GSH1 Gene Research Articles

Investigation of Dependency of Boric Acid and Lithium Metaborate Induced Yeast Toxicity on the Expressions of Antioxidative and Apoptotic Genes

ABSTRACT Boric acid and its less-known derivative lithium metaborate are inorganic acids used in a wide and diverse array of industrial and clinical applications. In particular, boric acid is accepted as a natural antimicrobial agent due to its activity on many different microbial species. We have examined the possible dependency of boric acid and lithium metaborate-induced yeast toxicity on the deletion of antioxidative CTT1, TRR2 and GSH2 genes and the expressions of certain important pro- and anti-apoptotic genes. While the investigated antioxidative genes have no function in the protection against boric acid mediated toxicity, the cell proliferation percentages of △GSH2 and △TRR2 mutants significantly decreased to approximately 57% and 75%, respectively, after 0.25% LMB treatment for 12 h compared to the wild yeast strain. The relative expressions of the AIF and NDI genes were generally induced for the highest concentrations of the drugs compared to the control. Similarly, anti-apoptotic BIR1 was also generally induced compared to the control, which indicates the importance of this gene in the resistance of yeast cells against BA and its derivatives mediated toxicity. These new findings are significant in terms of adding new data to the literature on understanding the toxic effect mechanisms of boron compounds.

Redox perturbations in yeast cells lacking glutathione reductase

Cellular redox homeostasis has a major effect on cell functions and its maintenance is supported by glutathione and protein thiols which serve as redox buffers in cells. The regulation of the glutathione biosynthetic pathway is a focus of a lot of scientific research. However, still little is known about how complex cellular networks influence glutathione homeostasis. In this work was used an experimental system based on an S. cerevisiae yeast mutant with a lack of the glutathione reductase enzyme and allyl alcohol as a precursor of acrolein inside the cell to determine the cellular processes influencing glutathione homeostasis. The absence of Glr1p slows down the growth rate of the cell population, especially in the presence of allyl alcohol, but does not lead to complete inhibition of the cell's reproductive capacity. It also amends the GSH/GSSG ratio and the share of NADPH and NADP+ in the total NADP(H) pool. The obtained results show that potential pathways involved in the maintenance of redox homeostasis are based from one side on de novo synthesis of GSH as indicated by increased activity of γ-GCS and increased expression of GSH1 gene in the Δglr1 mutant, from the other hand, on increased the level of NADPH. This is because the lower ratio of GSH/GSSG can be counterbalanced with the NADPH/NADP+ alternative system. The higher level of NADPH can be used by the thioredoxin system and other enzymes requiring NADPH to reduce cytosolic GSSG and maintain glutathione redox potential.

Effects of silicon and titanium dioxide nanoparticles on arsenic accumulation, phytochelatin metabolism, and antioxidant system by rice under arsenic toxicity.

Arsenic (As) is known to be one of the most toxic metalloids for humans and plants; however, little is known about the use of silicon (Si) and titanium dioxide (TiO2) nanoparticles (NPs) in reducing As toxicity in rice (Oryza sativa L.). The experiment was conducted to examine the effects of Si-NPs (50 and 100mg/L), TiO2-NPs (25 and 50mg/L) and As (50µM) on growth, photosynthetic pigments, antioxidant defense system, glyoxalase system, expression of Si/As transporters, and genes involved in As sequestration in rice under hydroponic conditions. The results revealed that Si- and TiO2-NPs by upregulating the activity of antioxidant enzymes and glyoxalase cycle reduced hydrogen peroxide, methylglyoxal, malondialdehyde, and electrolyte leakage, and thus protected the photosynthetic apparatus and improved plant growth under As stress. By increasing the expression of GSH1, PCS, and ABC1 genes, Si- and TiO2-NPs increased leaf and root accumulation of glutathione and phytochelatins and sequestered As in vacuoles, which protected plant cells from As toxicity. Si-NPs diminished As uptake and increased Si uptake in As-exposed rice plants by modulating the expression of Si/As transporters (Lsi1, Lsi2, and Lsi6). The results depicted that 100mg/L Si-NPs treatment had the highest positive effect on plant growth and tolerance under As stress compared to other treatments. In general, Si- and TiO2-NPs augmented the growth of rice under As stress through different strategies, which can be used to design effective fertilizers to enhance the crop growth and yield in areas contaminated with toxic metals.

