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The EHL1/2/3 genes were identified by whole-genome sequencing of Kyokai No. 7 (K7), which is a well-known representative Japanese sake yeast Saccharomyces cerevisiae. The genes are present in K7, but not in laboratory strain S288C. Although the genes were presumed to encode epoxide hydrolase based on homology analysis, their effect on cellular metabolism in sake yeast has not yet been clarified. We constructed ehl1/2/3 mutants harboring a stop codon in each gene using the haploid yeast strain H3 as the parental strain, which was derived from K701, and investigated the physiological role and effects of the EHL1/2/3 genes on sake quality. Metabolome analysis and vitamin requirement testing revealed that the EHL1/2/3 genes are partly responsible for the synthesis of pantothenate. For fermentation profiles, ethanol production by the ehl1/2/3 mutant was comparable with that of strain H3, but succinate production was decreased in the ehl1/2/3 mutant compared to strain H3 when cultured in yeast malt (YM) medium containing 10% glucose and during sake brewing. Ethyl hexanoate and isoamyl acetate levels in the ehl1/2/3 mutant strain were decreased compared to those of strain H3 during sake brewing. Thus, the EHL1/2/3 genes did not affect ethanol production but did affect the production of organic acids and aromatic components during sake brewing.

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Improving grain quality is a primary objective in contemporary rice breeding. Japanese modern rice breeding has developed two different types of rice, eating and sake-brewing rice, with different grain characteristics, indicating the selection of variant gene alleles during the breeding process. Given the critical importance of promptly and efficiently identifying genes selected in past breeding for future molecular breeding, we conducted genome scans for divergence, genome-wide association studies, and map-based cloning. Consequently, we successfully identified two genes, OsMnS and OsWOX9D, both contributing to rice grain traits. OsMnS encodes a mannan synthase that increases the white core frequency in the endosperm, a desirable trait for sake brewing but decreases the grain appearance quality. OsWOX9D encodes a grass-specific homeobox-containing transcription factor, which enhances grain width for better sake brewing. Furthermore, haplotype analysis revealed that their defective alleles were selected in East Asia, but not Europe, during modern improvement. In addition, our analyses indicate that a reduction in grain mannan content during African rice domestication may also be caused a defective OsMnS allele due to breeding selection. This study not only reveals the delicate balance between grain appearance quality and nutrition in rice but also provides a new strategy for isolating causal genes underlying complex traits, based on the concept of "breeding-assisted genomics" in plants.

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Although sake yeast mainly produces the taste of sake, sake brewery-inhabiting (kuratsuki) bacteria affect the taste of sake. Thus, kuratsuki bacteria may alter the metabolism of sake yeast through interactions between kuratsuki bacteria and sake yeast. This study aimed to confirm the effects of the combination of kuratsuki Kocuria TGY1127_2 and different sake yeast strains, AK25, K901, and K1801 on the taste of sake. Although the Brix and acidity during sake production using AK25 differed between sake with and without kuratsuki Kocuria, those using K901 and K1801 did not differ. Thus, sake yeast AK25 interacted with kuratsuki Kocuria and changed its characteristics of ethanol fermentation. In addition, the taste intensity changes, measured with a taste sensor TS-5000Z, showed that the effects of adding kuratsuki Kocuria varied among different sake yeasts. Thus, each sake yeast strain interacted with the kuratsuki bacterium and produced different metabolites, resulting in a change in the taste of sake. The findings of this study can lead to the brewing of sake using different types of kuratsuki bacteria which can affect the taste of sake.

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This study investigated the interactions of four bacteria strains isolated from Yamahai-shubo, the source of yeast used to produce a Japanese traditional rice wine, Yamahai-shikomi sake. The bacterial strains were nitrate-reducing Pseudomonas sp. 61-02, Leuconostoc mesenteroides LM-1, Lactiplantibacillus plantarum LP-2, and Latilactobacillus sakei LS-4. We examined fermentation factors for Yamahai-shubo and Yamahai-shikomi sake samples to compare the suitability of their bacterial combination (16 variations). As a result of principal component analysis, we found that two major groups were formed; one containing strain LP-2 and the other containing strain LS-4, and that strains LP-2 and LS-4 were important in the Yamahai-shikomi sake in the presence of strains 61-02 and LM-1. Then, we investigated the effects of strains LP-2 and LS-4 on the concentration of organic acids (pyruvic acid, citric acid, succinic acid, malic acid, and lactic acid) in Yamahai-shikomi sake. Only in lactic acid, a tendency to decrease with a smaller proportion of LS-4 strains in Yamahai-shubo was observed. Subsequently, their effect on the concentration of diacetyl, crucial for aroma, was investigated between the LP-2 and LS-4 strains. The sample prepared in the absence of strain LS-4 exhibited the lowest concentration of diacetyl. This result was supported by the statistical analysis for the sensory scores performed for aroma of each Yamahai-shikomi sake sample. In conclusion, strain LP-2 plays a more significant role in improving Yamahai-shikomi sake quality with strains LM-1 and 61-02 rather than strain LS-4 in Yamahai-shubo preparation and Yamahai-shikomi sake brewing.

