Methods for Oxygenation of Continuous Cultures of Brewer’s Yeast, Saccharomyces cerevisiae

Maintaining steady-state, aerobic cultures of yeast in a bioreactor depends on the configuration of the bioreactor system as well as the growth medium used. In this paper, we compare several conventional aeration methods with newer filter methods using a novel optical sensor array to monitor dissolved oxygen, pH, and biomass. With conventional methods, only a continuously stirred tank reactor configuration gave high aeration rates for cultures in yeast extract peptone dextrose (YPD) medium. For filters technologies, only a polydimethylsiloxan filter provided sufficient aeration of yeast cultures. Further, using the polydimethylsiloxan filter, the YPD medium gave inferior oxygenation rates of yeast compared to superior results with Synthetic Complete medium. It was found that the YPD medium itself, not the yeast cells, interfered with the filter giving the low oxygen transfer rates based on the volumetric transfer coefficient (KLa). The results are discussed for implications of miniaturized bioreactors in low-gravity environments.

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Replicative Aging in Pathogenic Fungi

Journal of Fungi ◽

10.3390/jof7010006 ◽

2020 ◽

Vol 7 (1) ◽

pp. 6

Author(s):

Somanon Bhattacharya ◽

Tejas Bouklas ◽

Bettina C. Fries

Keyword(s):

Cell Division ◽

Candida Glabrata ◽

Asymmetric Cell Division ◽

Pathogenic Fungi ◽

Yeast Cells ◽

Candida Auris ◽

Replicative Aging ◽

Yeast Saccharomyces Cerevisiae ◽

Phenotypic Variations ◽

Systemic Infections

Candida albicans, Candida auris, Candida glabrata, and Cryptococcus neoformans are pathogenic yeasts which can cause systemic infections in immune-compromised as well as immune-competent individuals. These yeasts undergo replicative aging analogous to a process first described in the nonpathogenic yeast Saccharomyces cerevisiae. The hallmark of replicative aging is the asymmetric cell division of mother yeast cells that leads to the production of a phenotypically distinct daughter cell. Several techniques to study aging that have been pioneered in S. cerevisiae have been adapted to study aging in other pathogenic yeasts. The studies indicate that aging is relevant for virulence in pathogenic fungi. As the mother yeast cell progressively ages, every ensuing asymmetric cell division leads to striking phenotypic changes, which results in increased antifungal and antiphagocytic resistance. This review summarizes the various techniques that are used to study replicative aging in pathogenic fungi along with their limitations. Additionally, the review summarizes some key phenotypic variations that have been identified and are associated with changes in virulence or resistance and thus promote persistence of older cells.

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Pulsed Electric Field (PEF) Enhances Iron Uptake by the Yeast Saccharomyces cerevisiae

Biomolecules ◽

10.3390/biom11060850 ◽

2021 ◽

Vol 11 (6) ◽

pp. 850

Author(s):

Karolina Nowosad ◽

Monika Sujka ◽

Urszula Pankiewicz ◽

Damijan Miklavčič ◽

Marta Arczewska

Keyword(s):

Saccharomyces Cerevisiae ◽

Electric Field ◽

Metal Ion ◽

Treatment Time ◽

Control Sample ◽

Pulsed Electric Field ◽

Yeast Cells ◽

Iron Ions ◽

Metal Ion Binding ◽

Yeast Saccharomyces Cerevisiae

The aim of the study was to investigate the influence of a pulsed electric field (PEF) on the level of iron ion accumulation in Saccharomyces cerevisiae cells and to select PEF conditions optimal for the highest uptake of this element. Iron ions were accumulated most efficiently when their source was iron (III) nitrate. When the following conditions of PEF treatment were used: voltage 1500 V, pulse width 10 μs, treatment time 20 min, and a number of pulses 1200, accumulation of iron ions in the cells from a 20 h-culture reached a maximum value of 48.01 mg/g dry mass. Application of the optimal PEF conditions thus increased iron accumulation in cells by 157% as compared to the sample enriched with iron without PEF. The second derivative of the FTIR spectra of iron-loaded and -unloaded yeast cells allowed us to determine the functional groups which may be involved in metal ion binding. The exposure of cells to PEF treatment only slightly influenced the biomass and cell viability. However, iron-enriched yeast (both with or without PEF) showed lower fermentative activity than a control sample. Thus obtained yeast biomass containing a high amount of incorporated iron may serve as an alternative to pharmacological supplementation in the state of iron deficiency.

