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     

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.     

Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Screening of drug‐resistant mutants of sake yeast strains has been a major method for creation of superior strains. We attempted to create a valproic acid (VPA)‐resistant mutant strain from sake yeast Kyokai No. 7 (K7). VPA is a branched‐chain fatty acid and is an inositol synthesis inhibitor in mammals and yeast. We succeeded in isolating a mutant of strain K7 that can survive long‐term in a VPA‐containing medium. This strain, K7‐VPA^LS, is significantly more resistant to not only VPA‐induced cell death but also ethanol in comparison with the parent strain. Further experiments showed that the new strain is likely to have a deficiency in inositol and/or phosphatidylinositol synthesis. The major characteristics of sake brewed by strain K7‐VPA^LS (compared with K7) were lower amino acidity, higher isoamyl acetate content without an increase in the isoamyl alcohol level and changes in constituent organic acids, particularly higher malate and succinate but lower acetate concentrations. In addition, taste sensor analysis revealed that K7‐VPA^LS‐brewed sake has milder sourness and higher saltiness or richness than K7‐brewed sake does. High isoamyl acetate production may be related to a deficiency in phosphatidylinositol because this compound directly inhibits alcohol acetyltransferase, an enzyme responsible for isoamyl acetate synthesis. Strain K7‐VPA^LS grew more rapidly than the parental strain did in a medium containing acetate as a sole carbon source, indicating that K7‐VPA^LS effectively assimilates acetate and converts it to malate and succinate through the glyoxylate cycle. Thus, strain K7‐VPA^LS shows improved characteristics for brewing of high‐quality sake. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

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.     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

高生物量富硒酵母菌的选育

以酿酒酵母PT为出发菌株,采用梯度浓度筛选的方法对其进行驯化,得到1株生物量较高和对亚硒酸钠抗性较高的菌株GB-1。对其进行紫外诱变处理,当致死率达91.85%时,获得多株突变株。通过多次平板初筛和摇瓶复筛,得到1株高生物量富硒酵母UV-PT。采用响应面法对富硒酵母UV-PT发酵条件进行了优化。借助于SAS软件,首先利用Plackett-Burman试验设计筛选出影响富硒的3个主要因素,即转速、温度、初始pH值。在此基础上,再利用Box-Behnken试验设计及响应面分析法对发酵条件进行优化,确定最佳发酵条件:转速为203r/min,发酵温度为30.3℃,初始pH值为4.52。结果表明,优化后富硒酵母的生物量和含硒量分别为10.62g/L、1003.26μg/g,硒总含量为10654.62μg/L,为出发菌株的1.62倍。     

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.     

富硒酵母的选育及其发酵条件的优化试验

通过对一株耐硒酵母进行UV和60Co逐级诱变,获得一株产量高的突变株Y7,发酵液的硒总含量达19mg/L,是出发菌株的1.58倍,经多次传代试验,证明其稳定性良好。通过单因素、L16(37)正交试验对其发酵培养基和培养条件进行优化,试验表明富硒酵母发酵培养基的最佳配方是:蔗糖6%,牛肉膏1%,蛋白胨1%,K2HPO4 0.15%,亚硒酸钠35μg/L;最佳培养条件:初始pH为4.0,温度32℃,装液量60mL/250mL摇瓶,转速200r/min,10%(v/v)接种量,培养48h。富硒酵母的硒总含量达23mg/L以上,是出发菌株的1.92倍。     

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.     

Isolation of a Copper‐resistant Sake Yeast Mutant with Improved Flavour Compound Production

The sake (traditional Japanese alcoholic beverage) yeast mutant A1 was previously isolated as a strain resistant to an isoprenoid analog. This strain is used for industrial sake brewing because of its increased production of isoamyl acetate. In this study, a physiological event was identified which was closely related to the elevation of alcohol acetyltransferase (AATase) activity in strain A1. This finding was applied for the isolation of another mutant with an improved capacity for flavour compound production. Strain A1 revealed an additional phenotype showing resistance to Cu²⁺, as seen from its growth and isoamyl acetate production, even in a medium with the copper ion at 6 mM. Mutant strains were successfully isolated with increased isoamyl acetate production capacity from sake yeast strain 2NF on the basis of a Cu²⁺‐resistant phenotype at a high yield. Among them, strain Cu7 was characterized by its ability to produce isoamyl acetate at the highest concentration under condition where isoamyl alcohol (its precursor) was accumulated to the lowest extent. Such a phenotype of strain Cu7 is applicable for the practical production of an alcoholic beverage of excellent quality in terms of flavour.     

