A database of microsatellite genotypes for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A system for genotyping Saccharomyces cerevisiae is described based on a multiplex of ten microsatellite loci and the MAT locus. A database of genotypes has been developed for 246 yeast strains, including a large set of commercial wine yeasts, as well as 35 sequenced natural isolates currently being sequenced. The latter allow us, for the first time, to make direct comparisons of the relationship between DNA sequence data and microsatellite-based genotypes. The genotyping system provides a rapid and valuable system for strain identification as well as studying population genetics of S. cerevisiae.     

A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors

Saccharomyces cerevisiae is the most widely used yeast in industrial/commercial food and beverage production and is even consumed as a nutritional supplement. Various cases of fungemia caused by this yeast species in severely debilitated traumatized or immune-deficient patients have been reported in recent years, suggesting that this species could be an opportunistic pathogen in such patients. To determine whether the industrial S. cerevisiae strains can be included in this virulent group of strains, we carried out a comparative study between clinical and industrial yeasts based on the various phenotypic traits associated with pathogenicity in two other yeast species (Candida albicans and Cryptococcus neoformans). The majority of the clinical isolates were found to secrete higher levels of protease and phospholipase, grow better at 42°C and show strong pseudohyphal growth relative to industrial yeasts. However three industrial yeast strains, one commercial wine strain, baker’s yeast and one commercial strain of S. cerevisiae (var. boulardii), were exceptions and based on their physiological traits these yeasts would appear to be related to clinical strains.     

Transfer of the flocculation property to the Baker's yeastSaccharomyces cerevisiae by conventional genetic manipulation

Summary An haploid MAT strain derived from a non flocculent commercial Baker's yeastSaccharomyces cerevisiae was mated with aSaccharomyces cerevisiae 1209 FLO₁ strain (flocculation degree F₅) and a diploid and extremely flocculent strain was selected. After sporulation and separation of single spore clones of the diploid hybrid, both MAT FLO₁ and MAT FLO₁ haploid strains were isolated and aSaccharomyces cerevisiae diploid strain FLO₁/FLO₁ carrying a great part of the genome of Baker's yeast was obtained by mating these.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

Yeast strains for concentrated substrates

Summary The screening of twenty yeast strains for ethanol productivity at high osmotic pressure at temperatures ranging from 32°C to 45°C is described. Shake flask fermentations of 30°, 40°, and 50° Bx cane molasses were performed. The effect of temperature on productivity at a non-inhibitory ethanol level is weakly pronounced. Most strains fermented poorly at 50° Bx molasses but two Schizosaccharomyces pombe and one commercial baker's yeast, Saccharomyces cerevisiae performed well at all concentrations of molasses. In an extended study with Schizosaccharomyces pombe (CBS 352) and Saccharomyces cerevisiae (SJAB, fresh yeast), simulating a continuous run it was shown that Schizosaccharomyces pombe was less sensitive to high DS than Saccharomyces cerevisiae. At 25% DS the productivity of Schizosaccharomyces pombe is almost twice that of Saccharomyces cerevisiae.     

接种发酵和自然发酵中酿酒酵母菌株多样性比较

[目的]探究自然发酵和接种发酵两种发酵方式,对霞多丽葡萄发酵中酵母菌种多样性和酿酒酵母菌株遗传多样性的影响.[方法]以霞多丽葡萄为原料,分别进行自然发酵和接种不同酿酒酵母菌株(NXU 17-26、UCD522和UCD2610)的发酵,利用26S rDNA D1/D2区序列分析和Interdelta指纹图谱技术分别进行酵...     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Characterization of industrial strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exhibiting filamentous growth induced by alcohols and nutrient deprivation

Summary The use of microorganisms in biotechnology is an important economic area of interest in Brazil, especially the use of Saccharomyces cerevisiae in the baking and alcohol fermentation industries. Dimorphism in S. cerevisiae (cell morphology alterations from budding cells to filamentous structures) has been observed in conditions of nitrogen and carbon deprivation and in the presence of fusel alcohols. This can be described as a defense mechanism that allows the yeast to forage for nutrients through cell elongation, hyphal formation and invasive growth. In this work fifteen industrial strains of S. cerevisiae (including haploid and diploid strains) isolated from the fermentative process for alcohol production were characterized for filamentation on solid culture media under growth conditions of carbon- and nitrogen-deprivation and in the presence of fusel alcohols. The majority of strains showed filamentation induced by isoamyl alcohol, butanol, isopropanol and isobutanol, but not by methanol. In rich medium (YEPD), both haploid and diploid strains showed invasive growth, although this kind of filamentous growth was more common in haploid strains. Similar results were observed when fructose or mannose was used as the sole carbon source. In nitrogen-deficient medium (SLAD) the strains did not filament. The results obtained indicate that the filamentation induced by higher alcohols and carbon deprivation (specially carbon) is a common process in industrial strains of S. cerevisiae contributing towards their maintenance/survival in adverse conditions.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

