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 (K701Δfaa1) 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 K701Δfaa1 was inferior to that of K701 but the production of ethyl caproate by K701Δfaa1 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.     

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.     

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.     

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.     

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.     

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.     

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28

We characterized a high malic acid production mechanism in sake yeast strain No. 28. No considerable differences in the activity of the enzymes that were involved in malic acid synthesis were observed between strain No. 28 and its parent strain, K1001. However, compared with strain K1001, which actively took up rhodamine 123 during staining, the cells of strain No. 28 were only lightly stained, even when cultured in high glucose concentrations. In addition, malic acid production by the respiratory-deficient strain of K1001 was 2.5-fold higher than that of the wild-type K1001 and wild-type No. 28. The findings of this study demonstrated that the high malic acid production by strain No. 28 is attributed to the suppression of mitochondrial activity.     

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.     

Molecular cloning and application of a gene complementing pantothenic acid auxotrophy of sake yeast Kyokai no. 7

Kyokai no. 7 is the most widely used yeast in sake brewing. This yeast is a pantothenic acid auxotroph at 35 degrees C, and this phenotype has been used to distinguish Kyokai no. 7 from other sake yeasts. We cloned a DNA fragment complementing the pantothenic acid auxotrophy from a genomic library of a Saccharomyces cerevisiae laboratory strain. DNA sequence analysis revealed that the DNA fragment encodes ECM31, the deletion of which had previously been identified as a calcofluor white-sensitive mutation. The ECM31 product is similar to the Escherichia coli ketopantoate hydroxymethyltransferase. Disruption of ECM31 in a laboratory S. cerevisiae strain resulted in pantothenic acid auxotrophy, indicating that ECM31 is also involved in pantothenic acid synthesis in yeast. A hybrid of a Kyokai no. 7 haploid and the ecm31 disruptant required pantothenic acid at 35 degrees C for its growth, suggesting that Kyokai no. 7 possesses a temperature-sensitive allele of ECM31. Thus, the ECM31 gene can be used as a selective marker in the transformation of Kyokai no. 7.     

Biofortification of folates in white wheat bread by selection of yeast strain and process

We here demonstrate that folate content in yeast fermented food can be dramatically increased by using a proper (i) yeast strain and (ii) cultivation procedure for the selected strain prior to food fermentation. Folate levels were 3 to 5-fold higher in white wheat bread leavened with a Saccharomyces cerevisiae strain CBS7764, cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation (135-139 microg/100 dry matter), compared to white wheat bread leavened with commercial Baker's yeast (27-43 microg/100 g). The commercial Baker's yeast strain had been industrially produced, using a fed-batch process, thereafter compressed and stored in the refrigerator until bakings were initiated. This strategy is an attractive alternative to fortification of bread with synthetically produced folic acid. By using a high folate producing strain cultured a suitable way folate levels obtained were in accordance with folic acid content in fortified cereal products.     

异常汉逊酵母发酵乙酸乙酯条件优化和代谢研究

以高粱和大曲为原料,用一株异常汉逊酵母NZ-19-1为发酵剂,通过正交试验优化了其发酵原料配比及发酵条件。结果表明,该酵母菌在葡萄糖含量27%的高粱水解液中,辅以4%的大曲,初始pH值为5.0,装料系数0.25,25℃下采用静置方法发酵13天,可获得富含乙酸乙酯含量的酒醅,初步建立了一种乙酸乙酯含量较高的勾兑用白酒的生产工艺。通过对这株高产菌特征性代谢产物和醇酰基转移酶的研究,得出该菌株合成乙酸乙酯的途径是:先利用葡萄糖生成乙酸和乙醇,乙酸在HSCoA的作用下激活为乙酰-CoA,与乙醇在醇酰基转移酶的催化作用下合成乙酸乙酯。这一结果可为今后将该菌株运用于富含乙酸乙酯的勾兑用白酒的生产提供技术参考。     

Permanence of yeast inocula in the winery ecosystem and presence in spontaneous fermentations

