Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.     

Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26

Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation.     

利用人工锌指文库选育高乙醇耐受性工业酵母菌株

选育高乙醇耐性的酿酒酵母菌株对提高燃料乙醇的发酵效率具有重要意义.锌指蛋白广泛存在于多种生物中,对基因的转录和翻译起重要的调节作用.利用人工设计的锌指蛋白可定向设计锌指序列及其排列顺序,实现对细胞内多个基因的全局调控.由于与环境胁迫反应相关的基因很多,因此可利用人工锌指蛋白技术获得耐受性提高的微生物重组菌.文中将人工锌指文库转入到酿酒酵母模式菌株S288c,选育了具有高乙醇耐受性的重组菌株M01,并分离了与乙醇耐受性提高相关的人工锌指蛋白表达载体pRS316ZFP-M01,转入工业酿酒酵母Sc4126,在含有不同浓度乙醇的平板上,工业酵母Sc4126的重组菌株表现出显著的耐受性提高.在高糖培养基(250 g/L)条件下进行乙醇发酵,发现重组菌的乙醇发酵效率明显快于野生型,发酵时间提前24 h,且发酵终点乙醇浓度提高6.3％.结果表明人工锌指文库能够提高酵母的乙醇耐受性,为构建发酵性能优良的酵母菌种奠定了基础.     

The stress response is repressed during fermentation in brewery strains of yeast

Yeast cells encounter a variety of environmental stresses during brewing and must respond to ensure cell survival. Cells can respond to stress by inducing a Heat Shock Response in which heat shock proteins (Hsps) are synthesized. In laboratory strains of Saccharomyces cerevisiae, the heat shock protein, Hsp104, plays a major role in the acquisition of tolerance to a variety of stresses such as heat, ethanol and sodium arsenite, and as such acts as an excellent stress indicator. The induction of Hsp104 in bottom-and top-fermenting brewery strains was examined when grown under laboratory and industrial fermentation conditions, and it was found that each brewing strain exhibits its own unique pattern of Hsp104 expression. During industrial fermentations, brewery strains are capable of mounting a stress response at the early stages of fermentation. However, as the fermentation proceeds, the response is repressed. The results suggest that conditions experienced in industrial brewing prevent the activation of the stress response. This study increases our understanding of alterations in gene expression patterns during the brewing process, and yields information that will aid in the definition of best practice in yeast management.     

The role of lager beer yeast in oxidative stability of model beer

Aims: In this study, we investigated the relationship between the ability of lager brewing yeast strains to tolerate oxidative stress and their ability to produce oxidative stable model beer. Methods and Results: Screening of 21 lager brewing yeast strains against diamide and paraquat showed that the oxidative stress resistance was strain dependent. Fermentation of model wort in European Brewing Convention tubes using three yeast strains with varying oxidative stress resistances resulted in three model beers with different rates of radical formation as measured by electron spin resonance in forced ageing experiments. Interestingly, the strain with the lowest oxidative stress resistance and lowest secretion of thioredoxin, as measured by Western blotting, resulted in the highest uptake of iron, as measured by inductively coupled plasma‐mass spectrometry, and the slowest formation of radicals in the model beers. Conclusions: A more oxidative stable beer is not obtained by a more‐oxidative‐stress‐tolerant lager brewing yeast strain, exhibiting a higher secretion of thioredoxin, but rather by a less‐oxidative‐stress‐tolerant strain, exhibiting a higher iron uptake. Significance and Impact of the Study: To obtain lager beers with enhanced oxidative stability, yeast strains should be screened for their low oxidative stress tolerance and/or high ability to take up iron rather than for their high oxidative stress tolerance and/or high ability to secrete thioredoxin.     

Analysis of the stress resistance of commercial wine yeast strains

Alcoholic fermentation is an essential step in wine production that is usually conducted by yeasts belonging to the species Saccharomyces cerevisiae. The ability to carry out vinification is largely influenced by the response of yeast cells to the stress conditions that affect them during this process. In this work, we present a systematic analysis of the resistance of 14 commercial S. cerevisiae wine yeast strains to heat shock, ethanol, oxidative, osmotic and glucose starvation stresses. Significant differences were found between these yeast strains under certain severe conditions, Vitilevure Pris Mouse and Lalvin T73 being the most resistant strains, while Fermiblanc arom SM102 and UCLM S235 were the most sensitive ones. Induction of the expression of the HSP12 and HSP104 genes was analyzed. These genes are reported to be involved in the tolerance to several stress conditions in laboratory yeast strains. Our results indicate that each commercial strain shows a unique pattern of gene expression, and no clear correlation between the induction levels of either gene and stress resistance under the conditions tested was found. However, the increase in mRNA levels in both genes under heat shock indicates that the molecular mechanisms involved in the regulation of their expression by stress function in all of the strains.     