Antioxidant activity assessment of Yingjisha sweet almond oil

SummarySweet almond is one of the main cash crops in Yingjisha, Xinjiang Autonomous Region, China. The composition of sweet almond oil (SAO) was analysed by gas chromatography‐mass spectrometry (GC‐MS). SAO’s antioxidant activities were investigated by measuring its scavenging ability with 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), 2,2′‐Azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), hydroxyl radical scavenging assay and 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (Trolox) equivalent antioxidant capacity (TEAC) assay. Furthermore, in vivo antioxidant capacity was assessed using Saccharomyces cerevisiae model stimulated by hydrogen peroxide (H2O2) and carbon tetrachloride (CCl4). The results showed that SAO was mainly composed of methyl oleate (21.28%), methyl stearate (18.55%) and methyl palmitate (14.1%). In vitro antioxidant test results showed that compared with the scavenging three free radicals capacity of 1 mmol L−1 Trolox, the SAO needed to reach 5 mg mL−1, 20 mg mL−1 and less than 20 mg mL−1, respectively. In vivo experiments showed that SAO rescued the survival and inhibited intracellular oxidation and lipid peroxidation of Saccharomyces cerevisiae exposed to H2O2 and CCl4. Additionally, our results showed that the catalase encoded by the Gsh1 gene may be involved in the mechanism of action of SAO against intracellular oxidation. These findings suggest that SAO possesses antioxidant capacities, and it is recommended to develop dietary antioxidants based on SAO.

Bioprocess optimization of glutathione production by Saccharomyces boulardii: biochemical characterization of glutathione peroxidase.

The well-known probiotic GRAS Saccharomyces boulardii (CNCM I-745) was used for the first time to produce glutathione (GSH). The culture conditions affecting GSH biosynthesis were screened using a Plackett-Burman design (PBD). Analyzing the regression coefficients for 12 tested variables, yeast extract, glucose, peptone, cysteine, temperature and agitation rate had a positive significant effect on GSH production with a maximum yeild 192mg/L. The impact of kinetics of adding cysteine was investigated in 19 experiments during the growth time course (0-36h), and the maximum yield of glutathione (235mg/L) was obtained by addition of cysteine after 8h post-inoculation. The most significant variables were further explored at five levels using central composite rotatable design (CCRD), giving a maximum production of GSH (552mg/L). Using baffled flasks, the yield of GSH was increased to 730mg/L, i.e., 1.32-fold increment. The two rate-limiting genes of GSH biosynthesis "γ-glutamyl cysteine synthetase (GSH1) and GSH-synthetase (GSH2)" were amplified and sequenced to validate the GSH biosynthetic potency of S. boulardii. The sequences of genes showed 99% similarity with GSH1 and GSH2 genes of S. cerevisiae. Glutathione peroxidase was purified and characterized from S. boulardii with molecular mass and subunit structure of 80kDa and 35kDa as revealed from native and SDS-PAGE, ensuring its homodimeric identity. The activity of GPx was reduced by 2.5-fold upon demetallization confirming its metalloproteinic identity. The GPx was strongly inhibited by hydroxylamine and DTNB, ensuring the implication of surface lysine and cysteine residues on the enzyme active site domains.

Genomic insights into the glutathione metabolism of the non-conventional wine yeast <i>Starmerella bacillaris</i>

Glutathione (GSH) is an antioxidant molecule of great technological interest due to its wide range of applications in the food and beverage industry. In winemaking, although glutathione is produced during fermentation, its addition is possible for the control of oxidative spoilage of wine. Recently, to improve wine quality, mixed fermentation has been proposed by introducing a selection of non-Saccharomyces yeasts as complementary starters to the oenological species S. cerevisiae. Among them, Starmerella bacillaris, an osmophilic and high glycerol producer yeast, has been extensively studied. In the present study, the genomes of two S. bacillaris strains were compared with S. cerevisiae to identify the GSH metabolic pathway. The results showed that GSH biosynthesis includes the GSH1 and GSH2 genes in both species. The identification of a new transcription factor which binds sites in the promoter region of these genes underlined differences in the transcriptional regulation of both species. Additionally, between S. bacillaris strains, a high number of polymorphisms was found in genes involved in GSH redox balance. Preliminary laboratory scale fermentations revealed marked differences in the cell glutathione content of the two S. bacillaris strains. By comparing genomes, it was possible to gain a better understanding of the genes involved in the GSH metabolism pathway in S. bacillaris.

Transcription factor Hap5 induces gsh2 expression to enhance 2-phenylethanol tolerance and production in an industrial yeast Candida glycerinogenes.

