Regulation of sulphate assimilation in Saccharomyces cerevisiae

We examined how the activity of O-acetylserine and O-acetylhomoserine sulphydrylase (OAS/OAH) SHLase of Saccharomyces cerevisiae is affected by sulphur source added to the growth medium and genetic background of the strain. In a wild-type strain, the activity was repressed if methionine, cysteine or glutathione was added to the growth medium. However, in a strain deficient of cystathionine γ-lyase, cysteine and glutathione were repressive, but methionine was not. In strains deficient of serine O-acetyltransferase (SATase), OAS/OAH SHLase activity was low regardless of sulphur source and was further lowered by cysteine and glutathione, but not by methionine. From these observations, we concluded that S-adenosylmethionine should be excluded from being the effector for regulation of OAS/OAH SHLase. Instead, we suspected that S. cerevisiae would have the same regulatory system as Escherichia coli for sulphate assimilation; i.e. cysteine inhibits SATase to lower the cellular concentration of OAS which is required for induction of the sulphate assimilation enzymes including OAS/OAH SHLase. Subsequently, we obtained data supporting this speculation.     

Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation.

Using a Saccharomyces cerevisiae strain having the activities of serine O-acetyl-transferase (SATase), O-acetylserine/O-acetylhomoserine sulphydrylase (OAS/OAH SHLase), cystathionine beta-synthase (beta-CTSase) and cystathionine gamma-lyase (gamma-CTLase), we individually disrupted CYS3(coding for gamma-CTLase) and CYS4 (coding for beta-CTSase). The obtained gene disruptants were cysteine-dependent and incorporated the radioactivity of (35)S-sulphate into homocysteine but not into cysteine or glutathione. We concluded, therefore, that SATase and OAS/OAH SHLase do not constitute a cysteine biosynthetic pathway and that cysteine is synthesized exclusively through the pathway constituted with beta-CTSase and gamma-CTLase; note that OAS/OAH SHLase supplies homocysteine to this pathway by acting as OAH SHLase. From further investigation upon the cys3-disruptant, we obtained results consistent with our earlier suggestion that cysteine and OAS play central roles in the regulation of sulphate assimilation. In addition, we found that sulphate transport activity was not induced at all in the cys4-disruptant, suggesting that CYS4 plays a role in the regulation of sulphate assimilation.     

Purification of properties of Saccharomyces cerevisiae cystathionine β-synthase

Cystathionine β-synthase (β-CTSase), which catalyses cystathionine synthesis from serine and homocysteine, was purified to homogeneity from Saccharomyces cerevisiae. The molecular mass of the enzyme was estimated to be 235 kDa by gel filtration and 55 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, indicating that it is a homotetramer. The N-terminal amino acid sequence of the enzyme perfectly coincided with that deduced from the nucleotide sequence of CYS4, except for the absence of initiation methionine. The purified β-CTSase catalysed cysteine synthesis from serine (or O-acetylserine) and H₂S. From this finding, we discuss the multifunctional nature and evolutionary divergence of S-metabolizing enzymes.     

Role of O-acetylhomoserine sulfhydrylase in sulfur amino acid synthesis in various yeasts

Mutants defective in O-acetylhomoserine sulfhydrylase (OAH-SHLase) were obtained in five yeast strains representative of different yeast genera: Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe and Trichosporon cutaneum. In vitro, in all five strains, the enzyme also had O-acetylserine (OAS) sulfhydrylase activity so it is a ‘bifunctional’ OAH/OAS-SHLase (Yamagata, 1989). The enzyme was only found to be essential in S. cerevisiae (OAH SHLase-negative mutants are auxotrophs). Its impairment in K. lactis caused a slower growth rate and a decrease of the sulfur amino acid pool. In T. cutaneum only the pool was affected whereas in Y. lipolytica and S. pombe the lesion caused no change in the growth rate nor in the pool. In all strains where OAH SHLase-negative mutants were prototrophs, a monofunctional OAS sulfhydrylase was detected. The results indicate that OAH SHLase may play different physiological roles in various yeasts.     

γ‐Glu‐Met synthesised using a bacterial glutaminase as a potential inhibitor of dipeptidyl peptidase IV

This study describes γ‐glutamyl dipeptides as competitive inhibitors of dipeptidyl peptidase‐IV (DPP‐IV), and the feasible synthesis of γ‐Glu‐Met through transpeptidation catalysed by a commercial glutaminase of Bacillus amyloliquefaciens. γ‐Glu‐Met, γ‐Glu‐Leu, γ‐Glu‐Phe, γ‐Glu‐Trp or γ‐Glu‐Tyr exhibited a competitive inhibitory effect on DPP‐IV. The yield of γ‐Glu‐Met was 26.16% under optimised conditions: 200 mm Gln, 20 mm Met, 0.1 U mL^?1 enzyme, pH 9.0, 37 °C and reaction time 3 h. For the first time, the side products containing characteristic sequences, that is poly‐γ‐glutamyl short chains with a terminal Met residue (γ‐Glu‐γ‐Glu‐Met and γ‐Glu‐γ‐Glu‐γ‐Glu‐Met) were identified. The superiority of the commercial glutaminase in the synthesis of DPP‐IV‐inhibitory peptides can enable the application of this novel process for manufacturing γ‐glutamyl‐peptides as potential functional ingredients in the type 2 diabetic diet.     

