DOI: 10.5061/dryad.9bv73
title: Data from: Potential application of the Oryza sativa monodehydroascorbate reductase gene (OsMDHAR) to improve the stress tolerance and fermentative capacity of Saccharomyces cerevisiae

description: Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is an important enzyme
 for ascorbate recycling. To examine whether heterologous expression of
 MDHAR from Oryza sativa (OsMDHAR) can prevent the deleterious effects of
 unfavorable growth conditions, we constructed a transgenic yeast strain
 harboring a recombinant plasmid carrying OsMDHAR (p426GPD::OsMDHAR).
 OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen
 peroxide by maintaining redox homoeostasis, proteostasis, and the
 ascorbate (AsA)-like pool following the accumulation of antioxidant
 enzymes and molecules, metabolic enzymes, and molecular chaperones and
 their cofactors, compared to wild-type (WT) cells carrying vector alone.
 The addition of exogenous AsA or its analogue isoascorbic acid increased
 the viability of WT and ara2Δ cells under oxidative stress. Furthermore,
 the survival of OsMDHAR-expressing cells was greater than that of WT cells
 when cells at mid-log growth phase were exposed to high concentrations of
 ethanol. High OsMDHAR expression also improved the fermentative capacity
 of the yeast during glucose-based batch fermentation at a standard
 cultivation temperature (30°C). The alcohol yield of OsMDHAR-expressing
 transgenic yeast during fermentation was approximately 25% (0.18 g·g-1)
 higher than that of WT yeast. Accordingly, OsMDHAR-expressing transgenic
 yeast showed prolonged survival during the environmental stresses produced
 during fermentation. These results suggest that heterologous OsMDHAR
 expression increases tolerance to reactive oxygen species-induced
 oxidative stress by improving cellular redox homeostasis and improves
 survival during fermentation, which enhances fermentative capacity.
description: Fig1Fig 1. Construction of
 an OsMDHAR-expressing yeast vector and the stress response of
 OsMDHAR-expressing yeast to hydrogen peroxide. (A) Schematic diagram of
 the p426GPD::OsMDHAR construct. The OsMDHAR gene (approximately 1.5 kbp)
 was subcloned to generate the p426GPD::OsMDHAR construct with OsMDHAR
 under the control of the constitutive GPD promoter. Semi-quantitative
 RT-PCR (B), immunoblotting (C), and an enzymatic assay (D) were performed
 to examine whether OsMDHAR is expressed in this yeast strain. PDA1 and
 tubulin (Tub) were used as housekeeping controls for RT-PCR and western
 blotting, respectively. The molecular size of the PCR product and
 molecular weight of the detected band were approximately 494 bp and 47
 kDa, respectively. Stress tolerance to hydrogen peroxide was evaluated by
 cell survival, growth kinetics, and spotting assays. (E) To monitor cell
 viability, yeast cells precultured in YPD medium were inoculated into
 fresh YPD medium and exposed to different concentrations of H2O2 for 16 h
 at 28°C. Then, the optical density at 600 nm (OD600) was measured.
 Circles, cells transformed with p426GPD-OsMDHAR (TC cells); squares,
 wild-type (WT) cells transformed with an empty vector. (F) For the growth
 kinetics assay, precultured yeast cells were inoculated into YPD medium
 containing 5 mM H2O2, and the OD600 was measured at 2-h intervals for 36
 h. A streaking assay was also performed, in which mid-log phase yeast
 cells (OD600 ≈ 2.0) were streaked onto YPD agar plates supplemented with 5
 mM H2O2. WT (squares) and TC (circles) cells in the absence of 5 mM H2O2;
 WT (upward triangles) and TC (diamonds) cells in the presence of 5 mM
 H2O2. (G) Mid-log phase yeast cells were exposed to 20 mM H2O2 for 1 h
 with shaking, and serially diluted with YPD medium. A 5-µL aliquot of each
 dilution was spotted onto YPD agar plates.Fig2Fig 2. Analyses of cell
 rescue proteins, redox state, and protein oxidation under oxidative
 conditions. (A) Expression changes in antioxidant and metabolic enzymes in
 mid-log phase yeast cells exposed to 20 mM H2O2 for 1 h with shaking.
