Yeast population dynamics during the fermentation and biological aging of sherry wines

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of "fino" sherry wine making. The four races of "flor" Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-flor S. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations of S. cerevisiae strains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.     

Correlation between acetaldehyde and ethanol resistance and expression of HSP genes in yeast strains isolated during the biological aging of sherry wines

In the production of sherry wines, the process of biological aging is essential for the development of their organoleptic properties. This process involves velum formation by "flor" yeasts. Several of these yeast strains have been isolated and characterized with regard to their genetic, physiological and metabolic properties. In this work, we studied their resistance to cold-, osmotic-, oxidative-, ethanol- and acetaldehyde-stress, and found, in most cases, a correlation between resistance to acetaldehyde stress and ethanol stress and isolation from "soleras." Moreover, gene expression analysis revealed induction of the heat shock protein (HSP) genes HSP12, HSP82, and especially HSP26 and HSP104, under acetaldehyde stress in most of the strains. In strain C, there was a clear correlation between resistance to ethanol and acetaldehyde, the high induction of HSP genes by these compounds and its presence as the predominant strain in most levels of several soleras.     

Yeast Population Dynamics during the Fermentation and Biological Aging of Sherry Wines

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of “fino” sherry wine making. The four races of “flor” Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-flor S. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations of S. cerevisiae strains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.     

Acetaldehyde and ethanol are responsible for mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) in flor yeasts

Flor yeasts grow and survive in fino sherry wine where the frequency of respiratory-deficient (petite) mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. However, restriction fragment length polymorphism (RFLP) of mtDNA from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of sherry wine on mtDNA. mtDNA RFLP increases as the concentration of these compounds also increases, but is followed by a total loss of mtDNA in petite cells. Yeasts with functional mitochondria (grande) are target of continuous variability, so that flor yeast mtDNA can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas petite mutants are eventually eliminated because metabolism in sherry wine is oxidative.     

RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces: a fast method for species identification and the differentiation of flor yeasts

The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus × S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.     

Genetic differentiation of the sherry yeasts Saccharomyces cerevisiae

Molecular genetic study of the yeast Saccharomyces cerevisiae isolated at various stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from the primary winemaking yeasts according to several physiological and molecular markers. All sherry strains independently of the place and time of their isolation carry a 24-bp deletion in the ITS 1 region of ribosomal DNA, whereas the yeasts of the primary winemaking lack this deletion. Molecular karyotypes of the sherry yeast from different populations were found very similar.     

Genetic Differentiation of the Sherry Yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Molecular and genetic studies of the yeast Saccharomyces cerevisiae isolated at distinct stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from primary winemaking yeasts according to several physiological and molecular markers. All sherry strains, regardless of the place and time of their isolation, carry a 24-bp deletion in the ITS1 region of ribosomal DNA, whereas the yeasts of primary winemaking lack this deletion. Molecular karyotypes of sherry yeasts from different populations were found to be very similar.     

Retention of Browning Compounds by Yeasts Involved in the Winemaking of Sherry Type Wines

Wine model solutions were used to study the ability of dehydrated yeasts to retain the brown products formed in the reaction between (+)-catechin and acetaldehyde. Saccharomyces cerevisiae races capensis and bayanus, two typical flor yeasts involved in the biological aging of sherry wines, had a higher capacity to retain coloured compounds than S. cerevisiae fermentative yeast. Of the flor yeasts, capensis exhibited a higher colour reduction capacity than bayanus. Such differences may account for the different rate at which browning compounds are removed at different times of year during the biological aging of wines.     

Changes in volatile compounds and aromatic series in sherry wine with high gluconic acid levels subjected to aging by submerged flor yeast cultures

Volatile compounds of sherry wine containing gluconic acid under aging by submerged flor yeast cultures were analyzed. The aroma profile was obtained by grouping the compounds in nine aromatic series. The balsamic, fatty, herbaceous and empyreumatic series increased significantly as consequence of the increase of pantolactone, acids (butanoic, 2-methylbutanoic and 3-methylbutanoic), methionol and gamma-butyrolactone compounds, respectively. The decrease of higher alcohols promoted solvent series diminished. These changes are consistent with those observed in the production of commercial sherry wine using traditional biological aging.     