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Koji is made using steamed rice and a koji mold, which plays an essential role in sake brewing. We challenge to build a new sake brewing method using the kuratsuki bacteria that have inhabited each sake brewery. In this paper, effects of the kuratsuki Kocuria strain TGY1127_2 were estimated on sake brewing in different koji conditions. Sake was produced by incubation of a mixture solution of koji, water, and sake yeast (strain K1401) with and without the kuratsuki Kocuria TGY1127_2. The effects of the kuratsuki Kocuria on the taste of the sake differed among different koji. The kuratsuki Kocuria led to an increase in ethanol concentration. Additionally, the sugar content (Brix) and acidity of the sake increased in proportion to the amount of koji. These results strongly suggest that the kuratsuki Kocuria does not adversely affect the fermentation activity of the sake yeast. Thus, the kuratsuki Kocuria had different effects on the taste of sake among different koji but the fermentation activity of the sake yeast was maintained.

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In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR+ ] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR+ ] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR+ ] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR+ ] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR+ ] cells in the industrial sake strains but not in the kimoto strains.

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The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.

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Sake (Japanese rice wine) was fermented in pottery for more than a millennium before wooden barrels were adopted to obtain a greater brewing capacity. Although a recently conducted analysis of sake brewed in pottery indicated that sake brewed in unglazed pottery contains more ethanol than that brewed in glazed pottery, little is known about the characteristics of sake brewed in pottery. In this study, we used two types of ceramic containers of identical size, one glazed and one unglazed, for small-scale sake brewing to evaluate the effects of glazing on fermentation properties. The following parameters were measured continuously in the sake samples over 3 weeks of fermentation: temperature, weight, ethanol concentration, and glucose concentration in sake mash. Taste-sensory values, minerals, and volatile components were also quantified in the final fermented sake mash. The results show that, in the unglazed containers, the temperature of the sake mash was lower and the weight loss was higher compared to the sake mash in the glazed containers. The quantity of ethanol and the levels of Na+, Fe3+, and Al3+ tended to be higher in the sake brewed in the unglazed pottery. A taste-sensory analysis revealed that umami and saltiness were also higher in the samples brewed in the unglazed pottery. These results suggest that glazing affects multiple fermentation parameters and the flavor of sake brewed in pottery. They may also suggest that the materials of the containers used in sake brewing generally affect the fermentation properties.

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Sake is a traditional Japanese alcoholic beverage made from rice and water, fermented by the filamentous fungi Aspergillus oryzae and the yeast Saccharomyces cerevisiae. Yeast strains, also called sake yeasts, with high alcohol yield and the ability to produce desired flavor compounds in the sake, have been isolated from the environment for more than a century. Furthermore, numerous methods to breed sake yeasts without genetic modification have been developed. The objectives of breeding include increasing the efficiency of production, improving the aroma and taste, enhancing safety, imparting functional properties, and altering the appearance of sake. With the recent development of molecular biology, the suitable sake brewing characteristics in sake yeasts, and the causes of acquisition of additional phenotypes in bred yeasts have been elucidated genetically. This mini-review summarizes the history and lineage of sake yeasts, their genetic characteristics, the major breeding methods used, and molecular biological analysis of the acquired strains. The data in this review on the metabolic mechanisms of sake yeasts and their genetic profiles will enable the development of future strains with superior phenotypes.

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Sake is a traditional Japanese alcoholic beverage brewed by the yeast Saccharomyces cerevisiae. Since the consumption and connoisseurship of sake has spread around the world, the development of new sake yeast strains to meet the demand for unique sakes has been promoted. Phenylalanine is an essential amino acid that is used to produce proteins and important signaling molecules involved in feelings of pleasure. In addition, phenylalanine is a precursor of 2-phenylethanol, a high-value aromatic alcohol with a rose-like flavor. As such, adjusting the quantitative balance between phenylalanine and 2-phenylethanol may introduce value-added qualities to sake. Here, we isolated a sake yeast mutant (strain K9-F39) with phenylalanine accumulation and found a missense mutation on the ARO80 gene encoding the His309Gln variant of the transcriptional activator Aro80p involved in the biosynthesis of 2-phenylethanol from phenylalanine. We speculated that mutation of ARO80 would decrease transcriptional activity and suppress the phenylalanine catabolism, resulting in an increase of intracellular phenylalanine. Indeed, sake brewed with strain K9-F39 contained 60% increase in phenylalanine, but only 10% less 2-phenylethanol than sake brewed with the parent strain. Use of the ARO80 mutant in sake brewing may be promising for the production of distinctive new sake varieties. ONE-SENTENCE SUMMARY: The ARO80 mutant is appropriate for controlling the content of phenylalanine and 2-phenylethanol.

