Influence of nitrogen on the biological aging of sherry wine

Nitrogen compounds are essential to the growth and metabolism of yeasts. The uptake and metabolism of nitrogen compounds by Saccharomyces cerevisiae depend not only on the strain and its physiological condition, but also on the chemical and physical properties of its environment. The effect of the addition of different amino acids (L ‐proline, L ‐threonine, L ‐arginine, L ‐glutamic acid, L ‐leucine and L ‐valine) to nitrogen‐depleted natural or nitrogen‐free synthetic wine on the cell growth, flor velum formation and sherry wine compound production was investigated under controlled biological aging by S. cerevisiae var. capensis strain G1 a typical flor yeast. The formation of flor velum was dependent on particular amino acid, oxygen availability and the composition of wine. Consumption of glycerol was related with the cell growth; in contrast, acetaldehyde tended to be released. Amino acid supplementation resulted in the release to wine of amino acids, esters and higher alcohols. The amino acid which was released in nearly all cases was L ‐leucine. Addition of L ‐glutamic acid resulted in the release mainly of ethyl acetate, in the case of L ‐leucine isoamyl alcohols were released, and for L ‐valine isobutanol. In the three cases, 1,1‐diethoxyethane was released in large quantities. The findings might indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential. Copyright © 2006 Society of Chemical Industry     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Efficient production of L‐lactic acid by Crabtree‐negative yeast Candida boidinii

Industrial production of L ‐lactic acid, which in polymerized form as poly‐lactic acid is widely used as a biodegradable plastic, has been attracting world‐wide attention. By genetic engineering we constructed a strain of the Crabtree‐negative yeast Candida boidinii that efficiently produced a large amount of L ‐lactic acid. The alcohol fermentation pathway of C. boidinii was altered by disruption of the PDC1 gene encoding pyruvate decarboxylase, resulting in an ethanol production that was reduced to 17% of the wild‐type strain. The alcohol fermentation pathway of the PDC1 deletion strain was then successfully utilized for the synthesis of L ‐lactic acid by placing the bovine L ‐lactate dehydrogenase‐encoding gene under the control of the PDC1 promoter by targeted integration. Optimizing the conditions for batch culture in a 5 l jar‐fermenter resulted in an L ‐lactic acid production reaching 85.9 g/l within 48 h. This productivity (1.79 g/l/h) is the highest thus far reported for L ‐lactic acid‐producing yeasts. DDBJ/EMBL/GenBank nucleotide database with Accession Nos. AB440630 and AB440631. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full‐length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l ‐cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l ‐cystine and some other cysteine conjugates, but not l ‐cystathionine or l ‐methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l ‐cysteine as a nitrogen source, and that overexpression of the gene results in increased H₂S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S‐ethyl‐l ‐cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

Tolerance to thermal and reductive stress in Saccharomyces cerevisiae is amenable to regulation by phosphorylation-dephosphorylation of ubiquitin conjugating enzyme 1 (Ubc1) S97 and S115

Ubiquitin conjugating enzyme 1 (Ubc1) is a member of the E2 family of enzymes that conjugates ubiquitin to damaged proteins destined for degradation by the ubiquitin proteasomal system. It is necessary for stress tolerance and is essential for cell survival in Saccharomyces cerevisiae. Ubc1 has five serine residues that are potential substrates for phosphorylation by kinases. However, no data are available to indicate that Ubc1 function or stress tolerance in S. cerevisiae is regulated by serine phosphorylation of Ubc1. We demonstrate that Ubc1 is phosphorylated in serine residue(s). Furthermore, expression of Ubc1 mutants that are ‘constitutively phosphorylated’ or ‘dephosphorylated’ in mitogen‐activated protein (MAP) kinase serine residues (S97 and S115) affected tolerance to thermal and reductive stress in S. cerevisiae. Specifically, expression of Ubc1S97A and S115D increased thermo‐tolerance in both BY4741 and TetO₇‐UBC1ura3Δ cells. Serine phosphorylation of Ubc1 was decreased in BY4741 cells following exposure at 40 °C. Tolerance to reductive stress in the same strains correlated with the expression of Ubc1S97A. Ubc1 phosphorylation did not show significant alteration under similar conditions. Both hog1Δ and slt2Δ cells expressing Ubc1S115D and Ubc1S115A were rendered tolerant to thermal and reductive stress respectively. Ubc1 phosphorylation was higher in BY4741 cells compared to hog1Δ cells at 30 °C and was significantly reduced in BY4741 cells upon exposure at 40 °C. Taken together, the cell survival assays and Ubc1 phosphorylation status in strains and under conditions as described above suggest that tolerance to thermal and reductive stress in S. cerevisiae may be regulated by MAP kinase‐mediated phosphorylation of Ubc1S97 and S115. Copyright © 2011 John Wiley & Sons, Ltd.     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature‐sensitive fission yeast strains, rrs1‐D14/22G and rrs1‐L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1‐84 (D22/30G) and rrs1‐124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two‐hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts. Copyright © 2015 John Wiley & Sons, Ltd.     