Aquaporin expression correlates with freeze tolerance in baker's yeast,and overexpression improves freeze tolerance in industrial strains

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.     

Genome-wide expression profile of sake brewing yeast under shaking and static conditions

To identify the genes responsible for characteristics, that are different as between sake brewing yeasts and laboratory yeast strains, we used a DNA microarray to compare the genome-wide gene expression profiles of a sake yeast, Saccharomyces cerevisiae K-9 (kyokai 9), and a laboratory yeast, S. cerevisiae X2180-1A, under shaking and static conditions.The genes overexpressed in K-9 more than in X2180-1A were related to C-metabolism, including the HXT, ATP, and COX genes, ergosterol biosynthesis, ERG genes, and thiamine metabolism, THI genes. These genes may contribute to higher growth rates and fermentation ability and the ethanol tolerance of sake yeast.The genes underexpressed in K-9 more than in X2180-1A were CUP1-1 and CUP1-2, PHO genes, which may explain the low copper tolerance and low acid phosphatase activity of sake yeast. These underexpressed genes agree with the features and the alteration of the genome structure of sake yeast.     

Analysis of adaptation to high ethanol concentration in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using DNA microarray

In industrial process, yeast cells are exposed to ethanol stress that affects the cell growth and the productivity. Thus, investigating the intracellular state of yeast cells under high ethanol concentration is important. In this study, using DNA microarray analysis, we performed comprehensive expression profiling of two strains of Saccharomyces cerevisiae, i.e., the ethanol-adapted strain that shows active growth under the ethanol stress condition and its parental strain used as the control. By comparing the expression profiles of these two strains under the ethanol stress condition, we found that the genes related to ribosomal proteins were highly up-regulated in the ethanol-adapted strain. Further, genes related to ATP synthesis in mitochondria were suggested to be important for growth under ethanol stress. We expect that the results will provide a better understanding of ethanol tolerance of yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chromosomal Integration and Expression of Two Bacterial alpha-Acetolactate Decarboxylase Genes in Brewer's Yeast

A bacterial gene encoding alpha-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the alpha-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the alpha-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.     

核桃JrGRAS2基因响应热胁迫的表达及功能分析

GRAS转录因子在植物响应逆境中起重要作用。为更好的了解核桃（Juglans regia）在逆境胁迫下的适应机制,本研究从‘香玲’核桃转录组中克隆获得一条GRAS基因（命名为JrGRAS2）,对其在不同高温胁迫下的表达进行分析,并将该基因插入酵母表达载体pYES2中构建重组载体pYES2-JrGRAS2,将pYES2-JrGRAS2转入酿酒酵母（Saccharomyces cerevisiae）INVSCI,同时以转化pYES2的重组酵母作为阴性对照,在酵母表达系统中研究该基因的抗热胁迫功能。结果显示,该基因开放读码框（ORF）全长1296bp,拟推导的蛋白分子量为47405.83Da,含有氨基酸数为431,理论等电点为5.66。在热胁迫下,JrGRAS2基因被显著诱导,特别是在36℃胁迫0.5h的茎内,其表达相对于对照被上调了335.5倍。对两种酵母进行热胁迫,发现转JrGRAS2基因酵母表现出较对照更高的生存活性。表明JrGRAS2基因具有响应热胁迫的能力,且能提高酵母的抗性,JrGRAS2基因可作为核桃逆境应答的重要候选基因。     

絮凝酵母海藻糖合成酶基因TPS1启动子区的克隆和乙醇胁迫下启动子活性的变化

提高生物能源生产菌株对各种胁迫因素的耐受性对于提高生产过程的经济性和高效生产生物能源具有重要的意义。对酿酒酵母乙醇耐性的分子机制的研究,可揭示影响其耐受性的关键基因,并通过代谢工程操作定向提高酵母菌的乙醇耐受性,从而提高燃料乙醇的生产效率。海藻糖对酵母菌在多种环境胁迫下的细胞活性具有保护作用,但其对乙醇耐性分子机制的研究还不够深入。克隆了自絮凝酵母Saccharomyces cerevisiae flo的海藻糖-6-磷酸合成酶基因TPS1的启动子区域,利用pYES2.0载体骨架,构建了PTPS1启动绿色荧光蛋白EGFP标记基因的报告载体,并转化酿酒酵母ATCC4126。对酵母转化子在含有7%和10%乙醇的生长培养基中的EGFP的表达情况进行相对荧光定量分析,发现PTPS1活性在7%乙醇存在下受到强烈诱导。EGFP表达量对高温和高糖胁迫无明显差别,显示了TPS1启动子对乙醇浓度的特异响应。研究结果表明,絮凝酵母海藻糖的合成是对乙醇胁迫的保护性反应。