The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

Abstract

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N-and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.Sake is a Japanese traditional alcoholic beverage made from steamed rice by parallel fermentation with Aspergillus oryzae, which is a source of saccharification enzymes, and sake yeasts, which produce ethanol from glucose. Sake yeasts are classified as Saccharomyces cerevisiae and are known to produce more than 18% (vol/vol) ethanol in sake mash (16). Almost all sake yeasts form a thick foam layer on sake mash during vigorous fermentation. This foam formation has been used as an indicator of fermentation progress: the foam rises when fermentation becomes strong and it disappears when fermentation becomes weak. However, this characteristic sometimes reduces the efficiency of sake fermentation because a large part of the fermentation tank is occupied by a thick foam layer. Thus, an absence of foam formation during fermentation is a preferable property.Ouchi and Akiyama (23) developed a method to screen nonfoaming mutants from foaming sake yeast using cell affinity for bubbles, and using this method they isolated a nonfoaming mutant from an industrial sake yeast. This mutant had almost the same characteristics as the parental strain except for its nonfoaming property in fermentation. Thus, this method of screening for nonfoaming mutants was applied to various strains of sake yeasts and the resultant nonfoaming yeasts are now widely used in commercial sake brewing. Comparison of the nonfoaming mutant with its parent revealed that the cell surface of the former is less hydrophobic than that of the latter, suggesting that cell surface hydrophobicity is related to foaming ability (25). However, the detailed molecular mechanism of foaming ability of sake yeast is still unknown. In this study, we have cloned a gene from a foamin...

FLO11-Based Model for Air-Liquid Interfacial Biofilm Formation bySaccharomyces cerevisiae

“…One recognized difference is that the FLO11 transcriptional activator FLO8 harbors a nonsense mutation in S288C-derived laboratory strains (21). It is of interest that Shimoi et al (31) have reported that foam-formation by a sake strain of S. cerevisiae is dependent on a different hydrophobic cell surface protein, Awa1, that is absent in the laboratory strain S288C. Similarly, Alexandre et al (1), described an unidentified but abundant 49-kDa cell wall-associated mannoprotein with relative hydrophobic character, present in the film-forming wine yeast P3 isolated from a French sherry-like wine (vin jaune).…”

Systematic identification in silico of covalently bound cell wall proteins and analysis of protein-polysaccharide linkages of the human pathogen Candida glabrata

“…The nonpathogenic S. cerevisiae contains only 11 known adhesive proteins, including agglutinins, Awa1p-like proteins, which mediate the foam-forming capacity of sake yeast (Shimoi et al, 2002), and flocculins. The human pathogens C. glabrata and C. albicans seem to contain a much larger number of putative adhesive proteins.…”

Identification and Characterization of a Novel Biotin Biosynthesis Gene inSaccharomyces cerevisiae

“…Although they are classified as S. cerevisiae strains (37), sake yeasts have many characteristics that differ from those of other yeast strains, such as foam formation in mash (25), tolerance of high concentrations of ethanol, strong fermentation at low temperatures, and rich flavors (12,15,16,38). Another remarkable characteristic of sake yeasts is that they are prototrophic for biotin (29,30), in contrast to other S. cerevisiae strains.…”