted quantitatively. This suggested a somewhat altered regulation of this major metabolic pathway in HD tissue. We then used the mathematical model to develop a hypothesis for a new regulatory interaction that might account for the observed changes; in HD, glyceraldehyde-3-phosphate dehydrogenase may be in closer proximity (perhaps because of the binding of glyceraldehyde-3-phosphate dehydrogenase to huntingtin) with aldolase and engage in channelling for glyceraldehyde-3-phosphate. By contrast to most of the speculation in the literature, our results suggest that the neuronal damage in HD tissue may be associated with increased energy metabolism at the tissue level leading to modified levels of various intermediary metabolites with pathological consequences.     

Aerobic adaptation in yeast. II. Changes in enzyme profiles during a step-down anaerobic-aerobic transfer.

The levels of various enzymes and components of the glycolytic and respiratory pathways of the yeast Saccharomyces cerevisiae have been determined during a step-down, anaerobic-to-aerobic transition. These activities were determined as an adjunct to the respective metabolite data reported in the first paper in this series. It is clear from the data that anaerobic conditions induce an environment conducive to express glycolytic enzyme activities, while manifesting a differential induction/repression effect on oxidative enzymes. An NAD/NADH mediated mechanism is proposed to explain this difference. Of the enzymes assayed only cytochrome c oxidase shows any direct response to oxygen challenge and consequently it is suggested that the assembly of this enzyme is the trigger mechanism and rate-limiting step in aerobic adaptation.     

Exploring the genetic control of glycolytic oscillations in Saccharomyces Cerevisiae

ABSTRACT: BACKGROUND: A well known example of oscillatory phenomena is the transient oscillations of glycolytic intermediates in Saccharomyces cerevisiae, their regulation being predominantly investigated by mathematical modeling. To our knowledge there has not been a genetic approach to elucidate the regulatory role of the different enzymes of the glycolytic pathway. RESULTS: We report that the laboratory strain BY4743 could also be used to investigate this oscillatory phenomenon, which traditionally has been studied using S. cerevisiae X2180. This has enabled us to employ existing isogenic deletion mutants and dissect the roles of isoforms, or subunits of key glycolytic enzymes in glycolytic oscillations. We demonstrate that deletion of TDH3 but not TDH2 and TDH1 (encoding glyceraldehyde-3-phosphate dehydrogenase: GAPDH) abolishes NADH oscillations. While deletion of each of the hexokinase (HK) encoding genes (HXK1 and HXK2) leads to oscillations that are longer lasting with lower amplitude, the effect of HXK2 deletion on the duration of the oscillations is stronger than that of HXK1. Most importantly our results show that the presence of beta (Pfk2) but not that of alpha subunits (Pfk1) of the hetero-octameric enzyme phosphofructokinase (PFK) is necessary to achieve these oscillations. Furthermore, we report that the cAMP-mediated PKA pathway (via some of its components responsible for feedback down-regulation) modulates the activity of glycoytic enzymes thus affecting oscillations. Deletion of both PDE2 (encoding a high affinity cAMP-phosphodiesterase) and IRA2 (encoding a GTPase activating protein- Ras-GAP, responsible for inactivating Ras-GTP) abolished glycolytic oscillations. CONCLUSIONS: The genetic approach to characterising the glycolytic oscillations in yeast has demonstrated differential roles of the two types of subunits of PFK, and the isoforms of GAPDH and HK. Furthermore, it has shown that PDE2 and IRA2, encoding components of the cAMP pathway responsible for negative feedback regulation of PKA, are required for glycolytic oscillations, suggesting an enticing link between these cAMP pathway components and the glycolysis pathway enzymes shown to have the greatest role in glycolytic oscillation. This study suggests that a systematic genetic approach combined with mathematical modelling can advance the study of oscillatory phenomena.     

Three aldo-keto reductases of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae is an industrially important yeast, which is also used extensively as a model eukaryote. The S. cerevisiae genome has been sequenced in its entirety and therefore represents an ideal organism in which to carry out functional analysis of genes. We have identified several open reading frames in the S. cerevisiae genome which show significant similarity to members of the aldo-keto reductase superfamily. The physiological roles of these gene products have not been previously determined, but their similarity to other enzymes suggests they may perform roles in carbohydrate metabolism and detoxification pathways. Cloning and expression of three of these enzymes has allowed their substrate specificities to be determined. Expression profiling and gene disruption analysis will allow potential roles for these enzymes within the cell to be examined.     

