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.     

Cystathionine γ-lyase of Saccharomyces cerevisiae: Structural gene and cystathionine γ-synthase activity

Purification of Saccharomyces cerevisiae cystathionine γ-lyase (γ-CTLase) was hampered by the presence of a protein migrating very close to it in various types of column chromatography. The enzyme and the contaminant were nevertheless separated by polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that they are coded for by CYS3(CYI1) and MET17(MET25), respectively, leading to the conclusion that CYS3 is the structural gene for γ-CTLase and that the contaminant is O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase). Based on these findings, we purified γ-CTLase by the following strategy: (1) extraction of OAS/OAH SHLase from a CYS3-disrupted strain; (2) preparation of antiserum against it; (3) identification of a strain devoid of the OAS/OAH SHLase protein using this antiserum; and (4) extraction of γ-CTLase from this strain. Purified γ-CTLase had cystathionine γ-synthase (γ-CTSase) activity if O-succinylhomoserine, but not O-acetylhomoserine, was used as substrate. From this notion we discuss the evolutional relationship between S. cerevisiae γ-CTLase and Escherichia coli γ-CTSase.     

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.     

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.     

A preliminary study on the effect of Lactobacillus casei expressing cystathionine lyase1/cystathionine lyase2 on Cheddar cheese and the formation of sulphur-containing compounds

Model Cheddar cheeses were manufactured from pasteurised milk with various Lactobacillus casei adjuncts. The added L. casei FAM18101, L. casei FAM18108, and L. casei FAM18149 possessed genetic variants of a gene cluster that is involved in the conversion of methionine to cysteine. The gene cluster comprises a cystathionine lyase (ctl) previously shown to degrade cysteine, homocysteine, cystathionine and methionine to volatile sulphur compounds such as hydrogen sulphide and methanethiol. A strain that does not possess the gene cluster was used as a control. An inhibitory effect of the adjuncts on starter culture activity was not observed. Expression analysis of the genetic variant ctl1 showed that the gene was expressed in cheese. The development of cysteine, methionine and volatile sulphur compounds was analysed during ripening. The cheeses containing the L. casei adjuncts possessing the gene cluster had markedly higher concentrations of cysteine, hydrogen sulphide and methanethiol at the end of ripening.     

Meaty flavour compounds formation from the submerged liquid culture of Pleurotus ostreatus

The capacity of oyster mushroom (Pleurotus ostreatus) mycelium to produce meaty flavour compounds in the presence of amino acids and sugars was studied. The submerged liquid culture of P. ostreatus mycelium along with base medium made of defatted soybean powder, sucrose, potassium phosphate, and magnesium sulphate was incubated for 3 days at 25 °C. Cysteine, glutamine, or methionine and fructose, galactose, ribose, or xylose were added to the base medium to study the effect of amino acids and sugars on meaty flavour formation by trained panelists. The flavour compounds were isolated and identified by GC–MS and GC retention time of authentic compounds. The base medium required P. ostreatus, cysteine, ribose, and heat treatment to produce meaty flavour. The sulphur containing heterocyclic compounds such as 2,5‐diethylthiophene, 2‐formyl‐5‐methylthiophene, 3‐ethyl‐2‐formylthiophene, and dimethylformyl thiophene were responsible for meaty flavour. These compounds were formed by non‐enzymatic browning reaction between ribose and cysteine during heat treatment.     

METHIONINE AND SULPHATE AS COMPETING AND COMPLEMENTARY SOURCES OF SULPHUR FOR YEAST DURING FERMENTATION

Study of the uptake of sulphate and methionine by an ale yeast from a range of media showed that utilisation of sulphate was fairly strictly controlled but assimilation of methionine was not. Cells never took up more than about 0.3 mMol sulphate per litre whilst methionine, up to an initial concentration of 10 mMol per litre, was completely absorbed. Sulphate-grown cells had low intracellular pools of amino acids and methinonine was never detected. Methionine-grown cells contained methionine in both cytosol and vacuole and the concentration of several other amino acids also increased in such a way to suggest that methionine catabolism was occurring. With mixed sulphur sources methionine prevented uptake of sulphate when the concentration of sulphate was high but not when it was low suggesting the presence of two sulphate transporters with different control properties. Sulphate did not influence uptake of methionine. Addition of other amino acids to the medium did reduce the rate and extent of methionine uptake but not the intracellular pool sizes. Pilot plant studies suggested that SO₂ production in a brewery is more likely to be a reflection of the overall nutritive status of the wort rather than be connected to the initial methionine concentration .     