2-Phenylethanol (2-PE) is an important flavor compound but also impairs cell growth severely, which in turn blocks its bioproduction. However, the molecular mechanism of 2-PE tolerance is unclear. In this study, a superb 2-PE stress-tolerant and producing yeast, Candida glycerinogenes, was selected to uncover the underlying mechanism of 2-PE tolerance. We discovered that Hap5 is an essential regulator to 2-PE resistance, and its induction by 2-PE stress occurs at the post-transcriptional level, rather than at the transcriptional level. Under 2-PE stress, Hap5 is activated and imported into the nucleus rapidly. Then, the nuclear Hap5 binds to the glutathione synthetase (gsh2) promoter via CCAAT box, to induce the expression of gsh2 gene. The increased gsh2 expression contributes to enhanced cellular glutathione content, and consequently alleviates ROS accumulation, lipid peroxidation, and cell membrane damage caused by 2-PE toxicity. Specifically, increasing the expression of gsh2 is effective in improving not just 2-PE tolerance (33.7% higher biomass under 29mM 2-PE), but also 2-PE production (16.2% higher). This study extends our knowledge of 2-PE tolerance mechanism and also provides a promising strategy to improve 2-PE production.

Melatonin promotes metabolism of bisphenol A by enhancing glutathione-dependent detoxification in Solanum lycopersicum L

Bisphenol A (BPA), a widely distributed organic compound, is toxic to animals and plants. Here we show the mechanism of BPA detoxification by melatonin (MEL) in tomato, which is otherwise poorly understood in plants. BPA treatment decreased the quantum yield of photosystem II (Fv/Fm) and increased the membrane lipid peroxidation and reactive oxygen species (ROS) accumulation dose-dependently, whereas exogenous MEL alleviated the BPA effects on Fv/Fm, lipid peroxidation, ROS accumulation and BPA uptake. Furthermore, BPA elevated the glutathione (GSH) content, activities of glutathione S-transferase (GST), and glutathione reductase (GR), and the transcript levels of GSH1, GR1, GST1 and MEL biosynthesis genes (COMT, T5H, and SNAT), whereas BPA + MEL showed even a more profound induction. Silencing GSH1, GR1 and GST1 genes compromised the BPA detoxification potential of tomato plants as revealed by an increased level of ROS, lipid peroxidation and BPA uptake, and a decreased Fv/Fm and GST activity; these changes were alleviated by MEL application. Under in vitro conditions, BPA was glutathionylated by GSH, which was further catalyzed by GST to cysteine and N-acetylcysteine conjugates. These findings suggest a crucial role for MEL in BPA detoxification via GSH and GST, and can be useful to reduce BPA residue for food safety.

Nickel Oxide (NiO) Nanoparticles Induce Loss of Cell Viability in Yeast Mediated by Oxidative Stress.

The present work aimed to elucidate whether the toxic effects of nickel oxide (NiO) nanoparticles (NPs) on the yeast Saccharomyces cerevisiae were associated with oxidative stress (OS) and what mechanisms may have contributed to this OS. Cells exposed to NiO NPs accumulated superoxide anions and hydrogen peroxide, which were intracellularly generated. Yeast cells coexposed to NiO NPs and antioxidants (l-ascorbic acid and N- tert-butyl-α-phenylnitrone) showed quenching of reactive oxygen species (ROS) and increased resistance to NiO NPs, indicating that the loss of cell viability was associated with ROS accumulation. Mutants lacking mitochondrial DNA (ρ0) displayed reduced levels of ROS and increased resistance to NiO NPs, which suggested the involvement of the mitochondrial respiratory chain in ROS production. Yeast cells exposed to NiO NPs presented decreased levels of reduced glutathione (GSH). Mutants deficient in GSH1 ( gsh1Δ) or GSH2 ( gsh2Δ) genes displayed increased levels of ROS and increased sensitivity to NiO NPs, which underline the central role of GSH against NiO NPs-induced OS. This work suggests that the increased levels of intracellular ROS (probably due to the perturbation of the electron transfer chain in mitochondria) combined with the depletion of GSH pool constitute important mechanisms of NiO NPs-induced loss of cell viability in the yeast S. cerevisiae.

Gene of the transcriptional activator MET4 is involved in regulation of glutathione biosynthesis in the methylotrophic yeast Ogataea (Hansenula) polymorpha.

Glutathione is the most abundant cellular thiol and the low molecular weight peptide present in cells. The methylotrophic yeast Ogataea (Hansenula) polymorpha is considered as a promising cell factory for the synthesis of glutathione. In this study, a competitive O. polymorpha glutathione producer was constructed by overexpression of the GSH2 gene, encoding γ-glutamylcysteine synthetase, the first enzyme involved in glutathione biosynthesis, and the MET4 gene coding for central regulator of sulfur metabolism. Overexpression of MET4 gene in the background of overexpressed GSH2 gene resulted in 5-fold increased glutathione production during shake flask cultivation as compared to the wild-type strain, reaching 2167 mg L-1. During bioreactor cultivation, glutathione accumulation by obtained recombinant strain was 5-fold increased relative to that by the parental strain with overexpressed only GSH2 gene, on the first 25 h of batch cultivation in mineral medium. Obtained results suggest involvement of Met4 transcriptional activator in regulation of GSH synthesis in the methylotrophic yeast O. polymorpha.

Accession-specific life strategies affect responses in leaves of Arabidopsis thaliana plants exposed to excess Cu and Cd

The natural accession Columbia (Col-0) is considered as the reference genome of the model plant Arabidopsis thaliana. Nonetheless, Col-0 plants are more sensitive to excess copper (Cu) and cadmium (Cd) than other widely used accessions such as Wassilewskija (Ws) plants. In the current study, this accession-specific metal sensitivity is further explored by comparing the responses in leaves of Col-0 and Ws plants exposed to excess Cu and Cd.Our results suggest that different life strategies favored by both accessions under physiological conditions affect their response to metal exposure. While Col-0 plants mainly invest in metal detoxification, Ws plants center on nutrient homeostasis. In particular, the higher expression of genes related to Cu homeostasis genes in non-exposed conditions indicates that Ws plants possess a constitutively efficient metal homeostasis. On the other hand, oxidative stress-related MAPK signaling appears to be boosted in leaves of Col-0 plants exposed to excess Cu. Furthermore, the upregulation of the glutathione (GSH) biosynthesis GSH2 gene and the increased GSH concentration after Cd exposure suggest the activation of detoxification mechanisms, such as phytochelatin production, to counteract the more severe Cd-induced oxidative stress in leaves of Col-0 plants. Exposure to Cd also led to a more pronounced ethylene signaling response in leaves of Col-0 as compared to Ws plants, which could be related to Cd-induced GSH metabolism. In conclusion, accession-specific life strategies clearly affect the way in which leaves of A. thaliana plants cope with excess Cu and Cd.

Overexpression of EcGSH1 induces glutathione production and alters somatic embryogenesis and plant development in Hevea brasiliensis

Oxidative stress occurring during in-vitro culture is detrimental for clonal propagation by somatic embryogenesis in particular in Hevea brasiliensis. In previous work, overexpression of the gene encoding the cytosolic reactive oxygen species detoxification enzyme HbCuZnSOD led to reduce somatic embryo regeneration in Hevea. In this study, the role of antioxidant was tested by overexpression of the E.coli GSH1 gene involved in glutathione biosynthesis in rubber embryogenic callus lines. Transgenic lines were successfully established and some plants were regenerated. Overexpression of EcGSH1 gene led to glutathione over-accumulation, and affected dramatically the somatic embryogenesis process and plant development. Upon a water deficit treatment, these plants displayed the greatest drop in photosynthetic nitrogen use efficiency, higher proline content, and higher glutathione reductase activity. Changes induced in transgenic lines overexpressing HbCuZnSOD and EcGSH1 were discussed as well as possible applications on plant material propagation, overcoming loss of natural rubber production through Tapping Panel Dryness, and tropical soil remediation.

Loss-of-function mutations in the APX1 gene result in enhanced selenium tolerance in Arabidopsis thaliana.

It is generally recognized that excess selenium (Se) has a negative effect on the growth and development of plants. Numerous studies have identified key genes involved in selenium tolerance in plants; however, our understanding of its molecular mechanisms is far from complete. In this study, we isolated an Arabidopsis selenium-resistant mutant from the mutant XVE pool lines because of its increased root growth and fresh weight in Se stress, and cloned the gene, which encodes the cytosolic ascorbate peroxidase (APX1). Two other APX1 gene knockout allelic lines were also selenium resistant, and the APX1-complementary COM1 restored the growth state of wild type under Se stress. In addition, these APX1 allelic lines accumulated more Se than did wild-type plants when subjected to Se stress. Further analysis revealed that the APX1-mediated Se tolerance was associated, at least in part, with the enhanced activities of antioxidant enzymes catalase, glutathione peroxidase and glutathione reductase. Moreover, enhanced Se resistance of the mutants was associated with glutathione (GSH), which had the higher expression level of GSH1 gene involved in GSH synthesis and consequently increased GSH content. Our results provide genetic evidence indicating that loss-of-function of APX1 results in tolerance to Se stress.