 Tubulin (Tub) was used as a loading control. (B) Hydroperoxide levels in
 TC cells in the absence (red bar) and presence (green bar) of 20 mM H2O2
 were assessed using FOX reagent and were calculated relative to that in WT
 cells grown under normal conditions, which was set to 100%. (C) Mid-log
 phase yeast were exposed to 20 mM H2O2 for 1 h at 28°C with shaking. Redox
 state was analyzed by measuring DCFHDA oxidation as an indicator of
 cytosolic ROS. (D) Sensitivity of mutants (sod1Δ, tsa1Δ, por1Δ, and por2Δ)
 to oxidative stress. Yeast cells (OD600 ≈ 1.0) were exposed to 10 mM H2O2
 for 1 h at 28°C with shaking, serially diluted with YPD medium, spotted
 onto YPD agar plates, and incubated for 2–3 days. (E) Expression changes
 in molecular chaperones in mid-log phase yeast cells exposed to 20 mM H2O2
 for 1 h with shaking. Tubulin (Tub) was used as a loading control. (F)
 Protein carbonylation in yeast cells exposed to 20 mM H2O2 for 1 h was
 calculated relative to that in WT cells under normal conditions, which was
 set to 100%. Red bar, normal conditions; green bar, H2O2 treatment; WT,
 yeast cells with an empty vector; TC, OsMDHAR-expressing yeast cells; N,
 normal conditions; S, H2O2 treatment.Fig3Fig 3. Stress response
 related to ascorbate (AsA)-like molecules. (A) AsA-like content in yeast
 cells exposed to 20 mM H2O2 for 1 h was analyzed and is shown as nmol per
 mg protein. The ratio shown is that of the reduced form to oxidized form.
 (B) ARA2 expression was evaluated by semi-quantitative RT-PCR. PDA1 was
 used as a control. (C) Oxidative stress response of yeast cells in the
 absence and presence of ARA2. Mid-log phase cells were serially diluted,
 and 5 µL of the diluted solutions were spotted onto YPD agar plates
 containing 4 mM H2O2 (upper panels). Mid-log phase cells were treated with
 20 mM H2O2 for 1 h with shaking, diluted with YPD medium, and spotted onto
 YPD agar plates. The plates were incubated for 2–3 days and photographed.
 (D) Stress sensitivity of ara2Δ yeast cells, in which the erythroascorbate
 (EAA) biosynthesis gene was deleted. Yeast cells (A600 ≈ 1.0) were exposed
 to 10 mM H2O2 for 1 h at 28°C with shaking, serially diluted with YPD
 medium, spotted onto YPD agar plates, and incubated for 2–3 days. (E) The
 redox state of ara2Δ yeast cells under oxidative conditions. Yeast cells
 (OD600 ≈ 1.0) were exposed to 10 mM H2O2 for 1 h after DCFHDA and DHAR 123
 treatment for 30 min and washed twice with phosphate-buffered saline
 (PBS). Probe intensity was observed by fluorescence microscopy. BY,
 wild-type cells without an empty vector; ara2Δ, cells with a deletion of
 the EAA biosynthetic gene ARA2; WT, wild-type yeast cells with an empty
 vector; TC, yeast cells with p426GPD::OsMDHAR; WA, ara2Δ yeast cells with
 an empty vector; TA, ara2Δ yeast cells with p426GPD::OsMDHAR; N, normal
 conditions; S, in the presence of H2O2.Fig4Fig 4. Fermentative
 capacity and the survival of OsMDHAR-expressing yeast cells during batch
 fermentation. (A) Fermentative capacity was analyzed by measuring the
 alcohol (AC) and residual glucose (RG) concentrations in YG medium after
 fermentation for 72 h at 30°C. Upward triangles, AC of WT cells; circles,
 AC of TC cells; squares, RG of WT cells; diamonds, RG of TC cells. (B)
 Time-dependent OsMDHAR expression during batch fermentation was evaluated
 by western blotting. Tubulin (Tub) was used as a loading control. (C)
 Growth kinetics during fermentation was assessed by measuring the OD600 at
 2-h intervals for the indicated time. Squares, WT cells; circles, TC
 cells. (D) Cell viability during fermentation at 30°C was assessed by a
 spotting assay. Cells were harvested after 24 h (upper panel), 48 h
 (middle panel), and 72 h (lower panel) of fermentation and serially
 diluted to 10−9. A 5-µL aliquot of each diluted solution was spotted onto
 YPD agar plates. After incubation for 3 days, the plates were
 photographed. (E) Stress response to ethanol. Mid-log phase yeast cells
 (OD600 ≈ 2.0) were exposed to different concentrations of ethanol (0, 15%,
 and 20%) for 1 h, serially diluted, and spotted onto YPD agar plates. WT,
 yeast cells with an empty vector; TC, OsMDHAR-expressing yeast
 cells.S1_FigSI Fig. Stress
 sensitivity and redox state of por1△ yeast cells under oxidative stress.
