Development of a glutathione production process from proteinaceous biomass resources using protease-displaying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.     

Molecular modeling of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> glutamate cysteine ligase and investigation of its interactions with glutathione

Trypanosoma cruzi glutamate cysteine ligase (TcGCL) is considered a potential drug target to develop novel antichagasic drugs. We have used a variety of computational methods to investigate the interactions between TcGCL with Glutathione (GSH). The three-dimensional structure of TcGCL was constructed by comparative modeling methods using the Saccharomyces cerevisiae glutamate cysteine ligase as template. Molecular dynamics simulations were used to validate the TcGCL model and to analyze the molecular interactions with GSH. Using RMSD clustering, the most prevalent GSH binding modes were identified paying attention to the residues involved in the molecular interactions. The GSH binding modes were used to propose pharmacophore models that can be exploited in further studies to identify novel antichagasic compounds.     

Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic

The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2–2.5-fold upon addition of either arsenate (As^V) or arsenite (As^III). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3′-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation.     

Glutathione: a review on biotechnological production

This Mini-Review summarizes the historic developments and technological achievements in the biotechnological production of glutathione in the past 30 years. Glutathione is the most abundant non-protein thiol compound present in living organisms. It is used as a pharmaceutical compound and can be used in food additives and the cosmetic industries. Glutathione can be produced using enzymatic methods in the presence of ATP and its three precursor amino acids (l-glutamic acid, l-cysteine, glycine). Alternatively, glutathione can be produced by direct fermentative methods using sugar as a starting material. In the latter method, Saccharomyces cerevisiae and Candida utilis are currently used to produce glutathione on an industrial scale. At the molecular level, the genes gshA and gshB, which encode the enzymes -glutamylcysteine synthetase and glutathione synthetase, respectively, have been cloned from Escherichia coli and over-expressed in E. coli, S. cerevisiae, and Lactococcus lactis. It is anticipated that, with the design and/or discovery of novel producers, the biotechnological production of glutathione will be further improved to expand the application range of this physiologically and medically important tripeptide.     

The <Emphasis Type="Italic">S</Emphasis>-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase 2 is reduced by interaction with glutathione peroxidase 3 in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glutathione peroxidases (Gpxs) are the key anti-oxidant enzymes found in Saccharomyces cerevisiae. Among the three Gpx isoforms, glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and modulates the activities of redox-sensitive thiol proteins involved in various biological reactions. By using a proteomic approach, glyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH2; EC 1.2.1.12) was found as a candidate protein for interaction with Gpx3. GAPDH, a key enzyme in glycolysis, is a multi-functional protein with multiple intracellular localizations and diverse activities. To validate the interaction between Gpx3 and GAPDH2, immunoprecipitation and a pull-down assay were carried out. The results clearly showed that GAPDH2 interacts with Gpx3 through its carboxyl-terminal domain both in vitro and in vivo. Additionally, Gpx3 helps to reduce the S-nitrosylation of GAPDH upon nitric oxide (NO) stress; this subsequently increases cellular viability. On the basis of our findings, we suggest that Gpx3 protects GAPDH from NO stress and thereby contributes to the maintenance of homeostasis during exposure to NO stress.     

Characterization of Antimicrobial Activity of the Lysosomes Isolated from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The antimicrobial activity of lysosomes, a cell organelle, against a range of test microorganisms was examined in this study. The lysosomes isolated from Saccharomyces cerevisiae showed antimicrobial activity to Escherichia coli that positively correlated with the pH of the phosphate buffer as a dissolving solvent. The lysosomes from S. cerevisiae exhibited optimal activity at a concentration of 40%, at pH 4.0 of phosphate buffer, and at broad range temperature, except of over 50°C. It was also found that the lysosomes have antimicrobial activity against seven different microorganisms including E. coli. In addition, S. cerevisiae were exposed by a treatment with H₂O₂ and lysosomes were isolated from H₂O₂ exposed S. cerevisiae. We found that fluorescent intensities of each isolated lysosomes were increased depending on the increment of treated H₂O₂ concentration, and the lysosomes from 20 mM H₂O₂ treated S. cerevisiae showed higher antimicrobial activity than those from normal S. cerevisiae. Therefore, it suggests that lysosomes isolated from S. cerevisiae can be used as an antimicrobial agent. In addition, lysosomes activated by H₂O₂ enhanced its antimicrobial activity.     

