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PubMed2006

Population genomic analysis of outcrossing and recombination in yeast.

The budding yeast Saccharomyces cerevisiae has been used by humans for millennia to make wine, beer and bread. More recently, it became a key model organism for studies of eukaryotic biology and for genomic analysis. However, relatively little is known about the natural lifestyle and population genetics of yeast. One major question is whether genetically diverse yeast strains mate and recombine in the wild. We developed a method to infer the evolutionary history of a species from genome sequences of multiple individuals and applied it to whole-genome sequence data from three strains of Saccharomyces cerevisiae and the sister species Saccharomyces paradoxus. We observed a pattern of sequence variation among yeast strains in which ancestral recombination events lead to a mosaic of segments with shared genealogy. Based on sequence divergence and the inferred median size of shared segments (approximately 2,000 bp), we estimated that although any two strains have undergone approximately 16 million cell divisions since their last common ancestor, only 314 outcrossing events have occurred during this time (roughly one every 50,000 divisions). Local correlations in polymorphism rates indicate that linkage disequilibrium in yeast should extend over kilobases. Our results provide the initial foundation for population studies of association between genotype and phenotype in S. cerevisiae.

Crosses, Genetic↗

PubMed2006

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.

Fermentation↗

PubMed1995

Genetically-modified brewing yeasts for the 21st century. Progress to date.

Academic studies and traditional breeding of yeasts depend upon their sporulation lifestyle. The strains used have been specially selected to sporulate readily and to mate producing new yeast types. Unfortunately brewing yeast strains do not behave in this way. They sporulate poorly, any spores which are formed are usually non-viable and any haploid strains produced are invariably non-maters. Only in recent years, with the development of recombinant-DNA techniques, has the specific breeding of new brewing yeast strains become widespread. Strains have been produced with the ability to ferment a wider range of carbohydrates, with altered flocculation properties and which produce beers with modified flavours. Many have been tested on the pilot scale and one, an amylolytic brewing yeast, has received approval for commercial use.

Carbohydrate Metabolism↗

PubMed1985

Improvement of a Wine Saccharomyces cerevisiae Strain by a Breeding Program.

Hybridization by spore conjugation was used to develop new and improved wine yeasts of Saccharomyces cerevisiae. The procedure was achieved with diploid, homothallic strains with high sporulation frequency and high spore viability. The method was verified by crossing flocculent and non-H(2)S-forming strains. Single-spore descendants of the hybrids were studied by tetrad analysis with regard to the aforementioned characters and the other two winemaking traits, i.e., ethanol production and fermentation rate. A highly flocculent, non-H(2)S-forming wine yeast strain with a high fermentation rate and high ethanol production was obtained.

Journal Article↗

PubMed1992

[Characteristics of obtaining protoplasts from 2 strains of Tolypocladium inflatum subsp. Blastosporum].

The optimal conditions for preparing protoplasts with high yields by using the cells of two (low and high potent) isogenic cyclosporine-producing Tolypocladium strains were developed. A specific medium containing 0.5 per cent yeast autolysate (by dry weight) and 3 per cent glucose was used. When grown on this medium the cells of the highly potent strain 847 acquired a yeast-like shape. High yields of protoplasts prepared from the low potent strain 43 mycelium were obtained via prior incubation with 0.01 M dithiothreitol followed by treatment with a complex of enzymes from Helix pomatia for 1.5 to 2 hours was used. For preparation of the protoplasts with employing the highly potent strain 847 cells the prior incubation with dithiothreitol was not required, but it was necessary to employ a mixture of the enzyme complex (Helix pomatia), drizilase (Irpex lacteus) and chitinase (Streptomyces griseus) for 18 hours. The electron microscopic data on the two isogenic strains and their protoplasts are presented. The protoplasts proved to be a suitable initial material for investigating bioenergetic processes at the subcellular level and further genetic improvement of the strains.

Cell Count↗

PubMed2005

The use of yeast mitochondria to study the properties of wild-type and mutant human mitochondrial ornithine transporter.