Ultradian metabolic oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide, a population synchronizer, is produced by sulphite reductase

We have reported that the consecutive cyclic production of H(2)S resulted in population synchrony of ultradian metabolic oscillation (Sohn et al., 2000). In order to understand the origin of H(2)S and its nature of periodic production, changes of sulphur compounds concentration and responsible enzymes were investigated. The concentrations of extracellular sulphate, intracellular glutathione and cysteine oscillated during metabolic oscillation but only the oscillation of sulphate concentration was out of phase with H(2)S production. The sulphate concentration in culture directly affected the amplitude and the period of metabolic oscillation: (a) the period of metabolic oscillation shortened from 50 min to 30 min when sulphate concentration in the medium was reduced from 46 mM to 2.5 mM; (b) the metabolic oscillation disappeared under sulphate-depletion conditions and arose again by the addition of sulphate. Pulse injection of sulphite (10 microM) perturbed metabolic oscillation with a burst production of H(2)S, while thiosulphate (up to 500 microM) was without apparent effect. Furthermore, addition of S-adenosyl methionine (100 microM) or azoxybacilin (3 mg/kg) decreased H(2)S production with perturbation of metabolic oscillation. The results presented here suggest that H(2)S, a population synchronizer, is produced by sulphite reductase in the sulphate assimilation pathway, and dynamic regulation of sulphate uptake plays an important role in ultradian metabolic oscillation.     

Isolation of glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae and their use in baker's yeast production

 Glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae 0511 were obtained by mutation under specific conditions. A total of 3388 strains were isolated and among them were found 46 mutants sensitive to methylglyoxal. The intracellular glutathione concentration of mutant strain S. cerevisiae 3033 was 0.0172 g/g dry weight, which was a decrease of >76% compared to that of the parent. The growth of mutant strains S. cerevisiae 3033 and S. cerevisiae 1116 in the medium with glutathione present and absent was compared to that of the parent strain. The sensibility of the baker's yeast strains studied to antifoaming agents, butanol and acetic acid was also investigated. The relationship between glutathione presence in the cell and the sensitivity of strain S. cerevisiae 3033 to antifoaming agents and butanol was ascertained, while such a connection with the presence of acetic acid in the molasses medium used for baker's yeast cultivation was not observed. The higher sensitivity of strain S. cerevisiae 3033 to some chemical compounds in the molasses nutrition medium was shown. Received: 2 November 1999 / Revised version: 15 February 2000     

Effects of the addition of sulphur compounds to the diet on utilisation of protein in young growing rats

A number of sulphur-containing compounds were added to a 10% casein diet in order to indicate their ability to replace a supplement of methionine. Net protein utilisation (NPU) was determined. Cysteine, homocysteine and methionine hydroxy analogue produced as great an increase in NPU as did methionine. Methionine sulphoxide increased the NPU to a lesser extent. Methionine sulphoximine, methyl methionine sulphonium hydrogen sulphate and methionine sulphone caused reductions in NPU. Other organic and all inorganic sulphur compounds showed no methionine sparing action.     

Influence of yeast strain on binding of sulphur dioxide in wines,and on its formation during fermentation

The amounts of sulphur dioxide bound by acetaldehyde, pyruvic acid and α-ketoglutaric acid during fermentation of three grape juices by eight wine yeasts (Saccharomyces sp.) are reported. These constituents accounted for 49–83 % (mean 69) of the measured bound SO₂, depending on the yeast strain and juice. the maximum range of concentrations of the binding components for individual wines were 10–48 ppm for acetaldehyde, 9–77 ppm for pyruvic acid and 5–63 ppm for α-ketoglutaric acid, depending on yeast strain and grape juice. the validity of the calculations was verified by an experiment with SO₂ and the three binding compounds in a multicomponent model system. The acetaldehyde content was related to the total SO₂ present, which itself was determined by the strain of yeast. SOz bound in the wines after a further SO₂ addition was correlated significantly with pyruvic and α-ketoglutaric acids, but not with acetaldehyde. Certain yeasts produced SO₂ during fermentation in grape juice and in synthetic media with defined sulphur sources. More SO₂ was produced at pH 3.6 than 3.0 in the absence of added sulphate in grape juice. Sulphate was the best sulphur source for SO₂ production in synthetic media, although some yeasts were able to produce smaller amounts of SO₂ from l-cysteine and reduced glutathione.     

