Sake Yeast Research Articles

Brewing characteristics of haploid strains isolated from sake yeast Kyokai No. 7

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai No. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small-scale sake-brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups, indicating fermentation ability and sake flavour.

QTL analysis of a sake yeast

QTL analysis of a sake yeast

Inhibition of mitochondrial fragmentation during sake brewing causes high malate production in sake yeast

We previously demonstrated the presence and fragmentation of mitochondria during alcohol fermentation. Here, we show that Fis1p induces mitochondrial fragmentation, and inhibition of mitochondrial fragmentation causes higher malate production during sake brewing. These findings indicate that mitochondrial morphology affects the metabolism of constituents, providing a breeding strategy for high-malate-producing yeasts.

Direct ethanol production from barley β-glucan by sake yeast displaying Aspergillus oryzae β-glucosidase and endoglucanase

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.

Common industrial sake yeast strains have three copies of the AQY1–ARR3 region of chromosome XVI in their genomes

Genomic analysis of industrial yeast strains is important for exploitation of their potential. We analysed the genomic structure of the most widely used sake yeast strain, Kyokai no. 7 (K7), by DNA microarray. Since the analysis suggested that the copy number of the AQY1-ARR3 region in the right arm of chromosome XVI was amplified, we performed Southern blot analysis using the AQY1 gene as a probe. The probe hybridized to three bands in the widely used sake strains derived from K7, but only to one band of 1.4 kb in the laboratory strains. Since the extra two bands were not observed in old sake strains, or in other industrial strains, the amplification of this region appeared to be specific for the widely used sake strains. The copy number of the AQY1-ARR3 region appeared to have increased by chromosomal translocation, since chromosomal Southern blot analysis revealed that the AQY1 probe hybridized to chromosomes IV and XIII, in addition to chromosome XVI, in which AQY1 of the laboratory strain is encoded. The chromosomal translocation was also confirmed by PCR analysis using primers that amplify the region containing the breakpoint. Cloning and sequencing of cosmids that encode the AQY1-ARR3 region revealed that this region is flanked by TG(1-3) repeats on the centromere-proximal side in chromosomes IV and XIII, suggesting that amplification of this region occurred by homologous recombination through TG(1-3) repeats. These results demonstrate the genomic characteristics of the modern widely used sake strains that discriminate them from other strains.

Efficient and Direct Fermentation of Starch to Ethanol by Sake Yeast Strains Displaying Fungal Glucoamylases

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.

Traditional healthful fermented products of Japan

A variety of fermentation products, such as foods containing probiotic bacteria, black rice vinegar (kurosu), soy sauce (shoyu), soybean-barley paste (miso), natto and tempeh, are sold in food stores in Japan. These fermented food products are produced by traditional methods that exploit mixed cultures of various non-toxic microorganisms. These microorganisms include lactic acid bacteria, acetic acid bacteria, sake yeast, koji molds and natto bacteria. Many traditional fermented foods have been studied and their effects on metabolism and/or immune system have been demonstrated in animal and/or human cells. This review summarizes the scientific basis for the effects of these traditional food products, which are currently produced commercially in Japan.

清酒もろみからの多酸性清酒酵母の分離とその特性

We isolated a sake yeast from sake mash unique for its organic acid productivity throughout five years of brewing. Results were that 9 strains, which s howed the high productivity of malate over 2 times than the parent, were obtained from sake mash using the K 9 derivative strain. The resistance to cycloheximide and dimethyl succinate was similar to the parental strains, and the assimilation of maltose and glycerol fell in comparison with the parent. The high malate-producing strains obtained exhibited lower acetate productivity than the parent. Thus, we proposed the following mechanism for acquiring high malate-production: During the fermentation period, sake yeast was mutated in order to activate the malate biosynthetic pathway from acetyl-CoA made from the acetate and glyoxylate, and then the high malate-producing yeast acquired malate high productivity.

きょうかい清酒酵母使用蔵からの質問とその対策

[in Japanese]

低温高発酵性清酒酵母の分離

[in Japanese]

きょうかい1801号を用いた混合仕込

[in Japanese]

Elevated expression of genes under the control of stress response element (STRE) and Msn2p in an ethanol-tolerance sake yeast Kyokai No. 11

Elevated expression of genes under the control of stress response element (STRE) and Msn2p in an ethanol-tolerance sake yeast Kyokai No. 11

Elevated expression of genes under the control of stress response element (STRE) and Msn2p in an ethanol-tolerance sake yeast Kyokai no. 11

