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.     

Significantly improved Escherichia coli tolerance and accumulation of Cd2+, Zn2+ and Cu2+ expressing Streptococcus thermophilus StGCS-GS with high glutathione content

Streptococcus thermophilus γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) which synthesized glutathione (GSH) without limit feedback inhibition was over-expressed as a fusion protein of TrxA-StGCS-GS to analyze its possibly functional role in heavy metal tolerance of Escherichia coli (BL21). For comparative analyses, Arabidopsis γ-glutamylcysteine synthetase (AtGCS) and glutathione synthetase (AtGS) were introduced into Escherichia coli (E. coli) in the same manner, respectively. The results showed that the growth and survivability of E. coli over-expressing TrxA-StGCS-GS were slightly influenced by 1 mM Cd²⁺, Zn²⁺ and Cu²⁺ toxicity, and it could withstand duration of these heavy metal stresses competently. In contrast, the two strains over-expressing TrxA-AtGCS and TrxA-AtGS were impacted apparently; the BL21 empty strain was even almost suppressed. Meanwhile, a much higher bioaccumulation of Cd²⁺, Zn²⁺, Cu²⁺ ions and glutathione content were observed in the strain over-expressing TrxA-StGCS-GS than in the other comparison strains. It could be concluded that over-expression of StGCS-GS offered a more significant enhancement of heavy metal tolerance to E. coli with superior GSH content to accumulate considerable heavy metal.     

Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response.     

Cell Surface Display of MerR on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for Biosorption of Mercury

The metalloregulatory protein MerR which plays important roles in mer operon system exhibits high affinity and selectivity toward mercury (II) (Hg²⁺). In order to improve the adsorption ability of Saccharomyces cerevisiae for Hg²⁺, MerR was displayed on the surface of S. cerevisiae for the first time with an α-agglutinin-based display system in this study. The merR gene was synthesized after being optimized and added restriction endonuclease sites EcoR I and Mlu I. The display of MerR was indirectly confirmed by the enhanced adsorption ability of S. cerevisiae for Hg²⁺ and colony PCR. The hydride generation atomic absorption spectrometry was applied to measure the Hg²⁺ content in water. The engineered yeast strain not only showed higher tolerance to Hg, but also their adsorption ability was much higher than that of origin and control strains. The engineered yeast could adsorb Hg²⁺ under a wide range of pH levels, and it could also adsorb Hg²⁺ effectively with Cd²⁺ and Cu²⁺ coexistence. Furthermore, the engineered yeast strain could adsorb ultra-trace Hg²⁺ effectively. The results above showed that the surface-engineered yeast strain could adsorb Hg²⁺ under complex environmental conditions and could be used for the biosorption and bioremediation of environmental Hg contaminants.     

Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1

Thin stillage (TS) is a waste residue that remains after bioethanol production, and its disposal reflects the high costs of bioethanol production. Thus, the development of cost-effective ways to process TS is a pending issue in bioethanol plants. The aim of this study was to evaluate the utilization of TS for the production of the valuable chemical, 1,3-propanediol (1,3-PDO), by Lactobacillus panis PM1. Different fermentation parameters, including temperature, pH and strains [wild-type and a recombinant strain expressing a NADPH-dependent aldehyde reductase (YqhD) gene] were tested in batch and fed-batch cultivations. The highest 1,3-PDO concentration (12.85 g/L) and yield (0.84 g/g) were achieved by batch fermentation at pH-4.5/30 °C by the YqhD recombinant strain. Furthermore, pH-controlled batch fermentation reduced the total fermentation period, resulting in the maximal 1,3-PDO concentration of 16.23 g/L and yield of 0.72 g/g in TS without an expensive nutrient or nitrogen (e.g., yeast extract, beef extract, and peptone) supplementation. The addition of two trace elements, Mg²⁺ and Mn²⁺, in TS increased 1,3-PDO yield (0.74 g/g) without 3-hydroxypropionaldehyde production, the only intermediate of 1,3-PDO biosynthetic pathway in L. panis PM1. Our results suggest that L. panis PM1 can offer a cost-effective process that utilizes the TS to produce a value-added chemical, 1,3-PDO.     

