Carbon starvation can induce energy deprivation and loss of fermentative capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 micro mol/g, while nitrogen-starved cells still contained approximately 6 micro mol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.     

Carbon Starvation Can Induce Energy Deprivation and Loss of Fermentative Capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 μmol/g, while nitrogen-starved cells still contained approximately 6 μmol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.     

Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast

High-cell-density fed-batch processes for bakers' yeast production will involve a low-average-specific growth rate due to the limited oxygen-transfer capacity of industrial bioreactors. The relationship between specific growth rate and fermentative capacity was investigated in aerobic, sucrose-limited fed-batch cultures of an industrial bakers' yeast strain. Using a defined mineral medium, biomass concentrations of 130 g dry weight/L were reproducibly attained. After an initial exponential-feed phase (mu = 0.18 h(-1)), oxygen-transfer limitation necessitated a gradual decrease of the specific growth rate to ca. 0.01 h(-1). Throughout fed-batch cultivation, sugar metabolism was fully respiratory, with a biomass yield of 0.5 g biomass/g sucrose(-1). Fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions with excess glucose) showed a strong positive correlation with specific growth rate. The fermentative capacity observed at the end of the process (mu = 0.01 h(-1)) was only half that observed during the exponential-feed phase (mu = 0.18 h(-1)). During fed-batch cultivation, activities of glycolytic enzymes, pyruvate decarboxylase and alcohol dehydrogenase in cell extracts did not exhibit marked changes. This suggests that changes of fermentative capacity during fed-batch cultivation were not primarily caused by regulation of the synthesis of glycolytic enzymes.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.     

Mixed and diverse metabolic and gene-expression regulation of the glycolytic and fermentative pathways in response to a HXK2 deletion in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the HXK2 gene, which encodes the glycolytic enzyme hexokinase II, is involved in the regulatory mechanism known as 'glucose repression'. Its deletion leads to fully respiratory growth at high glucose concentrations where the wild type ferments profusely. Here we describe that deletion of the HXK2 gene resulted in a 75% reduction in fermentative capacity. Using regulation analysis we found that the fluxes through most glycolytic and fermentative enzymes were regulated cooperatively by changes in their capacities (Vmax) and by changes in the way they interacted with the rest of the metabolism. Glucose transport and phosphofructokinase were regulated purely at the metabolic level. The reduction of fermentative capacity in the mutant was accompanied by a remarkable resilience of the remaining capacity to nutrient starvation. After starvation, the fermentative capacity of the hxk2Delta mutant was similar to that of the wild type. Based on our results and previous reports, we suggest an inverse correlation between glucose repression and the resilience of fermentative capacity towards nutrient starvation. Only a limited number of glycolytic enzyme activities changed upon starvation of the hxk2Delta mutant and we discuss to what extent this could explain the stability of the fermentative capacity.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Starvation response of Saccharomyces cerevisiae grown in anaerobic nitrogen- or carbon-limited chemostat cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h(-1) at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of Saccharomyces cerevisiae and ethanol production

Growth of Saccharomyces cerevisiae and fermentative ethanol production in the presence of acetic and lactic acids was measured in whole corn mash. In this industrial medium, as compared to glucose minimal medium, the yeast had increased tolerance to organic acid stress. It was concluded that the increased buffering capacity of whole corn mash, resulting in decreased concentration of undissociated acid, was responsible for this phenomenon.     

Relationship between sugar uptake kinetics and total sugar consumption in different industrialSaccharomyces cerevisiae strains during alcoholic fermentation

Summary The sugar transport, fermentative alcohol dehydrogenase (ADH) and protease activities of different industrial strains ofSaccharomyces cerevisiae were measured during batch alcoholic fermentation.These strains exhibited different apparent loss of activity of sugar transport, which seemed to be characteristic of each one. A good correlation was found systematically between the integration of sugar transport activity along fermentation and the maximum amount of sugar consumed during fermentation. In all strains sugar transport activity exhibit a lower half-time than fermentative ADH activity. These progressive declines of both sugar uptake and ADH activities ofSaccharomyces cerevisiae during batch fermentation seemed to not result from an increase in the protease activity.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Carbohydrate Uptake and Utilization byClostridium beijerinckiiNCIMB 8052

