Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

Analysis of homothallic Saccharomyces cerevisiae strain mating during must fermentation

Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (Saccharomyces cerevisiae), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus MAT to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.     

Analysis of Homothallic Saccharomyces cerevisiae Strain Mating during Must Fermentation

Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (Saccharomyces cerevisiae), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus MAT to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.     

Studies on strong and weak killer phenotypes of wine yeasts: production, activity of toxin in must, and its effect in mixed culture fermentation

R.A. MUSMANNO, T. DI MAGGIO and G. CORATZA.1999.Two different killer phenotypes were detected among K⁺ (killer) yeasts isolated from spontaneous wine fermentations using a plate bioassay. The two phenotypes differed in their degree of killer activity, and were designated as SK⁺(strong killer) and WK⁺(weak killer). Strains showing either phenotype were assayed for expression of killer activity under different growth conditions. Growth in must negatively affected expression of the killer activity of both phenotypes. The supernatant fluids from must cultures showed a lower killing effect than those from yeast phosphate dextrose broth (YPDB) cultures. The ability of the two K⁺ phenotypes to prevail on K-sensitive yeasts was studied in mixed-culture fermentation experiments. Under these conditions, only strains showing SK⁺ phenotype were able to prevail on the K-sensitive yeasts. These results suggest that the K⁺ phenotype could play a relevant role in spontaneous fermentations provided that the strain exhibits an SK⁺ phenotype, and that the latter phenotype should be preferred when selected K + strains are to be used as fermentation starters.     

The molecular genetic differentiation of cultured Saccharomyces strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that baker's yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae and S. bayanus var. uvarum hybrids. The molecular karyotyping of baker's, brewer's, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)5 can be used to study the populations of cultured S. cerevisiae strains.     

Physiological and genetic stability of hybrids of industrial wine yeasts Saccharomyces sensu stricto complex

Aims: The aim of this study was to examine the physiological and genetic stability of hybrids of industrial wine yeasts Saccharomyces sensu stricto complex subjected to acidic stress during fermentation. Methods and Results: Laboratory‐constructed yeast hybrids, one intraspecific Saccharomyces cerevisiae × S. cerevisiae and three interspecific S. cerevisiae ×Saccharomyces bayanus, were subcultured in aerobic or anaerobic conditions in media with or without l ‐malic acid. Changes in the biochemical profiles, karyotypes and mitochondrial DNA profiles of the segregates were assessed after 50–190 generations. All yeast segregates showed a tendency to increase the range of the tested compounds utilized as sole carbon sources. Interspecific hybrids were alloaneuploid and their genomes tended to undergo extensive rearrangement especially during fermentation. The karyotypes of segregates lost up to four and appearance up to five bands were recorded. The changes in their mtDNA patterns were even broader reaching 12 missing and six additional bands. These hybrids acquired the ability to sporulate and significantly changed their biochemical profiles. The alloaneuploid intraspecific S. cerevisiae hybrid was characterized by high genetic stability despite the phenotypic changes. l ‐malic acid was not found to affect the extent of genomic changes of the hybrids, which suggests that their demalication ability is combined with resistance to acidic stress. Conclusions: The results reveal the plasticity and extent of changes of chromosomal and mitochondrial DNA of interspecific hybrids of industrial wine yeast especially under anaerobiosis. They imply that karyotyping and restriction analysis of mitochondrial DNA make it possible to quickly assess the genetic stability of genetically modified industrial wine yeasts but may not be applied as the only method for their identification and discrimination. Significance and Impact of the Study: Laboratory‐constructed interspecific hybrids of industrial strains may provide a model for studying the adaptive evolution of wine yeasts under fermentative stress.     

