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PubMed2005

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity.

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.

Colony Count, Microbial↗

PubMed1981

Radioimmunoassay for yeast killer toxin from Saccharomyces cerevisiae.

A radioimmunoassay was developed for the K1 killer toxin from strain T158C/S14a of Saccharomyces cerevisiae. 125I-labeled toxin was made to a specific activity of 100 microCi/mg of protein (1 microCi = 37 kBq). Antibody to purified toxin was prepared in rabbits using toxin cross-linked to itself. These antibodies, partially purified by 50% ammonium sulfate precipitation and Sepharose CL-6B column chromatography, produced one precipitation band with killer toxin and bound 125I-labeled toxin in a radioimmunoassay. The antibody preparation also bound with the toxins from another K1 killer, A364A, and three chromosomal superkiller mutants derived from it.

Animals↗

PubMedDate not supplied

Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.

The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods.

Biological Assay↗

PubMed1978

Transformation of yeast by a replicating hybrid plasmid.

Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.

DNA, Recombinant↗

PubMed1994

[Future directions of antimycotic therapy].

There are presently numerous developments in the field of antifungal armamentarium. Amphotericin B has been considered as the standard treatment since the fifties for most invasive fungal infections. However, emergence of resistant strains and failures despite adequate treatment with amphotericin B are numerous and toxicity as well as side effects are limiting factors in many patients. Alternative modalities to administer amphotericin B using liposomal or lipid formulations are presently evaluated. Tremendous research has been performed in the development of new azoles including ketoconazole, fluconazole and itraconazole. The emergence of resistant strains of yeasts in patients treated with fluconazole requires attention. The problem of possible cross resistance for all azoles should be a matter of further investigation. Itraconazole is a new broad-spectrum antifungal agent. But due to its pharmacokinetic properties monitoring of serum concentrations is recommended in life-threatening situations. New formulations of itraconazole are currently under development and may be extremely useful. The role of azole antifungals for prophylactical administration or therapeutic purposes must be balanced carefully. Studies designed to evaluate adjuvant antifungal treatment based on G-CSF, M-CSF or GM-CSF in combination to antifungals would be useful. Future directions of antifungal treatments should include evaluation of combination of antifungal agents to each other. There is a tremendous need for pivotal clinical studies in the field of antifungal treatments which need international collaboration in order to collect rapidly significant numbers of data. Such comprehensive approaches are initiated by the EORTC Invasive Fungal Infections Cooperative Group, and all European clinicians taking care of mycosis patients are invited to cooperate.

Antifungal Agents↗

PubMed1996

Influence of the culture medium on the inhibitory activity of Arthrinium strains.

Modifications in the antibiotic capacity of Arthrinium strains when they were developed in culture media of various compositions were studied. The culture media used were 2% malt extract agar, Czapek-Dox agar, Sabouraud dextrose agar, oatmeal agar, Yoshimura's medium, mixed medium of salts, modified phytone agar, malt extract-yeast extract agar, potato dextrose agar, and Wickerham's medium. The inhibition values were compared with those obtained when the strains were developed in Wickerham's medium. The media which enhanced the production of inhibitory substances were potato dextrose agar and 2% malt extract agar.

Anti-Bacterial Agents↗

PubMed2003

[The genetic control of cell growth and development in yeast Saccharomyces cerevisiae. Disturbed sporulation in diploids with decreased activity of the Ras/cAMP signal transduction pathway].

Seven haploid strains (four with the MAT alpha mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast. Previous phenotypic analysis assigned six of these strains to a physiological group of strains with a lower activity of the Ras/cAMP signal transduction pathway. The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media. Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency). The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway. All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34 degrees C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose). Sporulation proved to depend on temperature and medium composition. The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.

Culture Media↗

PubMed2003

Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine.

AIMS: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. METHODS AND RESULTS: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected strains could be used as adjunct starters to make high quality Feta cheese.

Aminopeptidases↗

PubMed2006

Fermentative production of superoxide dismutase with Kluyveromyces marxianus.

This work sought to develop a fermentative process for the microbial production of superoxide dismutase (SOD), to overcome extraction from animal tissues. Twenty-eight wild-type yeast strains were screened for SOD productivity. Kluyveromyces marxianus L3 showed the highest SOD activity (62 U mg(-1)) and was used for process development. Oxidative stress conditions and parameters affecting oxygen transfer rate were exploited to improve production. The effects of dilution rate (0.067 vs 0.2 h(-1)), aeration pressure (0.3 vs 1.2 bar) and H(2)O(2) (0 vs 50 mM) were studied during chemostat experiments. Low dilution rate, high pressure and H(2)O(2) resulted in an increase in CuZn-SOD up to 475 U mg(-1). When a regulation of oxygen saturation was applied during batch cultures, CuZn-SOD was progressively higher at 60, 80 and 90% dissolved oxygen tension (DOT) (250, 330 and 630 U mg(-1), respectively). Furthermore, the highest growth rate and biomass yield were achieved at 90% DOT, this being therefore the best DOT condition for high overall productivity. Growth and productivity on different carbon sources were compared. Specific activity was higher on glycerol than on lactose or glucose (496, 454 and 341 U mg(-1), respectively). The highest biomass yield was achieved on lactose. It may be therefore the best substrate for SOD production.

Biotechnology↗

PubMed1995

Evolution in heterogeneous environments: genetic variability within and across different grains in Tribolium castaneum.

