Effect of the FAA1 gene disruption of sake yeast on the accumulation of ethyl caproate in sake mash

Fatty acid activation gene (FAA1) in sake yeast Kyokai no. 701 (K701) was disrupted to investigate the accumulation of ethyl caproate in sake mash. Ethyl caproate, recognized as an important apple-like flavor in sake, is generated by fatty acid synthesis in yeast cells. The disruptant for the FAA1 gene (K701deltafaa1) exhibited a reduced growth rate in a medium containing cerulenin and myristic acid or oleic acid compared with that of the parental strain (K701). In a sake brewing test in which the rice used was polished to 60% of its original size, the fermentation ability of K701deltafaa1 was inferior to that of K701 but the production of ethyl caproate by K701deltafaa1 was 1.6-fold higher than that by K701. These results suggest that the FAA1 gene in sake yeast plays an important role in sake brewing and the accumulation of ethyl caproate.     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

Isolation and characterization of sake yeast mutants deficient in gamma-aminobutyric acid utilization in sake brewing

Sake yeasts take up gamma-aminobutyric acid (GABA) derived from rice-koji in the primary stage of sake brewing. The GABA content in sake brewed with the UGA1 disruptant, which lacked GABA transaminase, was higher than that brewed with the wild-type strain K701. The UGA1 disruptant derived from sake yeast could not grow on a medium with GABA as the sole nitrogen source. We have isolated the sake yeast mutants of K701 that were unable to grow on a medium containing GABA as the sole nitrogen source. The growth defect of GAB7-1 and GAB7-2 mutants on GABA plates was complemented by UGA1, which encodes GABA transaminase, and UGA2, which encodes succinic semialdehyde dehydrogenase (SSADH), respectively. DNA sequence analysis revealed that GAB7-1 had a homozygous nonsense mutation in UGA1 and GAB7-2 had a heterozygous mutation (G247D) in UGA2. The GABA transaminase activity of GAB7-1 and the SSADH activity of GAB7-2 were markedly lower than those of K701. These GAB mutants displayed a higher intracellular GABA content. The GABA contents in sake brewed with the mutants GAB7-1 and GAB7-2 were 2.0 and 2.1 times higher, respectively, than that brewed with the wild-type strain K701. These results suggest that the reduced function of the GABA utilization pathway increases the GABA content in sake.     

Using promoter replacement and selection for loss of heterozygosity to generate an industrially applicable sake yeast strain that homozygously overproduces isoamyl acetate

By application of the high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake yeast (Kotaka et al., Appl. Microbiol. Biotechnol., 82, 387–395 (2009)), we constructed, from a heterozygous integrant, a homozygous diploid that overexpresses the alcohol acetyltransferase gene ATF2 from the SED1 promoter, without the need for sporulation and mating. Under the conditions of sake brewing, the homozygous integrant produced 1.4 times more isoamyl acetate than the parental, heterozygous strain. Furthermore, the homozygous integrant was more genetically stable than the heterozygous recombinant. Thus, the HELOH method can produce homozygous, recombinant sake yeast that is ready to be grown on an industrial scale using the well-established procedures of sake brewing. The HELOH method, therefore, facilitates genetic modification of this rarely sporulating diploid yeast strain while maintaining those characteristics required for industrial applications.     

Emergence of [GAR+] cells in yeast from sake brewing affects the fermentation properties

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.     

Amplified fragment length polymorphism of the AWA1 gene of sake yeasts for identification of sake yeast strains

Sake yeasts are used for sake brewing and have a crucial role in the quality of sake, since they produce not only ethanol but also various compounds that provide sake flavors. Therefore, the appropriate selection and monitoring of a strain used in sake mash is important. However, the identification of specific sake yeast strains has been difficult, because sake yeasts have similar characteristics in taxonomic and physiological analyses. We found amplified fragment length polymorphisms (AFLPs) in the PCR products of the AWA1 gene of sake yeast strains. The AWA1 gene encodes a cell wall protein that is responsible for foam formation in sake mash. This polymorphism of the AWA1 gene can be used for the identification of sake yeast strains.     

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. Copyright © 2008 John Wiley & Sons, Ltd.     

Isolation of sake yeast mutants producing a high level of ethyl caproate and/or isoamyl acetate

In the case of sake, ethyl caproate and isoamyl acetate are considered to be closely associated with flavor. Various mutant yeast strains producing a higher level of flavor compounds (ethyl caproate and/or isoamyl acetate) than the parent strain were isolated by ethyl methane sulfonate treatment followed by global selection. Two of the mutants obtained also showed a high malate productivity. These mutants would be promising for practical sake fermentation.     

Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of Δpdr5 and Δsnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity-1) × 1443] of the Δpdr5 and Δsnq2 strains rose faster than that of HL69. On the other hand, a Δyor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the Δyor1 slowed down compared with that of CTZ21. A Δyor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the Δpdr5 and Δsnq2 strains of CTZ21. In contrast, the Δyor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.     

Cloning and analysis of the AWA1 gene of a nonfoaming mutant of a sake yeast

Almost all sake yeasts form a thick foam layer on sake mash during fermentation. To reduce the amount of foam, nonfoaming mutants were bred from foam-forming sake yeasts. To elucidate the mechanism of this foam formation, we have cloned a gene from a foam-forming sake yeast that confers foam-forming ability to a nonfoaming mutant. This gene, named AWA1, encodes a glycosylphosphatidylinositol (GPI) anchor protein that is localized to the cell wall and is required for cell surface hydrophobicity. In this paper, we describe the genomic analysis of the AWA1 gene in a nonfoaming mutant strain K701 derived from a foam-forming sake yeast strain K7. K701-AWA1 was cloned in a cosmid and its sequence was compared with that of K7-AWA1. Although the 5' half of K701-AWA1 was identical to that of K7-AWA1, the 3' half of K701-AWA1 was different from that of K7-AWA1, resulting in a loss of the C-terminal hydrophobic sequence of Awa1p. Since this sequence is considered to be required for the anchoring of Awa1p to the cell wall, K7-Awa1p could not confer both cell surface hydrophobicity and foam-forming ability to strain K701 cells. Since the change found in K701-AWA1 was not a point mutation but a larger scale event, we analyzed chromosome rearrangement by pulsed-field gel electrophoresis Southern blot analyses. The results suggest that the left subtelomeric region of chromosome IX in strain K7 was translocated to the AWA1 gene in chromosome XV by a nonreciprocal recombination.     

Effect of NAD+-dependent isocitrate dehydrogenase gene (IDH1, IDH2) disruption of sake yeast on organic acid composition in sake mash

The ratio of organic acids in sake mash is a very important factor affecting the taste of alcoholic beverages. To alter the organic acid composition in sake and investigate the mechanism of producing organic acids in sake mash, we examined the effect of NAD+-dependent isocitrate dehydrogenase (IDH) activity deficiency in sake yeast by disrupting the IDH1 or IDH2 gene. Two haploid strains (MATa or MATa genotype) isolated from sake yeast Kyokai no. 701 (K701) were disrupted using the aureobasidin A resistant gene (AUR1-C) as a selection marker. These disruptants were defective in the activity of IDH and failed to grow on medium containing glycerol as a sole carbon source. Sake meter, alcohol concentration, and glucose consumption in sake brewed with the disruptants were reduced in comparison with those of the parental strains. The production of citrate (including isocitrate), malate, and acetate by the disruptants was increased, but succinate production was reduced to approximately half in comparison with the parental strains. These results indicate that approximately half the amount of succinate in sake mash is produced via the oxidative pathway of the TCA cycle in sake yeast. While the diploid strain constructed by mating haploid disruptants for the IDH gene exhibited stronger fermentation ability than the haploid disruptants, almost similar profiles of components in sake were obtained for both strains.     

Breeding of high malate‐producing diploid sake yeast with a homozygous mutation in the VID24 gene

Malate is an important taste component of sake (a Japanese alcoholic beverage) that is produced by the yeast Saccharomyces cerevisiae during alcoholic fermentation. A variety of methods for generating high malate‐producing yeast strains have been developed to date. We recently reported that a high malate‐producing strain was isolated as a mutant sensitive to dimethyl succinate (DMS), and that a mutation in the vacuolar import and degradation protein (VID) 24 gene was responsible for high malate productivity and DMS sensitivity. In this work, the relationships between heterozygous and homozygous mutants of VID24 and malate productivity in diploid sake yeast were examined and a method was developed for breeding a higher malate‐producing strain. First a diploid yeast was generated with a homozygous VID24 mutation by genetic engineering. The homozygous integrants produced more malate during sake brewing and grew more slowly in DMS medium than wild‐type and heterozygous integrants. Thus, the genotype of the VID24 mutation influenced the level of malate production and sensitivity to DMS in diploid yeast. Then a homozygous mutant from a heterozygous mutant was obtained without genetic engineering by ultraviolet irradiation and culturing in DMS with nystatin enrichment. The non‐genetically modified sake yeast with a homozygous VID24 mutation exhibited a higher level of malate productivity than the parent heterozygous mutant strain. These findings provide a basis for controlling malate production in yeast, and thereby regulating malate levels in sake. Copyright © 2016 The Institute of Brewing & Distilling     

Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain

Succinate dehydrogenase (SDH) of Saccharomyces cerevisiae consists of four subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. We determined the effect of SDH deficiency on the productivity of organic acids in a sake yeast strain Kyokai no. 9. The SDH activity of single disruptants was retained at 30-90% of that of the wild-type strain, but the activity disappeared in double disruptants of the SDH1 and SDH2 or SDH1b (the SDH1 homologue) genes. Two double disruptants showed no growth on a medium containing glycerol as the sole carbon source, while the single disruptants could utilize glycerol. These results indicate that double disruption of the SDH1 and SDH2 or SDH1b genes is required for complete loss of SDH activity and that the SDH1b gene compensates for the function of the SDH1 gene. The sdh1 sdh1b disruptant showed a marked increase in succinate productivity of up to 1.9-fold along with a decrease in malate productivity relative to the wild-type strains under shaking conditions. Under both static and sake brewing conditions, the productivity of these organic acids in the disruptants was virtually unchanged from that in the wild-type strain. Furthermore, SDH activity was undetectable in the wild-type and the disrupted strains under static conditions. These results suggest that SDH activity contributes to succinate production under shaking conditions, but not under static and sake brewing conditions.     

Behaviour of ethyl caproate during the production and distillation of ethyl caproate‐rich rice shochu

To increase the popularity of rice shochu, a process was developed to produce ethyl caproate‐rich rice shochu by adding a cultured broth of a caproic acid‐producing bacterial (CAPB) consortium to the fermentation. When the CAPB consortium containing fermented mash was subjected to vacuum distillation, the distillation efficiency of ethyl caproate was up to ~300%, while the distillation efficiencies of the other flavour compounds and ethanol were <100%. The behaviour of ethyl caproate during the production and distillation of an ethyl caproate‐rich rice shochu was investigated and the results showed that ethyl caproate was synthesized by Saccharomyces cerevisiae during the shochu production process, and that some of the ethyl caproate synthesized was secreted into the medium. Ethyl caproate in the medium evaporated easily and was transferred to the distillate when distilled. The increase in ethyl caproate after distillation was mainly derived from the release of the intracellular ethyl caproate, with negligible input from the chemical esterification of caproic acid and ethanol. During vacuum distillation, although few yeast cells were disrupted, secretion of the intracellular ethyl caproate was more efficient owing to the increase in temperature, allowing the distillation efficiency of ethyl caproate to be >100%. Copyright © 2016 The Institute of Brewing & Distilling     

A role of Saccharomyces cerevisiae fatty acid activation protein 4 in palmitoyl-CoA pool for growth in the presence of ethanol

It is well known that sake yeast has a high tolerance for ethanol, as compared to baker's yeast. To investigate the relationship between the ethanol tolerance of sake yeast and the palmitoyl-CoA pool for protein modification, the growth of yeast cells with depletion of the palmitoyl-CoA pool was monitored in the presence of ethanol. The overexpression of SNC1 was used to achieve the depletion of the palmitoyl-CoA pool, because the loss of Snc palmitoylation does not affect the general growth characteristics of yeast and does not interfere with the secretory processes (Couve, A. et al., Proc. Natl. Acad. Sci. USA, 92, 5987-5991 (1995)). Although the sake yeast UT-1 exhibited much better growth in the presence of ethanol than laboratory strains, the overexpression of Snc1 was accompanied by sparse growth with increasing ethanol concentration. Exogenous palmitic acid rescued the poor growth caused by Snc1 overexpression, and the overexpression of Snc1(ser95) (which could not palmitoylated) had no effect on the growth characteristics of strain UT-1, suggesting that the poor growth with Snc1 overexpression was due to an overall increase in proteins in the unpalmitoylated form. To ascertain that fatty acid activation has a distinct role in the growth of yeast in the presence of ethanol, FAA genes encoding long chain acyl-CoA synthetases were overexpressed in combination with snc1 overexpression. Interestingly, the growth defect caused by snc1 overexpression was rescued by the overexpression of FAA4, but not of FAA1, which plays a predominant role in laboratory strains. On the contrary, disruption of faa1 led to faster growth in the presence of ethanol. These results suggest that Faa1p and Faa4p play reciprocal roles in regulating protein modification during growth in the presence of ethanol, since Faa1p and Faa4p both function to incorporate palmitic acid into phospholipids and neutral lipids. Moreover, Northern hybridization analysis revealed that faa1 mRNA was expressed strongly in a laboratory strain, and weakly in the sake yeast strain K-7 which exhibited good growth in the presence of ethanol. The combination of the disruption of faa1 and exogenously supplied palmitic acid was highly effective for growth in the presence of ethanol even under the normal snc1 expression level, implying that activation of exogenous palmitic acid by Faa4p is of particular importance in growth in ethanol.     

QTL mapping of sake brewing characteristics of yeast

A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.