Glutathione production by Escherichia coli cells with hybrid plasmid containing tandemly polymerized genes for glutathione synthetase

Summary A DNA fragment containing the structural and promoter regions of glutathione synthetase (GSH II) gene (gsh II) from Escherichia coli B were polymerized. The dimeric and trimeric DNA fragments obtained were inserted into Bam HI site of vector plasmid pBR325 and the resulting hybrid plasmids were designated pGS401-02 and pGS401-03, respectively. The GSH II activity of E. coli cells with these hybrid plasmids increased depending on the number of the genes (gsh II) contained. To construct hybrid plasmids useful for glutathione production, another DNA fragment with a gene (gsh I) for -glutamylcysteine synthetase (GSH I) from E. coli B was inserted into Pst I sites of pGS401-02 and pGS401-03 and the hybrid plasmids obtained (pGS501-12 and pGS501-13, respectively) were introduced into E. coli B cells. Although the glutathione-producing activities of the cells with these plasmids were little improved as compared with that of cells with the hybrid plasmid (pGS501-11) containing both gsh I and gsh II because of the low activity of GSH I, our method has brought to light a new type of gene amplification.     

Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the methylotrophic yeast Hansenula polymorpha

The Hansenula polymorpha GSH1/MET1 gene was cloned by complementation of glutathione-dependent growth of H. polymorpha gsh1 mutant isolated previously as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistant and cadmium ion sensitive clone. The H. polymorpha GSH1 gene was capable of restoring cadmium ion resistance, MNNG sensitivity, normal glutathione level and cell proliferation on minimal media without addition of cysteine or glutathione, when introduced into the gsh1 mutant cells. It was shown that the H. polymorpha GSH1 gene has homology to the Saccharomyces cerevisiae MET1 gene encoding S-adenosyl-L-methionine uroporphyrinogen III transmethylase, responsible for the biosynthesis of sulfite reductase cofactor, sirohaem. The H. polymorpha GSH1/MET1 gene deletion cassette (Hpgsh1/met1::ScLEU2) was constructed and corresponding null mutants were isolated. Crossing data of the point gsh1 and null gsh1/met1 mutants demonstrated that both alleles were located to the same gene. The null gsh1/met1 mutant showed total growth restoration on minimal media supplemented with cysteine or glutathione as a sole sulfur source, but not with inorganic (sulfate, sulfite) or organic (methionine, S-adenosylmethionine) sources of sulfur. Moreover, both the point gsh1 and null gsh1/met1 mutants displayed increased sensitivity to the toxic carbon substrate methanol, formaldehyde, organic peroxide and cadmium ions.     

GSH2, a gene encoding gamma-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorpha

The GSH2 gene, encoding Hansenula polymorpha gamma-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121.     

Development of glutathione-deficient embryos in Arabidopsis is influenced by the maternal level of glutathione

Glutathione (GSH) biosynthesis-deficient gsh1 and gsh2 null mutants of Arabidopsis thaliana have late embryonic-lethal and early seedling-lethal phenotypes, respectively, when segregating from a phenotypically wild-type parent plant, indicating that GSH is required for seed maturation and during germination. In this study, we show that gsh2 embryos generated in a partially GSH-deficient parent plant, homozygous for either the cad2 mutation in the GSH1 gene or homozygous for mutations in CLT1, CLT2 and CLT3 encoding plastid thiol transporters, abort early in embryogenesis. In contrast, individuals homozygous for the same combinations of mutations but segregating from heterozygous, phenotypically wild-type parents exhibit the parental gsh2 seedling-lethal phenotype. Similarly, homozygous gsh1 embryos generated in a gsh1/cad2 partially GSH-deficient parent plant abort early in development. These observations indicate that the development of gsh1 and gsh2 embryos to a late stage is dependent on the level of GSH in the maternal plant.     

具有高谷胱甘肽合成活性重组大肠杆菌的构建及合成反应过程

以野生型大肠杆菌E.coliⅡ为宿主细胞,转化带有编码谷胱甘肽合成酶系的基因gshⅠ和gshⅡ的质粒pGH501,获得了一株谷胱甘肽合成活性、质粒稳定性和传代稳定性俱佳,并且能够重复使用的重组大肠杆菌E.coliⅡ\|1。该菌株经过甲苯处理后,能够在胞外积累4g/L左右的谷胱甘肽(GSH)。在合成反应体系中,提高L谷氨酸浓度可促进GSH合成,但L半胱氨酸浓度增大到20mmol/L后会抑制GSH的合成。根据GSH合成反应中能量辅因子的变化情况,提出E.coliⅡ\|1细胞控制的GSH合成反应机理：由谷胱甘肽合成酶(GSHⅡ)控制的第二步反应的能量供体是ADP而非ATP,该反应是整个GSH合成反应的限速步骤,高浓度ADP可能会抑制GSHⅡ的活性。在GSH合成反应体系中添加100mmol/L的L丝氨酸-硼酸钾混合物,可以有效地防止GSH的进一步降解,反应3 h后,GSH产量达到230mmol/L(约71g/L)。     

