Convergent adaptation of Saccharomyces uvarum to sulfite,an antimicrobial preservative widely used in human-driven fermentations

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VII^XVI and XI^XVI chromosomal translocations generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments.     

Construction and evaluation of self‐cloning bottom‐fermenting yeast with high SSU1 expression

Aim: To construct a self‐cloning brewer’s yeast that can minimize the unfavourable flavours caused by oxidation and certain kinds of sulfur compounds. Methods and Results: DNA fragments of a high‐expression promoter from the TDH3 gene originating from Saccharomyces cerevisiae were integrated into the promoter regions of the S. cerevisiae‐type and Saccharomyces bayanus‐type SSU1 genes of bottom‐fermenting brewer’s yeast. PCR and sequencing confirmed the TDH3 promoter was correctly introduced into the SSU1 regions of the constructed yeasts, and no foreign DNA sequences were found. Using the constructed yeasts, the concentration of sulfite in fermenting wort was higher when compared with the parent strain. In addition, the concentrations of hydrogen sulfide, 3‐methyl‐2‐buten‐1‐thiol (MBT) and 2‐mercapto‐3‐methyl‐1‐butanol (2M3MB) were lower when compared with the parent strain. Conclusion: We successfully constructed a self‐cloning brewer’s yeast with high SSU1 expression that enhanced the sulfite‐excreting ability and diminished the production ability of hydrogen sulfide, MBT and 2M3MB. Significance and Impact of the Study: The self‐cloning brewer’s yeast with high SSU1 expression would contribute to the production of superior quality beer with a high concentration of sulfite and low concentrations of hydrogen sulfide, MBT and 2M3MB.     

Two alleles of the sulfite resistance genes are differentially regulated in Saccharomyces cerevisiae

The sulfite resistance gene, SSU1-R, is widely distributed in wine yeasts. This gene has an upstream region distinct from that of the allelic gene, SSU1 and SSU1-R is expressed at a much higher level than SSU1. We characterized the promoters of both of these genes by analysis of their activity using the LacZ gene as a reporter. FZF1, the activator gene of SSU1, was shown to regulate SSU1-R expression indirectly. SSU1-R expression was activated under microaerobic conditions, and four 76-bp repeats, present within the SSU1-R promoter region, was essential for high expression. These results indicate that SSU1-R expression is regulated in different manner from that of SSU1. By deletion analysis of the SSU1-R promoter region, we found that at least two of the 76-bp repeats are necessary for promoter activity, and that the number of 76-bp repeats influences the activity. Hence, it was suggested that the number of 76-bp repeats increases in wine yeasts that require strong sulfite resistance.     

QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite

Quantitative genetics and QTL mapping are efficient strategies for deciphering the genetic polymorphisms that explain the phenotypic differences of individuals within the same species. Since a decade, this approach has been applied to eukaryotic microbes such as Saccharomyces cerevisiae in order to find natural genetic variations conferring adaptation of individuals to their environment. In this work, a QTL responsible for lag phase duration in the alcoholic fermentation of grape juice was dissected by reciprocal hemizygosity analysis. After invalidating the effect of some candidate genes, a chromosomal translocation affecting the lag phase was brought to light using de novo assembly of parental genomes. This newly described translocation (XV-t-XVI) involves the promoter region of ADH1 and the gene SSU1 and confers an increased expression of the sulfite pump during the first hours of alcoholic fermentation. This translocation constitutes another adaptation route of wine yeast to sulfites in addition to the translocation VIII-t-XVI previously described. A population survey of both translocation forms in a panel of domesticated yeast strains suggests that the translocation XV-t-XVI has been empirically selected by human activity.     

Reidentification of chromosomal CUP1 translocations in the wine yeasts Saccharomyces cerevisiae

Reciprocal translocations between chromosomes XVI and VIII were revealed in eight Saccharomyces cerevisiae strains (mostly wine ones) using pulse-field electrophoresis of native chromosomal DNAs and their hybridizations with the CUP1 and GAL4 probes. New and reciprocal translocations of at least the gene CUP1 occur at the expense of crossing-over in the hybrids of such strains with the genetic lines of normal karyotype during meiosis. Relationship between these reciprocal translocations and the sulfite (Na₂SO₃) resistance gene SSU1-R is discussed.     