Yeast deletion models have general utility for the study of a variety of inherited metabolic disorders. Mutations in the mitochondrial ornithine transporter result in hyperammonemia, hyperornithinemia, homocitrullinuria syndrome, a disorder of the urea cycle. To study the effects of mutations in a model system that more closely resembles the in vivo environment, we have developed an expression system based on a yeast strain lacking its endogenous ornithine transporter homologue. Wild-type human ornithine transporter and a recurrent mutation, DeltaF 188, were expressed and characterized with this system. The wild-type transporter appeared to insert into yeast mitochondria in the same orientation as in mammalian mitochondria. It showed stereospecificity, strong antiport activity and ornithine transport was competed by citrulline and arginine. The DeltaF 188 mutant was not incorporated into the membrane to the same extent as wild type, but retained significant residual activity and lost stereospecificity. In these isolated mitochondria, samarium chloride was found to be a potent blocker of transport compared to previously reported sulfhydryl-based inhibitors. A low-affinity background transport activity that promoted the exchange of ornithine for either acidic or basic amino acids was observed. This yeast model can readily be extended to the study of protein:protein interactions. In this manner, the use of yeast deletion strains can serve as a general framework to perform metabolic pathway analysis.

Amino Acid Transport Systems, Basic↗

PubMed2000

The awesome power of yeast biochemical genomics.

A new genomic strategy for identifying the gene encoding any biochemical activity has recently been developed, in which an array of individual yeast strains expressing a genomic set of open reading frames fused to glutathione S-transferase can be assayed for a biochemical activity of interest. Designated 'biochemical genomics', this approach represents an innovative application of genomic information.

Genes, Fungal↗

PubMed1996

A new efficient gene disruption cassette for repeated use in budding yeast.

The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

Base Sequence↗

PubMed1999

A biochemical genomics approach for identifying genes by the activity of their products.

For the identification of yeast genes specifying biochemical activities, a genomic strategy that is rapid, sensitive, and widely applicable was developed with an array of 6144 individual yeast strains, each containing a different yeast open reading frame (ORF) fused to glutathione S-transferase (GST). For the identification of ORF-associated activities, strains were grown in defined pools, and GST-ORFs were purified. Then, pools were assayed for activities, and active pools were deconvoluted to identify the source strains. Three previously unknown ORF-associated activities were identified with this strategy: a cyclic phosphodiesterase that acts on adenosine diphosphate-ribose 1"-2" cyclic phosphate (Appr>p), an Appr-1"-p-processing activity, and a cytochrome c methyltransferase.

Adenosine Diphosphate Ribose↗

PubMed1972

Factors influencing the production of H and M antigens by Histoplasma capsulatum: development and evaluation of a shake culture.

Studies were undertaken to improve the production of histoplasmin for use in complement-fixation tests and in the determination of H and M antibodies. A shake culture method performed at 25 C was developed with a yeast-phase inoculum. Eight strains of Histoplasma were tested in three synthetic media to evaluate the effects of strain and medium on H and M antigen production. Intrastrain variation was negligible, and antigen production was reproducible. All of the strains produced H antigen; six strains produced both H and M antigens, and two produced only H antigen. The time of H and M antigen appearance varied with the medium; M antigen appearance was dependent upon the strain and medium used. Titers of M antigen appeared to be greater in stagnant culture.

Antibodies↗

PubMed2001

Ethanol-induced cell aggregation (flocculation) and its physiological background in Schizosaccharomyces pombe rive 4-2-1.

Cell aggregation (flocculation) of the yeast Schizosaccharomyces pombe strain RIVE 4-2-1 developed in glucose-containing medium, but only in the presence of ethanol. Cell surface proteins which participated in cell to cell interactions were characterised by the susceptibility of flocculation to different proteolytic enzymes, heat treatment, denaturing and thiol compounds and by the inhibition of flocculation by sugars and derivatives. It was shown that a galactose-specific lectin was involved in this new type of flocculation.

Carbohydrates↗

PubMed1983

The use of a killer factor in the selection of hybrid yeast strains.