Regulation of cystathionine gamma-lyase in Saccharomyces cerevisiae.

Regulation of the two enzymes in reverse trans-sulfuration was investigated in Saccharomyces cerevisiae. In wild-type strains, cystathionine gamma-lyase, but not cystathionine beta-synthase, was depressed nearly 15-fold if cells were starved for both inorganic and organic sulfur compounds. In a met17 strain which is defective of O-acetylserine and O-acetylhomoserine sulfhydrylase, the same enzyme was derepressed if organic sulfur compounds were limited; the repressive effect was in the order of glutathione greater than methionine greater than cysteine. The repressive effect of methionine was not observed, however, in a cys2 cys4 strain which is deficient of serine O-acetyltransferase and cystathionine beta-synthase, indicating that methionine itself is not the effector. The weak repressive effect of cysteine was attributed to inefficient uptake of this amino acid. Our observations indicate that cystathionine gamma-lyase is the target of regulation in reverse trans-sulfuration and that cysteine is very likely to be the effector of this regulation.     

SpOPT1, a member of the oligopeptide family (OPT) of the fission yeast Schizosaccharomyces pombe,is involved in the transport of glutathione through the outer membrane of the cell

A protein involved in the transport of glutathione has been identified, cloned and characterized from the fission yeast Schizosaccharomyces pombe. Database searches revealed the Sz. pombe ORF SPAC29B12.10c as a close homologue to several members of the OPT family, including the Saccharomyces cerevisiae high‐affinity glutathione transporter Hgt1p. The gene product of SPAC29B12.10c has been identified as a protein, named SpOPT1, localized within the plasma membrane, transporting the tripeptide glutathione. Disruption of SPAC29B12.10c led to strains inable to grow on media containing glutathione as a sole source of sulphur, due to the inability to internalize the tripeptide. Disruptants contained significantly less glutathione than wild‐type cells. Furthermore, ΔSpopt1 strains were non‐viable in a glutathione biosynthesis‐defective (Δgsh2) background. However, it was possible to complement the disruption of Spopt1 by overexpressing the intact ORF in the disrupted strain. Copyright © 2009 John Wiley & Sons, Ltd.     

Glutathione levels influence chronological life span of Saccharomyces cerevisiae in a glucose‐dependent manner

Diet plays a key role in determining the longevity of the organisms since it has been demonstrated that glucose restriction increases life span whereas a high‐glucose diet decreases it. However, the molecular basis of how diet leads to the aging process is currently unknown. We propose that the quantity of glucose that fuels respiration influences reactive oxygen species generation and glutathione levels, and both chemical species impact in the aging process. Herein, we provide evidence that mutation of the gene GSH1 in Saccharomyces cerevisiae diminishes glutathione levels. Moreover, glutathione levels were higher with 0.5% than in 10% glucose in the gsh1Δ and wild‐type strains. Interestingly, the chronological life span was lowered in the gsh1Δ strain cultured with 10% glucose but not under dietary restriction. The gsh1Δ strain also showed inhibition of the mitochondrial respiration in 0.5 and 10% glucose but only increased the H₂O₂ levels under dietary restriction. These results correlate well with the GSH/GSSG ratio, which showed a decrease in gsh1Δ strain cultured with 0.5% glucose. Together, these data indicate that glutathione exhaustion impact negatively both the electron transport chain function and the chronological life span of yeast, the latter occurring when a low threshold level of this antioxidant is reached, independently of the H₂O₂ levels.     