Yeast sequencing reports. Gcs1, a gene encoding γ-glutamylcysteine synthetase in the fission yeast Schizosaccharomyces pombe

By complementation of a mutant resistant to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) we have identified the gcs1 gene, encoding a putative γ-glutamylcysteine synthetase. The gene is possibly interrupted by two introns and has 49% identical and 80% similar amino acids compared with the homologous protein from rat. In comparison with the Saccharomyces cerevisiae homologue it possesses 41% identical and 74% similar amino acids. The gsc1 sequence appears in the EMBL database under Accession Number X 85017.     

Evaluating potential applications of indigenous yeasts and their β‐glucosidases

The potential applications of wild yeast strains with β‐glucosidase activity were investigated by assaying their enzymatic production under simulated oenological conditions, coupled with the exploration of the potential applications of the β‐glucosidases by studying the enzymatic activity and stability under similar oenological conditions. The assay of enzymatic locations revealed that the β‐glucosidase activities from these wild strains occurred in the extracellular fraction, and in whole and permeabilized cells. The effects of different oenological factors on β‐glucosidase production indicated that the F6 Trichosporon asahii strain had higher β‐glucosidase production than the other strains under low pH conditions. However, the F35 Hanseniaspora uvarum strain and the F30 Saccharomyces cerevisiae strain showed higher β‐glucosidase production under high‐sugar conditions. Furthermore, the influence of oenological factors on the activity and stability of the β‐glucosidases revealed that the enzyme from the F6 T. asahii strain had a stronger low‐pH‐value resistance than the other yeast β‐glucosidases. These results suggest that the F35 H. uvarum, F30 S. cerevisiae and the F6 T. asahii β‐glucosidases may have some potentially applicable values in the fermentation industry. Copyright © 2015 The Institute of Brewing & Distilling     

Glycosylation of human α1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts

Human α₁-antitrypsin (α₁-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α₁-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α₁-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α₁-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α₁-AT. The human α₁-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α₁-AT in the methylotrophic yeasts appeared to be relatively shorter than those of α₁-AT in S. cerevisiae. The α₁-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α₁-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.     

Secretion of Mouse α-Amylase from Kluyveromyces lactis

We constructed two mouse α-amylase secretion vectors for Kluyveromyces lactis using the well-characterized signal sequence of the pGKL 128 kDa killer precursor protein. Both PHO5 and PGK expression cassettes from Saccharomyces cerevisiae directed the expression of mouse α-amylase in YPD medium at a similar level of efficiency. K. lactis transformants secreted glycosylated and non-glycosylated α-amylase into the culture medium and both species were enzymatically active. The K. lactis/S. cerevisiae shuttle secretion vector pMI6 was constructed, and K. lactis MD2/1(pMI6) secreted about four-fold more α-amylase than S. cerevisiae YNN27 harboring the same plasmid, indicating that K. lactis is an efficient host cell for the secretion and production of recombinant proteins. © 1997 John Wiley & Sons, Ltd.     

α‐Glucosidase Inhibitory Activities of Fatty Acids Purified from the Internal Organ of Sea Cucumber Stichopus japonicas

α‐Glucosidase inhibitory activities of the various solvent fractions (n‐hexane, CHCl₃, EtOAc, BuOH, and water) of sea cucumber internal organ were investigated. 1,3‐Dipalmitolein (1) and cis‐9‐octadecenoic acid (2) with potent α‐glucosidase inhibitory activity were purified from the n‐hexane fraction of sea cucumber internal organ. IC₅₀ values of compounds 1 and 2 were 4.45 and 14.87 μM against Saccharomyces cerevisiae α‐glucosidase. These compounds mildly inhibited rat‐intestinal α‐glucosidase. In addition, both compounds showed a mixed competitive inhibition against S. cerevisiae α‐glucosidase and were very stable at pH 2 up to 60 min. The K_I values of compounds 1 and 2 were 0.48 and 1.24 μM, respectively. Therefore, the internal organ of sea cucumber might be a potential new source of α‐glucosidase inhibitors suitably used for prevention of obesity and diabetes mellitus.     

