Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a “Stress Model” Fermentation System for Fuel Ethanol Yeast Strains

During industrial scale fuel ethanol fermentations, yeast encounters a multitude of stress factors that impose constraints on growth and fermentative metabolism. These stresses include high sugar concentration, elevated temperature, high ethanol concentrations, low external pH and the weak organic acids lactic and acetic. Yeast strains which are tolerant to these stresses and able to synthesize high ethanol concentrations in their presence would be most desirable for use in industrial scale fuel ethanol production. In this study, a “stress model” fermentation system was developed as a tool to screen candidate yeast strains for relative stress resistance. The stress model was designed on the basis that the degree of ethanol produced by a particular strain would be indicative of the stress resistance of that particular strain. Eight strains of Saccharomyces cerevisiae, each with different backgrounds and fermentative capabilities, were screened for relative stress resistance using the stress model. The results obtained indicate that the sum of the stress factors in the stress model exceeded the tolerance level of most of the strains screened (approximately 40%). Two strains in particular, J006 and A007, displayed superior fermentative performance and produced significantly (P > 0.01) higher final ethanol concentrations when compared to the other strains.     

Requirement of glutathione for Sod1 activation during lifespan extension

It has been shown that the activation of cytosolic superoxide dismutase (Sod1) in Saccharomyces cerevisiae is only dependent on Ccs1, which is responsible for insertion of copper into the enzyme catalytic center, and that glutathione (GSH) is not necessary for this process. In this work, we addressed an important role of GSH in Sod1 activation by a Ccs1‐dependent mechanism during oxidative stress and its role in yeast lifespan. Exponential cells of Saccharomyces cerevisiae, treated or not with 0.5 mM menadione for 1 h, were used for evaluation of the effect of a mild oxidative stress pre‐treatment on chronological lifespan. The results showed that menadione induced a lifespan extension in the wild‐type (WT) strain but this adaptive response was repressed in gsh1 and in sod1 strains. Interestingly, menadione treatment increased SOD1 and CCS1 gene expression in both WT and gsh1 strains. However, while these strains showed the same Sod1 activity before treatment, only the WT presented an increase of Sod1 activity after menadione exposure. Glutathionylation seems to be essential for Sod1 activation since no increase in activity was observed after menadione treatment in grx1 and grx2 null mutants. Our results suggest that GSH and glutathionylation are fundamental to protect Sod1 sulfhydryl residues under mild oxidative stress, enabling Sod1 activation and lifespan extension. Copyright © 2010 John Wiley & Sons, Ltd.     

A spheroplast rate assay for determination of cell wall integrity in yeast

The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1–5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5–15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls. © 1998 John Wiley & Sons, Ltd.     

A study on Saccharomyces cerevisiae and Candida utilis cell wall capacity for binding magnesium

The capacity for binding magnesium by bakery's yeast strain Saccharomyces cerevisiae No. 102 (Pure Culture Collection, Faculty Food Technology, Warsaw) and fodder yeast strain Candida utilis (ATCC 9950) was investigated in media supplemented with that element. The capacities of C. utilis (ATCC 9950) and S. cerevisiae (No. 102) biomass for binding magnesium were not statistically different in the first 24 h. In the next 24 h of cultivation the cells of C. utilis (ATCC 9950) were still able to bind magnesium ions, whereas those of S. cerevisiae (No. 102) released a part of previously bound magnesium to the medium. The major part of magnesium bound by the cells of C. utilis (ATCC 9950) was accumulated in cytosole. It was opposite to the cells of bakery yeast S. cerevisiae (No. 102) that accumulated magnesium mainly in the cell wall. The cells of C. utilis (ATCC 9950) yeast were smaller and their cell walls were thinner as compared to those of S. cerevisiae (No. 102) yeast. The thickness of the external mannoprotein layers was similar in both strains analyzed.     

Use of FT-NIR and XPS techniques to distinguish cell hull fractions prepared by autolysis or HPH from Saccharomyces cerevisiae and Brettanomyces bruxellensis strains

Yeast hulls can be used to adsorb undesirable compounds such as volatile phenols that may be present in wine. To understand this adsorption process, the properties of the cell walls and their chemical composition need to be better understood. A study was conducted using four different yeast fractions of either autolysed or high-pressure homogeniser (HPH)-crushed yeast biomasses of Saccharomyces cerevisiae and Brettanomyces bruxellensis. Near-infrared spectroscopy (FT-NIR) coupled with X-ray photoelectron spectroscopy (XPS) was used and analysed by principal component analysis (PCA). The FT-NIR spectral region of Saccharomyces and Brettanomyces statistically analysed by PCA showed a clear discrimination accounting for 76% of the variation in the data for PC1; moreover, yeast hulls prepared from the same strain and subjected to two different treatments were also separated. These methods classify yeast cell hulls (YCH) according to strain, composition and treatment applied. Our results indicate that yeast hulls obtained by autolysis are less rich in proteins than those resulting from HPH treatment due to the high pressure that releases more proteins and exposes them on the surface of the cell wall. The composition of YCH at the extreme surface is similar to that found deeper in the wall.     