Authentication and identification of Saccharomyces cerevisiae‘flor’ yeast races involved in sherry ageing

Yeasts involved in velum formation during biological ageing of sherry wine have to date been classified into four races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, rouxii) according to their abilities to ferment different sugars. It has been proposed that race succession during biological ageing is essential for the development of the organoleptical properties of sherry wines. In this work we studied the physiological characteristics, the molecular differentiation and the phylogenetic relationships of the four races employing type and reference strains from culture collections and natural environments. Using restriction analysis of the ribosomal region that includes the 5.8S rRNA gene and internal transcribed regions (5.8S-ITS) we were able to differentiate 'flor' and non-'flor' S. cerevisiae yeast strains. However, no correlation between fermentation profile, mitochondrial DNA restriction analysis or chromosomal profiles and these races was found. Moreover, sequences of the D1/D2 domain of the 26S rRNA gene and the 5.8S-ITS region from these strains were analysed and no genetic differences were noted suggesting that 'flor' yeast cannot be grouped into four different races and the four races are identified as S. cerevisiae. Since the yeasts isolated from velum in sherry wine present a unique 5.8S rRNA pattern different from the rest of the Saccharomyces cerevisiae strains we propose that they should be included as a single race or variety inside the S. cerevisiae taxon.     

Continuous Production of Flor Sherry from New York State Wines

Flor sherry-like wines were produced continuously from New York State wines from Delaware or cold-pressed Concord grapes whose pigment contents were reduced with activated carbon. The course of flor fermentation was followed by total aldehyde analysis. Optimal flor production was observed at 18 to 20 C. Two continuous methods of fermentation were used. A glass column packed with ceramic saddles densely covered with yeasts gave good results, but required more careful management than the second method of submerged fermentation in a laboratory fermentor, which gave a higher sherry output and higher aldehyde production. With the laboratory fermentor, it was possible to obtain a sherry output of 22 liters per 24 hr with an aldehyde content of 300 to 500 mg per liter. The flor sherry produced by these methods required subsequent aging and fortification to the desired alcohol content.     

French Jura flor yeasts: genotype and technological diversity

Fifty-four Saccharomyces cerevisiae strains were isolated from Jura “Vin Jaune” velum and characterized by conventional physiological and molecular tests including ITS RFLP and sequence analysis, karyotyping and inter delta typing. ITS RFLP and sequence revealed a specific group of related strains different from the specific profile of Sherry flor yeast caused by a 24 bp deletion in the ITS1 region described by Esteve-Zarzoso et al. (Antonie Van Leeuwenhoek 85:151–158, 2004). Interdelta typing, the most discriminative method, revealed a high diversity of Jura flor yeast strains and gathered strains in clusters unequally shared between the northern and southern part of the Jura vineyard. The assessment of phenotypic diversity among the isolated strains was investigated for three wine metabolites (ethanal, acetic acid, and sotolon) from micro scale velum tests. Except at an early stage of ageing, the production of these metabolites was not correlated to the five genetic groups obtained by interdelta typing, but correlated to the cellar where strains had been isolated. The different strains isolated in a cellar produced mostly one type of velum (thin or thick, grey or white); but thin and grey velums, recognized as responsible for high quality wines, were obtained more frequently for one of the five groups of delta genotypes.     

Changes in gluconic acid, polyols and major volatile compounds in sherry wine during aging with submerged flor yeast cultures

The traditional biological process by which sherry wines are aged can be accelerated by using submerged Saccharomyces cerevisiae var. capensis (G1) strain cultures previously grown in glycerol. The used controlled shaking conditions raise the acetaldehyde, acetoin, and meso 2,3-butanediol contents in the wine, and increases the consumption of gluconic acid by flor yeast relative to traditional biological aging under flor yeast velum.     

Unveiling nonessential gene deletions that confer significant morphological phenotypes beyond natural yeast strains

Background

Phenotypes are variable within species, with high phenotypic variation in the fitness and cell morphology of natural yeast strains due to genetic variation. A gene deletion collection of yeast laboratory strains also contains phenotypic variations, demonstrating the involvement of each gene and its specific function. However, to date, no study has compared the phenotypic variations between natural strains and gene deletion mutants in yeast.