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A CONVENIENT METHOD OF OBTAINING ASCOSPORES FROM BAKERS' YEAST

Canadian Journal of Research ◽

10.1139/cjr49c-015 ◽

1949 ◽

Vol 27c (4) ◽

pp. 179-189 ◽

Cited By ~ 25

Author(s):

A. M. Adams

Keyword(s):

Convenient Method ◽

Sodium Acetate ◽

Solid Medium ◽

Genetic Research ◽

Yeast Cells ◽

Sporulation Medium ◽

Direct Transfer ◽

Yeast Cultures ◽

High Yields ◽

Solid Nutrient Medium

The superiority of methods involving the use of sporulation media containing acetate, first introduced by Stantial and Elder, over several commonly employed methods is established. A new method for obtaining ascospores from bakers' yeast cultures is recommended involving the direct transfer of vegetative cells from a solid nutrient medium to a solid medium containing acetate. High yields of ascospores are consistently produced after seven days' incubation. This method should lend itself particularly to use in the preparation of ascospores for instructional work, and for genetic research in yeast, and may also find application in yeast taxonomy. The technique recommended is as follows: vegetative yeast cells are multiplied on tomato juice agar or on dextrose nutrient agar, and are then transferred to a solid sporulation medium containing 0.04% dextrose, 0.14% anhydrous sodium acetate, and 2% agar.

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The Cloning and Mapping of ADR6, a Gene Required for Sporulation and for Expression of the Alcohol Dehydrogenase II Isozyme From Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/116.4.531 ◽

1987 ◽

Vol 116 (4) ◽

pp. 531-540

Author(s):

Aileen K W Taguchi ◽

Elton T Young

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcohol Dehydrogenase ◽

Single Gene ◽

Carbon Sources ◽

Transcription Unit ◽

Yeast Cells ◽

Recessive Mutation ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Activating Sequence ◽

A Site

ABSTRACT The alcohol dehydrogenase II (ADH2) gene of the yeast, Saccharomyces cerevisiae, is not transcribed during growth on fermentable carbon sources such as glucose. Growth of yeast cells in a medium containing only nonfermentable carbon sources leads to a marked increase or derepression of ADH2 expression. The recessive mutation, adr6-1, leads to an inability to fully derepress ADH2 expression and to an inability to sporulate. The ADR6 gene product appears to act directly or indirectly on ADH2 sequences 3' to or including the presumptive TATAA box. The upstream activating sequence (UAS) located 5' to the TATAA box is not required for the Adr6- phenotype. Here, we describe the isolation of a recombinant plasmid containing the wild-type ADR6 gene. ADR6 codes for a 4.4-kb RNA which is present during growth both on glucose and on nonfermentable carbon sources. Disruption of the ADR6 transcription unit led to viable cells with decreased ADHII activity and an inability to sporulate. This indicates that both phenotypes result from mutations within a single gene and that the adr6-1 allele was representative of mutations at this locus. The ADR6 gene mapped to the left arm of chromosome XVI at a site 18 centimorgans from the centromere.