The yeast Saccharomyces cerevisiae Pdr16p restricts changes in ergosterol biosynthesis caused by the presence of azole antifungals

Pdr16p belongs to the family of phosphatidylinositol transfer proteins in yeast. The absence of Pdr16p results in enhanced susceptibility to azole antifungals in Saccharomyces cerevisiae. In the major fungal human pathogen Candida albicans, CaPDR16 is a contributing factor to clinical azole resistance. The current study was aimed at better understanding the function of Pdr16p, especially in relation to azole resistance in S. cerevisiae. We show that deletion of the PDR16 gene increased susceptibility of S. cerevisiae to azole antifungals that are used in clinical medicine and agriculture. Significant differences in the inhibition of the sterol biosynthetic pathway were observed between the pdr16Δ strain and its corresponding wild‐type (wt) strain when yeast cells were challenged by sub‐inhibitory concentrations of the azoles miconazole or fluconazole. The increased susceptibility to azoles, and enhanced changes in sterol biosynthesis upon exposure to azoles of the pdr16Δ strain compared to wt strain, are not the results of increased intracellular concentration of azoles in the pdr16Δ cells. We also show that overexpression of PDR17 complemented the azole susceptible phenotype of the pdr16Δ strain and corrected the enhanced sterol alterations in pdr16Δ cells in the presence of azoles. Pdr17p was found previously to be an essential part of a complex required for intermembrane transport of phosphatidylserine at regions of membrane apposition. Based on these observations, we propose a hypothesis that Pdr16p assists in shuttling sterols or their intermediates between membranes or, alternatively, between sterol biosynthetic enzymes or complexes. Copyright © 2013 John Wiley & Sons, Ltd.     

Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pH_i) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (μ_max) of 0.58–1.50 h⁻¹. At pH 2.5, slow growth was observed for the isolates from mawè (μ_max of 0.06–0.80 h⁻¹), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pH_i homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.     

The Effect of Proanthocyanidins on Growth and Alcoholic Fermentation of Wine Yeast under Copper Stress

Proanthocyanidins (PAs) derived from the grape skin, as well as from grape seeds, grape stems, are an important group of polyphenols in wine. The aim of this study was to understand the effect of PAs (0.1, 1.0 g/L) on growth and alcoholic fermentation of 2 strains of Saccharomyces cerevisiae (commercial strain FREDDO and newly selected strain BH8) during copper‐stress fermentation, using a simple model fermentation system. Our results showed that both PAs and Cu²⁺ could pose significant inhibition effects on the growth of yeast cells, CO₂ release, sugar consumption, and ethanol production during the initial phase of the fermentation. Compared to PAs, Cu²⁺ performed more obvious inhibition on the yeast growth and fermentation. However, adding 1.0 g/L PAs increased in the vitality and metabolism activity of yeast cells at the mid‐exponential phase of fermentation in the mediums with no copper and 0.1 mM Cu²⁺ added, shortened the period of wine fermentation, and decreased the copper residues. It indicated that PAs could improve the ability of wine yeast to resist detrimental effects under copper‐stress fermentation condition, maintaining cells metabolic activity, and fermentation could be controlled by manipulating PAs supplementation.     

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.     

Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses

Trehalose is believed to play an important role in stress tolerance in the yeast Saccharomyces cerevisiae. In this research, the responses to various environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses, were investigated in a strain with deletion of the NTH1, NTH2, and ATH1 genes encoding trehalases that are involved in trehalose degradation and the triple deletion strains overexpressing TPS1 or TPS2, both of which encode trehalose biosynthesis enzymes in S. cerevisiae. The contents of trehalose constitutively accumulated in the TPS1- and TPS2-overexpressing triple deletion strains were higher than that in the original triple deletion strain. High trehalose accumulation and growth activity were observed in the TPS2-overexpressing triple deletion strain after ethanol stress induction. The same was also observed in the triple deletion and the TPS1- and TPS2-overexpressing triple deletion strains after heat stress induction. In case of freezing stress, all the recombinant strains with high constitutive trehalose content showed high tolerance. However, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicated that high trehalose accumulation can make yeast cells resistant to multiple stresses, but the importance of this accumulation before or after stress induction is varied depending on the type of stress.     

