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.     

Tolerance mechanism of the ethanol-tolerant mutant of sake yeast

Several ethanol-tolerant mutants have been bred from industrial sake yeasts, but the mechanism of ethanol tolerance in these mutants has not been elucidated. After the determination of the entire genome sequence of Saccharomyces cerevisiae, various methods to monitor the whole-gene expression of the yeast have been developed. In this study, we used a commercially available nylon membrane on which virtually every gene of S. cerevisiae was spotted to compare expression profiles between the ethanol-tolerant mutant and its parent sake yeast to investigate the mechanism of ethanol tolerance in this mutant. As a result, we found that several genes were highly expressed only in the ethanol-tolerant mutant but not in the parent strain. These genes were known to be induced in cells that were exposed to various stresses, such as ethanol, heat, and high osmolarity, or at the stationary-phase but not at the log-phase. In the ethanol-tolerant mutant, the expression level of these stress-responsive genes was further increased after exposure to ethanol. We also found that substances such as catalase, glycerol and trehalose that may have protective roles under stressful conditions were accumulated in high amounts in the ethanol-tolerant mutant. The ethanol-tolerant mutant also exhibited resistance to other stresses including heat, high osmolarity and oxidative stress in addition to ethanol tolerance. These results indicate that the mutant exhibits multiple stress tolerance because of elevated expression of stress-responsive genes, resulting in accumulation of stress protective substances.     

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.     

Sake yeast strains have difficulty in entering a quiescent state after cell growth cessation

Sake yeast strains produce a high concentration of ethanol during sake brewing compared to laboratory yeast strains. As ethanol fermentation by yeast cells continues even after cell growth stops, analysis of the physiological state of the stationary phase cells is very important for understanding the mechanism of producing higher concentrations of ethanol. We compared the physiological characteristics of stationary phase cells of both sake and laboratory yeast strains in an aerobic batch culture and under sake brewing conditions. We unexpectedly found that sake yeast cells in the stationary phase had a lower buoyant density and stress tolerance than did the laboratory yeast cells under both experimental conditions. These results suggest that it is difficult for sake yeast cells to enter a quiescent state after cell growth has stopped, which may be one reason for the higher fermentation rate of sake yeast compared to laboratory yeast strains.     

Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing

The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12-20% ethanol, while no difference in specific ethanol production rate in the presence of 0-18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H(+) extrusion activity. Furthermore, the plasma membrane H(+)-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H(+)-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H(+)-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H(+)-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H(+)-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not capable of increasing the cellular inositol level in the final stage, exhibited a significantly higher methylene blue-staining ratio than the parental strain. It was suggested that the yeast cellular inositol level is one of the important factors which contribute to the high ethanol tolerance implied by the increased cell viability in the presence of 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.     

High expression of unsaturated fatty acid synthesis gene OLE 1 in sake yeasts

Gene Filters and Northern blot analysis revealed that the sake yeast strain Kyokai no. 7 (K 7) showed a higher expression level of OLE 1, which encodes a Delta-9 fatty acid desaturase gene, compared with the laboratory yeast strain X 2180-1A. Other sake yeasts also showed a high expression level of OLE 1. Unsaturated fatty acid concentrations in strain K 7 are higher than that in strain X 2180-1A, suggesting that the higher expression level of OLE 1 in sake yeasts increases the unsaturated fatty acid content in the cell membrane. Experiments using OLE 1 promoter:lacZ fusion reporter genes revealed that both the cis element of the OLE 1 promoter and trans factors are involved in the increased expression of OLE 1 in sake yeasts.     

一株有氧条件下高产乙醇酵母的筛选、鉴定及其生物学特性研究

采用平板涂布法从老白干香型大曲中获得一株有氧条件下高产乙醇酵母菌株,命名为YF1914。通过菌落形态、细胞显微结构、生理生化特性和26S rDNA D1/D2区方法对其进行鉴定,借助液体培养方式考察其乙醇耐受性、葡萄糖耐受性、乙酸耐受性、生长温度和pH等生物学特性。结果表明,该菌株为酿酒酵母(Saccharomyces cerevisiae)属于乙醇高耐受性酵母,耐受乙醇最高体积分数为18%,同时还具有较高的NaCl和葡萄糖耐受性,最高耐受质量分数分别为15%和80%,在温度为20~50 ℃和pH1~10能够生长,具有宽广的温度和pH适应性。综上,该酵母这些优良特性有利于其在未来白酒酿造方式变化中发挥作用。     

果蔬酵素中优良酵母菌的分离、鉴定及耐受性研究

该研究采用传统培养分离技术从自然发酵的果蔬酵素中分离酵母菌,通过高糖、乙醇耐受性测定从中筛选优良菌株,采用形态观察、生理生化试验及分子生物学技术对其进行菌种鉴定,并对其低pH、高糖、乙醇及高温耐受性进行分析。结果表明,分离筛选得到一株优良酵母菌,编号为7-1-1,经鉴定,该菌株属于异常威克汉姆酵母（Wickerhamomyces anomalus）,其具有良好的耐受性,能在低pH（1.5）、高糖（900 g/L）的培养基上生长,可耐受体积分数为14%的乙醇和37 ℃的高温,可为果蔬发酵剂的开发奠定一定的基础。     

