Dynamics of orthophosphate in yeast cytoplasm

Summary Orthophosphate concentration of baker's yeast Saccharomyces cerevisiae was investigated during dynamic conditions. As an example for those dynamics in cell metabolism the transition from glucose limitation to glucose excess (Crabtree-effect) was choosen. As a result of the metabolic switch from complete to partial oxidative metabolism, the cytoplasmic phosphate concentration increased suddenly from 8.4 mM to a maximum of 17.5 mM and transiently decreased to a minimum of 7.0 mM.     

Immobilization of yeast cells in plant cell wall frameworks

Summary Cell-structured support materials (CSM) representing the cell framework of denaturated and extracted mosses, duckweeds or parenchyma tissue particles have been used for the immobilization of Saccharomyces cerevisiae cells. The method consists of inoculation by soaking the dehydrated materials in a yeast suspension and propagation of the yeast cells that reach the relatively closed inner volumes of the cell-structured particles (inter- or intracellular spaces). In spite of high cell densities (up to 2.5 × 10⁹ cells/g wet immobilizate) the velocity of microaerobic glucose consumption was little influenced by intraparticular diffusion resistances, when yeast loaded CSM made from Wolffia arrhiza was incubated in 100 mM glucose at room temperature.     

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.     

Creation of a novel peptide endowing yeasts with acid tolerance using yeast cell-surface engineering

The cell wall of Saccharomyces cerevisiae plays an essential role in the biophysical characteristics of the cell surface. The modification of the cell wall property is an important factor for cellular adaptation to a stressful environment. In this study, we randomly modified the cell wall by displaying combinatorial random peptides on the yeast cell surface, and by screening, we successfully obtained a novel peptide, Scr35, that endowed yeasts with acid tolerance. The yeast, surface-modified by Scr35, was able to grow well under acidic condition and low glucose condition and showed high glucose uptake activity. However, the growth of the modified yeast became inferior as extracellular pH became higher. This inferiority was rescued by decreasing glucose concentration in a medium. Our results suggest that the optimum pH of a medium becomes low when the newly created Scr35 affects glucose uptake activity through cell-surface modification. Therefore, such artificial modification of the cell surface has a great potential as a useful tool for breeding acid-tolerant yeasts for industrial applications of S. cerevisiae as a biocatalyst.     

Evaluation of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans

A new fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), which had been previously developed for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeast Candida albicans. Lineweaver-Burk plots showed the uptake of 2-NBDG to be competitively inhibited by d-glucose and not by l-glucose, which suggested the involvement of the glucose transporting system of C. albicans in the uptake of 2-NBDG. A good correlation was obtained between the yeast viability, determined by the plate-count method, and the 2-NBDG uptake activity of yeast cells (correlation constant: r=0.97). This is expected to lead to the development of a new fluorescent probe for the determination of yeast cell viability.     

Revisiting the Crabtree/Warburg effect in a dynamic perspective: a fitness advantage against sugar-induced cell death

The mechanisms behind the Warburg effect in mammalian cells, as well as for the similar Crabtree effect in the yeast Saccharomyces cerevisiae, are still a matter of debate: why do cells shift from the energy-efficient respiration to the energy-inefficient fermentation at high sugar concentration?

This review reports on the strong similarities of these phenomena in both cell types, discusses the current ideas, and provides a novel interpretation of their common functional mechanism in a dynamic perspective. This is achieved by analysing another phenomenon, the sugar-induced-cell-death (SICD) occurring in yeast at high sugar concentration, to highlight the link between ATP depletion and cell death.

The integration between SICD and the dynamic functioning of the glycolytic process, suggests that the Crabtree/Warburg effect may be interpreted as the avoidance of ATP depletion in those conditions where glucose uptake is higher than the downstream processing capability of the second phase of glycolysis. It follows that the down-regulation of respiration is strategic for cell survival allowing the allocation of more resources to the fermentation pathway, thus maintaining the cell energetic homeostasis.     

A fed-batch cultivation method for the simultaneous utilization of glucose and formate by yeasts

A fed-batch cultivation method was elaborated which 1. warrants simultaneous consumption of glucose and formate, 2. is easy to employ and provides reproducible results, 3. gives information about the role of formate in yeast growth, 4. results in almost identical conditions for parallel cultures on glucose and glucose/formate. The method was applied to Hansenula polymorpha and Saccharomyces cerevisiae. Relevant results were obtained and were comparable to those found previously under chemostatic conditions. Thus, the method is considered suitable for comparative investigations of yeast growth on glucose/formate to demonstrate the role of formate as an additional energy donor.     