Physiological studies on the effect of Ca2+ on the duration of the lag phase of Saccharomyces cerevisiae

Abstract Cell multiplication and growth of Saccharomyces cereviseae were followed in 2-ml test tubes containing Wickerham's synthetic medium or very dilute synthetic media supplemented in various ways. The ability of the cell cultures to leave the lag phase and enter the exponential phase of growth was investigated. Multiplication was assessed by microscopical observation. The results showed great differences in times required for the cultures to leave the lag phases and begin multiplication. In Wickerham's medium, all cultures grew well 6 h after inoculation. In the dilute medium, several days elapsed before all the cultures grew. These cultures went into exponential growth with approximately first order kinetics. In the unsupplemented medium, the 'half-lives' in the lag phase were about 28 h. Addition of either Ca²⁺ or Ca²⁺ plus A23187 (calcimycin) reduced the half-lives to 10 and 6 h, respectively. The doubling times in the exponential phases of growth were not shortened by these additions. We suggest that Ca²⁺ plays a crucial role as a signal to switch on the mode of cell proliferation in S. cerevisiae .     

Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae

The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S phase occupying the middle half of the cell cycle. In each growth condition, start appeared to correspond to the G1 phase/S phase boundary. Bud emergence did not occur until mid S phase. These results show that the rate of transit through all phases of the cell cycle can vary considerably when cell cycle length changes. When cells growing at different rates were arrested in G1, the following synchronous S phase were of the duration expected from the length of S in each asynchronous population. Cells transferred from a poor nitrogen source to a good one after arrest in G1 went through the subsequent S phase at a rate characteristic of the better medium, indicating that cells are not committed in G1 to an S phase of a particular duration.     

A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P−) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 °C in supplemented phosphate-buffered saline, buffered peptone water , cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P− strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 °C, the lag phase duration of the P+ strain was significantly longer than P−. In all but the most selective medium, P+ strains had slower growth rates than P− strains at 37 °C, probably due to the increased metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P−. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phases than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 °C. No significant differences were observed between the length of lag phases of P+ and P− strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one or more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.     

Comparative study of some parameters of energy metabolism in two strains of Saccharomyces cerevisiae

A comparative study of energy metabolism in two strains Saccharomyces cerevisiae (the initial strain N 73 and laser-irradiated mutant strain Y-503) was performed. In all growth phases, the rates of oxygen consumption by cells of Y-503 were higher than in the initial strain. The maximum (threefold) increase in the rate of oxygen consumption was observed in the linear phase. The effects of respiratory chain inhibitors rotenone, antimycin A, and cyanide on cellular and mitochondrial respiration were identical. There are two sites of energy coupling in the respiratory chain of mitochondria in S. cerevisiae N 73 and Y-503, and electron flow mainly is mainly mediated by cytochrome oxidase. The data suggest that a higher respiratory activity of S. cerevisiae Y-503 cells in comparison with N 73 is associated with greater amounts of mitochondria and total surface area of coupling mitochondrial membranes, which appears to be a factor contributing to a high physiological and biochemical activity of this strain.     

Codon-optimized bacterial genes improve L-Arabinose fermentation in recombinant Saccharomyces cerevisiae

Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomyces cerevisiae, commonly used in bioethanol production, can utilize pentose sugars like l-arabinose or d-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial l-arabinose utilization pathway consisting of l-arabinose isomerase from Bacillus subtilis and l-ribulokinase and l-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. l-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on l-arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the l-arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved l-arabinose conversion rates. With this rational approach, the ethanol production rate from l-arabinose could be increased more than 2.5-fold from 0.014 g ethanol h(-1) (g dry weight)(-1) to 0.036 g ethanol h(-1) (g dry weight)(-1) and the ethanol yield could be increased from 0.24 g ethanol (g consumed l-arabinose)(-1) to 0.39 g ethanol (g consumed l-arabinose)(-1). These improvements make up a new starting point for the construction of more-efficient industrial l-arabinose-fermenting yeast strains by evolutionary engineering.     

Expression and activity of type 1 NAD(P)H dehydrogenase at different growth phases of the cyanobacterium, Synechocystis PCC 6803

The expression and activity of type 1 NAD(P)H dehydrogenase (NDH-1) were investigated in Synechocystis PCC 6803 cells during different growth phases (i.e. lag, logarithmic, stationary and decline phases). The relative amount of NDH-1, estimated by Western blot analysis using antibodies against NdhH, NdhI and NdhK, increased more than two-fold during growth from the lag to the logarithmic phase and then decreased after the logarithmic phase to reach lowest levels after 15 days (decline phase). The activity of light-dependent NADPH oxidation and cyclic electron flow around photosystem I (PSI) changed nearly in parallel with the amount of NdhH, NdhI and NdhK in cells across the growth phases. In contrast, the activity of photosynthetic O₂ evolution and respiratory O₂ uptake was not significantly different across phases of growth; the fluctuation of the activity at different phases was within 40%. These results suggested that the activity of light-dependent NADPH oxidation and PSI-cyclic electron flow are restricted by the amount of NDH-1 and that other factor(s) are limiting the rates of photosynthesis and respiration.