Cloning of the GSH1 and GSH2 genes complementing the defective biosynthesis of glutathione in the methylotrophic yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the gamma-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh- phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24 with the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme gamma-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

Glutathione (GSH) synthetase (Gsh2) catalyzes the ATP-dependent synthesis of GSH from gamma-glutamylcysteine (gamma-Glu-Cys) and glycine. GSH2, encoding the Saccharomyces cerevisiae enzyme, was isolated and used to construct strains that either lack or overproduce Gsh2. The identity of GSH2 was confirmed by the following criteria: 1) the predicted Gsh2 protein shared 37-39% identity and 58-60% similarity with GSH synthetases from other eukaryotes, 2) increased gene dosage of GSH2 resulted in elevated Gsh2 enzyme activity, 3) a strain deleted for GSH2 was dependent on exogenous GSH for wild-type growth rates, and 4) the gsh2 mutant lacked GSH and accumulated the dipeptide gamma-Glu-Cys intermediate in GSH biosynthesis. Overexpression of GSH2 had no effect on cellular GSH levels, whereas overexpression of GSH1, encoding the enzyme for the first step in GSH biosynthesis, lead to an approximately twofold increase in GSH levels, consistent with Gsh1 catalyzing the rate-limiting step in GSH biosynthesis. In contrast to a strain deleted for GSH1, which lacks both GSH and gamma-Glu-Cys, the strain deleted for GSH2 was found to be unaffected in mitochondrial function as well as resistance to oxidative stress induced by hydrogen peroxide, tert-butyl hydroperoxide, and the superoxide anion. Furthermore, gamma-Glu-Cys was at least as good as GSH in protecting yeast cells against an oxidant challenge, providing the first evidence that gamma-Glu-Cys can act as an antioxidant and substitute for GSH in a eukaryotic cell. However, the dipeptide could not fully substitute for the essential function of GSH in the cell as shown by the poor growth of the gsh2 mutant on minimal medium. We suggest that this function may be the detoxification of harmful intermediates that are generated during normal cellular metabolism.     

A novel cis-acting cysteine-responsive regulatory element of the gene for the high-affinity glutathione transporter of Saccharomyces cerevisiae

We cloned a DNA fragment from Saccharomyces cerevisiae that complemented the deficiency in high-affinity glutathione transport activity conferred by a gsh11 mutation, and found that the ORF responsible was YJL212c, which had already been designated as OPT1 and HGT1 by others. Northern analysis clearly demonstrated that this ORF, now referred to as OPT1/ HGT1/ GSH11, was induced by sulfur starvation and repressed by adding cysteine to the growth medium. Reporter gene assays showed that a segment spanning the region between positions -371 and -355 was essential for the regulation of this gene. A sequence of 9 nt, CCGCCACAC (from -364 to -356), in this region was shown to be required for protein binding, using an electrophoretic mobility shift assay. Based on these results, we propose that CCGCCACAC comprises the core of a cis-acting element involved in cysteine-responsive gene regulation in S. cerevisiae.     

The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain

A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.     

Cloning of the GSH1 and GSH2 Genes Complementing the Defective Biosynthesis of Glutathione in the Methylotrophic Yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh⁺ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the -glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh^– phenotype of the gsh2 mutant. The Gsh⁺ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme -glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

一种适用于产朊假丝酵母的基因敲除系统及其在gsh1基因敲除中的应用

报道一种适用于产朊假丝酵母Candida utilis的基因敲除系统,利用该敲除系统获得gsh1基因敲除杂合突变株。根据不同种属酵母菌γ-谷氨酰半胱氨酸合成酶(γ-GCS)蛋白质的保守序列,克隆C.utilis SZU 07-01的gsh1基因;以商品化质粒pPICZalpha A为基础,构建gsh1基因的敲除载体pPICZalpha A-kan 3,其中,kan基因的启动子TEF被替换为来自于C.utilis SZU 07-01的GAP启动子(pGAP:kan)。质粒电转化C.utilis,获得gsh1基因敲除杂合突变株C.utilis GSH-6。结合发酵培养得到的数据进行分析,突变株的γ-GCS酶活比出发菌株降低17.5%,GSH合成量降低61%,细胞干重降低18.5%。所构建敲除组件pGAP:kan的成功应用为从分子水平研究C.utilis中谷胱甘肽(GSH)的生理功能提供了一种新借鉴。     

Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor

Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in gamma-l-glutamyl-l-cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, gamma-glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that gamma-glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4-dependent expression of GSH1 and elevating the cellular levels of gamma-glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4-dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4-regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1-specific mechanisms exist that respond to GSH availability.     