The oenological practice of systematic inoculation with active dried yeasts is commonly used by many wineries around the world. However, the use of these yeasts is not free from controversy, since this practice has occasionally been described as having a negative effect on the biodiversity of natural yeast present in the wineries. The purpose of this study is to analyse the presence of commercial yeasts used as inocula in the ecosystem of three D.O.Ca. (“Qualified” Designation of Origin) Rioja wineries. It studies the permanence of these yeasts in winery equipment and their participation in spontaneous fermentations where they have not been used for inoculation. The results indicate that the presence of the active dry yeasts used in the wineries was scarce or non-existent, both in the ecosystem of each winery and in the spontaneous fermentations where they had not been added. So, repeated inoculation with active dry yeasts allowed a high presence and development of autochthonous (Saccharomyces and non-Saccharomyces) yeasts, both in equipment and in the spontaneous fermentations carried out.     

Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation

We analyzed the variability of volatile acidity and glycerol production by Saccharomyces cerevisiae on a large sample of high sugar musts. The production of volatile acidity was inversely correlated with the maximum cell population and the assimilable nitrogen concentration. The higher the nitrogen concentration, the less volatile acidity was produced. An approach to minimize volatile acidity production during high sugar fermentations by adjustment of assimilable nitrogen in musts was investigated in terms of both quantity and addition time. It was found that the optimal nitrogen concentration in the must is 190 mgN.l(-1). The best moment for nitrogen addition was at the beginning of fermentation. Addition at the end of the growth phase had less effect on volatile acidity reduction. We suggest that by stimulating cell growth, nitrogen addition provides NADH in the redox-equilibrating process, which in turn reduces volatile acidity formation.     

Changes in the Lipid Composition of Saccharomyces cerevisiae Race Capensis (G-1) During Alcoholic Fermentation and Flor Film Formation

The cellular lipid composition of one flor-forming strain of Saccharomyces cerevisiae during fermentation and the subsequent period of film formation with different oxygen levels was studied. Irrespective of fermentation conditions, only those yeasts which came into contact with oxygen after fermentation formed a flor film. After the fermentation, these yeasts entered an adaptation phase in which the percentage of oleic acid increased considerably at the expense of other long-chain fatty acids. Their phospholipid contents remained high, as well as the unsaturation index of their fatty acids and the ergosterol/phospholipids ratio was maintained below 1. These changes allowed an increased viability of yeasts in the wine of up to 80% and the acquisition of sufficient hydrophobicity and floatability to reach the surface and form flor film.     

Fruit wine produced from cagaita (Eugenia dysenterica DC) by both free and immobilised yeast cell fermentation

The aims of this work were to elaborate a fruit wine from cagaita (Eugenia dysenterica DC) pulp and to compare the fermentations conducted with free and with Ca-alginate immobilised cells. Two strains of Saccharomyces cerevisiae (UFLA CA11 and CAT-1) were tested and four fermentation batches were performed, in triplicate, at 22 °C for 336 h: UFLA CA11 in free and immobilised cells and CAT-1in free and immobilised cells. Fermentation time and ethanol production were influenced by the yeast strain and by the cell status, with immobilised cells of UFLA CA11 and CAT-1 fermenting faster (4 days and 8 days, respectively) than UFLA CA11 and CAT-1 free cells (10 days and 12 days, respectively). Ethanol content (g/L) was slightly higher when the fermentation was conducted with free cells (94.63 and 94.94 for UFLA CA11 and CAT-1, respectively) than with immobilised cells (86.82 and 87.21 for UFLA CA11 and CAT-1, respectively). The beverage from CAT-1 free cells showed the highest concentration of higher alcohols (82,086.12 ??/L), whereas the lowest concentration (37,812.17 ??/L) was found in the beverage from immobilised UFLA CA11. The ethyl ester concentration ranged from 1511.42 ??/L (CAT-1 free cells) to 2836.34 ??/L (UFLA CA11 free cells). According to the sensory evaluation, the fruit wine acceptability was greater than 70% for colour, flavour and taste for all cagaita beverages.