A breeding strategy to harness flavor diversity of Saccharomyces interspecific hybrids and minimize hydrogen sulfide production

Industrial food-grade yeast strains are selected for traits that enhance their application in quality production processes. Wine yeasts are required to survive in the harsh environment of fermenting grape must, while at the same time contributing to wine quality by producing desirable aromas and flavors. For this reason, there are hundreds of wine yeasts available, exhibiting characteristics that make them suitable for different fermentation conditions and winemaking practices. As wine styles evolve and technical winemaking requirements change, however, it becomes necessary to improve existing strains. This becomes a laborious and costly process when the targets for improvement involve flavor compound production. Here, we demonstrate a new approach harnessing preexisting industrial yeast strains that carry desirable flavor phenotypes - low hydrogen sulfide (H(2) S) production and high ester production. A low-H(2) S Saccharomyces cerevisiae strain previously generated by chemical mutagenesis was hybridized independently with two ester-producing natural interspecies hybrids of S.?cerevisiae and Saccharomyces kudriavzevii. Deficiencies in sporulation frequency and spore viability were overcome through use of complementary selectable traits, allowing successful isolation of several novel hybrids exhibiting both desired traits in a single round of selection.     

Selection and hybridization of wine yeasts for improved winemaking properties: Fermentation rate and aroma productivity

Three strains out of thirty-one wine yeasts (Saccharomyces cerevisiae) were selected for their good winemaking properties (fermentation rate, tolerance of sulfur dioxide, aroma productivity, wine quality) and genetic markers (KHR killer activity, galactose assimilation), and used for hybridization by spore to spore mating. Of the twelve hybrids produced, two, Hy17-108 (RIFY 1001 × RIFY 1067) and Hy41-308 (RIFY 1001 × RIFY 1065), were selected on the basis of a fermentation test. In experimental winemaking the two hybrids demonstrated improved aroma productivity for higher alcohols, aromatic esters and/or fatty acids, while their fermentation rate was nearly the same as that of the parental strains.     

Monitoring of killer yeast populations in mixed cultures: influence of incubation temperature of microvinifications samples

Killer yeasts are frequently used to combat and prevent contamination by wild-type yeasts during wine production and they can even dominate the wine fermentation. Stuck and sluggish fermentations can be caused by an unbalanced ratio of killer to sensitive yeasts in the bioreactor, and therefore it is important to determine the proportion of both populations. The aim of this study was to provide a simple tool to monitor killer yeast populations during controlled mixed microvinifications of killer and sensitive Saccharomyces cerevisiae. Samples were periodically extracted during vinification, seeded on Petri dishes and incubated at 25 and 37?°C; the latter temperature was assayed for possible inactivation of killer toxin production. Colonies developed under the described conditions were randomly transferred to killer phenotype detection medium. Significant differences in the killer/sensitive ratio were observed between both incubation temperatures in all microvinifications. These results suggest that 37?°C seems a better option to determine the biomass of sensitive yeasts, in order to avoid underestimation of sensitive cells in the presence of killer yeasts during fermentations. Incubation at a toxin-inhibiting temperature clearly showed the real ratio of killer to sensitive cells in fermentation systems.     

Evolutionary Role of Interspecies Hybridization and Genetic Exchanges in Yeasts

Summary: Forced interspecific hybridization has been used in yeasts for many years to study speciation or to construct artificial strains with novel fermentative and metabolic properties. Recent genome analyses indicate that natural hybrids are also generated spontaneously between yeasts belonging to distinct species, creating lineages with novel phenotypes, varied genetic stability, or altered virulence in the case of pathogens. Large segmental introgressions from evolutionarily distant species are also visible in some yeast genomes, suggesting that interspecific genetic exchanges occur during evolution. The origin of this phenomenon remains unclear, but it is likely based on weak prezygotic barriers, limited Dobzhansky-Muller (DM) incompatibilities, and rapid clonal expansions. Newly formed interspecies hybrids suffer rapid changes in the genetic contribution of each parent, including chromosome loss or aneuploidy, translocations, and loss of heterozygosity, that, except in a few recently studied cases, remain to be characterized more precisely at the genomic level by use of modern technologies. We review here known cases of natural or artificially formed interspecies hybrids between yeasts and discuss their potential importance in terms of genome evolution. Problems of meiotic fertility, ploidy constraint, gene and gene product compatibility, and nucleomitochondrial interactions are discussed and placed in the context of other known mechanisms of yeast genome evolution as a model for eukaryotes.     

