Wine yeast fermentation vigor may be improved by elimination of recessive growth-retarding alleles

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.

Section: Yeastmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Analysis of Homothallic Saccharomyces cerevisiae Strain Mating during Must Fermentation

Ambrona

Ramírez

2007

“…However, we recommend that homozygous single-spore clones from heterozygous hybrids with the desired technological properties (27) be selected, since these homozygotes should not incur the sudden phenotypic changes that can ruin an industrial yeast strain. This process could eliminate recessive growth-retarding alleles from industrial yeast populations and improve fermentation vigor (28).…”

Section: Discussionmentioning

confidence: 99%

Genetic Instability of Heterozygous, Hybrid, Natural Wine Yeasts

Ramírez¹,

Vinagre²,

Ambrona³

et al. 2004

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2 R /cyh2 R mutants resistant to high levels of cycloheximide can be directly isolated from cyh2 S /cyh2 S wine yeasts. Heterozygous cyh2 R /cyh2 S hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2 R /cyh2 R homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.

“…For enology, the natural variability of Saccharomyces cerevisiae strains has been exploited by means of the isolation of wild yeast strains from grapes, musts, or successful fermentations; characterization of the isolates; and selection of the most suitable strains for each particular wine-making process or production area. Classical genetic methods such as the isolation of spontaneous or induced mutants or sexual crosses have been seldom used for the improvement of S. cerevisiae strains used for wine making (2,19,23,26,37), probably because of the difficulties in the design of selection procedures and because of the fact that, very often, the progeny of an excellent wine-making strain performs poorer than the parent strain (18). During the last 15 years, several attempts to construct genetically engineered yeast strains have been published, and very interesting improvements in the wine-making process or the quality of the wine obtained have been reported, including improved primary and secondary flavors, malic acid decarboxylation by yeast, increased resveratrol, lactic acid, or glycerol contents, and improved survival properties under technological conditions (6,11,16,17,20,27,32,34,38,43,46,47).…”

mentioning

confidence: 99%

Comparison of Two Alternative Dominant Selectable Markers for Wine Yeast Transformation

Cebollero

González

2004

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10 3 transformants per g of DNA for the three wine yeast strains tested.During the past century, the selection of microbial strains has been one of the main sources of improvement for traditional fermentation processes, including the production of fermented foods such as wine or dairy products. For enology, the natural variability of Saccharomyces cerevisiae strains has been exploited by means of the isolation of wild yeast strains from grapes, musts, or successful fermentations; characterization of the isolates; and selection of the most suitable strains for each particular wine-making process or production area. Classical genetic methods such as the isolation of spontaneous or induced mutants or sexual crosses have been seldom used for the improvement of S. cerevisiae strains used for wine making (2,19,23,26,37), probably because of the difficulties in the design of selection procedures and because of the fact that, very often, the progeny of an excellent wine-making strain performs poorer than the parent strain (18). During the last 15 years, several attempts to construct genetically engineered yeast strains have been published, and very interesting improvements in the wine-making process or the quality of the wine obtained have been reported, including improved primary and secondary flavors, malic acid decarboxylation by yeast, increased resveratrol, lactic acid, or glycerol contents, and improved survival properties under technological conditions (6,11,16,17,20,27,32,34,38,43,46,47). However, because wild yeast strains are usually prototrophic, most of the strains obtained for these works contained antibiotic resistance genes which were used for selection of the recombinant strains and would be unacceptable for marketing. Puig et al. (35) published an interesting method for obtaining recombinant wine yeast strains that are free of antibiotic selection markers. In this method, a ura3⌬ strain is first constructed by sequential gene replacement of the wild-type URA3 gene, and the kanamycin resistance cassette used to this end is removed by homologous recombination. The resulting auxotrophic strain can then be ...