The Candida albicans 14-3-3 gene, BMH1, is essential for growth.

The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMP-dependent RAS/MAPK and rapamycin-sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR. Unlike other yeasts, C. albicans has only one 14-3-3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1 Delta/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis.     

Genome-wide search of Schizosaccharomyces pombe genes causing overexpression-mediated cell cycle defects

Genetic studies in yeasts enable an in vivo analysis of gene functions required for the cell division cycle (cdc genes) in eukaryotes. In order to characterize new functions involved in cell cycle regulation, we searched for genes causing cell division defects by overexpression in the fission yeast Schizosaccharomyces pombe. By using this dominant genetic strategy, 26 independent clones were isolated from a Sz. pombe cDNA library. The cloned cDNAs were partially sequenced and identified by computer analysis. The 26 clones isolated corresponded to 21 different genes. Among them, six were genes previously characterized in Sz. pombe, 11 were homologues to genes identified and characterized in other organisms, and four represented genes with unknown functions. In addition to known cell cycle regulators encoding inhibitory protein kinases (wee1, pka1) and DNA checkpoint proteins (Pcna, rad24), we have identified genes that are involved in a number of cellular processes. This includes protein synthesis (ribosomal proteins L7, L10, L29, L41, S6, S11, S17 and the PolyA-Binding Protein PABP), protein degradation (UBI3), nucleolar rRNA expression (fib, imp1, dbp2), cell cytoskeleton (act1) and glycolysis (pfk1). The interference caused in the cell cycle by overexpression of these genes may elucidate novel mechanisms coupling different cellular processes with the control of the cell division. The effect caused by some of them is described in more detail.     

The phosphorylation and characterisation of soybean isolate and its β‐conglycinin component by casein kinase II

A commercial soybean isolate was phosphorylated using casein kinase II purified from the yeast Yarrowia lipolytica. Both major reserve proteins, β‐conglycinin and glycinin, were phosphorylated in a sequential way. The soybean isolate incorporated up to 0.7 mol phosphate per mole in 2 h. It was found that the phosphoester bonds were stable over time. The solubility of the phosphorylated isolate with respect to pH was not dramatically increased in comparison with the native one. However, counting the radioactivity of ³²P incorporated into the proteins (only the solubility of the phosphorylated proteins was measured in this case) showed that the solubility of the proteins was dramatically improved (up to 90% solubility for phosphorylated β‐conglycinin at pH 4). β‐Conglycinin became more soluble in the presence of CaCl₂ upon phosphorylation; this was not the case for the isolate. The iron‐binding capacity of the soy isolate and β‐conglycinin was significantly improved after phosphorylation (two and six times respectively). © 1999 Society of Chemical Industry     

Comparative study on cytochrome P-450 of yeasts using specific antibodies to cytochromes P-450alk and P-450(14DM)

The occurrence of cytochrome P-450(14DM) (lanosterol 14 alpha-demethylase) and cytochrome P-450alk (long-chain alkane terminal hydroxylase) in various yeast strains was determined with immunological procedures. Cytochrome P-450(14DM), which is constitutive or housekeeping enzyme playing an essential role in ergosterol biogenesis, was found in all yeast strains so far tested. Cytochromes P-450(14DM) from different species of yeast were immunologically different, although they may have had a few common antigenic sites. In contrast, cytochrome P-450alk was detected only in the alkane-assimilating yeasts.     

