Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

The Arabidopsis Ethylene-Insensitive 2 gene is required for lead resistance

The Arabidopsis Ethylene-Insensitive 2 (EIN2) gene has been shown to be involved in the regulation of abiotic and biotic stresses, including ozone stress, high salt, oxidative stress and disease resistance. However, little is known about the role of EIN2 gene in lead (Pb) resistance in Arabidopsis. In this study, we showed that EIN2 gene is required for Pb(II) resistance in Arabidopsis. EIN2 gene was induced by Pb(II) treatment, and the ein2-1 mutant showed enhanced sensitivity to Pb(II). A higher Pb content was detected in ein2-1 plants than in wild-type plants when subjected to Pb(II) treatment, which was associated, at least in part, with reduction in expression of AtPDR12 gene, a pump excluding Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Moreover, the ein2-1 mutation also impaired glutathione (GSH)-dependent Pb(II) resistance, which was related to constitutive reduction of express of GSH1 gene involved in GSH synthesis and consequently reduced GSH content. Taken together, all these results suggest that EIN2 gene mediates Pb(II) resistance, at least in part, through two distinct mechanisms, a GSH-dependent mechanism and a GSH-independent AtPDR12-mediated mechanism.     

Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: Alterations in the activities and protein expression of glutathione-related enzymes

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM.     

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.     

Enzymatic synthesis of glutathione using engineered Saccharomyces cerevisiae

Two single gene cassettes, each containing one of the individual gene (γ-glutamylcysteine synthetase gene GSH1 or glutathione synthetase gene GSH2), were constructed under the control of alcohol dehydrogenase (ADH1) promoter and their respective native terminators. The recombinant plasmids constructed with Kan ^r or Hyg ^r as the selective markers and were transformed into Saccharomyces cerevisiae separately and jointly. Three engineered strains, GSH1-enhanced strain S.TS013/GSH1, GSH2-enhanced strain S.TS013/GSH2 and GSH1+GSH2 double-enhanced strain S.TS013/GSH1+GSH2, were constructed. Glutathione production using the recombinant strains to improve was then determined. By the cell dosage proportions of two engineered strains (S.TS013/GSH1, S.TS013/GSH2) and a two-stage reaction, GSH productivity increased by 84 and 59 % over that of the host strain and the S.TS013/GSH1+GSH2 strain, respectively.     

Involvement of the Azotobacter vinelandii Rhodanese-Like Protein RhdA in the Glutathione Regeneration Pathway

The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g. increased levels of lipid hydroperoxides) are provided. Starting from the evidence that glutathione was depleted in MV474, and using both in silico and in vitro approaches, here we studied the interaction of wild-type RhdA and Cys²³⁰Ala site-directed RhdA mutant with glutathione species. We found that RhdA was able to bind in vitro reduced glutathione (GSH) and that RhdA-Cys²³⁰ residue was mandatory for the complex formation. RhdA catalyzed glutathione-disulfide formation in the presence of a system generating the glutathione thiyl radical (GS^•, an oxidized form of GSH), thereby facilitating GSH regeneration. This reaction was negligible when the Cys²³⁰Ala RhdA mutant was used. The efficiency of RhdA as catalyst in GS^•-scavenging activity is discussed on the basis of the measured parameters of both interaction with glutathione species and kinetic studies.     

Jasmonic acid regulates the ascorbate–glutathione cycle in <Emphasis Type="Italic">Malus baccata</Emphasis> Borkh. roots under low root-zone temperature

Jasmonic acid (JA), which is an important phytohormone, plays a key role in plant growth, development and stress responses. Here, Malus baccata Borkh. seedlings were used to study the mechanism by which JA alleviates the oxidative damage induced by low root-zone temperature (5 °C) through regulating the ascorbate–glutathione (AsA–GSH) cycle. The roots of M. baccata Borkh. were subjected to three treatments [5 °C, 5 °C + JA, and 5 °C + ibuprofen (IBU)] for 0, 12, 24, and 48 h. The results showed that treatment with low root-zone temperature could modulate the non-enzymatic and enzymatic components of the AsA–GSH cycle, significantly inducing the accumulation of MDA and H₂O₂. Additionally, the endogenous JA content changed dramatically, and the expression levels of the related genes [lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC)] showed different trends. In plants pretreated with JA, the endogenous JA content increased at 24 h, and the gene expression levels of LOX, AOS, and AOC were upregulated. We also found a marked increase in the activities of antioxidant enzymes [ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR)], a decrease in oxidized glutathione (GSSG) and an increased GSH/GSSG ratio, which resulted in lower MDA and H₂O₂ contents. Thus, the oxidative stress was alleviated. Plants pretreated with IBU experienced an opposite effect on the function of the AsA–GSH cycle and the gene expression in the JA synthesis route relative to those subjected to exogenous JA treatment, indicating that endogenous JA can alleviate oxidative damage by regulating the function of the AsA–GSH cycle under low root-zone temperature.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

