High-hydrostatic-pressure treatment impairs actin cables and budding in Saccharomyces cerevisiae

We previously reported that the postgrowth of Escherichia coli K-12 after high-hydrostatic-pressure treatment (HPT) as moderate as 75 MPa for 30 min at 37 degrees C induced the formation of elongated cells due to an HPT-induced disorder in FtsZ ring formation, which is essential for cell division. Because an FtsZ ring is known as a bacterial cytoskeleton, we examined the effect of HPT on a eukaryotic cytoskeleton, such as actin cables (long bundles of actin filaments), of Saccharomyces cerevisiae. We found that actin cables disappeared after HPT (100 MPa) and were not reorganized until 3.5 h of growth after HPT. As long as actin cables disappeared, budding did not start. We also demonstrated that the in vitro polymerization of actin monomers was highly sensitive to HPT.     

Effects of Saccharomyces cerevisiae culture and Saccharomyces cerevisiae live cells on in vitro mixed ruminal microorganism fermentation

The objective of this study was to examine the effects of a Saccharomyces cerevisiae live cell product and a S. cerevisiae culture product on the in vitro mixed ruminal microorganism fermentation of ground corn, soluble starch, alfalfa hay, and Coastal bermudagrass hay. In the presence of ground corn, neither concentration (0.35 or 0.73 g/L) of S. cerevisiae culture nor live cells had any effect on final pH, H2, CH4, propionate, or butyrate. The S. cerevisiae culture had no effect on acetate, but both concentrations of S. cerevisiae live cells decreased acetate and the acetate:propionate ratio. When soluble starch was the substrate, both concentrations of S. cerevisiae live cells and 0.73 g/L of S. cerevisiae culture decreased the acetate:propionate ratio. Although the treatment effects were not statistically significant, both concentrations of live cells and 0.73 g/L of the culture decreased lactate concentrations compared with the control incubations. When alfalfa hay served as the substrate, neither the S. cerevisiae culture nor the live cells had an effect on propionate, butyrate, or the acetate:propionate ratio. Both concentrations of S. cerevisiae culture decreased the final pH and in vitro dry matter disappearance, and the 0.73 g/L treatment decreased the amount of acetate. However, both treatments of S. cerevisiae live cells increased final pH and decreased acetate and in vitro dry matter disappearance. Neither yeast treatment had much effect on the Coastal bermudagrass hay fermentations. In general, both S. cerevisiae supplements seemed to have similar effects on the mixed ruminal microorganism fermentation.     

Understanding the mechanism of heat stress tolerance caused by high trehalose accumulation in Saccharomyces cerevisiae using DNA microarray

DNA microarray analysis was performed to examine the stress tolerance mechanism of a Saccharomyces cerevisiae recombinant strain exhibiting high trehalose accumulation and heat stress tolerance. Results suggest that the upregulation of sugar transporter genes is one of the key events for heat stress tolerance of the recombinant strain.     

Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation

In order to establish an efficient bioethanol production system from rice straw, a new strategy to ferment the mixture of glucose and xylose by a sequential application of Saccharomyces cerevisiae and Pichia stipitis was developed, in which heat inactivation of S. cerevisiae cells before addition of P. stipitis was employed. The results showed that heating at 50°C for 6h was sufficient to give high xylose fermentation efficiency. By application of the inactivation process, 85% of the theoretical yield was achieved in the fermentation of the synthetic medium. At the same time, the xylitol production was reduced by 42.4% of the control process. In the simultaneous saccharification and fermentation of the lime-pretreated and CO(2)-neutralized rice straw, the inactivation of S. cerevisiae cells enabled the full conversion of glucose and xylose within 80 h. Finally, 21.1g/l of ethanol was produced from 10% (w/w) of pretreated rice straw and the ethanol yield of rice straw reached 72.5% of the theoretical yield. This process is expected to be useful for the ethanol production from lignocellulosic materials in the regions where large-scale application of recombinant microorganisms was restricted.     

Simple procedure for fatty acid analysis of glycerophospholipids in Escherichia coli and Saccharomyces cerevisiae

Rapid, convenient methods have been developed for fatty acid analysis of membrane glycerophospholipids in microorganisms. Fatty acid methyl esters derived from glycerophospholipids have been prepared directly from wet pellets of Escherichia coli cells or Saccharomyces cerevisiae spheroplasts without lipid extraction and fractionation in high yields under mild temperature conditions for analysis by gas chromatography.     

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28

We characterized a high malic acid production mechanism in sake yeast strain No. 28. No considerable differences in the activity of the enzymes that were involved in malic acid synthesis were observed between strain No. 28 and its parent strain, K1001. However, compared with strain K1001, which actively took up rhodamine 123 during staining, the cells of strain No. 28 were only lightly stained, even when cultured in high glucose concentrations. In addition, malic acid production by the respiratory-deficient strain of K1001 was 2.5-fold higher than that of the wild-type K1001 and wild-type No. 28. The findings of this study demonstrated that the high malic acid production by strain No. 28 is attributed to the suppression of mitochondrial activity.     

Optimization of honey-must preparation and alcoholic fermentation by Saccharomyces cerevisiae for mead production

Mead fermentation is a time-consuming process, often taking several months to complete. Despite of the use of starter cultures several problems still persist such as lack of uniformity of the final products, slow or premature fermentation arrest and the production of off-flavors by yeast. Thus the aim of this study was to optimize mead production through the use of an appropriate honey-must formulation to improve yeast performance alcoholic fermentation and thereby obtain a high quality product. Honey-must was centrifuged to reduce insoluble solids, pasteurized at 65 °C for 10 min, and then subjected to different conditions: nitrogen supplementation and addition of organic acids. Although the addition of diammonium phosphate (DAP) reduced fermentation length, it did not guarantee the completeness of the fermentation process, suggesting that other factors could account for the reduced yeast activity in honey-must fermentations. Sixteen yeast-derived aroma compounds which contribute to the sensorial quality of mead were identified and quantified. Global analysis of aromatic profiles revealed that the total concentration of aroma compounds in meads was higher in those fermentations where DAP was added. A positive correlation between nitrogen availability and the levels of ethyl and acetate esters, associated to the fruity character of fermented beverages, was observed whereas the presence of potassium tartrate and malic acid decreased, in general, their concentration.This study provides very useful information that can be used for improving mead quality.     

The effect of citric acid and pH on growth and metabolism of anaerobic Saccharomyces cerevisiae and Zygosaccharomyces bailii cultures

The effects of citric acid at pH values of 3.0, 4.0, and 4.5 on growth and metabolism of anaerobic Saccharomyces cerevisiae and Zygosaccharomyces bailii cultures were investigated. S. cerevisiae and Z. bailii exhibited similar tolerances to citric acid, as determined by growth measurements, at all three pH values investigated. The citric-acid-induced growth inhibition of both yeast species increased with increasing pH values, indicating that the antimicrobial mechanism of citric acid differs from that of classical weak-acid preservatives. In S. cerevisiae, citric acid shifted the primary energy metabolism towards lower ethanol production and higher glycerol production, thus resulting in lower ATP production. These metabolic changes in S. cerevisiae were pH-dependent; i.e. the higher the pH, the lower the ATP production, and they may explain why growth of S. cerevisiae is more inhibited by citric acid at higher pH values. In Z. bailii, citric acid also caused an increased glycerol production, although to a lesser extent than in S. cerevisiae, but it caused virtually no changes in ethanol and ATP production.     

Novel screening system for protein-protein interactions by bimolecular fluorescence complementation in Saccharomyces cerevisiae

For high-throughput screening of protein-protein interactions, we have developed a novel yeast screening system using Bimolecular fluorescence complementation (BiFC). Two yeast plasmids, in which genes of heterodimerized peptides LZA and LZB were each fused with those of non-fluorescent half fragments of Kusabira-Green mutant (mKG2), were transformed into a- and α-type yeast, respectively. Mating of them gave a library, which was screened by following green fluorescence resulted from LZA-LZB interaction. The method showed potential ability to detect the positive clones from a model library, in which green-fluorescent and non-fluorescent yeast was mixed in a ratio of 1:675.     

Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.     

Proteolytic activity of Saccharomyces cerevisiae strains associated with Italian dry-fermented sausages in a model system

Strains of Saccharomyces cerevisiae isolated from Italian salami were screened for proteolytic activity in a model system containing sarcoplasmic (SMS) or myofibrillar (MMS) proteins, at 20 °C for 14 days, to evaluate the possible influence on the proteolysis of fermented sausages.SDS-PAGE revealed that 14 of the most osmotolerant strains were responsible for the extensive hydrolysis of the main myofibrillar proteins, while only one strain was able to hydrolyze sarcoplasmic proteins. Free amino acids (FAA) accumulated during proteolysis were strain-dependent with different patterns from sarcoplasmic or myofibrillar protein fraction. In general, proteolysis lead Cys, Glu, Lys and Val as the most abundant FAA in the inoculated MMS samples. Volatile compound analysis, determined by SPME-GC-MS, evidenced 3-methyl butanol in MMS, and 2-methyl propanol and 3-methyl-1-butanol in SMS as major compounds. Our findings highlight that S. cerevisiae could influence the composition in amino acids and volatile compounds in fermented sausages, with a strain-dependent activity.     

Oleic acid and ergosterol supplementation mitigates oxidative stress in wine strains of Saccharomyces cerevisiae

During fermentation of high-sugar-containing medium lacking lipid nutrients, wine yeasts undergo oxidative stress and oxidative damage to cell membranes and proteins. Considering that cell membranes are important stress sensors, and that under hypoxic conditions wine yeasts modulate cell membranes composition by incorporating lipids available in the growth medium, in the present work, the effects of lipid nutrition on wine yeast oxidative stress response were evaluated on two strains of Saccharomyces cerevisiae. Biomarkers of oxidative stress, oxidative damage and antioxidant response were evaluated together with viability and acetic acid production during fermentation of a synthetic must lacking lipid nutrients as compared to added oleic acid and ergosterol. The results show that the availability of lipid nutrients causes a significant reduction in the intracellular content of reactive oxygen species and in the oxidative damage to membranes and proteins, as indicated by flow cytometry of cells stained with dihydroethidum (DHE) and propidium iodide (PI) and by Western blot of protein carbonyls. Accordingly, lipid nutrients feeding results in the increase in cell viability and superoxide activity, and the reduction in trehalose accumulation, proteinase A activity and production of acetic acid. In summary, these results are compatible with the hypothesis that the supplementation of lipid nutrients mitigates oxidative stress and oxidative damage in wine strains of S. cerevisiae during growth under unfavourable conditions.     

Stable disruption of ethanol production by deletion of the genes encoding alcohol dehydrogenase isozymes in Saccharomyces cerevisiae

We analyzed the effects of the deletions of genes encoding alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae. The decrease in ethanol production by ADH1 deletion alone could be partially compensated by the upregulation of other isozyme genes, while the deletion of all known ADH isozyme genes stably disrupted ethanol production.     

Comparative evaluation of some oenological properties in wine strains of Candida stellata, Candida zemplinina, Saccharomyces uvarum and Saccharomyces cerevisiae

Due to the recent changes in yeast taxonomy, a novel wine-related species Candida zemplinina as well as a “reinstated” species Saccharomyces uvarum have been accepted in addition to Candida stellata, Saccharomyces bayanus and Saccharomyces cerevisiae, and the use of the different taxon names has been inconsistent in the literature of food microbiology. The aim of this work is to make an exact comparison of genetically identified strains of these species, under oenological conditions. Dynamics and some important products of alcoholic fermentation were investigated in laboratory fermentations. The results show that C. zemplinina and C. stellata are similar in their strong fructophilic character. C. stellata produces more glycerol and fare more ethanol, which is comparable with that produced by S. uvarum. Strains of the latter species differed from S. cerevisiae mainly in low acetic acid production and lower ethanol yield. Revision of the oenological traits of these yeasts provides new data for consideration in the control of fermentation, with special regard to botrytized sweet wines, where they are frequently found in mixed population.