Brachyspira (Serpulina) hyodysenteriae gyrB Mutants and Interstrain Transfer of Coumermycin A1 Resistance

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a λZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A₁-resistant (Cn^r) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 μg of coumermycin A₁/ml. The coumermycin A₁ MICs were 25 to 100 μg/ml for the resistant strains and 0.1 to 0.25 μg/ml for strain B204. Four Cn^r strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly₇₈ to Ser (two strains), Gly₇₈ to Cys, and Thr₁₆₆ to Ala. When Cn^r strain 435A (Gly₇₈ to Ser) and Cm^r Km^r strain SH (ΔflaA1::cat Δnox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56°C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A₁, and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn^r Km^r Cm^r strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn^r Km^r Cm^r cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.     

Alcoholic fermentation by wild-type <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> versus recombinant strains with an elevated level of intracellular glutathione

The ability of baker’s yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.     

Physiological and enzymatic comparison between Pichia stipitis and recombinant Saccharomyces cerevisiae on xylose fermentation

In order to better understand the differences in xylose metabolism between natural xylose-utilizing Pichia stipitis and metabolically engineered Saccharomyces cerevisiae, we constructed a series of recombinant S. cerevisiae strains with different xylose reductase/xylitol dehydrogenase/xylulokinase activity ratios by integrating xylitol dehydrogenase gene (XYL2) into the chromosome with variable copies and heterogeneously expressing xylose reductase gene (XYL1) and endogenous xylulokinase gene (XKS1). The strain with the highest specific xylose uptake rate and ethanol productivity on pure xylose fermentation was selected to compare to P. stipitis under oxygen-limited condition. Physiological and enzymatic comparison showed that they have different patterns of xylose metabolism and NADPH generation.     

Live Recombinant Salmonella Typhi Vaccines Constructed to Investigate the Role of rpoS in Eliciting Immunity to a Heterologous Antigen

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (χ9639 and χ9640) were derived from the rpoS mutant strain Ty2 and one (χ9633) from the RpoS⁺ strain ISP1820. In χ9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS⁺ vaccines induced a balanced Th1/Th2 immune response while the RpoS⁻ strain χ9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS⁺ strain χ9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, χ9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts.     

Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: Chemical nature of cellular metal

Copper-sensitive (Cu^s) and copper-resistant (Cu^r) strains of Pseudomonas aeruginosa were characterized in terms of Cu²⁺ sensitivity, uptake and its compartmentalization in the possible cell sectors. Minimum inhibitory concentrations (MICs) of Cu²⁺ for the Cu^r strain (3.2 mM and 0.12 mM in enriched- and in minimal-medium, respectively) were almost 5-fold higher over that of its sensitive counterpart. While Cu^s strain accumulated Cu²⁺ to a maximum of 1.8 mol mg^–1 protein, Cu^r strain increased it to 2.37 mol mg^–1 protein. Both the strains also demonstrated energy- and pH-dependent Cu²⁺ uptake through the broad-substrate range divalent cation (Zn²⁺, Mg²⁺, Co²⁺) uptake system as well as through the system specific for Cu²⁺. Cell-fractionation study revealed that in Cu^r strain, periplasm and membrane are the main Cu²⁺ binding sites, whereas, in case of Cu^s strain, it is the cytoplasm. The overall observations indicate that the Cu^r strain restricted Cu²⁺ sequestration exterior to the cytoplasm as the possible strategy for Cu-resistance. The chemical nature of Cu²⁺ deposition in the respective strains was also ascertained by X-ray powder diffraction analysis.     

Tepidanaerobacter acetatoxydans sp. nov., an anaerobic, syntrophic acetate-oxidizing bacterium isolated from two ammonium-enriched mesophilic methanogenic processes

