Two-Dimensional protein map of Saccharomyces cerevisiae: Construction of a gene–protein index

This publication marks the beginning of the construction of a gene–protein index that relates proteins which are resolved on the two-dimensional protein map of Saccharomyces cerevisiae with their corresponding genes. We report the identification of 36 novel polypeptide spots on the yeast protein map. They correspond to the products of 26 genes. Together with the polypeptide spots previously identified, this raises to 41 the number of genes whose products have been identified on the protein map. The proteins identified here are concerned with four major areas of yeast cellular physiology: carbon metabolism, heat shock, amino acid biosynthesis and purine biosynthesis. Given the molecular weight and isoelectric point of the identified proteins, and the codon-usage bias of the corresponding genes, it can be estimated that 25 to 35% of all the soluble yeast proteins are detectable under the labelling and running gel conditions used in this study.     

Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co‐expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5′‐gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast‐enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast‐enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon‐optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest. Copyright © 2009 John Wiley & Sons, Ltd.     

An improved method for whole protein extraction from yeast Saccharomyces cerevisiae

A new method for protein extraction from yeast Saccharomyces cerevisiae cells is described. This method involves the use of LiAc and NaOH to enhance the permeability of yeast cell wall prior to protein extraction with SDS-PAGE sample buffer. It was safe and efficient compared to other methods reported so far in the literature. The proteins extracted with this new method retained their immunoreactive properties and are suitable for most applications in molecular biology studies.     

保宁醋醋曲中产香酵母的鉴定及挥发性成分研究

从保宁醋醋曲中筛选出4株产香较强的酵母,通过真菌r DNA-ITS区PCR扩增及构建系统发育树,确定四株产香酵母Y7和Y9、Y10、Y18分别为异常威克汉姆酵母（Wickerhamyces anomalus）和口津假丝酵母（Candida melibiosica）,采用GC-MS技术检测产香酵母发酵产物中的挥发性风味物质的种类和含量。结果表明:Y7、Y9、Y10、Y18风味物质种类数依次为26、30、21、22,其中,Y9、Y10、Y18的醇类物质种类较多,相对含量较高的为乙醇和苯乙醇,而Y7酯类物质种类最多,主要为具有浓郁果香的乙酸乙酯,其相对含量达到25.238%。产香酵母的风味物质种类及相对含量不同,可赋予食醋不同类型的香气,对于食醋的增香有着重要的意义。      

Identification of yeast strains using the polymerase chain reaction

Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.     

酵母蛋白饮料的研究

报导了酵母蛋白饮料的生产方法，乳化剂和稳定剂的选择，以及酵母蛋白饮料的质量标准。     

一株鸭梨酒酵母菌的鉴定

从市售鸭梨果皮中分离出1株酵母菌，经形态与培养特征、酵母假菌丝的观察、酵母菌子囊孢子的观察和生理生化试验，初步鉴定将酵母菌株GY定为酵母属的酿酒酵母(Saccharomyces cerevisive)。(孙悟)     

四株野生酵母菌株耐受性的研究

酿酒酵母对于葡萄酒风味形成起着重要的作用,使用产区酵母酿造葡萄酒可以突出产区的典型特色,从而避免我国葡萄酒在品种和风格上的单一。本实验利用WL培养基从采自攀枝花的葡萄自然发酵液中分离了酿酒酵母,结合显微镜下的细胞形态,及26S r DNA D1/D2区的序列分析和比对,成功分离得到四株酿酒酵母。进一步对这四株菌株的耐受性(高渗透压、高温、营养饥饿、高糖浓度、高浓度酒精、低p H)进行了研究,结果发现四株菌在上述耐受性方面有一定差异,以菌株C1和M1相对较佳。      

Relatedness of medically important strains of Saccharomyces cerevisiae as revealed by phylogenetics and metabolomics

Ten medically important Saccharomyces strains, comprising six clinical isolates of Saccharomyces cerevisiae and four probiotic strains of Saccharomyces boulardii, were characterized at the genetic and metabolic level and compared with non-medical, commercial yeast strains used in baking and wine-making. Strains were compared by genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis, by ribosomal DNA ITS1 sequencing and by metabolic footprinting using both direct injection mass spectrometry (DIMS) and gas chromatography-time of flight-mass spectrometry (GC-ToF-MS). Overall, the clinical isolates fell into different groupings when compared with the non-medical strains, with good but not perfect correlation amongst strains at both the genetic and metabolic levels. Probiotic strains of S. boulardii that are used therapeutically to treat human gastro-intestinal tract disorders showed tight clustering both genetically and metabolically. Metabolomics was found to be of value both as a taxonomic tool and as a means to investigate anomalous links between genotype and phenotype. Key discriminatory metabolites were identified when comparing the three main groups of clinical, probiotic and non-medical strains and included molecules such as trehalose, myo-inositol, lactic acid, fumaric acid and glycerol 3-phosphate. This study confirmed the link between a subset of clinical isolates and baking or probiotic strains but also highlighted that in general the clinical strains were more diverse at both the genomic and metabolic levels.     

Systematic analysis of yeast strains with possible defects in lipid metabolism

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild‐type strains, among them FY1679, CEN.PK2‐1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. Copyright © 1999 John Wiley & Sons, Ltd.     

Genetic analysis of the karyotype instability in natural wine yeast strains.