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Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.442-451.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 442-451

Author(s):

M Nishizawa ◽

R Araki ◽

Y Teranishi

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Pyruvate Kinase ◽

Transcriptional Activation ◽

Regulatory Elements ◽

Noncoding Region ◽

Yeast Cells ◽

Kinase Gene ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Activating Sequence

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

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Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3799-3805.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3799-3805

Author(s):

P J Schatz ◽

G E Georges ◽

F Solomon ◽

D Botstein

Keyword(s):

Amino Acids ◽

Variable Region ◽

Yeast Cells ◽

Meiotic Spindle ◽

Nuclear Movement ◽

Yeast Saccharomyces Cerevisiae ◽

Alpha Tubulin ◽

Essential Components ◽

Altered Proteins

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

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INTERCONVERSION OF YEAST MATING TYPES II. RESTORATION OF MATING ABILITY TO STERILE MUTANTS IN HOMOTHALLIC AND HETEROTHALLIC STRAINS

Genetics ◽

10.1093/genetics/85.3.373 ◽

1977 ◽

Vol 85 (3) ◽

pp. 373A-393

Author(s):

James B Hicks ◽

Ira Herskowitz

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Yeast Cells ◽

Mating Types ◽

Yeast Saccharomyces Cerevisiae ◽

Type Locus ◽

Mating Ability ◽

Mating Type Locus ◽

Regulatory Information ◽

Ho Gene

ABSTRACT The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HM a and hmα loci are blocs of unexpressed α regulatory information, and HMα and hm a loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.

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The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.5010-5019.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 5010-5019 ◽

Cited By ~ 1

Author(s):

J Heitman ◽

A Koller ◽

J Kunz ◽

R Henriquez ◽

A Schmidt ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Cyclosporin A ◽

Secretory Pathway ◽

Yeast Cells ◽

Amino Acid Transporters ◽

Yeast Saccharomyces Cerevisiae ◽

Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

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GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5101-5112.1991 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5101-5112

Author(s):

J S Flick ◽

M Johnston

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Interactions ◽

Tandem Repeats ◽

Glucose Repression ◽

Molecular Data ◽

Primary Response ◽

Yeast Cells ◽

Cell Fractionation ◽

Protein Protein Interactions ◽

Yeast Saccharomyces Cerevisiae

Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.

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Differentiation of Yeast Cultures in Ruminant Feedingstuffs Using a Rapid DNA Fingerprinting Technique

Proceedings of the British Society of Animal Production (1972) ◽

10.1017/s0308229600026489 ◽

1994 ◽

Vol 1994 ◽

pp. 101-101

Author(s):

Elizabeth Moore ◽

Denis R. Headon

Keyword(s):

Sodium Dodecyl ◽

Yeast Cells ◽

Microbial Populations ◽

Yeast Culture ◽

Yeast Cultures ◽

Yeast Strains ◽

Genetic Sequences ◽

Polymerase Chain ◽

Different Strains ◽

Triton X

Research indicates that certain yeast strains are beneficial in their capacity to stimulate key microbial populations. This stimulation is strain specific with similar yeast strains exerting their effect on totally different microbial populations. Future yeast culture supplements may contain mixtures of different strains designed to suit specific diets. This, therefore, requires the development of a rapid sensitive technique to differentiate among taxonomically similar yeast strains in animal diets. This technique, termed the Randomly Amplified Polymorphic DNA (RAPD) assay, is based upon the use of randomly designed short polynucleotide primers to amplify genetic sequences from the DNA of the desired yeast strain. Our objective involves the development of this technique to distinguish between closely related yeast strains present in feed. The feed sample investigated was a standard cattle ration containing three strains of Saccharomyces cerevisiae (1026, 2045 and 2020) and Candida utilis 3001 at a concentration of 106 CFU/g respectively. Isolation of single colonies of yeast strains present was achieved by feed extraction in dilution buffer followed by plating a series of dilutions on rose-bengal agar. Thirty randomly selected colonies were cultured in YPD (1% yeast extract, 2% peptone, 2% glucose) broth for 24 - 30 hours at 30°C. Genomic DNA was isolated from yeast cells by standard methods based on subjection of the cells to vortex mixing in the presence of glass beads, triton X-100, sodium dodecyl sulphate, phenol and chloroform. Isolated DNA from randomly selected colonies was amplified by Polymerase Chain Reaction (PCR) for 45 cycles of 1 min at 94°C, 1 min at 36°C and 1 min at 72°C using randomly designed 10 bp primers.

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