Fermentation Characteristics and Aromatic Profile of Plum Wines Produced with Indigenous Microbiota and Pure Cultures of Selected Yeast

The aim of this study was to assess and compare fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of different selected yeast. Experiments were carried out with plum (Prunus domestica L.) varieties of different fruit ripening times (?a?anska rana, ?a?anska lepotica, and Po?ega?a). Wine fermentations were conducted by the activity of indigenous microbiota, commercially available Saccharomyces cerevisiae and Saccharomyces bayanus yeast strains and joint activity of Schizosaccharomyces pombe and S. cerevisiae (sequential inoculation). Statistically significant differences in fermentative characteristics and the content of certain volatile compounds were observed as a result of metabolic activity of various indigenous and/or selected yeasts during fermentation of plum pomace. Minimal duration of fermentation (4 to 5 d) and fastest ethanol production rate (from 12.3 to 15.5 g/L/d) were the characteristics of the studied S. cerevisiae strains. Isobutanol, 3‐methyl‐1‐butanol, 1‐heptanol, and 1‐octanol were the most prevalent higher alcohols in the tested plum wine samples. The predominant ester in plum wines was ethyl acetate, ethyl lactate, amyl acetate, isoamyl acetate, and ethyl palmitate, esters responsible for the floral and fruity olfactory tones, were also present in large amounts. Also, the use of S. cerevisiae strains resulted in the production of plum wines with better sensory characteristics than ones produced with other investigated yeasts. Obtained results are significant since there is limited data on the compounds responsible for the unique flavor of plum wine, as well as on the impact of different yeast starter cultures application on the overall quality of fruit wines.     

Multi‐gene phylogenetic analysis reveals that shochu‐fermenting Saccharomyces cerevisiae strains form a distinct sub‐clade of the Japanese sake cluster

Shochu is a traditional Japanese distilled spirit. The formation of the distinguishing flavour of shochu produced in individual distilleries is attributed to putative indigenous yeast strains. In this study, we performed the first (to our knowledge) phylogenetic classification of shochu strains based on nucleotide gene sequences. We performed phylogenetic classification of 21 putative indigenous shochu yeast strains isolated from 11 distilleries. All of these strains were shown or confirmed to be Saccharomyces cerevisiae, sharing species identification with 34 known S. cerevisiae strains (including commonly used shochu, sake, ale, whisky, bakery, bioethanol and laboratory yeast strains and clinical isolate) that were tested in parallel. Our analysis used five genes that reflect genome‐level phylogeny for the strain‐level classification. In a first step, we demonstrated that partial regions of the ZAP1, THI7, PXL1, YRR1 and GLG1 genes were sufficient to reproduce previous sub‐species classifications. In a second step, these five analysed regions from each of 25 strains (four commonly used shochu strains and the 21 putative indigenous shochu strains) were concatenated and used to generate a phylogenetic tree. Further analysis revealed that the putative indigenous shochu yeast strains form a monophyletic group that includes both the shochu yeasts and a subset of the sake group strains; this cluster is a sister group to other sake yeast strains, together comprising a sake‐shochu group. Differences among shochu strains were small, suggesting that it may be possible to correlate subtle phenotypic differences among shochu flavours with specific differences in genome sequences. Copyright © 2017 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.     

