Role of trehalose and glycogen in the survival of aging Saccharomyces cerevisiae cells

The role of the storage carbohydrates trehalose and glycogen in the survival of aging Saccharomyces cerevisiae cells was studied. Culture aging for one week did not reduce cell viability. During this period, the cells accumulated the storage carbohydrates and raised the activity of the glycolytic enzymes hexokinase and phosphofructokinase. However, further aging led to a drastic drop in cell viability and to a decrease in the cellular content of trehalose and glycogen and in the activity of hexokinase and phosphofructokinase. The possible reasons for these changes are discussed.     

Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in Saccharomyces cerevisiae

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO₂ production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.     

Comparative analysis of glycogen and trehalose accumulation in methylotrophic and nonmethylotrophic yeasts

Trehalose and glycogen accumulate in certain yeast species when they are exposed to unfavorable growth conditions. Accumulations of these reserve carbohydrates in yeasts provide resistance to stress conditions. The results of this study indicate that certain Pichia species do not accumulate high levels of glycogen and trehalose under normal growth conditions. However, depending on the Pichia species, both saccharides accumulate at high levels when the Pichia cells are exposed to unfavorable or stress-inducing growth conditions. Growth in glycerol or methanol medium mostly led to trehalose accumulation in Pichia species tested in this study. It was shown that the metabolic pathways for glycogen and trehalose biosynthesis are present in Pichia species. However, it appears that the biosynthesis of trehalose and glycogen may be regulated in different manners in Pichia species than in the yeast S. cerevisiae. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 6, pp. 737–741. The text was submitted by the author in English.     

Effect of glucose starvation on germ-tube production byCandida albicans

By incubating starved and unstarved yeast cells in synthetic media with a pH of 4.5 or 6.7 at 37°C the effect of a 3 hours' glucose starvation on germ-tube production byCandida albicans was evaluated. In addition the endocellular content of total carbohydrates, glycogen, trehalose and proteins after and before the starvation were dosed. The most interesting result was the overcoming of the pH-regulated dimorphism, thanks to the starvation treatment. Infact the starved cultures produced germtubes indifferently in neutral or acid media, whereas the filamentation of the unstarved cultures was more copious in pH 6.7 medium. The endocellular content of trehalose and protein was unchanged, whereas total carbohydrates and glycogen showed a shortage after the 3 hours' glucose starvation. The possible involvements of these metabolic changes in the regulation of dimorphic transition are discussed.     

Effect of hypoxia on carbohydrate metabolism in scorpion tissues

The levels of glycogen, glucose, lactate, as well the activities of ten enzymes of carbohydrates metabolism in brain, liver and white muscle of sea scorpion have been investigated. Metabolite concentrations didn't change in brain and the levels of glycogen and lactate were constant in the rest tissues investigated. Glucose concentration decreased in the liver and increased in muscle. In brain hypoxia decreased the activity of hexokinase and increased one of pyruvate kinase, phosphoglucoisomerase and fructoso-1,6-bisphosphatase. In liver most of the enzymes showed the tendency to decrease of their activities. In muscle the activities of phosphofructokinase and phosphoglucoisomerase decreased. Mechanisms of carbohydrates metabolism regulation under hypoxia are discussed.     

Gaseous environments modify reserve carbohydrate contents and cell survival in the brewing yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The use of H₂, He and O₂ during batch fermentation of Saccharomyces cerevisiae BRAS291 increased the final intracellular glycogen contents of the cells from 2-fold to 10-fold compared with a gas-free condition, and this depended on the gas applied. Differently, the intracellular trehalose contents increased from 2-fold to 10-fold in reducing conditions compared with more oxidizing conditions. During storage at 4°C, the viability of cells cultivated with gas was twice that of cells cultivated without gas. These results could be explained by the intracellular carbohydrate contents as well as yeast ultrastructural modifications observed previously.     

Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source-dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1-H223Y allele (glc6-1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild-type TPS1 because, unlike the wild-type enzyme, TPS activity in tps1-H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high-copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose-6-phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.     