Engineered Saccharomyces cerevisiae that produces 1,3‐propanediol from d‐glucose

Aims: Saccharomyces cerevisiae is a safe micro‐organism used in fermentation industry. 1,3‐Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensively chemical synthesis. The aim of this study is to engineer a S. cerevisiae strain that can produce 1,3‐propanediol at low cost. Methods and Results: By using d ‐glucose as a feedstock, S. cerevisiae could produce glycerol, but not 1,3‐propanediol. In this study, we have cloned two genes yqhD and dhaB required for the production of 1,3‐propanediol from glycerol, and integrated them into the chromosome of S. cerevisiae W303‐1A by Agrobacterium tumefaciens‐mediated transformation. Both genes yqhD and dhaB functioned in the engineered S. cerevisiae and led to the production of 1,3‐propanediol from d ‐glucose. Conclusion: Saccharomyces cerevisiae can be engineered to produce 1,3‐propanediol from low‐cost feedstock d ‐glucose. Significance and Impact of the Study: To our knowledge, this is the first report on developing S. cerevisiae to produce 1,3‐propanediol by using A. tumefaciens‐mediated transformation. This study might lead to a safe and cost‐efficient method for industrial production of 1,3‐propanediol.     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.     

Role of Oxidative Phosphorylation in Histatin 5-Induced Cell Death in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The succeptibility of Saccharomyces cerevisiae to the anti-microbial peptide, histatin 5, was tested after pre-growth in fermentable and non-fermentable carbon sources and in the absence or presence of the uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP). S. cerevisiae was more resistant to histatin 5 when grown on a fermentable carbon source compared to growth on a non-fermentable carbon source, indicating an important role for oxidative phosphorylation in histatin 5-induced cell death. Oxidative phosphorylation is a pre-requisite for histatin 5-induced cell death in Candida albicans but this is not the case in S. cerevisiae. Incubation of CCCP-treated S. cerevisiae cells with histatin 5 still resulted in cell death. These results suggest that histatin 5-induced cell death in S. cerevisiae differs from that in C. albicans.Revisions received 28 September 2004     

Nystatin‐enhanced glutathione production by Saccharomyces cerevisiae depends on γ‐glutamylcysteine synthase activity and K+

Glutathione (GSH), an important tripeptide compound, is widely used as a therapeutic and in the food and cosmetic industries. To improve its production yield, we added the antibiotic nystatin to a batch fermentation of Saccharomyces cerevisiae, at different concentrations and at various times. Based on the results that nystatin can effectively stimulate GSH accumulation but at the same time inhibits cell growth, a three‐point addition strategy (0.05 mg/L at 8 h, 0.25 mg/L at 16 h, and 0.5 mg/L at 20 h) was developed to maximize GSH production. As a result, a maximum yield of 237.8 mg/L was obtained, which was by 50.6% higher than without the addition of nystatin. When combining this strategy with cysteine addition, the GSH yield increased to 278.9 mg/L. Subsequently, the γ‐glutamylcysteine synthetase (γ‐GCS) activity and K⁺ concentration were analyzed to investigate the possible mechanism involved in the increased production. It was found that the nystatin‐induced increase in the GSH yield was associated with a higher γ‐GCS activity and K⁺ concentration.     