Yeast strains isolated from the wild may show high rates of changes in their karyotypes during vegetative growth. We analysed over 500 karyotypes from mitotic and meiotic derivatives of strain DC5, which has a chromosome rearrangement rate of 8.2 x 10(-3) changes/generation. About 70% of the meiotic derivatives of DC5 had low rearrangement rates, with an average of 5.8 x 10(-4) changes/generation, suggesting that karyotype instability behaved as a dominant phenotype. Diploid derivatives with low karyotype variability in mitosis also had low rates of chromosomal rearrangement during meiosis, suggesting that the two phenotypes may be linked. DC5 and some of its meiotic derivatives (both with high and low karyotype variability) had chromosome XII hypervariable bands. Their distribution among the meiotic products indicates that they are not indicators for genetic instability. To our knowledge, data in this paper are the first to indicate that karyotypically unstable yeast strains may give stable progeny at high rates. Understanding of the relevant mechanism(s) may allow the design of genetic strategies to stabilize karyotypes from natural and/or industrial wine yeasts with unacceptable karyotype rearrangement rates.     

Identification of polypeptides of the carbon metabolism machinery on the two-dimensional protein map of Saccharomyces cerevisiae. Location of 23 additional polypeptides

Using a modification of the basic two-dimensional polyacrylamide gel electrophoresis technique, we have undertaken a systematic identification of the polypeptides of the protein map of Saccharomyces cerevisiae corresponding to components of the carbon metabolism machinery. To the previous location of nine glycolytic enzyme polypeptides on the yeast protein map we add the location of 23 polypeptides. Ten of them were identified as corresponding to cytoplasmic enzymes of the carbon metabolism machinery and 13 were characterized as mitochondrial proteins. The criteria used to establish the identification of these polypeptides spots include migration with purified proteins, immunodetection, overproduction by plasmid–carrying strains and physiological behaviour.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

宁夏玉泉营地区酿酒葡萄酵母菌的分离筛选及分子鉴定

为进一步探究宁夏玉泉营地区酿酒葡萄酵母菌的多样性,更好地开发利用本土葡萄酒产区酵母菌资源,从宁夏玉泉营地区的葡萄园土壤、葡萄果实表皮以及葡萄自然发酵过程中进行酵母菌的分离筛选,对得到的22株酵母菌株运用WL营养琼脂培养基进行初步聚类分类,并采用26S rDNA D1/D2区序列分析法进行菌株鉴定。结果表明,供试菌株共鉴定为6种,分别为大隐球酵母（Cryptococcus magnus）、美极梅奇酵母（Metschnikowia pulcherrima）、葡萄酒有孢汉生酵母（Hanseniaspora vineae）、酿酒酵母（Saccharomyces cerevisiae）、克鲁维毕赤酵母（Pichia kluyveri）、东方伊萨酵母（Issatchenkia orientalis）。     

Nutritional and toxicological evaluation of yeast (Saccharomyces cerevisiae) biomass and a yeast protein concentrate

Brewer's yeast was prepared by alkaline treatment for debittering, cell wall rupture and dehydration by spray drying. Yeast protein concentrate was prepared by centrifugation of the ruptured cell suspension, treatment of the supernatant with sodium perchlorate, precipitation of the protein at isoelectric pH (4.2) and neutralisation of the isoelectric protein to pH 6.5 with sodium hydroxide, prior to lyophylisation. Chemical characterisation was performed on the biomass and protein concentrate. Amino acid scores were 98.1 and 87.2% for the whole biomass and protein concentrate respectively, based on available lysine and compared with the FAO/WHO/UNU reference standard. The growth‐promoting property of the yeast biomass protein was roughly 85% of casein and was significantly better than for the yeast protein concentrate. No difference in growth was found between 15 and 30% dietary protein for all three sources, ie casein, whole yeast biomass and yeast protein concentrate. When tested for subchronical toxicity at 15 and 30% protein concentration, no evidence of toxicity was found for the whole yeast biomass, compared with casein, after 45 and 90 days of feeding. Retarded growth and discrete liver steatosis were observed in rats fed the yeast protein concentrate at both dietary levels. © 2000 Society of Chemical Industry     

煎饼发酵面糊中酵母菌的鉴定及保护剂配方的优化

从煎饼发酵面糊中分离筛选出具有当地特色及自然发酵煎饼风味的酵母菌菌株。其中Y8生长迅速且耐酸性强,采用VITEC-2 compact微生物鉴定仪对其进行鉴定,并对其制备冻干菌发酵剂的保护剂进行筛选和优化。结果表明,煎饼发酵面糊中酵母菌为酿酒酵母菌,保护剂优化配方:甘油4.0%、海藻糖1.3%、脱脂乳12.7%,在此优化条件下,酿酒酵母菌的存活率可达82%,为传统食品的连续化、工业化生产提供依据。      

富硒酵母中硒蛋氨酸的GC-MS/MS测定

建立酵母中硒蛋氨酸含量的气相色谱——串联质谱法(GC-MS/MS)分析方法。比较3种富硒酵母中硒蛋氨酸检测样品的提取方法,优化硒蛋氨酸的酶解提取条件;以氯甲酸乙酯为衍生化试剂,2-氯苯丙氨酸为内标物,采用选择离子模式对衍生物进行GC/MS/MS检测。结果表明,硒蛋氨酸的回收率为90.0%～97.0%,检测限为4μg/L,方法精密度(RSD)为7.8%。该方法简单快捷、定量准确、灵敏度高。     

白酒酒曲中9株酵母菌的分离与特性研究

从本地白酒酒曲中成功分离出9株酵母菌,依次命名为Y-1～Y-9。比较了这9个菌株的生理生化特性、耐高温能力和耐酒精能力等特性。结果表明:分离的9株酵母菌上述特性差别较大,其中,Y-4各方面均表现突出,该菌株能利用木糖在内的多种糖源,在45℃下生长良好,能耐14%的酒精度。对橘子汁为原料进行的发酵实验表明,上述菌株均能以橘子汁为原料生产酒精,其中,Y-4发酵能力最强,发酵84h后,CO2失重为12.913g。综上所述,Y-4是一株极具潜力的燃料乙醇生产工程菌株。