 (A) Growth kinetics was observed in YPD medium containing 3.5 mM H2O2 by
 monitoring optical density at 600 nm at 2-h intervals for 32 h. Square, BY
 cells; circle, por1Δ cells. (B) Cell survival by streaking (upper panel)
 and spotting (lower panel) assays in both BY and por1Δ cells. For
 streaking assay, yeast cells were cultured until reaching early log-phase
 (A600 ≈ 1.0) and streaked onto YPD agar plates supplemented with 3.5 mM
 H2O2. For spotting assay, yeast cells (A600 ≈ 1.0) were exposed to 10 mM
 H2O2 for 1 h at 28ºC with shaking, serially diluted to 10−4 with YPD
 medium, and spotted onto YPD agar plates. (C) Cytosolic and mitochondrial
 redox states were analyzed using the indicator probes for DCFHDA and DHR
 123, respectively. Yeast cells were pretreated with the probes for 20 min,
 washed twice with PBS, and treated with 10 mM H2O2 for 1 h with shaking.
 Probe intensity was visualized by fluorescence microscopy. (D) Stress
 sensitivity of por1Δ yeast cells to physiochemical stressors. Yeast cells
 (A600 ≈ 1.0) were streaked onto YPD agar plates containing various
 stressors as mentioned in Materials and Methods. BY, wild-type yeast cells
 without an empty vector; por1Δ, yeast cells in which the POR1 gene had
 been deleted.S2_FigS2 Fig. Exogenous effect
 of ascorbate and its analogue on the stress sensitivity of ara2Δ yeast
 cells. (A) Monitoring of the effect was performed by spotting assay. Yeast
 cells pretreated with 10 mM AsA and 10 mM IAA for 1 h were exposed to 20
 mM H2O2 for 1 h. Five microliters of diluted solutions were spotted onto
 YPD agar plates. Exogenous AsA- and IAA-treated cells showed better
 survival compared to cells under normal conditions, suggesting that their
 concentrations were sufficient for detoxifying endogenous and exogenous
 ROS and enhanced cell proliferation. WT, yeast cells transformed an empty
 vector; TC, OsMDHAR-expressing yeast cells. (B) Stress sensitivity of
 ara2Δ yeast cells to oxidative stress. Yeast cells were treated with
 abiotic stressors for 1 h with shaking and spotted onto YPD agar plates.
 For heat shock, yeast cells were incubated for 5 min at 55ºC, and then
 spotted as described above. BY, wild-type yeast cells without an empty
 vector; ara2Δ, yeast cells in which the ARA2 gene had been
 deleted.S3_FigS3 Fig. Response of
 OsMDHAR-expressing yeast cells to abiotic stressors. Yeast cells (A600 ≈
 2.0) were challenged with various stressors, including 0.4 mM MD, 15 mM
 t-BOOH, 20 mM CuSO4, 40 mM FeCl2, 10 mM CdCl2, and 1% SDS, for 1 h at 28ºC
 with shaking. Stressed cells were serially diluted to 10−4 with YPD
 medium, spotted onto YPD agar plates, and then incubated for 3 days at
 28ºC. WT, yeast cells transformed an empty vector; TC, OsMDHAR-expressing
 yeast cells.S1
 TableS1 Table.
 Oligonucleotide sequence used in this study.S2
 TableS2 Table. Strains and
 plasmids used in this study.