Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact

A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of Saccharomyces cerevisiae. Introduction of a tyrosine-insensitive allele of ARO4 (encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive ARO4 allele in combination with the Tyr-insensitive ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.     

An altered ribosomal protein in an edeine-resistant mutant of Saccharomyces cerevisiae

Summary The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO₄ system in the second.With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains. However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one. Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain.The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.     

Turnover of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae: differences between in vivo and in vitro degradation

Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with ¹⁴C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2–1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.Participant in the UNESCO Postgraduate Course On Modern Problems in Biology and Microbial Technology.     

Action of tryptophan analogues in Saccharomyces cerevisiae

In an analysis of the effects of various tryptophan and indole analogues in Saccharomyces cerevisiae we determined the mechanisms by which they cause growth inhibition: 4-Methyltryptophan causes a reduction in protein synthesis and a derepression of the tryptophan enzymes despite of the presence of high internal levels of tryptophan. This inhibition can only be observed in a mutant with increased permeability to the analogue. These results are consistent with but do not prove an interference of this analogue with the charging of tryptophan onto tRNA. 5-Methyltryptophan causes false feedback inhibition of anthranilate synthase, the first enzyme of the tryptophan pathway. This inhibits the further synthesis of tryptophan and results in results in tryptophan limitation, growth inhibition and derepression of the enzymes. Derepression eventually allows wild type cells to partially overcome the inhibitory effect of the analogue. 5-Fluoroindole is converted endogenously to 5-fluorotryptophan by tryptophan synthase. Both endogenous and externally supplied 5-fluorotryptophan are incorporated into protein. This leads to intoxication of the cells due to the accumulation of faulty proteins. 5-Fluorotryptophan also causes feedback inhibition of anthranilate synthase and reduces the synthesis of tryptophan which would otherwise compete with the analogues in the charging reaction. Indole acrylic acid inhibits the conversion of indole to tryptophan by tryptophan synthase. This results in a depletion of the tryptophan pool which, in turn, causes growth inhibition and derepression of the tryptophan enzymes.Abbreviations cpm counts per minute - OD optical density at 546 nm - TCA trichloro acetic acid - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for the corresponding tryptophan biosynthetic enzymes - trpl^– res. trp1^± refer to mutant strains synthesizing completely resp. partially defective enzymes     

Interaction of Saccharomyces cerevisiae with gold: toxicity and accumulation

This paper examines the effects of ionic gold on Saccharomyces cerevisiae, as determined by long-term (growth in gold-containing media) and short-term interactions (H⁺ efflux activity). An increasing gold concentration inhibited growth and at <0.2 mM Au, growth was not observed. Transmission electron microscopy revealed no differences in ultrastructure but fine electron dense particles were observed in unstained preparations from gold-containing medium. After glucose addition (to 10mM) to starved suspensions of S. cerevisiae, glucose-dependent reduction of external pH occurred as the cells extruded protons. In the presence of increasing gold concentrations, the lag time before proton extrusion did not change but the rate and duration decreased significantly with a marked influence on proton efflux rate being observed at 10 M. Extension of preincubation time of yeast cells in gold-containing medium resulted in a decreasing proton efflux rate and colloidal phase formation in the cell suspensions, the time between gold addition and the beginning of colloidal phase formation depending on the gold concentration used. Both Ca and Mg enhanced the inhibitory effect of gold on the yeast cells with Ca showing a stronger inhibitory effect than Mg.     

A potassium transport mutant of Saccharomyces cerevisiae

A mutant in Saccharomyces cerevisiae required one hundred times more K⁺ than wild type for the same half maximal growth rate. Mutant cells and wild type cells grown at millimolar K⁺ did not show significant differences in Rb⁺ transport. In the mutant, a rapid K⁺ loss induced by azide or incubation (4 h) in K⁺-free medium decreased the Rb⁺ transport K _m by one half; in the wild type, those treatments decreased the Rb⁺ K _m twenty and one hundred times, respectively. Mutant and wild type did not show significant differences in Na⁺ transport and in the Na⁺ inhibition of Rb⁺ transport, either in normal-K⁺ cells or in K⁺-starved cells. The results suggest that either two systems or one system with two interacting sites mediate K⁺ transport in S. cerevisiae.Abbreviations YPD yeast-peptone-dextrose medium     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains

The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S. cerevisiae X2180-1A and in a Kluyveromyces marxianus strain. We could classify these strains into four groups, which did not correlate with their geographical distribution. In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes. Two strains were found which, in contrast to strain X2180-1A, show derepression of gene TRP1. Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains.Non-standard abbreviations InGP Indole-3-glycerolphosphate - PRA N(5-phosphoribosyl)-anthranilate     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Heat Shock–Induced Changes in the Respiration of the Yeast Saccharomyces cerevisiae