A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1

Saccharomyces cerevisiae possesses a single essential gene (GLC7) encoding protein phosphatase 1 (PP1). Elevated expression of this gene from the GAL1 promoter is highly detrimental to the cell, causing a growth defect and aberrant bud morphology, which leads to cells exhibiting long, extended buds. By comparison, expression of GLC7 from the weaker MET3 promoter was without significant effect on either growth or morphology. However, repression of GLC7 expression from the MET3 promoter in cells where the MET3-GLC7 fusion was the sole source of PP1 resulted in a mitotic delay. Such cultures showed a massive decrease in the rate of proliferation in conjunction with a significant increase in the proportion of large, budded cells. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and anti-tubulin immunofluorescence analysis of these cells revealed that many were blocked in mitosis, with a short spindle and DAPI-stained material stretched between the mother and daughter cell within the bud neck. These results support a role for PP1 in the completion of mitosis in S. cerevisiae.     

VII. Yeast sequencing reports. The sequence of a 27 kb segment on the right arm of chromosome VII from Saccharomyces cerevisiae reveals MOL1, NAT2, RPL30B,RSR1, CYS4, PEM1/CHO2, NSR1 genes and ten new open reading frames

The DNA sequence of a 26 677 bp fragment from the right arm of chromosome VII from Saccharomyces cerevisiae reveals 18 open reading frames (ORFs) longer than 300 bp. Eight ORFs correspond to previously characterized genes. G6620 is the 3′ end of the MOL1 gene coding for a polypeptide similar to stress-inducible proteins from Fusarium; G6630 is the NAT2 gene which encodes a methionine N-acetyltransferase; G6635 is the RPL30B gene coding for the ribosomal protein L30; G6658 is RSR1 encoding a ras-related protein; G6667 is CYS4, the gene for cystathionine β-synthase; G6670 is identical to ORF2 located close to CYS4; G6673 is PEM1/CHO2 encoding a phosphatidylethanolamine methyltransferase; G7001 is the NSR1 gene coding for a nuclear signal recognition protein. G6664 shares significant homology with the ORF YKR076w from chromosome XI. The other nine ORFs show no significant homology to any protein sequence presently available in the public data bases. The sequence has been deposited in the EMBL data library under Accession Number X85807.     

Isolation of obligate anaerobic rumen bacteria capable of degrading the neurotoxin β‐ODAP (β‐N‐oxalyl‐L‐α,β‐diaminopropionic acid) as evaluated by a liquid chromatography/biosensor analysis system

Six pure strains of obligate anaerobes capable of degrading the toxin β‐N‐oxalyl‐L ‐α, β‐diaminopropionic acid (β‐ODAP) contained in grass pea (Lathyrus sativus) have been isolated from cow rumen. The new isolates were identified as Megasphaera elsdenii (five different genotypes) and Clostridium bifermentans using 16S rDNA analysis. The β‐ODAP degrading efficiency of the isolates was evaluated by measuring the amount of β‐ODAP in the growth medium, which contained β‐ODAP as the only carbon source, before and after incubation with the microbes. The method of analysis was liquid chromatography employing bioelectrochemical detection. The biosensor is based on co‐immobilising two enzymes, glutamate oxidase (GlOx) and horseradish peroxidase (HRP), on the end of a spectrographic graphite electrode. β‐ODAP is oxidised by GlOx to form H₂O₂, which in turn is bioelectrocatalytically reduced by HRP through a mediated reaction using a polymeric mediator incorporating Os^{2 + /3+} functionalities rapidly shuttling electrons with the electrode_giving rise to the analytical signal. On the basis of this analysis system, the new isolates are capable of utilising β‐ODAP as sole carbon source to a maximum of 90–95% within 5 days with concomitant increase in cell protein. Copyright © 2005 Society of Chemical Industry     

Structural and mechanical characterization of κ/ι-hybrid carrageenan gels in potassium salt using Fourier Transform rheology

The mechanical and structural properties of κ/ι-hybrid carrageenan gels obtained at various concentrations in the presence of 0.1 m KCl were studied with Fourier Transform rheology (FTR) and cryoSEM imaging. FTR data show that gels formed at concentration below 1.25 wt% exhibit a strain hardening behavior. The strain hardening is characterized by a quadratic increase of the scaled third harmonic with the strain and a third harmonic phase angle of zero degree. Both features are weakly depending on the concentration and conform to predictions from a strain hardening model devised for fractal colloidal gels. However, the phase angle of the third harmonic reveals that κ/ι-hybrid carrageenan gels obtained at higher concentrations show shear thinning behavior. Colloidal gel models used to extract structural information from the concentration scaling of gel equilibrium shear modulus G₀ and the strain dependence of FTR parameters suggest that κ/ι-hybrid carrageenan gels are built from aggregating rod-like strands (with fractal dimension x = 1.13) which essentially stretch under increasing strain. The mechanically relevant structural parameters fairly match the gel fractal dimension (d = 1.66) obtained from the cryoSEM analysis.     

