Characterization of Ccw7p cell wall proteins and the encoding genes of Saccharomyces cerevisiae wine yeast strains: relevance for flor formation.

The specific flavour of Sherry-type wines requires aromatic compounds produced as by-products of the oxidative metabolism of yeasts that are able to form a biofilm (flor) at the wine surface. A similar yeast pellicle develops on the surface of 'Tokaji Szamorodni', one of the traditional Hungarian botrytized wines, during maturation. In this work, patterns of biotinylated cell wall proteins extracted from film-forming and nonfilm-forming Saccharomyces cerevisiae strains were compared. It was found that all the tested 23 film-forming 'Szamorodni' yeast strains had a decreased size of the Ccw7/Hsp150 protein, one of the members of the Pir-protein family. Sequencing of the encoding genes revealed that the strains were lacking three out of the 11 repeating sequences characteristic to this protein family. One of the film-forming strains contained CCW7 alleles of different length, which was generated by intragenic tandem duplication of a sequence containing two repetitive domains. Unlike the film-forming strains, 16 nonfilm-forming wine yeasts isolated from a different botrytized wine, 'Tokaji Aszu', showed pronounced polymorphism of the CCW7 locus. It is highly probable that the modified Ccw7 protein does not contribute to the increased hydrophobicity of film-forming strains but it may influence molecular reorganization of the cell wall during stress adaptation.

Kovács M ，Stuparevic I ，Mrsa V ，Maráz A ... -  《FEMS YEAST RESEARCH》

FLO11 is essential for pellicle formation by wild pellicle-forming yeasts isolated from contaminated wines.

The Δflo11/Δflo11 disruptant strains constructed from three wild yeast strains, which were independently isolated from pellicles formed on the surface of contaminated stored wines, completely lost their ability to form pellicles. The FLO11 expression level of the pellicle-forming strains was much higher than that of non-pellicle-forming wine strains. Cell surface hydrophobicity of the Δflo11/Δflo11 disruptant strains was as high as that of the parental pellicle-forming strains. These findings indicate that FLO11 is essential for unfavorable pellicle formation of wild pellicle-forming yeasts by mechanisms other than increasing cell surface hydrophobicity.

Nakagawa Y ，Toda Y ，Yamamura H ，Hayakawa M ，Iimura Y ... -  《-》

HSP12 is essential for biofilm formation by a Sardinian wine strain of S. cerevisiae.

Sardinian sherry strains of S. cerevisiae form a biofilm on the surface of wine at the end of the ethanolic fermentation, when grape sugar is depleted and when further growth becomes dependent on access to oxygen. A point mutation in HSP12 or deletion of the entire gene results in inability to form this film. HSP12 encodes a heat-shock protein previously foundby others to be active during stationary phase, in cells depleted for glucose, and in cells metabolizing ethanol and fatty acids, all conditions associated with sherry biofilms.

Zara S ，Antonio Farris G ，Budroni M ，Bakalinsky AT ... -  《YEAST》

Btn2p is involved in ethanol tolerance and biofilm formation in flor yeast.

Flor yeasts are a particular kind of Saccharomyces cerevisiae strains involved in Sherry wine biological ageing. During this process, yeasts form a film on the wine surface and use ethanol as a carbon source, producing acetaldehyde as a by-product. Acetaldehyde induces BTN2 transcription in laboratory strains. Btn2p is involved in the control of the subcellular localization of different proteins. The BTN2 gene shows a complex expression pattern in wine yeast, increasing its expression by acetaldehyde, but repressing it by ethanol. A flor yeast strain transcribes more BTN2 than a first fermentation yeast during growth, but less under different stress conditions. BTN2 deletion decreases flor yeast resistance to high ethanol concentrations. Surprisingly, this effect is suppressed by the addition of high amounts of amino acids to the growth medium, indicating that the role of Btn2p protein in amino acid transport is important for ethanol resistance. Btn2p deletion increases the fermentative capacity of flor yeast and its overexpression prevents its growth on nonfermentable carbon sources. BTN2 deletion also affects the biofilm formation ability of flor yeast, and it increases its sliding motility, resulting in increased mat formation. This correlates with an increased transcription of the FLO11 gene, a gene essential for biofilm formation.

Espinazo-Romeu M ，Cantoral JM ，Matallana E ，Aranda A ... -  《FEMS YEAST RESEARCH》

Physiological and molecular characterization of flor yeasts: polymorphism of flor yeast populations.

Yeast strains which form velum on the surface of Sherry wine during the aging process have been isolated and characterized. According to their metabolic and molecular features most of the yeasts that were isolated belong to different races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis and rouxii). Due to the conditions under which these yeasts were isolated, all strains have in common the capacity to develop a film as an adaptive mechanism which allows them to grow and survive in 15.5% vol. ethanol. All strains were prototrophs for amino acids and most vitamins but they gave different responses to the killer factor. However, whereas their physiological features were similar, they showed a great heterogeneity with regards to the nuclear and mitochondrial genome (mtDNA): DNA content per cell was quite variable (1.3 to 2n), electrophoretic karyotypes of nuclear genomes indicated a main pattern with some variations, and polymorphism shown by the mtDNA was very high. Under extreme conditions such as Sherry wine with 15.5% vol. ethanol, no fermentable sugar and an exclusively oxidative metabolism, cells hardly grow and the maintenance of a live population depends on survival and respiration, which in turn depend on the mtDNA. At the same time these environmental conditions are mutagenic for the mtDNA, causing an increase in variation. Thus, the polymorphism observed might reflect the enormous variability induced by the ethanol followed by the selection of those mtDNA sequences which make the mitochondria metabolically active under these conditions.

Martínez P ，Codón AC ，Pérez L ，Benítez T ... -  《YEAST》