Functional genomics of commercial baker's yeasts that have different abilities for sugar utilization and high-sucrose tolerance under different sugar conditions

In the modern baking industry, high-sucrose-tolerant (HS) and maltose-utilizing (LS) yeast were developed using breeding techniques and are now used commercially. Sugar utilization and high-sucrose tolerance differ significantly between HS and LS yeasts. We analysed the gene expression profiles of HS and LS yeasts under different sucrose conditions in order to determine their basic physiology. Two-way hierarchical clustering was performed to obtain the overall patterns of gene expression. The clustering clearly showed that the gene expression patterns of LS yeast differed from those of HS yeast. Quality threshold clustering was used to identify the gene clusters containing upregulated genes (cluster 1) and downregulated genes (cluster 2) under high-sucrose conditions. Clusters 1 and 2 contained numerous genes involved in carbon and nitrogen metabolism, respectively. The expression level of the genes involved in the metabolism of glycerol and trehalose, which are known to be osmoprotectants, in LS yeast was higher than that in HS yeast under sucrose concentrations of 5-40%. No clear correlation was found between the expression level of the genes involved in the biosynthesis of the osmoprotectants and the intracellular contents of the osmoprotectants. The present gene expression data were compared with data previously reported in a comprehensive analysis of a gene deletion strain collection. Welch's t-test for this comparison showed that the relative growth rates of the deletion strains whose deletion occurred in genes belonging to cluster 1 were significantly higher than the average growth rates of all deletion strains.     

Molecular cloning and sequence analysis of the Zygosaccharomyces bailii HIS3 gene encoding the imidazole glycerolphosphate dehydratase

Zygosaccharomyces bailii is a spoilage yeast belonging to the Zygosaccharomyces genus. In recent years these yeasts, due to their exceptional resistance to several stresses, have become more and more interesting as model organisms to study the molecular basis of the said resistance. A Z. bailii cDNA library has been built and the 672 bp nucleotide sequence coding for the HIS3 gene was cloned by complementation of a Saccharomyces cerevisiae his3 mutant strain. The deduced 223 amino acid sequence shares a high degree of homology with His3p homologues in other non-conventional yeast species. The GeneBank Accession No. is AY050224.     

高产β-葡萄糖苷酶野生酵母的筛选及产酶能力差异性分析

为了比较分析野生酵母菌产β-葡萄糖苷酶的情况,筛选高产β-葡萄糖苷酶的酵母菌株,该研究采用七叶灵显色法和4-硝基苯基-β-D-吡喃葡萄糖苷（p-NPG）显色法对从宁夏贺兰山东麓分离的941株野生酵母的产β-葡萄糖苷酶能力进行筛选,通过WL培养基上的酵母菌落形态和26S rDNA D1/D2区的序列分析对所有酵母菌株进行分类鉴定,并且对酵母产β-葡萄糖苷酶能力进行差异性分析。 结果表明,941株酵母被鉴定为14个种,且筛选出了4株酵母菌高产β-葡萄糖苷酶：菌株SLY-4（98.51 U/L）、F2-24（76.93 U/L）、 F2-16（62.72 U/L）和HX-13（47.95 U/L）。 产酶能力差异性分析表明β-葡萄糖苷酶广泛分布于14种酵母,但是产酶能力表现出明显的种间和种内差异性。     

The mei3 region of the Schizosaccharomyces pombe genome

Expression of the mei3 gene is sufficient to induce meiosis in the fission yeast Schizosaccharomyces pombe. The mei3 gene is located 0.64 Mb from the telomere of the left arm of Sz. pombe chromosome II. We have sequenced and analysed 107 kb of DNA from the mei3 genomic region. The sequence includes 14 known genes (bag1-B, csh3, dps1, gpt1, mei3, mfm3, pac1, prp31, rpl38-1, rpn3, rti1, spa1, spm1 and ubc4) and 26 other open reading frames (ORFs) longer than 100 codons: a density of one protein-coding gene per 2.7 kb. Twenty-one of the 40 ORFs (53%) have introns. In addition there is one lone Tf1 transposon long terminal repeat (LTR), tRNA(Trp) and tRNA(Ser) genes and a 5S rRNA gene. 14 of the novel ORFs show sequence similarities which suggest functions of their products, including a coatomer alpha-subunit, a catechol O-methyltransferase, protein kinase, asparagine synthetase, zinc metalloprotease, acetyltransferase, phosphatidylinositol 4-kinase, inositol polyphosphate phosphatase, GTPase-activating protein, permease, pre-mRNA splicing factor, 20S proteasome component and a thioredoxin-like protein. One predicted protein has similarity to the human Cockayne syndrome protein CSA and one with human GTPase XPA binding protein XAB1. Three ORFs are likely to code for proteins because they have sequence similarity with hypothetical proteins, three encode predicted coiled-coil proteins and four are sequence orphans.     