The clostridia are a diverse group of obligately anaerobic bacteria with potential for the fermentative production of fuels, solvents and other chemicals. Several species exhibit a broad substrate range, but there have been few studies of the mechanisms involved in regulation of uptake and metabolism of fermentable carbohydrates.Clostridium beijerinckii(formerlyClostridium acetobutylicum) NCIMB 8052 exhibited transport activity for hexoses and hexitols. Glucose-grown cells transported glucose and fructose, but not galactose, glucitol (sorbitol) or mannitol, transport of which was induced by growth on the respective substrates. Phosphorylation of glucose, fructose, glucitol and mannitol by cell extracts was supported by phosphoenolpyruvate, indicating the involvement of a phosphotransferase system in uptake of these substrates. Fructose phosphorylation was also demonstrated by isolated membranes in the presence of fructose 1-phosphate, thus identifying this derivative as the product of the fructose phosphotransferase system. The presence of phosphotransferase activities in extracts prepared from cells grown on different carbon sources correlated with transport activities in whole cells, and the pattern of transport activities reflected the substrate preference of cells growing in the presence of glucose and another carbon source. Thus, glucose and fructose were co-metabolised, while utilization of glucitol was prevented by glucose, even in cells which were previously induced for glucitol metabolism. Of the substrates examined, only galactose appeared to be transported by a non-phosphotransferase mechanism, since a significant rate of phosphorylation of this sugar was supported by ATP rather than phosphoenolpyruvate.     

Control of exercise-stimulated muscle glucose uptake by GLUT4 is dependent on glucose phosphorylation capacity in the conscious mouse

Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during exercise because GLUT4 overexpression does not increase exercise-stimulated MGU. Instead of glucose transport, glucose phosphorylation is a primary limitation of exercise-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair exercise-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity was increased. Thus, C57BL/6J mice with hexokinase II (HKII) overexpression (HK(Tg)), a GLUT4 partial knock-out (G4(+/-)), or both (HK(Tg) + G4(+/-)) and wild-type (WT) littermates were implanted with carotid artery and jugular vein catheters for sampling and infusions at 4 months of age. After a 7-day recovery, 5-h fasted mice remained sedentary or ran on a treadmill at 0.6 mph for 30 min (n = 9-12 per group) and received a bolus of 2-deoxy[3H]glucose to provide an index of MGU (Rg). Arterial blood glucose and plasma insulin concentrations were similar in WT, G4(+/-), HKTg, and HKTg + G4(+/-) mice. Sedentary Rg values were the same in all genotypes in all muscles studied, confirming that glucose transport is a significant barrier to basal glucose uptake. Gastrocnemius and soleus Rg were greater in exercising compared with sedentary mice in all genotypes. During exercise, G4(+/-) mice had a marked increase in blood glucose that was corrected by the addition of HK II overexpression. Exercise Rg (micromol/100g/min) was not different between WT and G4(+/-) mice in the gastrocnemius (24 +/- 5 versus 21 +/- 2) or the soleus (54 +/- 6 versus 70 +/- 7). In contrast, the enhanced exercise Rg observed in HKTg mice compared with that in WT mice was absent in HKTg + G4(+/-) mice in both the gastrocnemius (39 +/- 7 versus 22 +/- 6) and the soleus (98 +/- 13 versus 65 +/- 13). Thus, glucose transport is not a significant barrier to exercise-stimulated MGU despite a 50% reduction in GLUT4 content when glucose phosphorylation capacity is normal. However, when glucose phosphorylation capacity is increased by HK II overexpression, GLUT4 availability becomes a marked limitation to exercise-stimulated MGU.     

Reversible loss of affinity induced by glucose in the maltose-H+ symport of Saccharomyces cerevisiae

Glucose represses and inactivates maltose transport in Saccharomyces cerevisiae. The inactivation has been described as an irreversible process involving proteolysis. We have studied the inactivation of the maltose-H+ symport in this yeast and have observed that the mechanism of inactivation depends on the physiological conditions. In resting cells there was a decrease in transport capacity. The rate of decrease was enhanced nonspecifically by the presence of a sugar, glucose being more effective than maltose. In growing cells, glucose induced a decrease in affinity of the H+-symport which could be recovered by starvation, even in the presence of cycloheximide; there was no loss in capacity or, if present, this loss could be explained fully by the dilution due to repression during growth on glucose. We submit that in growing cells inactivation consists in a reversible modification of the permease not involving proteolysis.     

Sexual dimorphism in primate aerobic capacity: a phylogenetic test

Male intrasexual competition should favour increased male physical prowess. This should in turn result in greater aerobic capacity in males than in females (i.e. sexual dimorphism) and a correlation between sexual dimorphism in aerobic capacity and the strength of sexual selection among species. However, physiological scaling laws predict that aerobic capacity should be lower per unit body mass in larger than in smaller animals, potentially reducing or reversing the sex difference and its association with measures of sexual selection. We used measures of haematocrit and red blood cell (RBC) counts from 45 species of primates to test four predictions related to sexual selection and body mass: (i) on average, males should have higher aerobic capacity than females, (ii) aerobic capacity should be higher in adult than juvenile males, (iii) aerobic capacity should increase with increasing sexual selection, but also that (iv) measures of aerobic capacity should co‐vary negatively with body mass. For the first two predictions, we used a phylogenetic paired t‐test developed for this study. We found support for predictions (i) and (ii). For prediction (iii), however, we found a negative correlation between the degree of sexual selection and aerobic capacity, which was opposite to our prediction. Prediction (iv) was generally supported. We also investigated whether substrate use, basal metabolic rate and agility influenced physiological measures of oxygen transport, but we found only weak evidence for a correlation between RBC count and agility.     

Reversibility of decreased insulin-stimulated glucose transport capacity in diabetic muscle with in vitro incubation. Insulin is not required

The mechanisms by which insulin deficiency affects muscle glucose transport were investigated. Epitrochlearis muscles from rats with streptozotocin-induced diabetes and from controls were incubated in vitro for 0.5-14 h. The incubation was shown not to impair muscle energy stores or tissue oxygenation. Diabetes decreased basal 3-O-methylglucose transport by 40% (p less than 0.01), and insulin-stimulated (20 milli-units/ml) glucose transport capacity by 70% (p less than 0.001). In vitro incubation gradually normalized insulin responsiveness (3.77 +/- 0.38 before versus 8.97 +/- 0.65 mumol X ml-1 X h-1 after 12 h of incubation). Basal glucose transport remained significantly reduced. The reversal of the insulin responsiveness did not require the presence of rat serum and, furthermore, took place even in the absence of insulin. In fact, insulin responsiveness was higher after incubation (14 h) with no insulin than with 100 microunits/ml insulin (9.85 +/- 0.59 versus 8.06 +/- 0.59 mumol X ml-1 X h-1, p less than 0.05). Glucose at 30 mM did not affect the normalization of the insulin-stimulated glucose transport capacity, whereas incubation in serum from diabetic rats resulted in a slightly (26%) blunted reversal (7.60 +/- 0.39 versus 8.89 +/- 0.45 mumol X ml-1 X h-1 with diabetic versus control serum for 14 h, p less than 0.05; before incubation the value was 3.87 +/- 0.40). Inhibition of protein synthesis by cycloheximide blocked the normalization by 80%. These results suggest the presence in diabetic serum of some labile factor that might inhibit the glucose transport system. The results indicate that the decreased insulin-stimulated glucose transport capacity, in the insulin-deficient diabetic muscle, is not a direct consequence of the lack of insulin or of high glucose concentrations.     

High capacity xylose transport in Candida intermedia PYCC 4715

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (mu(max)=0.5 h-1, 30 degrees C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km = 0.2 mM, Vmax = 7.5 mmol h-1 g-1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mM, Vmax = 11 mmol h-1 g-1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.     

Cloning of a second gene encoding 6-phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate

We have identified a new gene, PFK27, that encodes a second inducible 6-phosphofructo-2-kinase in the yeast Saccharomyces cerevisiae. Sequencing shows an open reading frame of 397 amino acids and 45.3 kDa. Amino acid sequence comparisons with other bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoenzymes of various organisms revealed similarities only to the kinase domains. Expression of PFK27 was induced severalfold by glucose and sucrose, but not by galactose or maltose, suggesting that sugar transport might be involved in triggering the induction signal. We have constructed a mutant strain devoid of any fructose-2,6-bisphosphate. The mutant strain grew well on several kinds and concentrations of carbon sources. The levels of hexose phosphates in the cells were increased, but flux rates for glucose utilization and ethanol production were similar to the wild-type strain. However, after the transfer of the mutant cells from respiratory to fermentative growth conditions, growth, glucose consumption and ethanol production were delayed in a transition phase. Our results show that fructose-2,6-bisphosphate is an important effector in vivo of the 6-phosphofructo-1-kinase/fructose-1,6-bisphospha-tase enzyme pair, and is involved in the initiation of glycolysis during the transition to a fermentative mode of metabolism. Nevertheless, it can be effectively replaced by other effectors and regulatory mechanisms during growth on glucose.