A Simple and Reliable Method for Hybridization of Homothallic Wine Strains of Saccharomyces cerevisiae

A procedure was developed for the hybridization and improvement of homothallic industrial wine yeasts. Killer cycloheximide-sensitive strains were crossed with killer-sensitive cycloheximide-resistant strains to get killer cycloheximide-resistant hybrids, thereby enabling hybrid selection and identification. This procedure also allows backcrossing of spore colonies from the hybrids with parental strains.     

Truth in wine yeast

Evolutionary history and early association with anthropogenic environments have made Saccharomyces cerevisiae the quintessential wine yeast. This species typically dominates any spontaneous wine fermentation and, until recently, virtually all commercially available wine starters belonged to this species. The Crabtree effect, and the ability to grow under fully anaerobic conditions, contribute decisively to their dominance in this environment. But not all strains of Saccharomyces cerevisiae are equally suitable as starter cultures. In this article, we review the physiological and genetic characteristics of S. cerevisiae wine strains, as well as the biotic and abiotic factors that have shaped them through evolution. Limited genetic diversity of this group of yeasts could be a constraint to solving the new challenges of oenology. However, research in this field has for many years been providing tools to increase this diversity, from genetic engineering and classical genetic tools to the inclusion of other yeast species in the catalogues of wine yeasts. On occasion, these less conventional species may contribute to the generation of interspecific hybrids with S. cerevisiae. Thus, our knowledge about wine strains of S. cerevisiae and other wine yeasts is constantly expanding. Over the last decades, wine yeast research has been a pillar for the modernisation of oenology, and we can be confident that yeast biotechnology will keep contributing to solving any challenges, such as climate change, that we may face in the future.     

Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: β-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Genetic studies on cycloheximide-resistant strains of Schizosaccharomyces pombe.

Cycloheximide-resistant mutants of Schizosaccharomyces pombe were isolated either as spontaneous mutants or after mutagenic treatment with nitrous acid, UV and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Twenty-three spontaneous mutants and 64 induced mutants were analysed genetically. Crosses revealed that at least four loci, designated cyh1, cyh2, cyh3 and cyh4 are responsible for resistance. Alleles of cyh1 show good growth on either high (100 mug/ml) or low (40 mug/ml) concentrations of cycloheximide whereas alleles at the cyh2, cyh3 and cyh4 loci gorw well on 40 mug/ml but poorly on 100 mug/ml. Some alleles at the cyh2 and cyh3 loci are also temperature sensitive (ts), the ts phenotype being conferred by the same gene as the resistance. In diploids, cyh1 and cyh4 are re-essive to wild type whereas cyh2 and cyh3 are semi-dominant. There was no intragenic complementation between three cyh1 alleles. Cross-resistance to trichodermin and anisomycin was shown by cyh2, cyh3 and cyh4 but not cyh1. Most cyh1 alleles, of spontaneous and UV origin only, were cold sensitive (cs) at 14 degrees and some of these were also cycloheximide dependent at the same temperature. It is suggested that the cyh1 and cyh4 genes are involved in ribosome formation or function and the other loci probably affect the uptake of cycloheximide by the cells.     

