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ABSTRACT: The MuS1 gene is highly homologous to many stress-related proteins in plants. Here, we characterized whether a new candidate gene, MuS1, is related to multiple stress tolerance in yeast as it is in plants. Transgenic yeast strain expressing MuS1 were more resistant to hydrogen peroxide, menadione, high salinity, metals (i.e., cadmium, copper, iron, and zinc), ethanol, and lactic acid than wild-type strain transformed with a vector alone. In addition, the alcohol yield of the transgenic yeast strain was higher than that of the wild-type strain during the batch fermentation process. These results show that MuS1-expressing transgenic yeast strain exhibits enhanced alcohol yield as well as tolerance to abiotic stresses, especially metal stress.
The Journal of Microbiology 06/2012; 50(3):544-6. · 1.10 Impact Factor
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ABSTRACT: To determine whether the exogenous expression of glutathione reductase (GR) from Brassica rapa subsp. pekinensis (BrGR) can reduce the deleterious effects of unfavorable conditions, we constructed a transgenic Saccharomyces cerevisiae strain bearing the GR gene cloned into the yeast expression vector, pVTU260. BrGR expression was confirmed by semi reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, immunoblotting analysis and an enzyme assay. Ectopic BrGR-expression improved cellular glutathione (GSH) homeostasis after higher GSH accumulation in the transgenic yeast than in the wild-type yeast under H(2)O(2)-induced oxidative stress. The BrGR-expressing yeast strain induced the activation of metabolic enzymes (Hxt, G6PDH, GAPDH and Ald), antioxidant systems (Gpx, Trx2, Trx3, Trr1, Tsa1 and porin) and molecular chaperones (Hsp104, Hsp90, Hsp70, Hsp42, Hsp26, Grp, Sti1 and Zpr1), which led to lower oxidative protein damage after a reduction in the level of cellular ROS in the BrGR-expressing yeast strain exposed to H(2)O(2) than in the wild-type yeast strain. BrGR-expression increased the ability to adapt and recover from H(2)O(2)-induced oxidative stress and various stressors, including heat shock, menadione, tert-butyl hydroperoxide, heavy metals, sodium dodecyl sulfate, ethanol and NaCl, but did not affect fermentation capacity. These results suggest that ectopic BrGR expression confers acquired tolerance by improving proteostasis and redox homeostasis through co-activation of various cell rescue proteins against ROS-induced oxidative stress in yeast cells.
MIRCEN Journal of Applied Microbiology and Biotechnology 05/2012; 28(5):1901-15. · 1.08 Impact Factor
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ABSTRACT: Cyclophilins contain the conserved activity of cis-trans peptidyl-prolyl isomerase that is implicated in protein folding and function as molecular chaperones. The yeast cyclophilin A gene (cpr1) was subcloned to the prokaryotic expression vector pKM260. It was found that the expression of Cpr1 drastically increased the cell viability of E. coli BL21 in the presence of abiotic stress conditions, such as cadmium, copper, hydrogen peroxide, heat, and SDS. Thus, this study illustrates the importance of Cpr1 as a molecular chaperone that improved cellular stress responses when E. coli cells were exposed to adverse conditions, and it also shows the possibility of increasing the stability of E. coli strains utilized for the production of recombinant proteins.
Journal of Microbiology and Biotechnology 06/2010; 20(6):974-7. · 1.38 Impact Factor
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ABSTRACT: Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. We found the expression of cyclophilin A, Cpr1, changes in response to exposure to yeast Saccharomyces cerevisiae to abiotic stress conditions. The effect of Cpr1 overexpression in stress responses was therefore examined. The CPR1 gene was cloned to the yeast expression vector pVTU260 under regulation of an endogenous alcohol dehydrogenase (ADH) promoter. The overexpression of Cpr1 drastically increased cell viability of yeast in the presence of stress inducers, such as cadmium, cobalt, copper, hydrogen peroxide, tert-butyl hydroperoxide (t-BOOH), and sodium dodecyl sulfate (SDS). The Cpr1 expression also enhanced the cell rescue program resulting in a variety of antioxidant enzymes including thioredoxin system (particularly, thioredoxin peroxidase), metabolic enzymes (glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase), and molecular chaperones (Hsp104, Hsp90, Hsp60 and Hsp42). Thus, our study illustrates the importance of Cpr1 as a molecular chaperone that improves cellular stress responses through collaborative relationships with other proteins when yeast cells are exposed to adverse conditions, and it also premises the improvement of yeast strains.
Molecules and Cells 05/2010; 29(6):567-74. · 2.18 Impact Factor
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ABSTRACT: Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H(2)O(2), menadione, and heavy metal (CdCl(2), ZnCl(2) and AlCl(2))-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H(2)O(2) stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.
Molecules and Cells 11/2009; 28(5):479-87. · 2.18 Impact Factor
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ABSTRACT: Maintaining redox balance is one of the crucial requirements for a cell to endure stress from the outside. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) plays an important role in the ascorbate-glutathione cycle; one of the major ROS scavenging systems in most known biological systems. A cDNA clone of the DHAR gene from Oryza sativa (OsDHAR) was isolated and overexpressed in Escherichia coli BL21 (DE3) strain from the pET-28a(+) expression vector. The OsDHAR transformed E. coli cells showed significantly higher DHAR activity and a lower level of ROS than the E. coli cells transformed by an empty pET-28a(+) vector. Also, the DHAR-overexpressing E. coli strain was more tolerant to oxidant- and heavy metal-mediated stress conditions than the control E. coli strain. The results suggest that the overexpressed rice DHAR gene effectively functions in a prokaryotic system and provide protection to various oxidative stresses.
Molecules and Cells 12/2008; 26(6):616-20. · 2.18 Impact Factor