Identification and characterization of a drug‐sensitive strain enables puromycin‐based translational assays in Saccharomyces cerevisiae

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C‐terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6?, pdr1? and pdr3? (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin‐based assays in yeast, which will enable new avenues of inquiry into protein production and maturation. Copyright © 2014 John Wiley & Sons, Ltd.     

Occurrence and growth of yeasts in processed meat products - Implications for potential spoilage

Spoilage of meat products is in general attributed to bacteria but new processing and storage techniques inhibiting growth of bacteria may provide opportunities for yeasts to dominate the microflora and cause spoilage of the product. With the aim of obtaining a deeper understanding of the potential role of yeast in spoilage of five different processed meat products (bacon, ham, salami and two different liver patés), yeasts were isolated, enumerated and identified during processing, in the final product and in the final product at the end of shelf life. Yeasts were isolated along the bacon production line in numbers up to 4.2 log (CFU/g). Smoking of the bacon reduced the yeast counts to lower than 1.0 log (CFU/g) or non-detectable levels. In general, yeasts were only isolated in low numbers during the production of salami, cooked ham and liver paté. In the final products yeasts were detected in low numbers in a few samples (3 out of 30) samples, 1.0-1.3 log (CFU/g). By the end of storage, yeasts were only detected in 1 out of 25 investigated samples 1.8 log (CFU/g). A combination of phenotypic and genotypic methods was used to identify the yeast microflora present during production of the processed meat products. The yeast microflora was complex with 4-12 different species isolated from the different production sites. In general, Candida zeylanoides, Debaryomyces hansenii and the newly described Candida alimentaria were found to be the dominant yeast species. In addition, three putatively previously undescribed yeast species were isolated. Fourteen isolates, representing seven different species isolated during the production of the processed meat products and one species isolated from spoiled, modified atmosphere packed, sliced ham, were screened for their ability to grow in a meat model substrate under a low oxygen/high carbon-dioxide atmosphere (0.5% O(2), 20% CO(2), 79.5% N(2)) at two different temperatures (5 and 8°C). Eleven out of the tested 14 strains were able to grow in the meat model substrate with C. zeylanoides, D. hansenii, Pichia guilliermondii and Candida sake reaching levels of 10(5)-5×10(6) log (CFU/g), where sensoryical changes appear.     

贵州产地刺梨果酒产酒精酵母的筛选及其发酵性能研究

选取贵州遵义、荔波、六盘水、都匀4个地区的新鲜刺梨为实验材料,采用孟加拉红培养基分离纯化、2,3,5-三苯基氯化四氮唑（TTC）培养基初筛产酒精能力强的酵母菌株,结合杜氏小管产气实验和耐受性实验复筛发酵性能优良的酵母菌株,然后采用复筛菌株酿造刺梨果酒,测定果酒理化指标,最后基于26S rDNA基因序列对复筛菌株进行分子生物学鉴定。结果表明,从刺梨中共分离得到35株酵母菌,并从中复筛3株优良酵母,编号分别为GL14、GP21、GP24,其产气快、凝聚性强,可耐受50%葡萄糖、18%vol乙醇、300 mg/L SO2、pH=2环境。菌株GL14、GP24发酵酿制的刺梨果酒在澄清度及酒精度方面优于菌株GP21,而菌株GP21在刺梨果酒的香气上更优,说明3株酵母菌均具有刺梨果酒酿造潜力。经鉴定,菌株GL14为异常威克汉姆酵母（Wickerhamomyces anomalus）,菌株GP21、GP24为热带假丝酵母（Candida tropicalis）。     

