Aneuploidy,copy number variation and unique chromosomal structures in bottom‐fermenting yeast revealed by array‐CGH

DNA microarray for comparative genome hybridization (CGH) of bottom‐fermenting yeast was performed based on our in‐house DNA sequence data. Aneuploidy, copy number variation and unique chromosomal structures were observed among bottom‐fermenting yeast strains. Our array experiments revealed a correlation between copy number variation and mRNA expression levels. Chromosomal structures in a Saccharomyces carlsbergensis‐type strain and in a S. monacensis‐type strain that both belong to S. pastorianus phylogenetically differed greatly from those in contemporary industrial bottom‐fermenting yeast strains. The knowledge gained in this study contributes to a more precise genomic characterization of bottom‐fermenting yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Exploiting heterozygosity in industrial yeasts to create new and improved baker's yeasts

The main aim of the work was to utilize heterozygosity of industrial yeast strains to construct new baker's yeast strains. Commercial baker's yeast strain ALKO 743, its more ethanol tolerant descendant ALKO 554 selected initially for growth over 300 generations in increasing ethanol concentrations in a glucose medium, and ALKO 3460 from an old domestic sour dough starter were used as starting strains. Isolated meiotic segregants of the strains were characterized genetically for sporulation ability and mating type, and the ploidy was determined physically. Heterozygosity of the segregant strains was estimated by a variety of molecular characterizations and fermentation and growth assays. The results showed wide heterozygosity and that the segregants were clustered into subgroups. This clustering was used for choosing distantly or closely related partners for strain construction crosses. Intrastrain hybrids made with segregants of ALKO 743 showed 16–24% hybrid vigour or heterosis. Interstrain hybrids with segregants of ALKO 743 and ALKO 3460 showed a wide variety of characteristics but also clear heterosis of 27–31% effects as assayed by lean and sugar dough raising. Distiller's yeast ALKO 554 turned out to be a diploid genetic segregant and not just a more ethanol tolerant mutant of the tetraploid parent strain ALKO 743.     

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.     

Flocculation in industrial strains of Saccharomyces cerevisiae: role of cell wall polysaccharides and lectin‐like receptors

Yeast flocculation is the reversible aggregation of yeast cells promoted by the interaction between lectin‐like protein receptors with mannose side chains on adjacent cell walls. Flocculation is governed by several physiological factors, including the type of nutrient sugar available to yeast. We grew four industrial strains of Saccharomyces cerevisiae , representing applications in the brewing, winemaking and bioethanol sectors, to late stationary phase and quantified the cellular content of mannans, glucans and lectin‐like proteins on yeast cell surfaces. Results indicated that brewing and champagne strains showed moderate to high flocculation ability when grown with glucose, fructose, maltose or galactose, whereas winemaking and fuel alcohol strains only showed moderate flocculation when grown on maltose and galactose. All yeast strains studied were weakly flocculent when grown on mannose. With regard to lectin‐like receptors, their number played a more important role in governing yeast flocculation than the mannan and glucan contents in yeast cell walls. We conclude that all the industrial strains of S. cerevisiae belonged to New‐Flo type on the basis of their flocculation behaviour observed when cultured on different sugars. Quantification of yeast cell wall polysaccharides and receptor sites indicates that mannan and glucan levels remain almost constant, irrespective of the strain under investigation. The main difference in flocculation characteristics in industrial yeast strains appears to be due to variations in concentrations of lectin‐like cell surface receptors. Our findings may benefit brewers, winemakers and other yeast‐based technologies in design of media to prevent premature flocculation during fermentation. Copyright © 2017 The Institute of Brewing & Distilling     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

A CONVENIENT DOMINANT SELECTION MARKER FOR GENE TRANSFER IN INDUSTRIAL STRAINS OF SACCHAROMYCES YEAST: SMRI ENCODED RESISTANCE TO THE HERBICIDE SULFOMETURON METHYL

The SMR1-410 gene of S. cerevisiae, encoding resistance to the herbicide sulfometuron methyl (SM), was used as a dominant selection marker in yeast replicating and yeast integrating vectors for the transformation of wild type strains of baking, brewing (ale and lager), distilling, wine and sake Saccharomyces yeasts. Transformation of lithium treated cells by a YEp vector resulted in transformation frequencies ranging from 200 to 8,000 transformants per 10 ug of DNA. Utilizing a yeast integrating vector with SMR1–410 as the only yeast DNA sequences, it was demonstrated that a single copy of SMR1–410 is sufficient to confer stably inherited SM resistance. Thus the SMR1–410 sequence has the unique ability to act as a selectable marker and to also provide a site for chromosomal integration. Since transformants were resistant to levels at least seven fold higher than wild type strains the resistance phenotype was clearly expressed and easily scored in all industrial strains tested. Unlike other selection markers derived from mammalian or bacterial cells, SMR1–410 is derived from S. cerevisiae. Thus industrial utilization of this marker as a means of genetically improving food and beverage strains of Saccharomyces yeasts by recombinant DNA technology is enhanced, as government regulatory agencies are likely to view it in a more favourable light.     

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.     

Isolation of a lager yeast with an increased copy number of the YCK1 gene and high fermentation performance

The selection of yeast with good fermentation characteristics is critical for producing beer with desirable qualities. A yeast population was selected with an enhanced fermentation rate, referred to as high‐fermentation yeast (HFY), which was derived from the wild‐type Sacchromyces pastorianus yeast population (WTY). To identify genes that contribute to the fermentation performance, we compared the genetic profiles of the WTY and HFY populations by next‐generation sequencing. Several chromosomal regions were found to exhibit markedly different sequence coverage, suggesting chromosomal duplications and deletions, which might have occurred during selection of the HFY population. Among the genes with altered coverage, the copy number of the Saccharomyces eubayanus‐type YCK1 (SeYCK1) gene was almost two times higher in the HFY population than in the WTY population. The gene which is involved in glucose sensing in Saccharomyces cerevisiae was at a higher level in the HFY population throughout fermentation. These findings suggest that the chromosomal duplication of a region including the SeYCK1 gene locus of the HFY population is at least partially responsible for the differences in the fermentation properties between the WTY and HFY populations. © 2018 The Institute of Brewing & Distilling     

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     

PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

Investigating flavour characteristics of British ale yeasts: techniques,resources and opportunities for innovation

Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour‐related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Susceptibility and resistance to ethanol in Saccharomyces strains isolated from wild and fermentative environments

In this work, we apply statistical modelling techniques to study the influence of increasing concentrations of ethanol on the overall growth of 29 yeast strains belonging to different Saccharomyces and non‐Saccharomyces species. A modified Gompertz equation for decay was used to objectively estimate the noninhibitory concentration (NIC) and minimum inhibitory concentration (MIC) for the assayed strains to ethanol, which are related to the susceptibility and resistance of yeasts to this compound, respectively. A first ANOVA analysis, grouping strains as a function of their respective Saccharomyces species, revealed that S. cerevisiae was the yeast with the highest, and statistically significant, ethanol resistance value. Then, a second factorial ANOVA analysis, using the origin of strains (wild or fermentative) and their taxonomic classification (S. cerevisiae, S. paradoxus or S. bayanus var. uvarum) as categorical predictor variables, showed that no significant differences for the NIC and MIC parameters were found between both ecological niches within the same species, indicative that these physiological characteristics were presumably not modified throughout the adaptation to human‐manipulated fermentative environments. Finally, differences among selected strains with respect to ethanol tolerance were correlated to the initial contents of unsaturated fatty acids, mainly oleic acid. Copyright © 2010 John Wiley & Sons, Ltd.