<i>Azospirillum brasilense</i> and <i>Saccharomyces cerevisiae</i> as Alternative for Decrease the Effect of Salinity Stress in Tomato (<i>Lycopersicon esculentum</i>) Growth

The salinity stress is one of the most relevant abiotic stresses that affects the agricultural production. The present study was performed to study the improvement of the salt tolerance of tomato plants which is known for their susceptibility to salt stress. The present study aimed to assess to what extent strain Azospirillum brasilense (N040) and Saccharomyces cerevisiae improve the salt tolerance to tomato plants treated with different salt concentration. The inoculant strain A. brasilense (N040) was previously adapted to survive up to 7% NaCl in the basal media. A greenhouse experiment was conducted to evaluate the effect of this inoculation on growth parameter such as: plant height, root length, fresh and dry weight, fruits fresh weight, chlorophyll content, proline and total soluble sugar in tomato plants under salt stress condition. The results revealed that co-inoculation of Azospirillum brasilense (N040) and Saccharomyces cerevisiae significantly increased the level of proline (8.63 mg/g FW) and total soluble sugar (120 mg/g FW) of leaves under salinity condition comparing to non-inoculated plants (2.3 mg/g FW and 70 mg/g FW, respectively). Plants co-inoculated with adapted strain of A. brasilense and S. cerevisiae showed the highest significant (p < 0.01) increase in fruit yield (1166.6 g/plant), plant high (115 cm) and roots length (52.6) compared whit un-inoculated control plants (42 g/pant, 43.3 cm and 29.6 cm, respectively). In contrast, Na⁺ ion content was significantly decreased in the leaves of salt stressed plants treated with the A. brasilense (N040) and S. cerevisiae. Finally, the results showed that dual benefits provided by both A. brasilense (N040) and S. cerevisiae can provide a major way to improve tomato yields in saline soils.     

<Emphasis Type="Italic">Pichia stipitis</Emphasis> xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)

Background  

Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21.     

Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae

Aims: Adenosine triphosphate (ATP) during the enzymatic production of glutathione is necessary. In this study, our aims were to investigate the reason for low glutathione production in Escherichia coli coupled with an ATP regeneration system and to develop a new strategy to improve the system. Methods and Results: Glutathione can be synthesized by enzymatic methods in the presence of ATP and three precursor amino acids (l ‐glutamic acid, l ‐cysteine and glycine). In this study, glutathione was produced from E. coli JM109 (pBV03) coupled with an ATP regeneration system, by using glycolytic pathway of Saccharomyces cerevisiae WSH2 as ATP regenerator from adenosine and glucose. In the coupled system, adenosine used for ATP regeneration by S. cerevisiae WSH2 was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03). As a consequence, S. cerevisiae WSH2 could not obtain enough adenosine for ATP regeneration in the glycolytic pathway in spite of consuming 400 mmol l^?1 glucose within 1 h. By adding adenosine deaminase inhibitor to block the metabolism from adenosine to hypoxanthine, glutathione production (8·92 mmol l^?1) enhanced 2·74‐fold in the coupled system. Conclusions: This unusual phenomenon that adenosine was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03) revealed that less glutathione production in the coupled ATP regeneration system was because of the poor efficiency of ATP generation. Significance and Impact of the Study: The results presented here provide a strategy to improve the efficiency of the coupled ATP regeneration system for enhancing glutathione production. The application potential can be microbial processes where ATP is needed.     

Comparative Kinetic Effects of Mn (II), Mg (II) and the ATP/ADP Ratio on Phosphoenolpyruvate Carboxykinases from <Emphasis Type="Italic">Anaerobiospirillum succiniciproducens</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The kinetic affinity for CO₂ of phosphoenolpyruvate PEP⁵ carboxykinase from Anaerobiospirillum succiniciproducens, an obligate anaerobe which PEP carboxykinase catalyzes the carboxylation of PEP in one of the final steps of succinate production from glucose, is compared with that of the PEP carboxykinase from Saccharomyces cerevisiae, which catalyzes the decarboxylation of oxaloacetate in one of the first steps in the biosynthesis of glucose. For the A. succiniciproducens enzyme, at physiological concentrations of Mn²⁺ and Mg²⁺, the affinity for CO₂ increases as the ATP/ADP ratio is increased in the assay medium, while the opposite effect is seen for the S. cerevisiae enzyme. The results show that a high ATP/ADP ratio favors CO₂ fixation by the PEP carboxykinase from A. succiniciproducens but not for the S. cerevisiae enzyme. These findings are in agreement with the proposed physiological roles of S. cerevisiae and A. succiniciproducens PEP carboxykinases, and expand recent observations performed with the enzyme isolated from Panicum maximum (Chen et al. (2002) Plant Physiology 128: 160–164).     