Bioactive Oxylipins in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae (including strains used in fermentation processes) produce short chain (mainly 8 carbon) oxylipins and not potent inflammatory long chain (20 carbon) oxylipins such as prostaglandins. When acetylsalicylic acid (aspirin) was added to cultures of Sacch. cerevisiae UOFS Y‐2330, flocculation was significantly inhibited as well as the production of 3‐hydroxy 8:0 thereby linking flocculation and this oxylipin. Furthermore, no traces of 3‐hydroxy 8:0 could be detected at the start of flocculation in this yeast. This research is based on (i) reports that yeasts in general can produce bioactive prostaglandins, (ii) findings suggesting a link between aspirin‐sensitive prostaglandins and biofilm formation by Candida albicans, (iii) the discovery that the addition of low concentrations of aspirin abolish yeast biofilm formation and sexual cell aggregation and (iv) the recent discovery of a novel potent aspirin‐sensitive pro‐inflammatory 3‐hydroxy prostaglandin E₂ synthesized by Candida albicans in conjunction with mammalian cells probably during candidiasis.     

Detection of β‐Glucosidase and Esterase Activities in Wild Yeast in a Distillery Environment

The study of β‐glucosidase and esterase in wild yeast, the enzymatic activities of which contribute to the distinctive flavours of grape‐derived alcoholic beverages, was the aim of this work. The study focused on wild yeast isolated from grape pomace and on identifying strains with interesting characteristics by examining their electrophoretic profiles. Zymograms revealed a high level of polymorphism. Some of these wild yeasts may be of interest for improving the quality of the distillate. This study also highlights the necessity of associating enzymatic properties to various environmental conditions, since these play an important role in the expression of wild yeast performance.     

黄酒生产中酵母菌发酵性能研究

随机分离黄酒生产用酒饼中的酵母菌菌株,比较不同来源的酵母菌细胞形态、细胞大小、发酵力、酵母悬浮细胞数、总降糖、酒精度、凝聚力、致死温度和耐酒精度,研究黄酒生产中酵母菌的发酵性能。结果表明,不同来源的7株酵母菌中,总降糖、酒精度、发酵力、凝聚力有显著差异,细胞形态、大小、致死温度、耐酒精度等没有明显差异。     

Genetically modified industrial yeast ready for application

Tremendous progress in the genetic engineering of yeast had been achieved at the end of 20th century, including the complete genome sequence, genome-wide gene expression profiling, and whole gene disruption strains. Nevertheless, genetically modified (GM) baking, brewing, wine, and sake yeasts have not, as yet, been used commercially, although numerous industrial recombinant yeasts have been constructed. The recent progress of genetic engineering for the construction of GM yeast is reviewed and possible requirements for their application are discussed. 'Self-cloning' yeast will be the most likely candidate for the first commercial application of GM microorganisms in food and beverage industries.     

2μm DNA PLASMID IN BREWERY YEASTS

Using an agarose gel screening procedure, 2 μm DNA plasmid was detected in all of 10 brewing strains of Saccharomyces yeast examined and in both of two non-brewing, dextrin-utilising strains. Plasmid DNA was identified in yeast grown with access to air in MYGP medium or in hopped wort, and in yeast harvested from 3-day wort fermentations. The yeast plasmid is a suitable self-cloning vector for the genetic manipulation of brewing yeasts by transformation.     

Adaptive evolution of wine yeast

Alcoholic fermentation is one of the main phases in wine production. It is usually conducted by yeasts belonging to the species Saccharomyces cerevisiae. Industrial S. cerevisiae strains are highly specialized organisms, which have evolved to utilize to their full potential the different environments or ecological niches. So, during the alcoholic fermentation, the yeast has been adapted to different kinds of stress conditions; this adaptation is call "domestication". In this review, we describe the different mechanisms involved in the adaptive evolution of wine yeast strains.