Fermentation characteristics of hybrids between the cryophilic wine yeast Saccharomyces bayanus and the mesophilic wine yeast Saccharomyces cerevisiae

The cryophilic wine yeasts Saccharomyces bayanus YM-84 and YM-126 were used for hybridization with the mesophilic wine yeast Saccharomyces cerevisiae OC-2. All six hybrids were stable in tetrad analysis and pulsed field gel electrophoresis, even after twenty subcultures over two years. The fermentabilities of these hybrids at a low temperature of 7°C were superior to the mesophilic wine yeast and the same as the cryophilic wine yeasts. Conversely, their fermentabilities at the intermediate temperatures of 28 and 35°C were similar to the mesophilic wine yeast. For laboratory-scale wine-making using Koshu grape juice at 10°C, the fermentability of these hybrids was superior to the mesophilic wine yeast. They also produced higher amounts of malic acid and flavor compounds such as higher alcohols, β-phenylethyl alcohol, isoamyl acetate and β-phenylethyl acetate, and lower amounts of acetic acid than those of OC-2. These results suggest that the cryophilic wine yeast S. bayanus is useful for improving the low temperature fermentation ability of wine yeast strains.     

Killer phenotype of indigenous yeasts isolated from Argentinian wine cellars and their potential starter cultures for winemaking

Of 31 yeasts, from different surfaces of two cellars from the northwest region of Argentina, 11 expressed killer activity against the sensitive strain Saccharomyces cerevisiae P351. Five of these killer yeasts were identified as S. cerevisiae by phenotypic tests and PCR-RFLP analysis. Two S. cerevisiae killer strains, Cf5 and Cf8, were selected based on their excellent kinetic and enological properties as potential autochthonous mixed starter cultures to be used during wine fermentation. They could dominate the natural microbiota in fermentation vats and keep the typical sensorial characteristics of the wine of this region.     

A Large Set of Newly Created Interspecific Saccharomyces Hybrids Increases Aromatic Diversity in Lager Beers

Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, “Saaz” and “Frohberg.” This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.     

Comorbidity of GJB2 and WFS1 mutations in one family

It is rarely reported that two distinct genetic mutations affecting hearing have been found in one family. We report on a family exhibiting comorbid mutation of GJB2 and WFS1. A four-generation Japanese family with autosomal dominant sensorineural hearing loss was studied. In 7 of the 24 family members, audiometric evaluations and genetic analysis were performed. We detected A-to-C nucleotide transversion (c.2576G>C) in exon 8 of WFS1 that was predicted to result in an arginine-to-proline substitution at codon 859 (R859P), G-to-A transition (c.109G>A) in exon 2 of GJB2 that was predicted to result in a valine-to-isoleucine substitution at codon 37 (V37I), and C-to-T transition (c.427C>T) in exon 2 of GJB2 that was predicted to result in an arginine-to-tryptophan substitution at codon 143 (R143W). Two individuals who had heterozygosity of GJB2 mutations and heterozygosity of WFS1 mutations showed low-frequency hearing loss. One individual who had homozygosity of GJB2 mutation without WFS1 mutation had moderate, gradual high tone hearing loss. On the other hand, a moderate flat loss configuration was seen in one individual who had compound heterozygosity of GJB2 and heterozygosity of WFS1 mutations. Our results indicate that the individual who has both GJB2 and WFS1 mutations can show GJB2 phenotype.     

Developmental instability in hybrids between Lychnis viscaria and Lychnis alpina (Caryophyllaceae)

Developmental instability shown by increased fluctuating asymmetry can be caused by either genetic or environmental stress. Genomic coadaptation and heterozygosity are the genetic factors that are commonly assumed to increase the level of developmental stability. Therefore, in hybrid populations the level of fluctuating asymmetry (FA) can be lower due to higher heterozygosity or higher due to disruption of coadapted gene complexes, depending on the degree of divergence between hybridizing taxa. Here I present data on FA in petals from hybrids between Lychnis viscaria (Caryophyllaceae) and Lychnis alpina and from parental species grown in a common garden environment. Petal asymmetry of hybrids was clearly higher than that of either parental species grown in common environment. Between the two parental species petal asymmetry did not differ. The mean size of the petals in hybrids was about the same as in L. viscaria, thus indicating no heterotic effect. Therefore, it seems that hybrids between L. viscaria and L. alpina do suffer from the disruption of coadapted gene complexes as indicated by higher developmental instability.