The incubation of Saccharomyces cerevisiaeat elevated temperature (45°C) stimulated the respiration of yeast cells and decreased their survival rate. The respiration-deficient mutant of this yeast was found to be more tolerant to the elevated temperature than the wild-type strain. At the same time, the cultivation of the wild-type strain in an ethanol-containing medium enhanced the respiration, catalase activity, and thermotolerance of yeast cells, as compared with their growth in a glucose-containing medium. It is suggested that the enhanced respiration of yeast cells at 45°C leads to an intense accumulation of reactive oxygen species, which may be one of the reasons for the heat shock–induced cell death.     

Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.     

Metabolism of lysine-chromium complex in Saccharomyces cerevisiae

Saccharomyces cerevisiae which cannot utilize lysine as a sole nitrogen source is shown to metabolize a Lysine 3 Cr³⁺ (1:1) complex synthesized, as a combined nitrogen and carbon source. It induces rapid uptake of lysine and prevents loss of viability, in contrast with free lysine. That complexation with trivalent chromium has the effect of profoundly influencing intracellular distribution and metabolism of the liganded amino acid is demonstrated.     

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare.The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus.

Subjects and methods

Vaginal samples were collected from a total of262 (asymptomaticandsymptomatic) women with vaginitis attending the centre of family planning of General hospital ofPiraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae.

Results

A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker’s yeast.

Conclusions

Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy.     

Electroinduced release of invertase from Saccharomyces cerevisiae

Invertase liberation from Saccharomyces cerevisiae was detected after application of series of rectangular millisecond electric pulses. Maximal yield (60% from the activity in crude extract) was achieved within 8 h after pulsation. As shown by staining SDS-PAGE for invertase activity, the main part of liberated enzyme is a high molecular weight periplasmic invertase.     

A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae

To produce an industrial strain of Saccharomyces cerevisiae that metabolizes xylose, we constructed a rDNA integration vector and YIp integration vector, containing the xylose-utilizing genes, XYL1 and XYL2, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and XKS1, which encodes xylulokinase (XK) from S. cerevisiae, with the G418 resistance gene KanMX as a dominant selectable marker. The rDNA results in integration of multiple copies of the target genes. The industrial stain of S. cerevisiae NAN-27 was transformed with the two integration vectors to produce two recombinant strains, S. cerevisiae NAN-127 and NAN-123. Upon transformation, multiple copies of the xylose-utilizing genes were integrated into the genome rDNA locus of S. cerevisiae. Strain NAN-127 consumed twice as much xylose and produced 39% more ethanol than the parent strain, while NAN-123 consumed 10% more xylose and produced 10% more ethanol than the parent strain over 94 h.     

The effect of growth factors on anoxic chemostat cultures of two Saccharomyces cerevisiae strains

In anoxic chemostat cultures of Saccharomyces cerevisiae ATCC 4126 and CBS 8066 grown in a medium containing yeast extract, a sharp increase in the steady-state residual glucose concentration occurred at relatively low dilution rates, contrary to the expected Monod kinetics. However, supplementation with vitamins and amino acids facilitated efficient glucose uptake. This enhanced requirement for growth factors under anoxic conditions and at high growth rates could explain the exceptionally high apparent k _s values for S. cerevisiae reported in the literature.     

Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides

Autolytic degradation of yeast RNA occurs in many foods and beverages and can impact on the sensory quality of the product, but the resulting complex mixture of nucleotides, nucleosides and nucleobases has not been properly characterised. In this study, yeast autolysis was induced by incubating cell suspensions of Saccharomyces cerevisiae at 30–60 °C (pH 7.0), and at pH 4.0–7.0 (40 °C) for 10–14 days, and the RNA degradation products formed during the process were determined by reversed-phase HPLC. Up to 95% of cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3′-, 5′- and 2′-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases. The rate of RNA degradation and the composition of the breakdown products varied with temperature and pH. RNA degradation was fastest at 50 °C (pH 7.0). Autolysis at lower temperatures (30 °C and 40 °C) and at pH 5.0 and 6.0 favoured the formation of 3′-nucleotides, whereas autolysis at 40 °C and 50 °C (pH 7.0) favoured 5′- and 2′-nucleotides. The best conditions for the formation of the two flavour-enhancing nucleotides, 5′-AMP and 5′-GMP, were 50 °C (pH 7.0) and pH 4.0 (40 °C), respectively.