发酵梨酒中产香酵母的分离、鉴定及生物学特性的研究

从自然发酵的梨酒中分离筛选出一株产香浓郁且糖利用率低的酵母菌,对其生物学特性进行了详细的研究,并通过26SrDNA D1/D2序列分析手段将该酵母菌鉴定到种。研究结果表明:该菌种在生长4h后进入对数生长期,16h后进入稳定生长期;最适生长温度为28℃,其范围为26~32℃;最适pH为5.5,范围为5.1~5.9;在发酵的第6d的香气最浓,其总酯含量达到0.38g/L。经过26SrDNA D1/D2序列分析鉴定为东方伊莎酵母(Issatchenkia orientalis),命名为Issatchenkia orientalisYS03。      

复合诱变选育耐高温产乙醇酵母菌的研究

为获得耐高温产乙醇菌株,对实验室保藏的菌株JZ进行分子生物学鉴定和紫外（UV）-硫酸二乙酯（DES）复合诱变,并通过单因素试验及响应面试验对筛选得到的诱变菌株进行发酵条件优化。结果表明,菌株JZ被鉴定为库德里阿兹威毕赤酵母（Pichia kudriavzevii）。经紫外（UV）-硫酸二乙酯（DES）复合诱变,得到诱变菌株JZ-2,且当紫外照射时间为80 s,硫酸二乙酯含量为5%时,在40 ℃条件下发酵6 d,乙醇产量可达4.8%vol。通过单因素试验及响应面试验确定最佳发酵条件为发酵温度40 ℃、初始pH值为4、初始糖度16 °Bx、接种量12%,静置发酵时间6 d。在此优化条件下,乙醇产量达5.6%vol,比优化前相比提高了17%。     

Co60诱变原生质体选育高产酒精酵母

将较高产酒精酵母制备成原生质体,经Co^60诱变后,采用四级筛选,得到高产酒精酵母菌株Co-158,其遗传性状稳定。以玉米粉糖化醪为基质发酵72h,成熟醪酒精体积分数为17～％（v／v）,残还原糖为0．0137％。Co-158菌;洙成熟醪酒精体积分数比出发菌株提高了16．34％,比ADY提高了24．48％,残还原糖含量亦远远低于出发菌株和ADY。     

凉州熏醋酿造过程产乙偶姻酵母菌筛选及其发酵培养基优化

乙偶姻是凉州熏醋特征风味成分四甲基吡嗪的前体。本实验从凉州熏醋发酵料醅中分离获得了一株产乙偶姻酵母菌T8,通过经典鉴定方法结合ITS基因测序的方法,确定其为葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）。为探明T8菌株生长及代谢产乙偶姻的营养需求,本实验对产乙偶姻酵母菌T8发酵培养基进行了优化,以期为传统食醋酿造工艺现代化改造过程中人工接种增香酵母种子的制备提供培养基。通过研究不同碳源、氮源、无机盐对T8菌株产乙偶姻的影响,确定了发酵培养基的最终组分:葡萄糖60 g/L,蛋白胨10 g/L,酵母膏10 g/L,（NH₄）₂HPO₄8 g/L,KH₂PO₄0.5 g/L,Mg SO₄·7H₂O 0.75 g/L,Mn SO₄0.6 g/L。培养基优化后,乙偶姻产量达到15.236 g/L,与初始发酵培养基相比提高了20.65%。人工接种T8菌株种子液所酿食醋中四甲基吡嗪的含量较对照提高了22.1%,增香效果明显,证明H.Uvarum T8菌株可以作为食醋酿造的增香酵母。