The course of adaptation to heterogeneous environments is influenced by the magnitude of genetic variation for ecologically important characters within each environment and the extent of genotype x environment interaction. Using the genetic correlation between the expression of characters in different environments as a measure of genotype x environment interaction is particularly useful for evolutionary interpretation. In this study, we estimated patterns of genetic variability and cross-environment genetic correlations for pupal weight and development time in two strains of the flour beetle Tribolium castaneum in five flours (wheat with brewer's yeast, wheat, rice, corn and oat). Wheat plus yeast is the standard medium in which the strains have been reared for hundreds of generations; other flours are novel environments. The results indicated moderate levels of genetic variation within the various flours for pupal weight but not for development time. Performance varied considerably across flours, with the highest performance for both strains found in the standard medium and the poorest in oat flour. The genetic correlations of pupal weight across flours in both strains were generally not significantly different from + 1. This suggests that evolution of body size in different flours cannot proceed independently, and that improved performance in the novel flours may produce declines in fitness in the standard environment.

Animals↗

PubMedDate not supplied

Engineered Saccharomyces cerevisiae strain BioS-OS1/2, for the detection of oxidative stress.

One of the major stress factors during space and high-altitude flight is the oxidative damage caused by the release of reactive oxygen intermediates (ROIs) in human tissues. ROIs are released in response to several stress factors including radiation in space. Since ROIs contribute to several pathological conditions, there has been a great interest in developing a biosensor that can monitor the impact of ROIs on biological systems. Toward this goal, we sought to engineer a yeast stain that can monitor oxidative stress and be easily integrated into a biosensor platform. Saccharomyces cerevisiae respond to hyperoxidative stress by activating the expression of many proteins including the transcription factor, Yap1. Activated Yap1 primarily binds to the Yap-1 response elements in the promoters of genes that combat oxidative stress. Based on these observations, we genetically altered the Yap-1 pathway in the YCR094W BY4742 strain of S. cerevisiae by fusing the YREs in the promoter region of TRX2 gene to a cDNA-insert encoding green fluorescent protein (GFP). Exposure of this engineered yeast strain BioS-OS1 to varying levels of oxidative stress, as generated by different concentrations of H(2)O(2) or diamide, elicits robust expression of GFP that can be monitored by the fluorescence of GFP by as early as 1 h. BioS-OS1 can detect a H(2)O(2) concentration from 300 microM onward. We also show that the signaling strength of the strain can be increased by engineering multiple YREs in the upstream of the cDNA-insert encoding GFP. Thus, the results presented here demonstrate that the engineered BioS-OS yeast strain can detect ROI-generating oxidative stress and validate the use of this prototypic strain for the development of a biosensor to detect and monitor oxidative stress factors during space and high altitude flights.

Biosensing Techniques↗

PubMed2004

Polynucleotide phosphorylase-based photometric assay for inorganic phosphate.

Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5'-diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5'-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.

Adenosine Diphosphate↗

PubMed1988

[The growth and protein A accumulation of Staphylococcus aureus A-676 on a medium made from nonfood raw material].

In this research the experimental analytical method of balancing culture media, based on the determination of the yield of the target product, i.e. staphylococcal protein A, has been used. The medium, developed in the course of this research work on the basis of a hydrolysate of protein-containing waste products of the tanning industry, makes it possible to achieve the growth of producer strains similar to that achieved in peptone-yeast medium. The parameters of the growth of S. aureus strain A-676 and the biological activity of protein A obtained in the newly developed medium have been studied.

Bacteriological Techniques↗

PubMed1995

Fermentation of whey and starch by transformed Saccharomyces cerevisiae cells.

Among the main agro-industrial wastes, whey and starch are of prime importance. In previous work we showed that strains of Saccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose. Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced/l in 1 h of fermentation). A yeast strain for the simultaneous utilization of lactose and starch has also been developed. Biotechnological perspectives are discussed.

Animals↗

PubMed2005

Efficient production of L-Lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene.

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.

Animals↗

PubMed2005

Genetic determinants of volatile-thiol release by Saccharomyces cerevisiae during wine fermentation.

Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.

Fermentation↗

PubMed2006

A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora.

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Deltamcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development.

Amino Acid Sequence↗

PubMed2005

Cloning and characterization of a dual-specificity kinase gene in rice (Oryza sative).

We identified and isolated a dual-specificity kinase gene,OsSTY kinase (O. sative serine/threonine/tyrosine kinase) gene, from rice. OsSTY kinase gene encoded a protein of 417 amino acids with calculated molecular weight 45926 Da and isoelectric point 7.689. OsSTY kinase is involved in the multiple stresses signaling pathway. Low- and high-temperature (4 degrees C and 37 degrees C) stresses significantly induce the expression of OsSTY kinase. The kinase is also up-regulated upon wounding (by cut), salicylic acid (SA) and ethophon (ET). Moreover,ectopic expression of OsSTY kinase gene in yeast Saccharomyces cerevisiae ste7/ste7 mutant partially suppressed the pseudohyphal development defect of the strain under the condition of nitrogen starvation. Ste7 is a serine/threonine/tyrosine kinase of Saccharomyces cerevisiae, which shares 32% identity and 50% similarity with OsSTY kinase in the kinase domains. This finding suggested that OsSTY kinase could correct, at least partially, the defect of ste7/ste7 mutant.

Amino Acid Sequence↗