Arrestment of carbohydrate metabolism during anaerobic dormancy and aerobic acidosis inArtemia embryos: determination of pH-sensitive control points

Summary Changes in concentrations of trehalose, glycogen, glycerol, some glycolytic intermediates and adenylate nucleotides that occur during aerobic development have been compared to those seen during anaerobic dormancy and aerobic acidosis in gastrula-stage embryos ofArtemia. The latter two incubation conditions are known to foster large drops in intracellular pH (Busa et al. 1982; Busa and Crowe 1983).During aerobic development, trehalose levels decline while glycogen and glycerol are synthesized (Fig. 1). These transitions are blocked during both anaerobic dormancy and aerobic acidosis, but are resumed by return of embryos to aerobic incubation (Fig. 1). Thus, it is concluded that carbohydrate catabolism in hydrated embryos is directly modulated by intracellular pH. Changes in metabolite levels (Figs. 2–4) reveal that this process is controlled primarily at the trehalase and hexokinase reactions with a less-pronounced negative crossover point noted at the phosphofructokinase step. Each of these reactions is shown to be nonequilibrium by comparing the mass action ratio to the equilibrium constant (Table 1).When embryos are placed under anaerobic conditions, ATP levels drop dramatically while AMP increases in concentration (Fig. 5). These changes are reflected in a drop in adenylate energy charge from a control value of 0.73 to 0.42 (Fig. 6). Aerobic acidosis only leads to a slight decrease in energy charge, emphasizing that shifts in adenylate poolsAbbreviations pH _i intracellular pH - glyceraldehyde 3-P glyeraldehyde 3-phosphate - DHAP dihydroxyacetone phosphate - glucose 6-P glucose 6-phosphate - fructose 6-P fructose 6-phosphate - fructose 1,6-P ₂ fructose 1,6-bisphosphate - HK hexokinase - PFK phosphofructokinase - MAR mass action ratio - K _eq equilibrium constant     

Activity and isoenzyme patterns of glycolytic enzymes during perinatal development of rat lung

The enzymes hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were studied in rat lung during development starting at day 16 of gestation (day-6) until 5 days after birth. During gestation, the activities of hexokinase type II, enolase and pyruvate kinase decreased and reached adult values at birth or shortly thereafter. Hexokinase type I remained relatively constant and the decrease of soluble type II hexokinase was compensated for by an increment of particle-bound hexokinase starting at day 20 of gestation until birth. In contrast, phosphofructokinase activity increased until day 20 of gestation followed by a rapid fall in activity until 2 days after birth. Except for hexokinase no isoenzyme shifts were observed in the period of observation. The results are discussed with respect to the proposed relationship between glycogen breakdown and surfactant synthesis during the perinatal period and suggest a regulatory role for phosphofructokinase in this process.     

Comparison of fermentation properties and specific enzyme activities of free and calcium-alginate-entrapped Saccharomyces cerevisiae

Summary Physiological properties have been determined for calcium-alginate-entrapped Saccharomyces cerevisiae in comparison to cells in suspension under identical culture conditions. Cells grown in the form of microcolonies in the alginate beads showed faster glucose uptake and ethanol productivity with simultaneously decreased product and cell yields. Increased specific hexokinase and phosphofructokinase activities could be determined in these cells. Immobilized single cells showed only slightly enhanced glucose turnover and no higher specific hexokinase activity. The significant alterations in physiology are apparently connected with growth of the cells in aggregates. Offprint requests to: H.-J. Rehm     

Enzymes of carbohydrate metabolism ofCotugnia digonopora and their activity in the presence of anthelmintics,in vitro

Cotugnia digonopora, a fowl cycllophyllidean cestode, was found to possess most of the enzymes, associated with the glycolytic sequence and phosphoenolpyruvate branch point, in the cytosol fraction. Enzymes of malate metabolism were predominantly mitrochondrial. Anthelmintic agents inhibited hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, fumarate reductase, and malic enzyme. In intact worms this effect was significantly reduced. However, the activities of glycogen Phosphorylase and pyruvate kinase were significantly enhanced. Communication No. 4113 from CDRI, Lucknow.     

Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.     

Behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel and performing alcoholic fermentation

Summary The behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel was studied during their continuous function in an ethanol-producing reactor. Polymerization destroys 40% to 80% of the cells, depending on their physiological state. A three day adaptation phase is required before ethanol production stabilizes and this phase corresponds to an increase in cell concentration in the gels and to protein synthesis. The amounts of DNA, glucan, glycogen and trehalose are different in entrapped and free cells. Microscopic observation shows that 75% to 85% of the cells lose their integrity and that the remainder appear to multiply normally. Within a gel particle, both viability and fermentation activity are heterogeneous. A high percentage of cells have low viability and low fermentation activity. A proportion of cells remains capable of forming colonies and these cells have higher fermentation activity and are preferentially localized at the surface of gel particles.     

Carbohydrate Composition during Germination and Outgrowth of Ascospores of Saccharomyces cerevisiae

Single spores of Saccharomyces cerevisiae were examined to distinguish changes in the synthesis and degradation of intracellular and wall carbohydrates during germination and outgrowth. Intracellular carbohydrate was fractionated into trehalose and glycogen. Trehalose degradation occurred during germination and outgrowth. The intracellular glycogen was degraded during germination and then synthesized during outgrowth. Wall carbohydrate was fractionated into glycogen, glucan and mannan. The wall glycogen and the KOH-soluble glucan were degraded during germination and then synthesized prior to and during outgrowth, respectively. The major component of the KOH-insoluble glucan in the wall is β-1,3-glucan. The glucan and mannan were synthesized during outgrowth.

The study revealed that the development of a vegetative cell from a spore follows rapid decreases in the amounts of trehalose, glycogen and KOH-soluble glucan during germination, and great increases in the amounts of glycogen, β-1,3-glucan and mannan during outgrowth.     

Effect of adrenaline, hydrocortisone, insulin and dibutyryl-cAMP on glycolysis and glycogenolysis in white rat liver slices]

Epinephrine, hydrocortisone, and dibutyril cAMP inhibited glycolysis and glucogenolysis. The inhibitory effect was also found when glucose-6-phosphate (G-6-P) was used as a glycolysis substrate, but not for fructose-1,6-diphosphate. This is the evidence of hexokinase activity inhibition by hormones and dibutyril cAMP, and presumably of phospholylase and phosphofructokinase as well. In the simulated cell-free system the hormones produced no effect, dibutyril cAMP inhibiting hexokinase alone. For the realization of hormones effect their interaction with the cell membrane is required. Inhibition of glycogen and G-6-P decomposition to lactic acid in the rat liver slices was not associated with the hormone action on phosphorylase and phosphofructokinase through cAMP and proteinkinase directly. The results obtained indicated the existence of a supplementary mechanism that modified cAMP effect on the activity of the said enzymes. Insulin was effective in any of the cases.     

Enhanced fermentative capacity of yeasts engineered in storage carbohydrate metabolism

During yeast biomass production, cells are grown through several batch and fed‐batch cultures on molasses. This industrial process produces several types of stresses along the process, including thermic, osmotic, starvation, and oxidative stress. It has been shown that Saccharomyces cerevisiae strains with enhanced stress resistance present enhanced fermentative capacity of yeast biomass produced. On the other hand, storage carbohydrates have been related to several types of stress resistance in S. cerevisiae. Here we have engineered industrial strains in storage carbohydrate metabolism by overexpressing the GSY2 gene, that encodes the glycogen synthase enzyme, and deleting NTH1 gene, that encodes the neutral trehalase enzyme. Industrial biomass production process simulations were performed with control and modified strains to measure cellular carbohydrates and fermentation capacity of the produced biomass. These modifications increased glycogen and trehalose levels respectively during bench‐top trials of industrial biomass propagation. We finally show that these strains display an improved fermentative capacity than its parental strain after biomass production. Modification of storage carbohydrate content increases fermentation or metabolic capacity of yeast which can be an interesting application for the food industry. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:20–24, 2015     