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Kocuria isolates collected from the sake brewing process have inhabited the Narimasa Sake Brewery in Toyama, Japan. To investigate the effect of these actinobacterial isolates on the growth and metabolism of sake yeast, co-cultivation of sake yeast and Kocuria isolates was performed in a medium containing tryptone, glucose and yeast extract (TGY), and a solution containing koji (steamed rice covered with Aspergillus oryzae) and glucose. In the TGY medium, the ethanol concentration and the number of living cells of each microorganism were measured. In the koji solution, the concentrations of ethanol and organic acids (citric acid, lactic acid and succinic acid) were measured. The results showed that in TGY media, the growth of each Kocuria isolate in the co-culture of the two Kocuria isolates was similar to that in each monoculture. However, the growth of both Kocuria isolates was inhibited in the co-cultures of sake yeast and Kocuria isolates. On the other hand, the growth and ethanol productivity of sake yeast did not differ between its monoculture and co-cultures with Kocuria isolates. In the koji solution, Kocuria isolates TGY1120_3 and TGY1127_2 affected the concentrations of ethanol and lactic acid, respectively. Thus, Kocuria isolates affected the microbial metabolism, but the effects were not identical between the two isolates. This strongly suggests that bacteria inhabiting a sake brewery may influence the flavor and taste of sake products of the brewery.

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Bacteria belonging to the genus Kocuria were identified as bacteria peculiar to a sake brewery in Toyama, Japan. Comparison of the 16S rRNA gene sequences revealed two groups of Kocuria isolates. Among known species, one group was similar to K. koreensis (Kk type), and the other, K. uropygioeca (Ku type). We determined complete genomic DNA sequences from two isolates, TGY1120_3 and TGY1127_2, which belong to types Kk and Ku, respectively. Comparison of these genomic information showed that these isolates differ at the species level with different genomic characters. Isolate TGY1120_3 comprised one chromosome and three plasmids, and the same transposon coding region was located on two loci on the chromosome and one locus on one plasmid, suggesting that the genetic element may be transferred between the chromosome and plasmid. Isolate TGY1127_2 comprised one chromosome and one plasmid. This plasmid encoded an identical transposase coding region, strongly suggesting that the genetic element may be transferred between these different isolates through plasmids. These four plasmids carried a highly similar region, indicating that they share a common ancestor. Thus, these two isolates may form a community and exchange their genetic information during sake brewing.

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Sake-brewing cultivars among varieties of Japanese rice (Oryza sativa L.) have traits adapted to the sake-brewing process, such as a high white-core expression rate (WCE). Our previous study detected putative quantitative trait loci (QTLs) associated with a high WCE derived from Yamadanishiki, a popular brewing rice cultivar. Because the occurrence of white-core grains depends on air temperature and the position of the grain on the panicle, phenotyping of WCE must consider these variable conditions. In this study, qWCE6, a QTL for the WCE on chromosome 6, was validated for the first time, and the phenotyping method examined for its suitability in fine-mapping. A clear tendency towards high WCE was observed in late-heading substituted lines which headed under low daily mean temperature at the experimental location. White-core grains were often expressed by the primary spikelets on the upper panicle, producing a high percentage of superior grains. The segregating population for qWCE6 in late heading revealed a distinct difference in WCE between the Koshihikari and Yamadanishiki homozygous alleles at qWCE6 as determined from that locality. Further, two insertion/deletion markers were developed for the marker-assisted selection of qWCE6. Our results will be useful for informing the breeding of sake-brewing rice cultivars.

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N-Acetyltransferase Mpr1 was originally discovered as an enzyme that detoxifies L-azetidine-2-carboxylate through its N-acetylation in the yeast Saccharomyces cerevisiae Σ1278b. Mpr1 protects yeast cells from oxidative stresses possibly by activating a novel L-arginine biosynthesis. We recently constructed a stable variant of Mpr1 (N203K) by a rational design based on the structure of the wild-type Mpr1 (WT). Here, we examined the effects of N203K on ethanol fermentation of the sake yeast S. cerevisiae strain lacking the MPR1 gene. When N203K was expressed in the diploid Japanese sake strain, its fermentation performance was improved compared to WT. In a laboratory-scale brewing, a sake strain expressing N203K produced more ethanol than WT. N203K also affected the contents of flavor compounds and organic acids. These results suggest that the stable Mpr1 variant contributes to the construction of new industrial yeast strains with improved fermentation ability and diversity of taste and flavor.

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The formation of guaiacol, a potent phenolic off-odor compound in the Japanese sake brewing process, was investigated. Eight rice koji samples were analyzed, and one contained guaiacol and 4-vinylguaiacol (4-VG) at extraordinarily high levels: 374 and 2433 µg/kg dry mass koji, respectively. All samples contained ferulic and vanillic acids at concentrations of mg/kg dry mass koji. Guaiacol forming microorganisms were isolated from four rice koji samples. They were identified as Bacillus subtilis, B. amyloliquefaciens/subtilis, and Staphylococcus gallinarum using 16S rRNA gene sequence. These spoilage bacteria convert vanillic acid to guaiacol and ferulic acid to 4-VG. However, they convert very little ferulic acid or 4-VG to guaiacol. Nine strains of koji fungi tested produced vanillic acid at the mg/kg dry mass koji level after cultivation. These results indicated that spoilage bacteria form guaiacol from vanillic acid, which is a product of koji cultivation in the sake brewing process.

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The relationship between the protein composition of rice and nitrogen compounds (amino acids and oligo-peptides) in the produced sake were investigated using endosperm protein mutant rice (LGC-1, LGC-Jun, Kx433, QA28), sake rice (Yamadanishiki) and cooking rice (Nipponbare, Nihonmasari, Koshihikari). The total nitrogen concentration, amino acid concentration and most peptide peak areas determined by RP-HPLC and gel filtration chromatography of the produced sake were lower when sake was made from a low glutelin content rice mutant compared with other rice varieties. The concentration of nitrogen compounds in the sake positively correlated with the glutelin content of the highly milled rice grains used for sake production. Sake produced using a low glutelin content rice mutant is generally evaluated as having a light taste. Our findings suggest that nitrogen compounds (oligo-peptides and amino acids) derived from rice glutelin significantly contribute to the taste of sake.

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Low protein content and sufficient grain rigidity are desired properties for the rice used in high-quality sake brewing such as Daiginjo-shu (polishing ratio of the rice, less than 50%). Two kinds of rice, sake rice (SR) and cooking rice (CR), have been used for sake brewing. Compared with those of SR, analyses of CR for high-quality sake brewing using highly polished rice have been limited. Here we described the original screening of late-maturing CR Sensyuraku (SEN) as rice with low protein content and characterization of its properties for high-quality sake brewing. The protein content of SEN was lower than those of SR Gohyakumangoku (GOM) and CR Yukinosei (YUK), and its grain rigidity was higher than that of GOM. The excellent properties of SEN with respect to both water-adsorption and enzyme digestibility were confirmed using a Rapid Visco Analyzer (RVA). Further, we confirmed a clear taste of sake produced from SEN by sensory evaluation. Thus, SEN has excellent properties, equivalent to those of SR, for high-quality sake brewing.

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The present study showed that the lysis of yeast cells and subsequent release of cell contents in sake mash accelerated dimethyl trisulfide (DMTS) formation. Among these, heat unstable and relatively high molecular weight compounds were assumed to be enzymes; thus, enzymatic reactions probably contribute to DMTS formation.

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Mitochondria are sites of oxidative respiration. During sake brewing, sake yeasts are exposed to long periods of hypoxia; the structure, role, and metabolism of mitochondria of sake yeasts have not been studied in detail. It was first elucidated that the mitochondrial structure of sake yeast transforms from filamentous to dotted structure during sake brewing, which affects malate metabolism. Based on the information of yeast mitochondria during sake brewing, practical technologies have been developed; (i) breeding pyruvate-underproducing sake yeast by the isolation of a mutant resistant to an inhibitor of mitochondrial pyruvate transport; and (ii) modifying malate and succinate production by manipulating mitochondrial activity. During the bread-making process, baker's yeast cells are exposed to a variety of baking-associated stresses, such as freeze-thaw, air-drying, and high sucrose concentrations. These treatments induce oxidative stress generating reactive oxygen species due to mitochondrial damage. A novel metabolism of proline and arginine catalyzed by N-acetyltransferase Mpr1 in the mitochondria eventually leads to synthesis of nitric oxide, which confers oxidative stress tolerance on yeast cells. The enhancement of proline and arginine metabolism could be promising for breeding novel baker's yeast strains that are tolerant to multiple baking-associated stresses. These new and practical methods provide approaches to improve the processes in the field of industrial fermentation technologies.

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