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full‐length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l ‐cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l ‐cystine and some other cysteine conjugates, but not l ‐cystathionine or l ‐methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l ‐cysteine as a nitrogen source, and that overexpression of the gene results in increased H₂S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S‐ethyl‐l ‐cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

CHILL PROOFING BY A STRAIN OF SACCHAROMYCES CEREVISIAE SECRETING PROTEOLYTIC ENZYMES

The attempted exploitation of a strain of yeast with extracellular protease activity in the fermentation of brewers' wort is described. S. cerevisiae strain YS01 was previously shown to secrete proteases with activity against a variety of substrates. It is shown here that these included some of the tannin precipitable proteins involved in the formation of chill haze in beer and consequently, more stable beers resulted from incubation with added YS01 cell-free culture supernatants. The proteases were also produced in an active form at low levels during fermentation of brewers' wort using established commercial conditions. Concomitantly, a marginally more stable beer resulted. Respiratory deficient, auxotrophic rare-mating was used to transfer the extracellular proteolytic characteristic (epr1.1) to a commercial ale yeast. Segregants of the recombinants obtained showed great instability in protease secretion and were unsuitable for brewing purposes. The potential advantages and drawbacks to this approach are discussed.     

Proximate composition,amino acid content and haemagglutinating and trypsin‐inhibiting activities of some Brazilian Vigna unguiculata (L) Walp cultivars

Seeds of Brazilian Vigna unguiculata (L) Walp cultivars (EPACE 10, EPACE 11, Pitiuba, TVu 1888, IPA 206 and Olho de Ovelha) were analysed to establish their proximate composition, amino acid content and presence of antinutritional and/or toxic factors. The seed protein, carbohydrate and oil contents ranged from 195 to 261 g kg⁻¹ dry matter, from 678 to 761 g kg⁻¹ dry matter and from 12 to 36 g kg⁻¹ dry matter respectively. EPACE 10, EPACE 11, Pitiuba, TVu 1888, IPA 206 and Olho de Ovelha cultivars are rich in glutamin/glutamic acid, asparagin/aspartic acid and phenylalanine + tyrosine. The essential amino acid profile compared with the FAO/WHO/UNU scoring pattern requirements for different age groups showed that these seeds have methionine + cysteine as the first limiting amino acid for 2–5‐year‐old children. However, only Pitiuba, IPA 206 and Olho de Ovelha are deficient in methionine + cysteine for 10–12‐year‐old children. The contents of threonine, valine, isoleucine, leucine and methionine + cysteine of all cultivars were lower than those of hen egg. Haemagglutinating activity measured against rabbit erythrocytes was found to be present in the six cultivars, but only after the red cells were treated with proteolytic enzymes. All cultivars displayed protease inhibitor activity which varied from about 12.0 to 30.6 g trypsin inhibited per kg flour. Urease and toxic activities were not detected in any of the studied cultivars. © 2000 Society of Chemical Industry     

THE PRODUCTION OF HYDROGEN SULPHIDE BY YEAST AND BY ZYMOMONAS ANAEROBIA

Radiochemical experiments indicated that most of the free hydrogen sulphide excreted by a brewing yeast in wort and by Zymomonas anaerobia in beer was derived from sulphate. Sulphate was also assimilated by the organisms but most of their cellular sulphur was derived from other sources. In a synthetic beer medium, excessive amounts of hydrogen sulphide were liberated by Z. anaerobia when pantothenate was deficient. Sulphate and zinc ions stimulated sulphide production by the bacterium.     

Role of the sulphate assimilation pathway in utilization of glutathione as a sulphur source by Saccharomyces cerevisiae.

Mutants unable to grow on medium containing glutathione as a sole source of sulphur (GSH medium) were isolated from Saccharomyces cerevisiae strains carrying met17(deficiency of O-acetylserine and O-acetylhomoserine sulphydrylase). They were defective in the high-affinity glutathione transport system, GSH-P1. Newly acquired mutations belonged to the same complementation group, gsh11. However, it became apparent that gsh11 conferred the mutant phenotype not by itself but in collaboration with met17. Moreover, mutations conferring the defect in sulphate assimilation made the cell unable to grow on GSH medium in collaboration with gsh11. From this finding, we propose that the sulphate assimilation pathway acts as a sulphur-recycling system and that this function is especially vital to the cell when the supply of glutathione is limited.