A new selection method of yeast cell hybrids, based on the application of yeast killer factors, has been developed in the model system of induced protoplast fusion of Saccharomyces cerevisiae superkiller strain T158C (alpha, his-) and different sensitive strains. The method is comparable in its efficiency to the currently used selection techniques based on the auxotrophy or respiration deficiency of the parental cells. Selection by means of a killer factor, when compared to other methods, has the following advantages: (1) Sensitivity to a killer factor is a current natural property of most of the yeast strains. (2) Killer strains of different species of yeast are available and the killer character can be easily transmitted from strain to strain. (3) Hybrid cells produce a killer factor (K+) and are resistant to it (R+). Both these properties are easily and independently testable. (4) Hybrid cells can be cured of the "killer character" by growth at elevated temperatures. Therefore the killer character can also serve as a temporary marker for the purposes of selection of hybrid cells and can be eliminated when no longer desired. On an example of interspecific fusion of strain T158C of S. cerevisiae and strain P9 of S. uvarum the procedure has been shown to be advantageous particularly for the construction of new industrial yeast strains because mutagenesis of the original industrial strain is not required.

Cell Survival↗

PubMed1999

A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains.

We have developed a simple, fast and reliable method for the analysis of genetic stability in budding yeast strains. The assay relies on our previous finding that cells expressing the green fluorescent protein (GFP) can be detected and counted by flow cytometric analysis (FACS) (Niedenthal et al., 1996). Expression of a gfp-carrying CEN-plasmid in a wild-type strain resulted in the emission of strong fluorescence from 80% of the cell population. Strong fluorescence and presence of the plasmid, determined by the presence of the URA3 genetic marker, was strictly correlated. Expression of this plasmid in 266 yeast strains, each carrying a complete deletion of a novel, non-essential gene identified in the S. cerevisiae sequencing project, pinpointed 12 strains with an increased level of mitotic plasmid loss. Finally we have shown that measurement of mitotic loss of artificial chromosome fragments equipped with the gfp expression cassette can be performed quantitatively using FACS.

Chromosome Segregation↗

PubMed1994

A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity.

A system has been developed for the release of heterologous proteins from Saccharomyces cerevisiae, based on the use of thermosensitive osmotic-remedial mutants, deficient in cell integrity, that lyse at the non-permissive temperature, thus releasing the bulk of intracellular proteins and leaving behind cell ghosts and debris. The strains developed combine the lyt2 mutation (which is allelic to gene SLT2/MPK1 coding for a MAP kinase homolog), with the disruption of genes PEP4 and PRB1 known to produce a protease-deficient background. Cells transformed with the appropriate bacterial gene, released about 70% of the heterologous protein chloramphenicol acetyl transferase (CAT) in bioreactor cultivation upon switching growth temperature to 37 degrees C, or by osmotic shock of the cells preincubated at 37 degrees C in the presence of 1 M sorbitol. It is suggested that our release system could be advantageous for obtaining large-scale protein preparations for downstream processing without any mechanical breakage of the cells, enzymatic treatment or chemical extraction.

Chloramphenicol O-Acetyltransferase↗

PubMed2006

Isolation of Saccharomyces cerevisiae strains producing higher levels of flavoring compounds for production of "cachaça" the Brazilian sugarcane spirit.

In Brazil, spontaneous fermentation and open vessels are still used to produce cachaça (the Brazilian sugarcane spirit) and this fermentation is characterized by mixed cultures with continuous succession of yeast species. This work shows the development of a methodology for isolation of yeasts, particularly Saccharomyces cerevisiae, used in the production of cachaça. According to the proposed strategy, the strains were selected for their ability to adapt to stress conditions encountered during fermentation of the sugarcane juice such as high sucrose concentration; high temperatures and high alcohol concentration; for their capacity to flocculate; and for their higher fermentative ability. For strains with such characteristics, specific procedures were employed to select for 5,5,5-trifluoro-DL-leucine (TFL) and cerulenin-resistant strains, since these characteristics are related to a higher capacity of production of the flavoring compounds isoamyl alcohol and caproic acid, respectively. The effectiveness of such a selection strategy was documented. Taken together, the results obtained present the development of a new strategy to isolate yeast strains with appropriated characteristics to be used in the cachaça industry. Moreover, the results obtained offer an explanation for the great variability in terms of chemical composition found in products obtained even in a single distillery.