DNA Profiling of Transgenes in Genetically Modified Plants

In silico sequence diversities of four orthologous plant gsh1 genes and their anino acid translates of GSH1 proteins (Glutathione Synthase) were compared to the non-orthologous prokaryotic gshI/GSHI gene/protein of E. coli (NCBI # X03954). Primer pair was designed and transgene detection was carried out in two types of gshI-transgenic poplar clones (Populus x canescens) of ggs11 (cyt-ECS) and lgl6 (chl-ECS). Usefulness of genetic modification technologies (GMO) is indicated.

Selenium tolerance of an Arabidopsis drought-resistant mutant csm1-1

We isolated the Arabidopsis drought-resistant mutant csm1-1 and cloned the CSM1 gene (previously called RCI3) which encodes a kind of peroxidase. In this study, we showed that the mutant csm1-1 was more tolerant to selenium (Se) stress than the wild-type, and that the mutant csm1-1 accumulated similar Se content compared with the wild-type when subjected to Se stress. Further analysis revealed that the Se resistance of mutant csm1-1 was associated, at least in part, with the enhanced activities of antioxidant enzymes GSH-POD (glutathione peroxidase) and POD (peroxidase). In addition, enhanced Se resistance of the mutant csm1-1 was partially GSH dependent, which was related to the higher expression level of GSH1 gene involved in GSH synthesis and consequently increased GSH content.

Транскрипційна регуляція гену GSH2 Hansenula polymorpha

In a previous study we cloned GSH2 gene, encoding gamma-glutamylcysteine synthetase (gammaGCS) in the yeast Hansenula polymorpha. In this study an analysis of molecular organisation of the H. polymorpha GSH2 gene promoter was conducted and the potential binding sites of Yap1, Skn7, Creb/Atf1, and Cbfl transcription factors were detected. It was established that full regulation of GSH2 gene expression in the response to cadmium and oxidative stress requires the length of GSH2 promoter to be longer than 450 bp from the start of translation initiation. To study the transcriptional regulation of H. polymorpha GSH2 gene recombinant strain, harbouring a reporter system, in which 1.832 kb regulatory region of GSH2 gene was fused to structural and terminatory regions of alcohol oxidase gene, was constructed. It was shown that maximum increase in H. polymorpha GSH2 gene transcription by 33% occurs in the rich medium under four-hour incubation with 1 microM concentration of cadmium ions. In the minimal medium the GSH2 gene expression does not correlate with the increased total cellular glutathione levels under cadmium ion treatment. We assume that the increased content of total cellular glutathione under cadmium stress in the yeast H. polymorpha probably is not controlled on the level of GSH2 gene transcription.

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.

Phytoextraction Potential of Wild Type and 35S-GSHI Transgenic Poplar Trees (Populus × Canescens) for Environmental Pollutants Herbicide Paraquat, Salt Sodium, Zinc Sulfate and Nitric Oxide In Vitro

Phytoextraction potentials of two transgenic (TR) poplar (Populus × canescens) clones TRggs11 and TRlgl6 were compared with that of wild-type (WT) following exposure to paraquat, zinc sulfate, common salt and nitric oxide (NO), using a leaf-disc system incubated for 21 days on EDTA-containing nutritive WPM media in vitro. Glutathione (GSH) contents of leaf discs of TRlgl6 and TRggs11 showed increments to 296% and 190%, respectively, compared with WT. NO exposure led to a twofold GSH content in TRlgl6, which was coupled with a significantly increased sulfate uptake when exposed to 10−3 M ZnSO4. The highest mineral contents of Na, Zn, Mn, Cu, and Mo was observed in the TRggs11 clone. Salt-induced activity of catalase enzyme increased in both TR clones significantly compared with WT under NaCl (0.75% and 1.5%) exposure. The in silico sequence analyses of gsh1 genes revealed that P. × canadensis and Salix sachalinensis show the closest sequence similarity to that of P. × canescens, which predicted an active GSH production with high phytoextraction potentials of these species with indication for their use where P. × canescens can not be grown.

Evaluation of the Role of Glutathione in the Lead-Induced Toxicity in Saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (L-glutamic acid, L-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.

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.