A yeast biosensor for glucose determination

Summary A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast Hansenula anomala is retained in calcium alginate gel on the surface of a glass electrode. This biosensor gives a Nernstian response in glucose concentration of 5·10^–4–5·10^–3 mol/l with a response time of 5 min and a life-time of at least 2 months. Mannose and fructose are the only significantly interfering substances. The biosensor was used for measurement of glucose concentration in urine with results comparable to those obtained by a photometric enzymatic method.     

Less is more or more is less: Implications of glucose metabolism in the regulation of the reproductive potential and total lifespan of the Saccharomyces cerevisiae yeast

Carbohydrates are dietary nutrients that have an influence on cells physiology, cell reproductive capacity and, consequently, the lifespan of organisms. They are used in cellular processes after conversion to glucose, which is the primary source of energy and carbon skeleton for biosynthetic processes. Studies of the influence of glucose on cellular parameters and lifespan of organisms are primarily concerned with the effect of low glucose concentration defined as calorie restriction conditions. However, the effect of high glucose concentration on cell physiology is also very important. Thus, a comparative analysis of the effects of low and high glucose concentration conditions on cell efficiency was proposed with regard to reproductive capacity and total lifespan of the cell. Glucose concentration determines the type of metabolism and biosynthetic capabilities, which in turn, through the regulation on the cell size, may affect the reproductive capacity of cells. This study was conducted on yeast cells of wild-type and mutant strains Δgpa2 and Δgpr1 with glucose signalling pathway impairment. Such an experimental model enabled testing both the role of glucose concentration in the regulation of metabolic changes and the extent to which these changes depend on the extracellular or intracellular glucose concentrations. It has been shown here that calorie/glucose excess connected with changes in cell metabolic fluxes increases biosynthetic capabilities of yeast cells. This leads to an increase in cell dry weight accompanied by the increase in cell size and a simultaneous decrease in the reproductive potential and the overall length of cell life.     

Correlation between glucose/fructose discrepancy and hexokinase kinetic properties in different <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeast strains

Grape juice contains about equal amounts of glucose and fructose, but wine strains of Saccharomyces cerevisiae ferment glucose slightly faster than fructose, leading to fructose concentrations that exceed glucose concentrations in the fermenting must. A high fructose/glucose ratio may contribute to sluggish and stuck fermentations, a major problem in the global wine industry. We evaluated wine yeast strains with different glucose and fructose consumption rates to show that a lower glucose preference correlates with a higher fructose/glucose phosphorylation ratio in cell extracts and a lower K _m for both sugars. Hxk1 has a threefold higher V _max with fructose than with glucose, whereas Hxk2 has only a slightly higher V _max with glucose than with fructose. Overexpression of HXK1 in a laboratory strain of S. cerevisiae (W303–1A) accelerated fructose consumption more than glucose consumption, but overexpression in a wine yeast strain (VIN13) reduced fructose consumption less than glucose consumption. Results with laboratory strains expressing a single kinase showed that total hexokinase activity is inversely correlated with the glucose/fructose (G/F) discrepancy. The latter has been defined as the difference between the rate of glucose and fructose fermentation. We conclude that the G/F discrepancy in wine yeast strains correlates with the kinetic properties of hexokinase-mediated sugar phosphorylation. A higher fructose/glucose phosphorylation ratio and a lower K _m might serve as markers in selection and breeding of wine yeast strains with a lower tendency for sluggish fructose fermentation.     

Purification and Some properties of Endo-β-1,6-Glucanase from Acinetobacter sp.

A kind of endo-β-1, 6-glucanase has been purified from the culture filtrate of Acinetobacter sp. grown in the medium containing baker’s yeast cells as a carbon source. A 100-fold purified preparation was obtained by DEAE-Sephadex A–50 column chromatography. The enzyme hydrolyzed pustulan giving a series of gentio-oligosaccharides and glucose. Gentiotriose and gentiotetraose were hydrolyzed by this enzyme yielding glucose and gentiobiose, and glucose, gentiobiose and gentiotriose, respectively. Gentiobiose was not hydrolyzed. Baker’s yeast glucans obtained from the isolated cell walls were also hydrolyzed by this enzyme giving a series of oligosaccharides and glucose. From the action patterns on these carbohydrates, we concluded the present enzyme being endo-β-1, 6-glucanase.     

Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants

Cell lysis is induced in Schizosaccharomyces pombe ?ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ?ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ?ura4 cells.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of β-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin. Three β-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these β-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit β-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying β-glucosidase.     

The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. II. Candida utilis

Candida utilis was grown in batch culture with and without oxygen control. The concentrations of A-, B-, and C-type cytochromes were found to vary with the initial glucose concentration, with the dissolved oxygen concentration, and with time. A-type was the most sensitive. After glucose was essentially exhausted, the yeast catabolized ethanol, if it had been growing in a relatively low initial glucose concentration, or non-glucose carbohydrate, including some of that previously accumulated within the cell, if it had been growing in a high initial glucose concentration. This difference in metabolic pattern could explain why cytochrome derepression was initiated soon after glucose uptake ceased only if initial glucose had been relatively low. The effects of glucose and dissolved oxygen concentrations on yeast cytochromes and respiratory activity are discussed.     

Oscillatory growth rate responses of S. Cerevisiae in continuous culture

The yeast S. cerevisiae was grown on dilute, chemically defined media in continuous culture with either glucose or ammonium sulfate as the growth-limiting ingredient. Changes in dilution rate or glucose concentration induced decaying oscillations in the numbers of yeast growing on ammonium sulfate-limited media. Spot checks indicated that Cell dry weight and Kjeldahl nitrogen followed the cell numbers during these oscillations. With glucose-limited media, there was no response to step changes in ammonium sulfate concentration, and dilution rate step changes gave non-oscillatory transient responses.     

Effect and potential mechanism of action of sea cucumber saponins on postprandial blood glucose in mice

Postprandial blood glucose control is the major goal in the treatment of diabetes. Here, we investigated the effect of sea cucumber saponins (SCSs) on postprandial blood glucose levels. SCS inhibited yeast as well as rat intestinal α-glucosidase activity in a dose-dependent manner and showed better inhibition of yeast α-glucosidases compared to the positive control. Further studies were performed using ICR mice treated with SCS and starch or SCS alone by oral gavage. Unexpectedly, SCS increased postprandial blood glucose levels a short time (1 h) after oral gavage. The serum corticosterone (CORT) level showed a consistent correlation with glucose levels. In vitro experiments confirmed that SCS treatment increased the secretion of CORT in the Y1 adrenal cell line. Overall, these studies demonstrated that SCS gavage could inhibit α-glucosidase activity but cannot attenuate postprandial blood glucose level within short time periods. The underlying mechanisms are probably related to increased serum CORT levels.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Characterization of optimal growth conditions and carotenoid production of strain Rhodotorula mucilaginosa HP isolated from larvae of Pieris rapae

Yeasts have been studied because of their production of a pigment known as carotenoid with potential application in food and feed supplements. A carotenoid‐producing yeast was isolated from the larvae of Pieris rapae, named HP. The strain HP was identified as Rhodotorula mucilaginosa classified by its carbohydrate fermentation pattern and physiological tests. Rhodotorula mucilaginosa HP produces several exogenous enzymes: alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, acid phosphatase and β‐glucosidase. Using response surface methodology, selected medium components (yeast extract, malt extract, peptone, glucose) were tested to find the optimum conditions for carotenoid production and the growth of R. mucilaginosa HP. Central composite design was used to control the concentrations of medium components. Peptone and glucose had the largest effects on carotenoid production and cell growth of R. mucilaginosa HP, respectively. The estimated optimal growth conditions of R. mucilaginosa HP were: yeast extract 3.23%, malt extract 2.84%, peptone 6.99% and glucose 12.86%. The estimated optimal conditions for carotenoid production were: yeast extract 2.17%, malt extract 2.11%, peptone 5.79% and glucose 12.46%. These results will assist in the formulation of an appropriate culture medium for optimal carotenoid production of R. mucilaginosa HP for commercial use.     

The use of plant cell vesicles for immobilization of yeast cells producing ethanol

The preparation of immobilized living yeast cells adsorbed into or onto delipided specimens of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) is reported. These yeast cell-plant cell conjugates were used for the repeated batch production of ethanol from glucose (143 to 246 g/l) or saccharose (150 g/l). Up to 25 fermentation cycles at 30°C were performed. The cycle time for complete substrate conversion to ethanol was reduced 10-fold by a 5-fold increase of the yeast cell Wolffia conjugate concentration (ε = 0.08 to ε =0.4) ε = volume of cell conjugate/totnl reaction volume. The corresponding ethanol production was 11.5 to 13.5 vol% and 9 vol% respectively. The reported results on the discontinuous ethanol fermentation with Wolffia-immobilized yeast cells open the field for their application in continuous ethanol production processes.