Simultaneous utilization of ammonia,free amino acids and peptides during fermentative growth of Saccharomyces cerevisiae

The efficiency of nitrogen use by yeast is one of the key determinants of the successful completion of alcoholic fermentations. In this work the growth of Saccharomyces cerevisiae S288c in a synthetic medium containing ammonia and free amino acids, supplemented with yeast hydrolysate, was studied. Experiments with ¹⁵NH₄Cl and ¹⁵N‐labelled yeast hydrolysate were carried out to gain insight into which of these three classes of assimilable nitrogen sources yeast cells prefer. Co‐consumption of all three sources was observed; approximately 40% of the total nitrogen in the yeast protein fraction originated from yeast hydrolysate, while free amino acids and ammonia contributed 40 and 20%, respectively. The results indicate that several amino acids are more readily obtained from peptides, most likely when the uptake of their free forms is competitively inhibited and/or repressed. During the second half of each fermentation, a decrease in the incorporation of yeast hydrolysate‐derived nitrogen was observed. These results highlight the nutritional role of peptides in various yeast fermentations. Copyright © 2016 The Institute of Brewing & Distilling     

Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.     

Relationship between growth of food‐spoilage yeast in high‐sugar environments and sensitivity to high‐intensity pulsed UV light irradiation

The relationship between prior growth of food‐spoilage yeast in high‐sugar environments and their subsequent survival postpulsed UV (PUV) irradiation was investigated. Test yeast were separately grown to early stationary phase in YPD broth containing increasing concentrations of glucose (1–50% w/v) and were flashed with ≤40 pulses of broad‐spectrum light at lamp discharge energy settings of 3.2, 7.2 and 12.8 J (equivalent to UV doses of 0.53, 1.09 and 3.36 μJ cm^?2, respectively) and their inactivation measured. Findings showed that prior growth in high‐sugar conditions (≥30% glucose w/v) enhanced the sensitivity of all nine representative strains of Zygosaccharomyces bailii, Z. rouxii and Saccharomyces cerevisiae yeast to PUV irradiation. Significant differences in inactivation amongst different yeast types also occurred depending on amount of UV dose applied, where the order of increasing sensitivity of osmotically stressed yeast to PUV irradiation was shown to be Z. rouxii, Z. bailii and >S. cerevisiae. For example, a 1.2‐log order difference in CFU mL^?1 reduction occurred between Z. bailii 11 486 and S. cerevisiae 834 when grown in 50% w/v sugar samples and treated with the uppermost test UV dosage of 3.36 μJ cm^?2, where these two yeast strains were reduced by 3.8 and 5.0 log orders, respectively, after this PUV treatment regime compared to untreated controls. The higher the UV dose applied the greater the reduction in yeast numbers. For example, a 1.0‐, 1.4‐ and 4.0‐log order differences in CFU mL^?1 numbers occurred for S. cerevisiae 834 grown in 15% w/v sugar samples and then treated with PUV dose of 0.53, 1.09 and 3.36 μJ cm^?2, respectively. These findings support the development of PUV for the treatment of high‐sugar foods that are prone to spoilage by osmotolerant yeast.     

Characteristics of yeast flora in Chinese strong‐flavoured liquor fermentation in the Yibin region of China

The yeast community in the Chinese strong‐flavoured liquor region of Yibin was investigated and the ethanol producing abilities and extracellular enzymes activities of the isolates were tested. A total of 110 yeast were isolated on Wallerstein Laboratory medium and through 26S rRNA D1/D2 region sequence analysis identified as 13 yeast species. These were Wickerhamomyces anomalus, Debaryomyces hansenii, Issatchenkia orientalis, Lodderomyces elongisporus, Clavispora lusitaniae, Saccharomyces cerevisiae, Pichia fermentans, Pichia manshurica, Pichia membranifaciens, Torulaspora delbrueckii, Trichosporon insectorum, Trichosporonoides megachiliensis, Zygosaccharomyces bailii, and one uncertain species. These yeast species, composed of various strains, formed the special yeast community in the Yibin region. Approximately 73.6% of the strains belong to the four dominant species: W. anomalus, D. hansenii, I. orientalis and L. elongisporus. The 110 yeast strains produced 0.6–9.0% (v/v) alcohol (average of 5.4%, v/v) in a grain medium, and 0.2–7.2% (v/v) alcohol (average value of 2.9%, v/v) in a yeast extract–peptone–dextrose medium. Furthermore, the 49 strains that produced pectinase, lipase, cellulase, amylase or protease generally showed better ethanol‐producing ability than those strains that do not produce extracellular enzymes. This work profiles the ethanol‐producing ability and the organic matter utilization of the yeast community in Chinese strong‐flavoured liquor produced in the Yibin region and provides a better understanding of Chinese strong‐flavoured liquor fermentation. Copyright © 2016 The Institute of Brewing & Distilling     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.