Co-expression of xylose reductase gene from <Emphasis Type="Italic">Candida shehatae</Emphasis> and endogenous xylitol dehydrogenase gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and the effect of metabolizing xylose to ethanol

The inability oft Saccharomyces cerevisiae to utilize xylose is attributed to its inability to convert xylose to xylulose. Low xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in S. cerevisiae are regarded as the reason of blocking the pathway from xylose to xylulose. We had found that Candida shehatae could also be another source for XR gene except Pichia stipitis in the previous study. In this study, we tried to investigate if the expressed XR from C. shehatae could work with the over-expressed endogenous XDH together to achieve the same goal of converting xylose to ethanol in S. cerevisiae. The XR gene (XYL1) from C. shehatae and endogenous XDH gene (XYL2) were both cloned and over-expressed in host S. cerevisiae cell. The specific enzyme activities of XR and XDH were measured and the result of fermentation revealed that the new combination of two enzymes from different sources other than P. stipitis could also coordinate and work with each other and confer xylose utilization ability to S. cerevisiae.     

Xylitol does not inhibit xylose fermentation by engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">xylA</Emphasis> as severely as it inhibits xylose isomerase reaction in vitro

Efficient fermentation of xylose, which is abundant in hydrolysates of lignocellulosic biomass, is essential for producing cellulosic biofuels economically. While heterologous expression of xylose isomerase in Saccharomyces cerevisiae has been proposed as a strategy to engineer this yeast for xylose fermentation, only a few xylose isomerase genes from fungi and bacteria have been functionally expressed in S. cerevisiae. We cloned two bacterial xylose isomerase genes from anaerobic bacteria (Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1) and introduced them into S. cerevisiae. While the transformant with xylA from B. longum could not assimilate xylose, the transformant with xylA from B. stercoris was able to grow on xylose. This result suggests that the xylose isomerase (BsXI) from B. stercoris is functionally expressed in S. cerevisiae. The engineered S. cerevisiae strain with BsXI consumed xylose and produced ethanol with a good yield (0.31 g/g) under anaerobic conditions. Interestingly, significant amounts of xylitol (0.23 g xylitol/g xylose) were still accumulated during xylose fermentation even though the introduced BsXI might not cause redox imbalance. We investigated the potential inhibitory effects of the accumulated xylitol on xylose fermentation. Although xylitol inhibited in vitro BsXI activity significantly (K _I = 5.1 ± 1.15 mM), only small decreases (less than 10%) in xylose consumption and ethanol production rates were observed when xylitol was added into the fermentation medium. These results suggest that xylitol accumulation does not inhibit xylose fermentation by engineered S. cerevisiae expressing xylA as severely as it inhibits the xylose isomerase reaction in vitro.     

Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

Comparative genetics of yeasts: A novel β-fructosidase gene <Emphasis Type="Italic">SUC8</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Molecular and genetic analyses revealed that the distillers race XII, which is an ancestor of Saccharomyces cerevisiae Peterhof and Gatchina genetic lines, has three polymeric β-fructosidase genes: SUC2, SUC5, and SUC8. The latter gene located on the X chromosome was identitied in this work for the first time. The presence of the single SUC2 gene in yeasts used in the international project on sequencing of the S. cerevisiae genome is discussed.     

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L^-1 glutathione with glutathione content and yield of 4.3% and 25.6 mg_glutathione/g_glucose, respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L^-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.