An electrophoretic karyotype for Candida albicans reveals large chromosomes in multiples

Summary Using field-inversion gel electrophoresis we defined an electrophoretic karyotype for the yeast, Candida albicans. The karyotype is distinct from other species of Candida and is species specific. A total of five distinct chromosomal mobility groups were observed, at least four of which are composed of a minimum of two fragments each. From the apparent sizes of these fragments relative to the large chromosomes of the morphologically related yeast Saccharomyces cerevisiae, together with the known genome size of this organism, we conclude that the karyotype is the result of the migration of intact chromosomes.     

Substitution of serine caused by a recessive lethal suppressor in yeast.

The SUP-RL1 suppressor in the yeast Saccharomyces cerevisiae causes lethality in haploid strains but not in diploid or aneuploid strains that are heterozygous for the suppressor locus. This recessive lethal suppressor acts on amber (UAG) nutritional markers, and can cause the production of approximately 50% of the normal amount of iso-1-cytochrome c in disomic strains that are heterozygous for the SUP-RL1 suppressor, and that contain the cyc1-179 allele which has an amber codon corresponding to amino acid position 9. The suppressed iso-1-cytochrome c contains a residue of serine at the position that corresponds to the site of the amber codon. SUP-RL1 was found to lie between thr4 and MAL2 on chromosome III, approximately 30 map units from the mating-type locus. It is suggested that the gene product of SUP-RL1 may be a species of serine transfer RNA that normally reads the serine codon UCG, and that is represented only once in the haploid genome.     

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.     

Fermentation behaviour of controlled mixed and sequential cultures of Candida cantarellii and Saccharomyces cerevisiae wine yeasts

Behaviour of Candida cantarellii and Saccharomyces cerevisiae strains during the fermentation of Syrah grape must using pure, mixed and sequential yeast cultures was studied. Different kinds of inocula have been tested according to the type of culture. Inocula proportions used in mixed C. cantarellii and S. cerevisiae strains reflect the population levels in natural grape microbiota. Biomass evolution of both strains was analysed in relation to different byproduct levels. Saccharomyces cerevisiae overcame C. cantarellii in the different co-culture assays at 48 h of fermentation. The final concentration of ethanol was similar in mixed and both sequential tests and higher (from 7.8 to 10.6%) than in S. cerevisiae pure culture. In mixed and sequential cultures, the glycerol content of the final products was 44.3 to 52.8% higher than the one obtained with pure S. cerevisiae fermentation. Wine analytical profiles of experiments that involved S. cerevisiae and C. cantarellii strains differed from the pure ones mainly in acetoin, propanol and succinic acid contents. From an enological point of view, analysed byproducts are relevant. Considering this, mixed assay and the inoculation of S. cerevisiae after 3 days of pure C. cantarellii fermentation appear to be the more appropriate options to develop the particular characteristics of Syrah wines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Amplification and loss of repeat units of the human minisatellite MS1 integrated in chromosome III of a haploid yeast strain

Minisatellites comprise arrays of tandemly repeated short DNA sequences which show extensive variation in repeat unit number. The mechanisms that underlie this length variation are not understood. In order to study processes influencing length changes of minisatellites, we integrated the human minisatellite MS1 into a haploid strain of the yeast Saccharomyces cerevisiae. Frequent spontaneous generation of MS1 alleles with new lengths were observed in this yeast strain. Hence it is concluded that recombination between members of a pair of homologous chromosomes is not a prerequisite for the generation of length changes in MS1 in yeast.     

Yeast population dynamics in spontaneous fermentations: Comparison between two different wine-producing areas over a period of three years

Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.     

Functional expression of amine oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> (AO-I) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.     

Taxonomical classification of yeasts isolated from kefir based on the sequence of their ribosomal RNA genes

Yeast strains present in 10 samples of kefir of different commercial and domestic origins have been isolated and classified taxonomically on the basis of the internal transcribed sequences (ITS) of their ribosomal RNA genes. A total of 18 yeast strains representing 10 different species have been characterized. Of the three commercial kefir samples analyed, two contained the well characterized yeast Kluyveromyces lactis while no yeast was found in the other one. A broader spectrum of yeast species was found among the home-made kefir samples, of which Issatchenkia orientalis, Saccharomyces unisporus, Saccharomyces exiguus and Saccharomyces humaticus were the most representative species.     

Identification and characterization of yeasts in brem, a traditional Balinese rice wine

Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer (ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae(35 strains), Candida glabrata(six strains), Pichia anomala(three strains) and Issatchenkia orientalis(seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.     

Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines

Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast.