Exo-β-glucanases in yeast

1. A number of yeast species were examined for the presence of β-glucanases. Extracts obtained by cell disruption of Saccharomyces cerevisiae, Fabospora fragilis and Hansenula anomala hydrolysed laminarin and pustulan with the production of glucose. Enzymic activities were also detected in the culture fluids of F. fragilis and H. anomala grown aerobically in buffered mineral medium with glucose as the carbon source. 2. F. fragilis and H. anomala possessed approximately sevenfold higher β-(1→3)-glucanase activity than S. cerevisiae. 3. Intracellular exo-β-glucanase from baker's yeast was purified 344-fold from the dialysed cell extract. 4. Exo-β-glucanase from F. fragilis was purified 114-fold from the dialysed culture fluid and 423-fold from the dialysed intracellular extract. The purified extracellular and intracellular enzymes had similar properties and essentially the same specific activity, 79 enzyme units/mg. of protein. 5. Extracellular exo-β-glucanase of H. anomala was purified 600-fold. 6. The optimum pH of the enzymes from F. fragilis, S. cerevisiae and H. anomala was 5·5 in each case. Chromatographic evidence indicated that the three enzymes remove glucosyl units sequentially from laminarin as well as pustulan. 7. The ratio of activities towards laminarin and pustulan remained constant during purification of the exo-β-glucanase obtained from the three species, suggesting a single enzyme. Additional evidence for its unienzymic nature are: (i) the two activities were destroyed at exactly the same rate on heating of the purified enzyme from F. fragilis at three different temperatures; (ii) the competitive inhibitor glucono-δ-lactone gave the same value of K_i when tested with either substrate; (iii) quantitative application of the `mixed-substrate' method with the purified enzyme of S. cerevisiae gave data that were in excellent agreement with those calculated on the assumption of a single enzyme. 8. The purified exo-β-glucanases of the different species of yeast had different kinetic constants. The ratios of maximal velocities and K_m values with laminarin and pustulan differed markedly. Comparison of V_max. and K_m values suggests that the rapid release of spores from asci in F. fragilis might be explained in terms of an enzyme with higher maximal velocity and higher affinity to the ascus wall than that present in baker's yeast. 9. The estimated molecular weights for exo-β-glucanases from F. fragilis, S. cerevisiae and H. anomala were 22000, 40000 and 30000 respectively.     

Programmed death in yeast as adaptation?

During recent years, several pieces of indirect evidence of a programmed death in yeast have been published. Among them there are observations that some mammalian pro- or anti-apoptotic proteins induce or prevent the death of yeast; some toxic compounds kill yeast at lower concentrations if protein synthesis is operative; this death, as well as the death due to certain mutations, shows some apoptotic markers. In April 2002, the yeast programmed death concept received direct support. Madeo et al. [Madeo et al., Mol. Cell 9 (2002) 911-917] disclosed a caspase which is activated by H(2)O(2) or aging and is required for the protein-synthesis-dependent death of yeast. Thus, a specific apoptosis-mediating protein was identified for the first time in Saccharomyces cerevisiae. Independently, Severin and Hyman [Severin, F.F., Hyman, A.A., Curr. Biol. 12 (2002) R233-R235] discovered that death of yeast, induced by a high level of a pheromone, is programmed. In particular, the death was found to be prevented by cycloheximide and cyclosporin A. It required mitochondrial DNA, cytochrome c and the pheromone-initiated protein kinase cascade. When haploids of opposite mating types were mixed, some cells died, the inhibitory pattern being the same as in the case of the killing by pheromone. Inhibition of mating proved to be favorable for death. Thus, pheromone not only activates mating but also eliminates yeast cells failing to mate. Such an effect should (i) stimulate switch of the yeast population from vegetative to sexual reproduction, and (ii) shorten the life span and, hence, accelerate changing of generations. As a result, the probability of appearance of new traits could be enhanced when ambient conditions turned for the worse.     

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.     

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.     

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.     

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.     

Which aging in yeast is “true”?

Two model systems, “replicative aging” and “chronological aging” (CA), which are used for gerontological research on the yeast Saccharomyces cerevisiae, are compared. In the first case, the number of daughter cells generated by an individual mother cell before cell propagation irreversibly stops is analyzed. This makes the model very similar to the well-known Hayflick model. In the case of CA, the survival of yeast cell population in the stationary phase of growth is studied. It is noted that the second model is similar to the “stationary phase aging” model, which is used in the author’s laboratory for cytogerontological studies on animal and human cells. It is assumed that the concept of cell proliferation restriction as the main cause of age-related accumulation in the cells of multicellular organisms of macromolecular defects (primarily DNA damage) leading to deterioration of tissue and organ functioning and, as a result, to an increase in the death probability allows explaining how the aging process proceeds in almost any living organisms. Apparently, in all cases, this process is initiated by the appearance of slow propagating (or not propagating at all) cells, which leads to the termination of “dilution,” with the help of new cells, of macromolecular defects accumulating at the level of whole cell population. It is concluded that data on the geropromoter or geroprotector activity of various factors obtained in tests on the yeast CA model can be used with a high reliability to understand the mechanisms of human aging and longevity.     

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.     

Conjugation in the yeast Saccharomycopsis capsularis schiönning

Cells of the yeast Saccharomycopsis capsularis fused in pairs after dissolving of part of the cross wall between them near the lateral wall. After nuclear migrations through the opening, the cross wall was closed again and the cells at both sides became asci. The wall of the ascospores developed from a prospore wall. Between the two unit membranes a very thin dark layer broadened to the dark layer of the wall and after that, the light inner layer developed. Immature spores in the strain studied had a ledge which disappeared during maturation.     

Inactivation of exogenous β-lactamase in transformed yeast Saccharomyces cerevisiae

Summary The stability of bacterial -lactamase in transformedSacharomyces cerevisiae grown on glucose was studied. A culture of a prototrophic strain showed marked inactivation shortly before the stationary phase. This was also observed in cells starved of nitrogen. The level of reserve carbohydrate was lower both in the stationary-phase culture of the auxotroph and in the glucose-starved culture of the prototroph, where less inactivation was observed. Such a close correlation suggests that inactivation may be triggered mainly in response to nitrogen-limitation which regulates reserve carbohydrate metabolism.     

A novel oxylipin-associated ‘ghosting’ phenomenon in yeast flocculation

Research on the distribution of oxylipins (3-hydroxy fatty acids) in flocculant strains of the yeast Saccharomyces cerevisiae led to the uncovering of a novel ghosting phenomenon observed during assumed lectin-mediated aggregation. We found that intracellular oxylipin-containing osmiophilic layers migrate through yeast cell walls in a ghostlike fashion without visually affecting the cell wall structure or the layers. This migration resulted in the binding of these layers to cell walls of adjacent cells. Consequently, ghosting seems a prerequisite for flocculation to occur. However, ghosting alone may not be sufficient to ensure flocculation.     

Flocculation gene variability in industrial brewer’s yeast strains

The brewer’s yeast genome encodes a ‘Flo’ flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmental-friendly technique to separate yeast cells from the final beer. FLO genes have the notable capacity to evolve and diverge many times faster than other genes. In actual practice, this genetic variability may directly alter the flocculin structure, which in turn may affect the flocculation onset and/or strength in an uncontrolled manner. Here, 16 ale and lager yeast strains from different breweries, one laboratory Saccharomyces cerevisiae and one reference Saccharomyces pastorianus strain, with divergent flocculation strengths, were selected and screened for characteristic FLO gene sequences. Most of the strains could be distinguished by a typical pattern of these FLO gene markers. The FLO1 and FLO10 markers were only present in five out of the 18 yeast strains, while the FLO9 marker was ubiquitous in all the tested strains. Surprisingly, three strongly flocculating ale yeast strains in this screening also share a typical ‘lager’ yeast FLO gene marker. Further analysis revealed that a complete Lg-FLO1 allele was present in these ale yeasts. Taken together, this explicit genetic variation between flocculation genes hampers attempts to understand and control the flocculation behavior in industrial brewer’s yeasts.     

Demonstrating β-glucan and yeast peptide clearance in biopharmaceutical downstream processes

The use of yeast- and plant-derived hydrolysates in cell culture production processes has sparked concerns over the potential immunogenicity risk posed by β-glucans and yeast peptides contained in these raw materials. This article utilizes a combination of in-process testing from large-scale manufacturing and scale-down spiking studies to demonstrate the clearance of β-glucans and yeast peptides through chromatographic steps in the downstream purification process for a monoclonal antibody. β-Glucans were found to flow through most all three modes of chromatography (Protein A, cation and anion exchange) without binding to the resins or the product. Protein A affinity chromatography was found to provide the best clearance factor. The efficacy of the resin sanitization and storage procedures to prevent carryover from one run to the next was also demonstrated. Yeast peptides were found to be metabolized during the cell culture process and were undetectable after the Protein A purification step. The data presented here serve to allay concerns about the use of hydrolysates in cell culture production. The methodology presented here provides a template to demonstrate clearance of β-glucans and yeast peptides through chromatographic steps in downstream processing.