FLO gene-dependent phenotypes in industrial wine yeast strains

Most commercial yeast strains are nonflocculent. However, controlled flocculation phenotypes could provide significant benefits to many fermentation-based industries. In nonflocculent laboratory strains, it has been demonstrated that it is possible to adjust flocculation and adhesion phenotypes to desired specifications by altering expression of the otherwise silent but dominant flocculation (FLO) genes. However, FLO genes are characterized by high allele heterogeneity and are subjected to epigenetic regulation. Extrapolation of data obtained in laboratory strains to industrial strains may therefore not always be applicable. Here, we assess the adhesion phenotypes that are associated with the expression of a chromosomal copy of the FLO1, FLO5, or FLO11 open reading frame in two nonflocculent commercial wine yeast strains, BM45 and VIN13. The chromosomal promoters of these genes were replaced with stationary phase-inducible promoters of the HSP30 and ADH2 genes. Under standard laboratory and wine making conditions, the strategy resulted in expected and stable expression patterns of these genes in both strains. However, the specific impact of the expression of individual FLO genes showed significant differences between the two wine strains and with corresponding phenotypes in laboratory strains. The data suggest that optimization of the flocculation pattern of individual commercial strains will have to be based on a strain-by-strain approach.     

Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

Adaptive evolution of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to generate strains with enhanced glycerol production

The development of new wine yeast strains with improved characteristics is critical in the highly competitive wine market, which faces the demand of ever-changing consumer preferences. Although new strains can be constructed using recombinant DNA technologies, consumer concerns about genetically modified (GM) organisms strongly limit their use in food and beverage production. We have applied a non-GM approach, adaptive evolution with sulfite at alkaline pH as a selective agent, to create a stable yeast strain with enhanced glycerol production; a desirable characteristic for wine palate. A mutant isolated using this approach produced 41% more glycerol than the parental strain it was derived from, and had enhanced sulfite tolerance. Backcrossing to produce heterozygous diploids revealed that the high-glycerol phenotype is recessive, while tolerance to sulfite was partially dominant, and these traits, at least in part, segregated from each other. This work demonstrates the potential of adaptive evolution for development of novel non-GM yeast strains, and highlights the complexity of adaptive responses to sulfite selection.     

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO₂-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

Comparison of two alternative dominant selectable markers for wine yeast transformation

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 microg of DNA for the three wine yeast strains tested.     

Phenotypic and genotypic diversity of wine yeasts used for acidic musts

The aim of this study was to examine the physiological and genetic stability of the industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum under acidic stress during fermentation. The yeasts were sub-cultured in aerobic or fermentative conditions in media with or without l-malic acid. Changes in the biochemical profiles, karyotypes, and mitochondrial DNA profiles were assessed after minimum 50 generations. All yeast segregates showed a tendency to increase the range of compounds used as sole carbon sources. The wild strains and their segregates were aneuploidal or diploidal. One of the four strains of S. cerevisiae did not reveal any changes in the electrophoretic profiles of chromosomal and mitochondrial DNA, irrespective of culture conditions. The extent of genomic changes in the other yeasts was strain-dependent. In the karyotypes of the segregates, the loss of up to 2 and the appearance up to 3 bands was noted. The changes in their mtDNA patterns were much broader, reaching 5 missing and 10 additional bands. The only exception was S. bayanus var. uvarum Y.00779, characterized by significantly greater genome plasticity only under fermentative stress. Changes in karyotypes and mtDNA profiles prove that fermentative stress is the main driving force of the adaptive evolution of the yeasts. l-malic acid does not influence the extent of genomic changes and the resistance of wine yeasts exhibiting increased demalication activity to acidic stress is rather related to their ability to decompose this acid. The phenotypic changes in segregates, which were found even in yeasts that did not reveal deviations in their DNA profiles, show that phenotypic characterization may be misleading in wine yeast identification. Because of yeast gross genomic diversity, karyotyping even though it does not seem to be a good discriminative tool, can be useful in determining the stability of wine yeasts. Restriction analysis of mitochondrial DNA appears to be a more sensitive method allowing for an early detection of genotypic changes in yeasts. Thus, if both of these methods are applied, it is possible to conduct the quick routine assessment of wine yeast stability in pure culture collections depositing industrial strains.     

Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway

Aims: An evolution‐based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically, molybdate and chromate resistance was used as selectable phenotype to select sulfate permease–deficient variants that unable to produce sulfites and hydrogen sulfide (H₂S). Methods and Results: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the conjugation of gametes. Then, the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty‐four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfite production (SO₂ <10 mg l^?1) and with an impaired H₂S production in grape must without added sulfites were selected. Conclusions: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly, resistance to toxic analogues of sulfate allowed us to detect strains that unable to assimilate sulfates. Significance and Impact of the Study: This strategy that combines the sexual recombination of spores and application of a specific selective pressure provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H₂S.