Results

The morphological variance was compared between 110 most distinct gene deletion strains and 36 typical natural yeast strains using a generalized linear model. The gene deletion strains had higher morphological variance than the natural strains. Thirty-six gene deletion mutants conferred significant morphological changes beyond that of the natural strains, revealing the importance of the genes with high genetic interaction and specific cellular functions for species conservation.

Conclusion

Based on the morphological analysis, we discovered gene deletion mutants whose morphologies were not seen in nature. Our multivariate approach to the morphological diversity provided a new insight into the evolution and species conservation of yeast.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-932) contains supplementary material, which is available to authorized users.     

Population Structure and Comparative Genome Hybridization of European Flor Yeast Reveal a Unique Group of Saccharomyces cerevisiae Strains with Few Gene Duplications in Their Genome

Wine biological aging is a wine making process used to produce specific beverages in several countries in Europe, including Spain, Italy, France, and Hungary. This process involves the formation of a velum at the surface of the wine. Here, we present the first large scale comparison of all European flor strains involved in this process. We inferred the population structure of these European flor strains from their microsatellite genotype diversity and analyzed their ploidy. We show that almost all of these flor strains belong to the same cluster and are diploid, except for a few Spanish strains. Comparison of the array hybridization profile of six flor strains originating from these four countries, with that of three wine strains did not reveal any large segmental amplification. Nonetheless, some genes, including YKL221W/MCH2 and YKL222C, were amplified in the genome of four out of six flor strains. Finally, we correlated ICR1 ncRNA and FLO11 polymorphisms with flor yeast population structure, and associate the presence of wild type ICR1 and a long Flo11p with thin velum formation in a cluster of Jura strains. These results provide new insight into the diversity of flor yeast and show that combinations of different adaptive changes can lead to an increase of hydrophobicity and affect velum formation.     

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.     

Genomics and biochemistry of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeast strains

Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term “unconscious” selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces. Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using “classical” and modern techniques for improving wine-making technology.     

Chemotaxis of Pseudomonas syringae subsp. savastanoi Virulence Mutants

Several mutants of Pseudomonas syringae subsp. savastanoi were tested for their ability to sense and respond to a chemotactic gradient in low concentrations of yeast extract. The mutants were deficient in one or both of the genes coding for the synthesis of the plant hormones indole-3-acetic acid (IAA) and isopentenyl adenosine. Mutations which resulted in the loss of IAA production were due to the loss of the entire plasmid containing the iaa operon or to an 18-kb deletion of the iaa region. Additional mutants tested were deficient in their ability to produce isopentenyl adenosine as a result of the loss of the ptz-bearing plasmid. In all cases, strains which had lost the ability to produce IAA exhibited enhanced motility of up to 2.5 times that of the wild type (IAA⁺) in medium containing 0.01% yeast extract. No differences in motility were observed on medium containing lower concentrations of yeast extract. The presence or absence of the cytokinin plasmid and the presence or absence of inorganic nitrogen in the medium had no effect on the relative mobility of the strains.     

Genome-wide amplifications caused by chromosomal rearrangements play a major role in the adaptive evolution of natural yeast

The relative importance of gross chromosomal rearrangements to adaptive evolution has not been precisely defined. The Saccharomyces cerevisiae flor yeast strains offer significant advantages for the study of molecular evolution since they have recently evolved to a high degree of specialization in a very restrictive environment. Using DNA microarray technology, we have compared the genomes of two prominent variants of S. cerevisiae flor yeast strains. The strains differ from one another in the DNA copy number of 116 genomic regions that comprise 38% of the genome. In most cases, these regions are amplicons flanked by repeated sequences or other recombination hotspots previously described as regions where double-strand breaks occur. The presence of genes that confer specific characteristics to the flor yeast within the amplicons supports the role of chromosomal rearrangements as a major mechanism of adaptive evolution in S. cerevisiae. We propose that nonallelic interactions are enhanced by ethanol- and acetaldehyde-induced double-strand breaks in the chromosomal DNA, which are repaired by pathways that yield gross chromosomal rearrangements. This mechanism of chromosomal evolution could also account for the sexual isolation shown among the flor yeast.