Preparation of 5′-GMP-rich yeast extracts from spent brewer's yeast

Spent brewer's yeast was autolysed and used as a raw material for the preparation of 5-GMP-rich yeast extracts. Malt rootlets were used as a source of 5-phosphodiesterase. The crude enzyme was extracted from malt rootlets and pretreated to inactivate 5-nucleotidase. The optimum pretreatment conditions were heating at 65 °C for 30 min or 70 °C for 7 min. The effects of autolysis time, phosphodiesterase concentration and incubation period on 5-GMP content were examined. The suitable autolysis time was 8 h. The preferable enzyme treatment period was in the range of 8–14 h. Longer autolysis and enzyme incubation periods caused a decrease in the 5-GMP content from 0.7–0.9% (w/w) to 0.2–0.4% (w/w). The 5-GMP content in extracts from debittered and non-debittered yeast was similar. The highest 5-GMP content in yeast extract was 0.93% (w/w), obtained with a phosphodiesterase concentration of 1.6unit/ml of yeast extract (5% solids content).     

Cosntruction of the physical map of yeast（Saccharomyces cerevisiae）chromosome V~1

There are about 17 chromosomes in yeast Saccharomycescerevisiae.A middle sized chromosome,chromosome V,waschosen in this work for studying and constructing the physi-cal maps.Chromosome V from strain A364a was isolatedby pulsed-field gradient gel electrophoresis(PFGE).Gelslices containing chromosome V DNA were digestedwith two rare cutting enzymes,NotⅠand SfiⅠ,and three6-Nt recognizing enzymes,SmaⅠ,SstⅡ and ApaⅠ.Several strategies-partial or complete digestions,digestion with different sets of two enzymes,and hybrid-ization with cloned genetically mapped probes(CAN1,URA3,CEN5,PRO3,CHO1,SUP19,RAD51,RAD3)——were used to align the restriction fragments.There are 9,9,15,17,and 20 sites for NotⅠ,SfiⅠ,SmaⅠ,SstⅡ and ApaⅠrespectively in the map of the A364a chromosome V.Itstotal length was calculated to be 620 Kb(Kilo-bases).Thedistributions of the cutting sites for these five enzymesthrough the whole chromosome are not uniform.A comp-arison between the physical map and the genetic map wasalso made.     

Glycogen--a physiological determinant of yeast flocculation?

Evidence is provided to extend earlier observations that glycogen and flocculence levels vary concurrently in brewing yeast. The use of glycogen mutants, the alterations of growth conditions specifically to inhibit glycogen storage, and observations on glycogen decreases during endogenous metabolism have verified the above. A mechanism by which glycogen might exert its effect on flocculation is suggested.     

Intracellular pH of baker’s yeast

The distribution of bromophenol blue between the cell and the medium was used to calculate the intracellular pH of yeast. In buffered media the intracellular pH exhibited a plateau at pH _i =5.8 for low external pH values and another at pH _i =7.6 for high external pH values. The production of H ions by the yeast during utilization of glucose is not accompanied by an alkalinization of the cell interior. The pH _i even decreases somewhat in the presence of glucose and K ions.

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   Внутриклеточный pH дрожжей вычисляли на основании распределения бромфеноловой сини между клетками и средой.     
   
   

β-glucanases of the yeast Pichia polymorpha

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl--d-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A₅₀ has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different -glucanases located in the periplasmic space is presented.     

Biosynthesis of β-lactam nuclei in yeast

We have engineered brewer's yeast as a general platform for de novo synthesis of diverse β-lactam nuclei starting from simple sugars, thereby enabling ready access to a number of structurally different antibiotics of significant pharmaceutical importance. The biosynthesis of β-lactam nuclei has received much attention in recent years, while rational engineering of non-native antibiotics-producing microbes to produce β-lactam nuclei remains challenging. Benefited by the integration of heterologous biosynthetic pathways and rationally designed enzymes that catalyze hydrolysis and ring expansion reactions, we succeeded in constructing synthetic yeast cell factories which produce antibiotic cephalosporin C (CPC, 170.1 ± 4.9 μg/g DCW) and the downstream β-lactam nuclei, including 6-amino penicillanic acid (6-APA, 5.3 ± 0.2 mg/g DCW), 7-amino cephalosporanic acid (7-ACA, 6.2 ± 1.1 μg/g DCW) as well as 7-amino desacetoxy cephalosporanic acid (7-ADCA, 1.7 ± 0.1 mg/g DCW). This work established a Saccharomyces cerevisiae platform capable of synthesizing multiple β-lactam nuclei by combining natural and artificial enzymes, which serves as a metabolic tool to produce valuable β-lactam intermediates and new antibiotics.     