Levels of trehalose and glycogen inArthrobacter globiformis under conditions of nutrient starvation and osmotic stress

Cells ofArthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-M_r carbohydrates (800 g/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-M_r carbohydrate levels increased to 1300 g/mg protein. Low-M_r pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of ,-trehalose. Levels of trehalose inArthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties ofArthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.     

松针瘿蚊越冬幼虫体内酶活性的时序变化

昆虫的越冬耐寒过程与糖酵解、磷酸己糖途径和抗冻保护性物质合成等一些中间代谢有关的酶有关。该文对松针瘿蚊Thecodiplosis japonensis老熟幼虫1998/1999越冬期间体内上述代谢酶活性的变化进行了研究。越冬期间体内糖原磷酸化酶活性明显地增加,糖酵解有关的酶（己糖激酶、乳酸脱氢酶和醛缩酶）活性较低,以保证更多的碳源（糖原）转化成海藻糖。越冬期间,体内葡萄糖-6-磷酸脱氢酶活性增高所产生的还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH),可为细胞在亚低温状态下发挥正常功能以及体内抗冻保护性物质的合成提供还原动力,同时通过调节体内海藻糖酶活性来维持越冬期间较高含量的海藻糖和移除春季体内累积的过多的海藻糖。     

Enhancing yeast cell viability after dehydration by modification of the lipid profile

In the present study, we analysed metabolite features during the dehydration-rehydration process for different yeast species genetically closely related to S. cerevisiae, in order to determine whether metabolites might play a role in cell viability. We ranked the species S. cerevisiae, S. paradoxus, S. kudriavzevii, L. kluyveri, N. castellii, S. mikatae, S. bayanus, and S. servazzii according to their viability rate after the dehydration-rehydration process, and showed that desiccation tolerance across the species did not correlate with the intracellular content of trehalose or glycogen. Cell lipid composition was also investigated during this process, to see whether the content of triacylglycerols and phosphatidylcholine showed significant variations across the species. The increase of phosphatidylcholine level increase in both S. paradoxus and S. bayanus cells grown in supplemented media enhanced both their cell viability after stress imposition and lipid storage.     

D2O effects on association behavior and binding properties of phospho- and dephospho-cytoplasmic malic dehydrogenase

Changes in the concentration of three major carbohydrates, e.g., glycogen, trehalose, and cellulose, were determined during differentiation of Dictyostelium discoideum in a stage study. These three carbohydrates consituted 50–63% of the total carbohydrates. Total carbohydrate content per cell aliquot did not change between the aggregation and sorocarp stages of differentiation. The isolation, purification, and characterization of cellulose is described. Cellulose consisted of an alkali-insoluble (alpha) and an alkali-soluble (beta) fraction. Total cellulose accumulated from very low amounts in late pseudoplasmodium cells to about 35% of carbohydrates in mature sorocarps at a rate of 0.07 μmole glucose equiv/min/ml packed cell volume. Purified alkali-insoluble cellulose constituted about 19% of total carbohydrates in mature sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Trehalose constituted 10–11% of the carbohydrates in sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Glycogen, comparing several methods of determination, was rapidly degraded between the culmination and sorocarp stages of differentiation at an average rate of 0.05 μmole glucose equiv/min/ml packed cell volume. The major portion of glycogen was soluble in TCA and constituted 35% of total carbohydrates in aggregated cells and about 11% in mature sorocarps. A minor fraction of glycogen, about 15% of total carbohydrates in aggregated cells, was solubilized by KOH from a TCA precipitate. A mild acidic treatment of solubilized cell constituents increased the glycogen content by 55%, as judged by an enzymatic assay.