Effect of cellular glutathione content on the induction of DNA double strand breaks by 25 MeV electrons

The effect of endogenous glutathione (GSH) on the induction of DNA double strand breaks (dsb) by 25 MeV electrons was investigated using stationary haploid yeast cells defective in gamma-glutamyl-cysteine-synthetase (gsh 1) containing less than 5 per cent of the normal GSH content. In gsh 1 cells the induction of dsb is increased by a factor of 1.5 under oxic and 1.8 under anoxic irradiation conditions: whereas the oxygen enhancement ratio was only slightly decreased (1.9) compared to wild-type cells (2.4).     

Yeast cys3 and gsh1 mutant cells display overlapping but non-identical symptoms of oxidative stress with regard to subcellular protein localization and CDP-DAG metabolism

In a screen for temperature-sensitive (37 degrees C) mutants of Saccharomyces cerevisiae that are defective in the proper localization of the Golgi transmembrane protein Emp47p, we uncovered a constitutive loss-of-function mutation in CYS3/STR1, the gene coding for cystathionine-gamma-lyase. We showed by immunofluorescence, sucrose-gradient analysis and quantitative Western analysis that the mutant mislocalized Emp47p to the vacuole at high temperature, while Golgi structures were apparently normal and biosynthetic routing of the vacuolar carboxypeptidase Y (CPY) and the plasma membrane GPI-anchored protein Gas1p were unaffected. The effect of high temperature on Emp47p localization, as well as the temperature sensitivity of the mutant strain on rich medium, appear to be caused by oxidative stress and are correlated with severe reductions in the intracellular levels of low-molecular-weight thiols. In accordance with this conclusion, cys3-2 mutant cells were more sensitive to the oxidizing agent 1-chloro-2,4-dinitrobenzene, which also aggravated the mislocalization of Emp47p observed at high temperature. Furthermore, all the phenotypes of the mutant were completely complemented by exogenous supply of the main low-molecular-weight thiol, glutathione (GSH) and, importantly, the thiol beta-mercaptoethanol reversed the temperature sensitivity of the mutant. A comparison of our mutant with a mutant defective in GSH synthesis showed that gsh1Delta cells were similar to wild-type cells under the stress conditions tested, with the exception of one novel oxidative stress-related phenotype that is observed in both cys3-2 and gsh1Delta mutant cells - a defect in CDP-DAG metabolism upon shift to the non-permissive temperature. As most of the stress-related phenotypes of cys3-2 mutant cells are more severe than those seen in gsh1Delta cells, we conclude that cysteine as such is required and sufficient to confer some degree of protection from oxidative stress in yeast cells.     

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.     

Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development

Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, γ-glutamate cysteine ligase (GSH1), responsible for synthesis of γ-glutamylcysteine (γ-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by γ-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of γ-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by γ-EC in vitro strongly suggests export of γ-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, γ-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.     

Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P. alba) overexpressing glutathione synthetase

The poplar hybrid Populus tremula X P. alba was transformed with the Escherichia coli gene for glutathione synthetase ( gsh II ) targetted to the cytosol. Leaves of five lines of transgenic plants exhibited glutathione synthetase activities 15- to 60-fold higher than those of wild-type plants. Total glutathione levels and GSH/GSSG ratios were similar in transgenic and wild-type plants. Precursor feeding experiments with cysteine and γ-glutamylcysteine suggest that glutathione synthesis in the cytoplasm is controlled by a multistep procedure that includes (i) the availability of cysteine, (ii) the availability of γ-glutamylcysteine, and (iii) regulation of the activities of both γ-glutamylcysteine synthetase and glutathione synthetase. However step (ii) may set an upper limit for the cellular glutathione content.     

A perturbation in glutathione biosynthesis disrupts endoplasmic reticulum morphology and secretory membrane traffic in Arabidopsis thaliana

To identify potentially novel and essential components of plant membrane trafficking mechanisms we performed a GFP-based forward genetic screen for seedling-lethal biosynthetic membrane trafficking mutants in Arabidopsis thaliana. Amongst these mutants, four recessive alleles of GSH2, which encodes glutathione synthase (GSH2), were recovered. Each allele was characterized by loss of the typical polygonal endoplasmic reticulum (ER) network and the accumulation of swollen ER-derived bodies which accumulated a soluble secretory marker. Since GSH2 is responsible for converting γ-glutamylcysteine (γ-EC) to glutathione (GSH) in the glutathione biosynthesis pathway, gsh2 mutants exhibited γ-EC hyperaccumulation and GSH deficiency. Redox-sensitive GFP revealed that gsh2 seedlings maintained redox poise in the cytoplasm but were more sensitive to oxidative challenge. Genetic and pharmacological evidence indicated that γ-EC accumulation rather than GSH deficiency was responsible for the perturbation of ER morphology. Use of soluble and membrane-bound ER markers suggested that the swollen ER bodies were derived from ER fusiform bodies. Despite the gross perturbation of ER morphology, gsh2 seedlings did not suffer from constitutive oxidative ER stress or lack of an unfolded protein response, and homozygotes for the weakest allele could be propagated. The link between glutathione biosynthesis and ER morphology and function is discussed.