Aggresome formation and segregation of inclusions influence toxicity of α-synuclein and synphilin-1 in yeast

PD (Parkinson's disease) is a neurodegenerative disorder, caused by a selective loss of dopaminergic neurons in the substantia nigra, which affects an increasing number of the elderly population worldwide. One of the major hallmarks of PD is the occurrence of intracellular protein deposits in the dying neurons, termed Lewy bodies, which contain different proteins, including aggregated α-synuclein and its interacting protein synphilin-1. During the last decade, a number of groups developed yeast models that reproduced important features of PD and allowed the deciphering of pathways underlying the cytotoxicity triggered by α-synuclein. Here, we review the recent contributions obtained with yeast models designed to study the presumed pathobiology of synphilin-1. These models pointed towards a crucial role of the sirtuin Sir2 and the chaperonin complex TRiC (TCP-1 ring complex)/CCT (chaperonin containing TCP-1) in handling misfolded and aggregated proteins.     

Inducibility of error-prone DNA repair in yeast?

Whereas some experimental evidence suggests that mutagenesis in yeast after treatment with DNA-damaging agents involves inducible functions, a general-acting error-prone repair activity analogous to the SOS system of Escherichia coli has not yet been demonstrated. The current literature on the problem of inducibility of mutagenic repair in yeast is reviewed with emphasis on the differences in the experimental procedures applied.     

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for -glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL₂ and C. albidus AL₃, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of -glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl--D-glucopyranoside and salicin. The time course of growth and -glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL₂ and C. albidus AL₃ synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.     

Kluyveromyces—A new yeast genus of theEndomycetales

Summary A new budding yeast has been isolated from soil. Its most striking feature is the formation, generally preceded by isogamous or heterogamous conjugation, of unusually large multispored asci. The organism possesses a strong fermentative ability, fermenting glucose, galactose, saccharose and raffinose for one-third. Nitrate is not assimilated. It forms a pellicle in malt extract. A pseudomycelium is also formed. Cytological examination showed the vegetative cells to be uninucleate. The relationship which this yeast shows with theDipodascaceae and, in particular, withDipodascus uninucleatus Biggs, is discussed. For the classification of the yeast the new genusKluyveromyces was created. For the species the nameKluyveromyces polysporus is proposed. The Latin diagnosis of both the genus and the species is given.     

Transport of ethanol in baker’s yeast

Ethanol is transported into various strains of baker's yeast by simple diffusion (no effect of inhibitors and a linear concentration dependence of the initial rate of uptake and final distribution in cells). It distributes itself in 96.6 +/- 16.2% of intracellular water.     

Crucial role of the Rcl1p–Bms1p interaction for yeast pre-ribosomal RNA processing

The essential Rcl1p and Bms1p proteins form a complex required for 40S ribosomal subunit maturation. Bms1p is a GTPase and Rcl1p has been proposed to catalyse the endonucleolytic cleavage at site A₂ separating the pre-40S and pre-60S maturation pathways. We determined the 2.0 Å crystal structure of Bms1p associated with Rcl1p. We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A₂. Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing. We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.     

Influence of glucose on the α-glucoside permease activity of yeast

Summary The change in the -glucoside permease activity of baker's yeast, Saccharomyces cerevisiae, has been followed in the presence of maltose and/or glucose in the medium. Three separate effects of glucose on the permease were distinguished: an immediate effect that apparently involves a conformational transformation of the permease, an inactivation of the permease before the initiation of growth, and a repression and derepression of the synthesis of permease. Conceivable mechanisms for regulation of the glucose effects are briefly discussed.     

Involvement of the yeast metacaspase Yca1 in ubp10Δ-programmed cell death

UBP10 encodes a deubiquitinating enzyme of Saccharomyces cerevisiae. Its inactivation results in a complex phenotype characterized by a subpopulation of cells that exhibits the typical cellular markers of apoptosis. Here, we show that additional deletion of YCA1, coding for the yeast metacaspase, suppressed the ubp10 disruptant phenotype. Moreover, YCA1 overexpression, without any external stimulus, had a detrimental effect on growth and viability of ubp10 cells accompanied by an increase of apoptotic cells. This response was completely abrogated by ascorbic acid addition.We also observed that cells lacking UBP10 had an endogenous caspase activity, revealed by incubation in vivo with FITC-labeled VAD-fmk. All these results argue in favour of an involvement of the yeast metacaspase in the active cell death triggered by loss of UBP10 function.     

Induction of isoenzymes of alcohol dehydrogenase in “flor” yeast

Summary Two isoenzymes of alcohol dehydrogenase (adh I and adh II) from Saccharomyces cheresiensis have been differentiated by thermal treatment of the crude extracts. The effect of pH on the stability and the K _m for ethanol are different for the two isoenzymes.The proportions in which they are present depend on the carbon source used by the yeast: adh I is the major component in cells grown on glucose, and adh II in those grown on ethanol. Cells grown on glucose plus ethanol show high levels of both isoenzymes, indicating that the synthesis of adh I is subjected to nutritional induction by glucose, and that of adh II by ethanol.The physiological roles of the two isoenzymes are discussed in relation with the nutritional characteristics of S. cheresiensis.     

Bioavailability of enterai yeast—selenium in preterm infants

There is no data or literature on the effects of supplementing infants with yeast selenium, although its intestinal absorption and bioavailability are higher in adults compared with other selenium compounds. The aim of the present investigation was to study the impact of selenium enriched yeast on the serum selenium concentration of preterm infants living in a low selenium area (Hungary). Twenty-eight preterm infants with mean ± SD birth weight of 962 ± 129 g and gestational age 27 ± 1 wk were randomized into two groups at birth with respect to selenium supplementation. In the supplemented group (n = 14) infants received 4.8 mg yeast selenium containing 5 μg selenium daily via nasogastric drip during the first 14 postnatal days. The nonsupplemented infants were used as a reference group. In the supplemented group, the serum selenium concentration increased from 32.1 ± 8.5 μg/L to 41.5 ± 6.5 μg/L and in the nonsupplemented group it decreased from 25.9 ± 6.8 μg/L to 18.2 ± 6.4 μg/L from birth in two weeks time. Compared with previous studies, our results suggest that the bioavailability of selenium in the form of yeast selenium is higher than that of other selenium compounds used for preterm infants. We did not observe any complications or side-effects owing to enterai yeast selenium supplementation. We conclude that selenium enriched yeast is a safe and an effective form of short-term enterai selenium supplementation for infants.     

Induction of β galactosidase in the yeast Kluyveromyces lactis

Summary The inductive effect of lactose, -methyl-thio-D-galactopyranoside, (TMG) and glucose on galactosidase synthesis in Kluyveromyces lactis has been studied. Whereas TMG gave a five fold stimulation of the rate of -galactosidase synthesis, lactose only gave a small stimulation. Glucose caused represssion at levels above 10^-3M but stimulated -galactosidase synthesis when added at lower concentrations.     

Condensation of α-ketobutyrate and acetyl-CoA in baker's yeast

Summary Dialyzed cell-free preparations of baker's yeast fortified with magnesium and potassium ions, CoA and ATP incorporate ¹⁴C-labeled acetate in the presence of unlabeled -ketobutyrate. This acetate-fixing reaction results in the formation of only one product that has been isolated by paper chromatography and is catalyzed by an enzyme which condenses in the absence of magnesium ions 1 mol of acetyl-CoA with 1 mol of -ketobutyrate. The new condensing enzyme is very active in the crude extracts and has been separated by ammonium sulfate fractionation from other enzymes previously reported to occur in baker's yeast, which condense acetyl-CoA with the following -ketoacids: glyoxylate, pyruvate, oxaloacetate, -ketoisovalerate, and -ketoglutarate.

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Zusammenfassung Dialysierte zellfreie Extrakte aus Bäckerhefe fixieren, mit Hilfe von Magnesium und Kaliumionen, Coenzyme A und ATP, ¹⁴C-markiertes Acetat in Gegenwart von unmarkiertem -Ketobutyrat. Diese Acetat-Fixierungsreaktion führt zur Bildung eines einzigen Produktes, das durch Papierchromatographie isoliert worden ist, und wird von einem Enzym katalysiert, das, in Abwesenheit von Magnesiumionen, 1 Mol Acetyl-CoA mit 1 Mol -Ketobutyrat kondensiert. Das neue kondensierende Enzym ist sehr aktiv in Rohextrakten und konnte durch Ammonsulfatfraktionierung von anderen schon beschriebenen Hefeenzymen, welche die Kondensation von Acetyl-CoA mit verschiedenen -Ketosäuren, nämlich Glyoxyl-, Brenztrauben-, Oxalessig-, -Ketoisovalerian- und -Ketoglutarsäure, durchführen, getrennt werden.

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This investigation was supported by Grant No. AM 06848-02 from the National Institutes of Health, United States Public Health Service.     

Effect of anticalmodulin drugs on the action of yeast α factor pheromone

Saccharomyces cerevisiae factor pheromone arrest growth of cells of the a mating type (MAT a) at the G1 phase of the cell cycle. When treatment of MAT a cells with factor was carried out in the presence of anticalmodulin drugs, trifluoperazine or chlorpromazine, the extent of cell growth arrest induced by factor was reduced or even became undetectable. These results lend support to the hypothesis that calmodulin plays a role as mediator in the action of factor on MAT a cells.Abbreviation MAT mating type     

Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast

α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.