Cloning and genetic characterization of a calcium- and phospholipid-binding protein from Saccharomyces cerevisiae that is homologous to translation elongation factor-1γ

We have isolated a gene (CAM1) from the yeast Saccharomyces cerevisiae that encodes a protein homologous to the translational cofactor elongation factor-1γ (EF-1γ) first identified in the brine shrimp Artemia salina. The predicted Cam1 amino acid sequence consists of 415 residues that share 32% identity with the Artemia protein, increasing to 72% when conservative substitutions are included. The calculated M_r of Cam1p (47 092 Da) is in close agreement with that of EF-1γ (M_r = 49 200 Da), and hydropathy plots of each protein exhibit strikingly similar profiles. Disruption of the CAM1 locus yields four viable meiotic progeny, indicating that under normal growth conditions the Cam1 protein is non-essential. Attempts to elicit a translational phenotype have been unsuccessful. Since EF-1γ participates in the regulation of a GTP-binding protein (EF-1α), double mutants with cam1 disruptions and various mutant alleles of known GTP-binding proteins were constructed and examined. No evidence was found for an interaction of CAM1 with TEF1, TEF2, SEC4, YPT1, RAS1, RAS2, CDC6, ARF1, ARF2 or CIN4. The possibility that Cam1p may play a redundant role in the regulation of protein synthesis or another GTP-dependent process is discussed.     

Cryogelation of cereal β-glucans: structure and molecular size effects

The effects of molecular size and fine structure of mixed-linkage cereal (1→3), (1→4) β- -glucans (β-glucans) on their cryogelation behavior were investigated. Values of apparent molecular weight (M_w) for oat β-glucans ranged between 65 and 200×10³, whereas the respective values for both barley and wheat β-glucan preparations were about 200×10³. The fine structure of cereal β-glucans, as assessed by high-performance anion-exchange chromatography of the cellulosic oligomers released by the action of lichenase, revealed differences in the relative amounts of 3-O-β-cellobiosyl- -glucose (DP3) and 3-O-β-cellotriosyl- -glucose (DP4) units only among the different genera of cereals; the weight percent of DP3 units estimated as 67.1, 63.3, and 55.3–55.8% for wheat, barley, and oat β-glucans, respectively. Aqueous β-glucan solutions (1–3% w/v) were subjected to 12 freezing (−18 °C for 24 h) and thawing (5 °C for 24 h) cycles. The phenomenological appearance of the gelled materials obtained after this process as well as the yield of cryostructurates were influenced by the initial solution concentration, the number of freeze–thaw cycles, as well as by the molecular features of the β-glucans. Such effects were unraveled by studying the cryogelation process with differential scanning calorimetry (DSC), small strain dynamic rheometry, and large deformation mechanical measurements. For the cereal β-glucan cryogels the storage modulus, G′, increased and the tan δ decreased with decreasing polysaccharide molecular size and with increasing initial solution concentration, number of freeze–thaw cycles, and trisaccharide segments in the polymeric chains. The apparent melting enthalpy values (ΔH) of β-glucan cryostructurates, as determined from the DSC endothermic peaks, increased with decreasing molecular size and with increasing amount of cellotriose units, but they were independent of the number of freeze–thaw cycles. The DSC melting temperature of the gel network was found to increase with the molecular size and amount of DP3 units of β-glucans. Moreover, large deformation mechanical tests (compression mode) revealed an increase in strength of cereal β-glucan cryogels with increasing molecular size and decreasing trisaccharide units in the polysaccharide preparation.     

Chemical Constituents of Gold‐red Apple and Their α‐Glucosidase Inhibitory Activities

Ten compounds were isolated and purified from the peels of gold‐red apple (Malus domestica) for the 1st time. The identified compounds are 3β, 20β‐dihydroxyursan‐28‐oic acid (1), 2α‐hydroxyoleanolic acid (2), euscaphic acid (3), 3‐O‐p‐coumaroyl tormentic acid (4), ursolic acid (5), 2α‐hydroxyursolic acid (6), oleanolic acid (7), betulinic acid (8), linolic acid (9), and α‐linolenic acid (10). Their structures were determined by interpreting their nuclear magnetic resonance and mass spectrometry (MS) spectra, and by comparison with literature data. Compound 1 is new, and compound 2 is herein reported for the 1st time for the genus Malus. α‐Glucosidase inhibition assay revealed 6 of the triterpenoid isolates as remarkable α‐glucosidase inhibitors, with betulinic acid showing the strongest inhibition (IC₅₀ = 15.19 μM). Ultra‐performance liquid chromatography‐electrospray ionization MS analysis of the fruit peels, pomace, flesh, and juice revealed that the peels and pomace contained high levels of triterpenes, suggesting that wastes from the fruit juice industry could serve as rich sources of bioactive triterpenes.