Adaptation, Physiological↗

PubMed2006

Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae.

Uncovering genetic control of variation in ethanol tolerance in natural populations of yeast Saccharomyces cerevisiae is essential for understanding the evolution of fermentation, the dominant lifestyle of the species, and for improving efficiency of selection for strains with high ethanol tolerance, a character of great economic value for the brewing and biofuel industries. To date, as many as 251 genes have been predicted to be involved in influencing this character. Candidacy of these genes was determined from a tested phenotypic effect following gene knockout, from an induced change in gene function under an ethanol stress condition, or by mutagenesis. This article represents the first genomics approach for dissecting genetic variation in ethanol tolerance between two yeast strains with a highly divergent trait phenotype. We developed a simple but reliable experimental protocol for scoring the phenotype and a set of STR/SNP markers evenly covering the whole genome. We created a mapping population comprising 319 segregants from crossing the parental strains. On the basis of the data sets, we find that the tolerance trait has a high heritability and that additive genetic variance dominates genetic variation of the trait. Segregation at five QTL detected has explained approximately 50% of phenotypic variation; in particular, the major QTL mapped on yeast chromosome 9 has accounted for a quarter of the phenotypic variation. We integrated the QTL analysis with the predicted candidacy of ethanol resistance genes and found that only a few of these candidates fall in the QTL regions.

Chromosome Mapping↗

PubMed1999

Yeast colonisation & fungaemia in preterm neonates in a tertiary care centre.

Seventy consecutive preterm neonates who stayed in the hospital for more than seven days between March and October 1996, were studied for colonisation at oral, umbilical, groin, and rectal areas and for fungaemia. Overall, 71.4 per cent of the neonates were colonised and colonisation occurred within 24 h in 38 per cent preterm neonates. Neonates weighing less than 1500 g were colonised more frequently at more than one site and had higher load of yeast. Candida albicans (19%), Pichia (Hansenula) anomala (17.5%), C. tropicalis (13.2%), C. parapsilosis (12.3%) and Trichosporon cutaneum (10.0%) were the predominant colonising yeasts. Fungaemia was detected in 22.8 per cent of preterm neonates with predominance of P. anomala fungaemia (62.5%). Prematurity, male sex, broad spectrum antibiotic therapy, intubation and higher colonising rate were identified as significant risk factors for development of fungaemia. Except one strain of C. tropicalis, all yeast strains were sensitive to commonly used systemic antifungal agents. Study highlights the importance of routine surveillance of yeast colonisation of preterm neonates with identifying possible risk factors.

Anti-Bacterial Agents↗

PubMed1993

[Utilization of molecular techniques for the characterization of wine yeasts and the study of the wine-making process].

The study of the fermentation process in the Alicante region allowed us to conclude that adverse climatic conditions could be responsible for deficient wine-making with serious problems arising fermentations, usually causing incomplete fermentation. In order to avoid these problems, we selected a Saccharomyces cerevisiae strain, namely T73, isolated in the same Alicante region, to be used to perform controlled fermentations. The use of selected strains in the wine-making process requires the development of characterization techniques that can clearly differentiate between the inoculated strain and the wild strains present in the musts. In order to differentiate strains present in the wine ecosystems, an extensive survey of different methods of yeast strains identification has been carried out. However, these techniques are very complex to be used in industry. For this reason, we have developed a new, simple, inexpensive and rapid method based on mitochondrial DNA restriction analysis. This technique was applied to the control of wine fermentations conducted by active dry yeasts. This molecular approach allows us to understand the role of the inoculated dry yeast strain and that of the natural S. cerevisiae flora during wine fermentation.

DNA, Fungal↗