Mutation of the CgPDR16 gene attenuates azole tolerance and biofilm production in pathogenic Candida glabrata

The PDR16 gene encodes the homologue of Sec14p, participating in protein secretion, regulation of lipid synthesis and turnover in vivo and acting as a phosphatidylinositol transfer protein in vitro. This gene is also involved in the regulation of multidrug resistance in Saccharomyces cerevisiae and pathogenic yeasts. Here we report the results of functional analysis of the CgPDR16 gene, whose mutation has been previously shown to enhance fluconazole sensitivity in Candida glabrata mutant cells. We have cloned the CgPDR16 gene, which was able to complement the pdr16Δ mutation in both C. glabrata and S. cerevisiae. Along with fluconazole, the pdr16Δ mutation resulted in increased susceptibility of mutant cells to several azole antifungals without changes in sensitivity to polyene antibiotics, cycloheximide, NQO, 5‐fluorocytosine and oxidants inducing the intracellular formation of reactive oxygen species. The susceptibility of the pdr16Δ mutant strain to itraconazole and 5‐fluorocytosine was enhanced by CTBT [7‐chlorotetrazolo(5,1‐c)benzo(1,2,4)triazine] inducing oxidative stress. The pdr16Δ mutation increased the accumulation of rhodamine 6G in mutant cells, decreased the level of itraconazole resistance caused by gain‐of‐function mutations in the CgPDR1 gene, and reduced cell surface hydrophobicity and biofilm production. These results point to the pleiotropic phenotype of the pdr16Δ mutant and support the role of the CgPDR16 gene in the control of drug susceptibility and virulence in the pathogenic C. glabrata. Copyright © 2013 John Wiley & Sons, Ltd.     

PGK1, the gene encoding the glycolitic enzyme phosphoglycerate kinase,acts as a multicopy suppressor of apoptotic phenotypes in S. cerevisiae

In a previous paper we reported the construction of a S. cerevisiae strain lacking the essential gene LSM4, which could survive by the introduction of a truncated form of the orthologous gene from Kluyveromyces lactis. This strain showed apoptotic hallmarks and other phenotypes, including an increased sensitivity to caffeine and acetic acid. The suppression of the latter phenotype by overexpressing yeast genes allowed the isolation of PGK1, the gene encoding the glycolytic enzyme phosphoglycerate kinase. This gene restored normal ageing, oxygen peroxide resistance and nuclear integrity in the mutant. Other phenotypes, such as caffeine sensitivity and glycerol utilization, were also suppressed. Copyright © 2009 John Wiley & Sons, Ltd.     

High levels of copper retard the growth of Saccharomyces cerevisiae by altering cellular morphology and reducing its potential for ethanolic fermentation

Previous research has shown that high levels of Cu²⁺ inhibit the growth of yeast and induce sluggish fermentation, resulting in a decrease in alcohol production. Little data are available on the effects of copper on the cell morphology, especially the surface elasticity of Saccharomyces cerevisiae. This work investigated the effects of Cu²⁺ (0.5, 1.0 and 1.5 mm ) on the growth, surface characteristics and elasticity of two strains of S. cerevisiae in a low sugar model synthetic medium. The results indicated that high levels of Cu²⁺ retarded the growth rate of S. cerevisiae and reduced the utilisation of reducing sugars. Cells showed intercellular adhesion, became small and deformed, and the surface was uneven and rough after the adsorption of Cu²⁺. The Young's modulus also decreased with an increase in the concentration of Cu²⁺, indicating that the cells had softened. This study reveals the response mechanism of S. cerevisiae under copper stress by altering cellular morphology and mechanical properties.     

Sweet Wine Production by Two Osmotolerant Saccharomyces cerevisiae Strains

The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts.     

A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation

Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of the Saccharomyces cerevisiae cell wall. These mechanical homogenization procedures often involve the manual use of devices such as the Dounce homogenizer, and so are very operator‐dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenizing yeast cells based on nitrogen cavitation. This has been optimized to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane‐bound compartments of S. cerevisiae. We present an integrated protocol that is fast, facile, robust and efficient and that will enable ‘omics’ studies of the subcellular compartments of S. cerevisiae and other yeasts. © 2014 The Authors. Yeast published by John Wiley & Sons Ltd.