Potential Application of the Oryza sativa Monodehydroascorbate Reductase Gene (OsMDHAR) to Improve the Stress Tolerance and Fermentative Capacity of Saccharomyces cerevisiae

Kim, Il-Sup; Kim, Young-Saeng; Kim, Yul-Ho; Park, Ae Kyung; Kim, Han-Woo; Lee, Jun-Hyuck; Yoon, Ho-Sung

doi:10.1371/journal.pone.0158841

Cited by 12 publications

(8 citation statements)

References 67 publications

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“…cDNA containing the open reading frame (ORF) of OsMDHAR and OsDHAR was subcloned into the yeast expression vector p426GPD (yeast 2 μ expression vector with URA3 marker; GenBank Accession No. DQ019861), which allows constitutive expression of a target gene under the control of the yeast GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter 36 . To measure cell viability, cells (1 × 10 6 cells per mL) grown at 28 °C overnight were inoculated in YPD medium (1% yeast extract, 2% peptone and 2% dextrose) and cultured for 8 h. Mid-log phase cells (OD 600 ≈ 3.0) were exposed to 20 mM H 2 O 2 for 1 h at 28 °C and serially diluted (10 0 to 10 −4 ) with YPD medium.…”

Section: Methodsmentioning

confidence: 99%

Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR, from Oryza sativa L. japonica

Park

Kim

et al. 2016

Sci Rep

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Ascorbic acid (AsA) maintains redox homeostasis by scavenging reactive oxygen species from prokaryotes to eukaryotes, especially plants. The enzyme monodehydroascorbate reductase (MDHAR) regenerates AsA by catalysing the reduction of monodehydroascorbate, using NADH or NADPH as an electron donor. The detailed recycling mechanism of MDHAR remains unclear due to lack of structural information. Here, we present the crystal structures of MDHAR in the presence of cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), and complexed with AsA as well as its analogue, isoascorbic acid (ISD). The overall structure of MDHAR is similar to other iron-sulphur protein reductases, except for a unique long loop of 63–80 residues, which seems to be essential in forming the active site pocket. From the structural analysis and structure-guided point mutations, we found that the Arg320 residue plays a major substrate binding role, and the Tyr349 residue mediates electron transfer from NAD(P)H to bound substrate via FAD. The enzymatic activity of MDHAR favours NADH as an electron donor over NADPH. Our results show, for the first time, structural insights into this preference. The MDHAR-ISD complex structure revealed an alternative binding conformation of ISD, compared with the MDHAR-AsA complex. This implies a broad substrate (antioxidant) specificity and resulting greater protective ability of MDHAR.

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Section: Methodsmentioning

confidence: 99%

Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR, from Oryza sativa L. japonica

Park

Kim

et al. 2016

Sci Rep

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“…The specific properties of yeast can be altered via genetic improvement through numerous approaches, including sexual breeding, parasexual hybridization, random mutagenesis, metabolically genetic engineering such as genome editing, and transformation using recombinant genes [37][38][39]. Using transgenic biotechnology, yeast is engineered to express foreign and exogenous genes to generate products of industrial interest, such as bioethanol-based biofuel and secondary metabolites [37,40,41]. Owing to their academic and industrial interests, studies on stress tolerance in yeast are of fundamental scientific importance [42].…”

Section: Introductionmentioning

confidence: 99%

Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Kim

Choi

Son

et al. 2021

Genes

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The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

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“…This carrying capacity also reflects the ability of yeast cells to respond to oxidative damage by producing viable offspring or survivors, which sometimes is called stress tolerance assays. These studies represent the capacity of the cells to counteract and resist the oxidative damage, representing cell survival [ 30 , 31 , 36 , 38 , 39 , 40 , 41 , 42 ]. Even showing statistical differences between applied doses, and being significantly different from completely stressed yeasts (OxDinOxD) and unstressed ones (YPD), our results indicate that tested compounds enhance biological functions enabling the complete recovery of cell biochemistry and metabolism Indeed, tested compounds counteract the oxidative damage induced by the used H 2 O 2 medium and increase significantly the oxidative protection rate when compared to resveratrol ( Figure 5 ).…”

Section: Resultsmentioning

confidence: 99%

Characterization of Novel Synthetic Polyphenols: Validation of Antioxidant and Vasculoprotective Activities

et al. 2020

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Antioxidant compounds, including polyphenols, have therapeutic effects because of their anti-inflammatory, antihypertensive, antithrombotic and antiproliferative properties. They play important roles in protecting the cardiovascular and neurological systems, by having preventive or protective effects against free radicals produced by either normal or pathological metabolism in such systems. For instance, resveratrol, a well-known potent antioxidant, has a counteracting effect on the excess of reactive oxygen species (ROS) and has a number of therapeutic benefits, like anti-inflammatory, anti-cancer and cardioprotective activities. Based on previous work from our group, and on the most frequent OH substitutions of natural polyphenols, we designed two series of synthetically accessible bis-polyhydroxyphenyl derivatives, separated by amide or urea linkers. These compounds exhibit high antioxidant ability (oxygen radical absorbance capacity (ORAC) assay) and interesting radical scavenging activity (RSA) values (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and α,α-diphenyl-β-picrylhydrazyl (DPPH) tests). Some of the best polyphenols were evaluated in two biological systems, endothelial cells (in vitro) and whole aorta (ex vivo), highly susceptible for the deleterious effects of prooxidants under different inflammatory conditions, showing protection against oxidative stress induced by inflammatory stimuli relevant in cardiovascular diseases, i.e., Angiotensin II and IL-1β. Selected compounds also showed strong in vivo antioxidant properties when evaluated in the model organism Saccharomyces cerevisiae.

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Potential Application of the Oryza sativa Monodehydroascorbate Reductase Gene (OsMDHAR) to Improve the Stress Tolerance and Fermentative Capacity of Saccharomyces cerevisiae

Cited by 12 publications

References 67 publications

Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR, from Oryza sativa L. japonica

Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR, from Oryza sativa L. japonica

Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Characterization of Novel Synthetic Polyphenols: Validation of Antioxidant and Vasculoprotective Activities

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