The Glutathione Reductase GSR-1 Determines Stress Tolerance and Longevity in Caenorhabditis elegans

Glutathione (GSH) and GSH-dependent enzymes play a key role in cellular detoxification processes that enable organism to cope with various internal and environmental stressors. However, it is often not clear, which components of the complex GSH-metabolism are required for tolerance towards a certain stressor. To address this question, a small scale RNAi-screen was carried out in Caenorhabditis elegans where GSH-related genes were systematically knocked down and worms were subsequently analysed for their survival rate under sub-lethal concentrations of arsenite and the redox cycler juglone. While the knockdown of γ-glutamylcysteine synthetase led to a diminished survival rate under arsenite stress conditions, GSR-1 (glutathione reductase) was shown to be essential for survival under juglone stress conditions. gsr-1 is the sole GSR encoding gene found in C. elegans. Knockdown of GSR-1 hardly affected total glutathione levels nor reduced glutathione/glutathione disulphide (GSH/GSSG) ratio under normal laboratory conditions. Nevertheless, when GSSG recycling was impaired by gsr-1(RNAi), GSH synthesis was induced, but not vice versa. Moreover, the impact of GSSG recycling was potentiated under oxidative stress conditions, explaining the enormous effect gsr-1(RNAi) knockdown had on juglone tolerance. Accordingly, overexpression of GSR-1 was capable of increasing stress tolerance. Furthermore, expression levels of SKN-1-regulated GSR-1 also affected life span of C. elegans, emphasising the crucial role the GSH redox state plays in both processes.     

Use of Monochlorobimane to Determine the In Vivo Effect of Cadmium,Copper and Lead on Glutathione Status in Mercenaria mercenaria Brown Cells

This study was undertaken to demonstrate the synthesis of glutathione (GSH) in Mercenaria mercenaria brown cells to test the hypothesis that failure to achieve 100% mortality with metal treatment is the result of high concentrations of GSH heterogeneously distributed in the brown cell population and to determine the effect of Cd²⁺, Cu²⁺, and Pb²⁺ on the GSH status in brown cells. The monochlorobimane (MCB) assay appeared to be selective for GSH in brown cells and a close relationship between the levels of GSH measured by MCB and a standard enzymatic method was found. The fluorescent GSH-bimane adduct, once formed within the cell, was not released from the cell. The technique was used to establish that GSH was synthesized in brown cells and was heterogeneously distributed in the brown cell population. Metal concentrations as high as 40 mM cadmium, 6.0 μM copper, or 20 mM lead did not deplete but caused decreases in brown cell GSH concentration that differed significantly (P < 0.05) from controls. The decrease caused by cadmium, copper, and lead was not in a dose dependent manner, whereas, the decrease caused by N-ethylmaleimide (NEM) was. It appears that the partial kill phenomenon associated with metal toxicity may be due to the heterogeneous distribution of GSH in the brown cell population.     

Glutathione in Intact Vacuoles: Comparison of Glutathione Pools in Isolated Vacuoles,Plastids, and Mitochondria from Roots of Red Beet

Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved methods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-performance liquid chromatography (HPLC), and spectrophotometry with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione (GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mitochondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione concentrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram protein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were 1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glutathione was higher than in other organelles.     

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems

A pathway for the synthesis of dimethyl seledine from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30 000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO₂ (93% inhibition by 10^?6 M arsenite in the presence of a 20 000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.     

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.     

Surface Display of a Bifunctional Glutathione Synthetase on Saccharomyces cerevisiae for Converting Chicken Feather Hydrolysate into Glutathione

The low economic profits of feather recycling lead that the large amount of feathers is currently discarded in China. To convert feather hydrolysates into GSH with high values, surface display of the bifunctional glutathione synthetase encoded by gcsgs from Streptococcus thermophilus on Saccharomyces cerevisiae and the potential in glutathione (GSH) production from feather hydrolysates were studied. The surface-displayed GCSGS could be used to convert feather hydrolysates into GSH. Results showed that 10 g/l of feather was converted into 321.8 mg/l GSH by the Trichoderma atroviride F6 and surface-displayed GCSGS in the study. Compared with production of intracellular GSH by S. cerevisiae from amino acids or feather hydrolysate, the concentration of GSH in the study was higher, and purification of GSH was more feasible. Due to the glycolytic pathway, the S. cerevisiae was used to generate ATP and cheap feather hydrolysate as precursors, the process for GSH production based on surface-displayed GCSGS is cheap and feasible. The process showed the potential to convert feather hydrolysates into GSH on an industrial scale.     

Glutathione Reductase-null Malaria Parasites Have Normal Blood Stage Growth but Arrest during Development in the Mosquito

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.