自然发酵剁辣椒中优良酵母菌的筛选及鉴定

为获得具有良好发酵特性的酵母菌株,对实验室前期于自然发酵剁辣椒中分离出的10株酵母菌进行筛选,通过比较分离菌株的产香、产酯、产醇能力以及对6% NaCl的耐受性,初步筛选出具有良好发酵性能和生长特性的酵母菌株;并对筛选菌株进行耐盐和耐酸等特性评价。结果表明,菌株Y-3、D-17、H-23、H-37的发酵性能和在6% NaCl中的耐受性表现良好;其中菌株Y-3在酸性环境和高盐环境中表现出了良好的生长优势;经形态学观察及分子生物学技术鉴定菌株Y-3为Hanseniaspora pseudoguilliermondii。菌株Y-3可应用于剁辣椒接种强化发酵生产。     

Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses

Trehalose is believed to play an important role in stress tolerance in the yeast Saccharomyces cerevisiae. In this research, the responses to various environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses, were investigated in a strain with deletion of the NTH1, NTH2, and ATH1 genes encoding trehalases that are involved in trehalose degradation and the triple deletion strains overexpressing TPS1 or TPS2, both of which encode trehalose biosynthesis enzymes in S. cerevisiae. The contents of trehalose constitutively accumulated in the TPS1- and TPS2-overexpressing triple deletion strains were higher than that in the original triple deletion strain. High trehalose accumulation and growth activity were observed in the TPS2-overexpressing triple deletion strain after ethanol stress induction. The same was also observed in the triple deletion and the TPS1- and TPS2-overexpressing triple deletion strains after heat stress induction. In case of freezing stress, all the recombinant strains with high constitutive trehalose content showed high tolerance. However, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicated that high trehalose accumulation can make yeast cells resistant to multiple stresses, but the importance of this accumulation before or after stress induction is varied depending on the type of stress.     

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.     

Effect of overexpression of LAS17 on stress tolerance and the stability of extrachromosomal DNA in Saccharomyces cerevisiae

The effects of the overexpression of LAS17/BEE1, which encodes a yeast protein exhibiting sequence homology to the Wiscott-Aldrich syndrome protein, on the cell growth of Saccharomyces cerevisiae were examined. Sake yeast strain UT-1 grows at a faster rate as a result of the overexpression of LAS17 than control cultures under various stresses such as high temperature, high ethanol concentration, and oxidative stress, and the tolerance to these stresses was increased compared with the control. Moreover, a high cell survival rate was attained with overexpression of LAS17, when cells in the stationary phase of the growth cycle were subjected to heat killing (48 degrees C) or ethanol killing (20% v/v). In addition, the rate of induction of rho- was markedly reduced by overexpression of LAS17 when serine, tyrosine, and aspartic acid were used as N sources and the yeast was cultured at 35 degrees C, while rho- strains in control cultures were induced at a high frequency. After the incubation of cells harboring a multicopy vector in YPD or synthetic complete medium, almost all of the cells inherited the vector at about 15 copies per cell as a result of the overexpression of LAS17, whereas the cells harboring the control vector accounted for only 15% of the total number of cells. These results suggest that Las17p might be a multifunctional protein involved in cell growth regulation, extrachromosomal DNA transportation and stress responses.     

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-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.     

高产酸本土非酿酒酵母菌株的筛选及发酵性能研究

以25株本土非酿酒酵母菌为研究对象,采用酵母浸出粉胨葡萄糖（YPD）10培养基及Triple M改良模拟汁初筛,并进行耐受性（乙醇、SO2、糖及pH）测定及葡萄汁发酵,筛选能够有效增加葡萄酒酸度的优良本土非酿酒酵母菌。结果表明,非酿酒酵母菌株LT1及HU4产酸性能较好,具有较好的乙醇、SO2、糖和pH耐受性,其中菌株LT1能耐受乙醇体积分数12%、糖400 g/L及pH 2.75,菌株HU4能耐受乙醇体积分数6%vol、糖250 g/L及pH 2.75。菌株LT1和HU4在葡萄汁中启酵时间较短,发酵旺盛期CO2质量损失速率均＞0.8 g/（L·h）,乳酸产量分别为0.93 g/L、1.14 g/L,乙酸产量分别为0.38 g/L、0.42 g/L,具备酿造增酸葡萄酒的潜力。     

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.     

TOR1基因缺失对酿酒酵母耐受性的影响

酿酒酵母是最常见且应用最为广泛的酵母菌种,是以糖质和淀粉质为原料的乙醇发酵最经典的菌株。在发酵过程中,有很多不可避免的胁迫环境如高温条件、高渗条件等出现,这些胁迫会阻碍细胞生长并降低细胞的发酵能力,给发酵行业带来一定的经济损失。因此,为改善菌种的耐受性,该研究主要以实验室现有菌株AY12a为亲本菌株,URA3基因作筛选标记,通过胞内同源重组,实现TOR1基因的敲除,最终成功构建突变株AY12a-tor1Δ。对酵母进行耐受性的测定,发现AY12a-tor 1Δ具有一定的耐高温性能,在高渗条件下也有一定的耐受性,同时具有一定的氧化环境耐受性。同时将突变株与AY12a进行模拟白酒发酵(玉米浓醪发酵),并对发酵完成后的酒度、残糖、48 h细胞存活率、CO 2失重及发酵时间进行测定。发酵数据显示突变株AY12a-tor 1Δ乙醇产量有所上升,残糖含量下降,48 h细胞存活率没有下降,发酵时间有所延长。