The sake yeast strain Kyokai no. 11 (K11) is an ethanol-tolerant mutant of strain Kyokai no. 7 (K7), which shows higher viability in an ethanol solution than strain K7. To clarify the mechanism underlying the ethanol tolerance of this strain, the gene expression profiles of K7 and K11 were analyzed using DNA microarrays. The results indicate that many genes induced by stresses were highly expressed in strain K11 not exposed to stresses. Analysis of HSP12, one of the most highly expressed genes in strain K11 compared with strain K7, revealed that a trans-acting factor of strain K11 was involved in the elevated expression of HSP12. Many of the highly expressed genes in strain K11 including HSP12 were under the control of a cis-acting factor called the stress response element (STRE). The addition of STRE sequences to a promoter region of a reporter gene resulted in constitutive high-level expression in strain K11. It was reported that transcription factors Msn2p and Msn4p bind to STRE sequences. DNA sequence analyses of MSN2 and MSN4 of strains K7 and K11 revealed that only Msn2p was functional in these strains. When two copies of MSN2 in strain K11 were disrupted, the expression level of the reporter gene under the control of STRE decreased to the level of strain K7, indicating that Msn2p is required for the elevated expression of the STRE-controlled genes in K11.

Intraspecies Diversity of the Industrial Yeast StrainsSaccharomyces cerevisiaeandSaccharomyces pastorianusBased on Analysis of the Sequences of the Internal Transcribed Spacer (ITS) Regions and the D1/D2 Region of 26S rDNA

We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions.

Construction and analysis of self-cloning sake yeasts that accumulate proline

We constructed self-cloning diploid sake yeast strains that accumulate proline. The appropriate proline level is important for its protective effect against ethanol stress in yeast cells. Sake brewed with the proline-accumulating strains contained two- to threefold more proline than the sake brewed with the parent strain. It was also suggested that intracellular proline does not affect overall fermentation profiles, but reduces fermentation time in terms of ethanol production rate.

Genome-Wide Expression Profile of Sake Brewing Yeast under Shaking and Static Conditions

To identify the genes responsible for characteristics, that are different as between sake brewing yeasts and laboratory yeast strains, we used a DNA microarray to compare the genome-wide gene expression profiles of a sake yeast, Saccharomyces cerevisiae K-9 (kyokai 9), and a laboratory yeast, S. cerevisiae X2180-1A, under shaking and static conditions. The genes overexpressed in K-9 more than in X2180-1A were related to C-metabolism, including the HXT, ATP, and COX genes, ergosterol biosynthesis, ERG genes, and thiamine metabolism, THI genes. These genes may contribute to higher growth rates and fermentation ability and the ethanol tolerance of sake yeast. The genes underexpressed in K-9 more than in X2180-1A were CUP1-1 and CUP1-2, PHO genes, which may explain the low copper tolerance and low acid phosphatase activity of sake yeast. These underexpressed genes agree with the features and the alteration of the genome structure of sake yeast.

RRNA遺伝子のスペーサー領域の塩基配列に基づく焼酎酵母の識別

Nucleotide sequences of the spacer regions between the small subunit rDNA and large subunit rDNA, and the spacer between the ribosomal gene clusters were analyzed to discriminate 11 shochu yeast strains. Clone libraries of the nontranscribed spacer (NTS) and the internal transcribed spacer (ITS) region of each strain were constructed and plural clones in each library were sequenced. Strain KF-3, which was constructed by protoplast fusion of shochu yeast and sake yeast, had a considerablequantity of unique sequences in both ITS and NTS regions compared with the other 10 strains. Although no difference was detected in the ITS region among the 10 strains other than KF-3, differences were detected in numerous sites in the NTS region. Shochu yeast strains investigated in this study were successfully discerned from each other according to the different nucletides in 11 sites in NTS region. The results suggested that the NTS region was highly variable and could be used to discriminate yeast strains with similar inheritance backgrounds.

口かみ「もろみ」に添加した清酒酵母の消長

Sake yeast with the fermentation ability for glucose, maltose and maltotriose was added to Kuchikami Rice Moromi. Increases and decreases of the microorganisms, the general composition, and the composition of saccharide were investigated, and the influence on the alcoholic production of the added sake yeast in Kuchikami Rice Moromi was examined. Though the added sake yeast increased in the early period of Kuchikami Rice Moromi, it decreased and disappeared about 5 days later. After that, though Saccharomyces sp.(wild type) appeared and increased, the number of cells was 106/ml. After the added sake yeast disappeared, the fermentation continued and the alcoholic production increased. This phenomenon was due to the alcoholic fermentation by Sacch. sp.(wild type), and was not influenced by the added sake yeast. Sacch. sp. and Lactic acid bacteria existed universally in Kuchikami Rice Moromi. The increase and decrease of those microorganisms was not constant as is Kimoto in sake production.

清酒酵母解析・開発におけるプロテオーム解析技術の応用

[in Japanese]

きょうかい1801号清酒酵母使用後のアンケート調査

きょうかい1801号清酒酵母使用後のアンケート調査