Four anaerobic syntrophic acetate-oxidizing bacteria, the thermotolerant strains Re1^T, Re2, T1 and T2, were isolated from two different mesophilic methanogenic systems. The strains originate from sludge of a continuously stirred laboratory-scale reactor containing high levels of ammonium and from a high ammonium enrichment culture. Comparative 16S rRNA gene sequence analysis confirmed that the strains belong to the Firmicutes-Clostridia class. The most closely related species to strains Re1^T, Re2, T1 and T2 was Tepidanaerobacter syntrophicus, with a 16S rRNA gene sequence identity of 96%. The DNA-DNA relatedness of strains Re2, T1 and T2 to strain Re1^T was 92, 102, 81%, respectively. The gene encoding the acetogen key enzyme formyltetrahydrofolate synthetase (FTHFS) was detected and partly sequenced from the strains. In pure culture the bacteria used different organic compounds as carbon and energy source, such as organic acids, alcohols, sugars and amino acids. Furthermore, acetate-oxidizing ability was observed during co-cultivation with a hydrogen-consuming Methanoculleus sp. The bacteria were found to be spore-forming, rod-shaped and motile, and possessed Gram-positive cell walls. The four strains were thermotolerant and grew at temperatures between 25 and 55 °C. Strain Re1^T had a DNA G + C content of 38.4% and the major fatty acids were C_18:1 w7c, C_18:1 w9c, anteiso-C_17:0, C_16:1 w7c and C_18:0. The genetic and phenotypic properties of strains Re1^T, Re2, T1 and T2 suggest classification as representatives of a novel species of the genus Tepidanaerobacter; the name Tepidanaerobacter acetatoxydans sp. nov. is suggested. The type strain of T. acetatoxydans is Re1^T (=DSM 21804^T = JCM 16047^T).     

Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette

A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and γ-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene ( ADH2 ) of Saccharomyces cerevisiae . The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1 + CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for γ-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21–22%, GSH content increased by 20–22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.     

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca²⁺-permeable channel currents by ABA or oscillation of the cytosolic free Ca²⁺ concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca²⁺ oscillation in ABA signal pathway of Arabidopsis guard cells.     

Secretion expression of SOD1 and its overlapping function with GSH in brewing yeast strain for better flavor and anti-aging ability

Superoxide dismutase (SOD) is a significant antioxidant, but unlike glutathione (GSH), SOD cannot be secreted into beer by yeast cells during fermentation, this directly leads to the limited application of SOD in beer anti-aging. In this investigation, we constructed the SOD1 secretion cassette in which strong promoter PGK1p and the sequence of secreting signal factor from Saccharomyces cerevisiae were both harbored to the upstream of coding sequence of SOD1 gene, as a result, the obtained strains carrying this cassette successfully realized the secretion of SOD1. In order to overcome the limitation of previous genetic modification on yeast strains, one new comprehensive strategy was adopted targeting the suitable homologous sites by gene deletion and SOD1 + GSH1 co-overexpression, and the new strain ST31 (Δadh2::SOD1 + Δilv2::GSH1) was constructed. The results of the pilot-scale fermentation showed that the diacetyl content of ST31 was lower by 42 % than that of the host, and the acetaldehyde content decreased by 29 %, the GSH content in the fermenting liquor of ST31 increased by 29 % compared with the host. Both SOD activity test and the positive and negative staining assay after native PAGE indicated that the secreted active SOD in the fermenting liquor of ST31 was mainly a dimer with the size of 32,500 Da. The anti-aging indexes such as the thiobarbituric acid and the resistance staling value further proved that the flavor stability of the beer brewed with strain ST31 was not only better than that of the original strain, but also better than that of the previous engineering strains. The multi-modification and comprehensive improvement of the beer yeast strain would greatly enhance beer quality than ever, and the self-cloning strain would be attractive to the public due to its bio-safety.     

Fumaric Acid Production in Saccharomyces cerevisiae by In Silico Aided Metabolic Engineering

Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L^–1 without any apparent change in growth in fed-batch culture. FT-IR and ¹H and ¹³C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L^–1 FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L^–1 FA in batch culture when the SFC1 gene encoding a succinate–fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering.     

Metabolic engineering of Bacillus amyloliquefaciens for poly-gamma-glutamic acid (γ-PGA) overproduction

We constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens strain with considerable γ-PGA production. It was carried out by double-deletion of the cwlO gene and epsA-O cluster, as well as insertion of the vgb gene in the bacteria chromosome. The final generated strain NK-PV elicited the highest production of γ-PGA (5.12 g l⁻¹), which was 63.2% higher than that of the wild-type NK-1 strain (3.14 g l⁻¹). The γ-PGA purity also improved in the NK-PV strain of 80.4% compared with 76.8% for the control. Experiments on bacterial biofilm formation experiment showed that NK-1 and NK-c (ΔcwlO) strains can form biofilm; the epsA-O deletion NK-7 and NK-PV strains could only form an incomplete biofilm.     

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicum l-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67 g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37 g/l 2-methyl-1-butanol and 2.76 g/l 3-methyl-1-butanol in defined medium within 48 h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.     

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.