Enzymatic improvement of mitochondrial thiol oxidase Erv1 for oxidized glutathione fermentation by Saccharomyces cerevisiae
© The Author(s) 2017
Received: 20 December 2016
Accepted: 7 March 2017
Published: 15 March 2017
Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes.
We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene.
This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.
KeywordsGlutathione Saccharomyces cerevisiae Thiol oxidase Mia40 Erv1
Glutathione is the most abundant thiol-containing tripeptide in all living organisms . Glutathione is widely used in the medical, food, and cosmetic industries [2, 3] due to its various physiological functions such as acting as an antioxidant, a detoxifier of xenobiotics, and an immune booster [4–9]. Thus the demand for glutathione has increased in recent years. Glutathione is industrially produced mainly by fermentation using Saccharomyces cerevisiae, which contains a high concentration of glutathione and is accepted as a food-producing microorganism.
Glutathione is biologically synthesized by γ-glutamylcysteine (γ-GC) synthetase (GCS, EC 184.108.40.206) encoded by GSH1 and by glutathione synthetase (GS, EC 220.127.116.11) encoded by GSH2 from three precursor amino acids. GCS catalyzes the reaction to form γ-GC from l-glutamic acid and l-cysteine. GS catalyzes the reaction to form glutathione from γ-GC and glycine. Other glutathione-related enzymes include thiol oxidase (TO, EC 18.104.22.168) encoded by ERV1, ERV2, and ERO1, and glutathione-disulfide reductase (GR, EC 22.214.171.124) encoded by GLR1. TOs catalyze the reaction to form oxidized glutathione (GSSG) from reduced glutathione (GSH) by oxidizing the thiols, and GR catalyzes the reaction to form GSH from GSSG by reducing a disulfide bond.
In many cases, reduced glutathione (GSH) primarily exists to respond to oxidative stress in living organisms , and thus many glutathione-related studies have focused on GSH [11–13]. However, GSSG is of interest in glutathione production. In industrial mass production of glutathione, GSSG is preferable due to its higher stability. Furthermore, enhancements of GSSG production help to avoid a negative feedback regulation by GSH1, and consequently increase total glutathione production by fermentation with S. cerevisiae [14, 15]. GSSG also has advantages in utilization; it has the same extent of antioxidant activity in the intestines after dietary intake as GSH , and promotes plant growth more potently than GSH [17, 18].
In our previous study, deletion of the GLR1 gene and over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for generating high GSSG production among three types of different thiol oxidase genes in S. cerevisiae cells . Therefore, in this study, we improved the enzymatic activity of Erv1 for GSH oxidation by amino acid replacements, and consequently enhanced GSSG production in S. cerevisiae by using mutant ERV1 genes.
Strains, media, and primers
Saccharomyces cerevisiae GCIΔGLR1, GSH1/GSH2 cocktail δ-integrated and GLR1 deleted YPH499 (ABC1193/NBRC 10505) strain was previously constructed  and used for glutathione production in this study. Saccharomyces cerevisiae GCIΔGLR1 derivative strains were aerobically grown at 30 °C in yeast extract-peptonedextrose (YPD) medium (10 g l−1 yeast extract, 20 g l−1 bacto-peptone, and 20 g l−1 glucose) supplemented with 0.5 mg l−1 aureobasidin A (Aba). Escherichia coli NovaBlue (Novagen, Darmstadt, Germany) strain was used for DNA manipulation. E. coli Rosetta™(DE3)pLysS (Novagen) strain was used to produce recombinant proteins. E. coli strains were aerobically grown at 37 °C in Luria–Bertani (LB) medium (10 g l−1 tryptone, 5 g l−1 yeast extract, and 5 g l−1 sodium chloride). Ampicillin (Amp; 50 mg l−1) and chloramphenicol (Cm; 50 mg l−1) were added as necessary. The primer sequences used in this study are summarized in Additional file 1: Table S1.
Construction of plasmids
The ERV1 gene was amplified by polymerase chain reaction (PCR) from complementary DNA (cDNA) of S. cerevisiae YPH499 using primers ERV1F1 and ERV1R1. The cDNA was prepared by reverse transcription PCR using a PrimeScrip RT-PCR Kit (Takara Bio, Otsu, Japan) from total RNA extracted from S. cerevisiae YPH499 cells using NucleoSpin RNA (Takara Bio). The PCR product was cloned between SphI and BamHI sites of pUC19 (Takara Bio). After the sequence was checked, the ERV1 gene was subcloned between NdeI and XhoI sites of pET-22b (Novagen) to give pET-ERV1. pET-ERV1 was used for Erv1 protein preparation. The plasmids for preparation of Erv1 variant proteins were constructed by inverse PCR using corresponding primer pairs and templates (Additional file 1: Table S1). For expression of ERV1 and its mutant genes in S. cerevisiae cells, the ERV1 gene was amplified by PCR from pET-ERV1 using primers ERV1F2 and ERV1R2. The PCR product was cloned between NheI and BamHI sites of pGK406 designed for target gene expression in S. cerevisiae . The mutant genes coding Erv1 variants were also amplified by the same primer pairs and cloned into pGK406.
Plasmid introduction into S. cerevisiae
Derivatives of pGK406 were introduced into S. cerevisiae cells using the lithium acetate method [21, 22]. Transformants were selected by uracil auxotrophy. Target gene insertion into the genomic DNA of each transformant was confirmed by PCR using the appropriate primer sets.
Preparation of recombinant proteins
The E. coli Rosetta™(DE3)pLysS strain harboring pET-ERV1 was aerobically grown in 5 ml of liquid LB medium supplemented with Amp and Cm at 37 °C for 18 h. The 1 ml of grown cells was inoculated into 100 ml of liquid LB medium supplemented with Amp, Cm, and 1% lactose, and aerobically grown at 20 °C for 48 h. The grown cells were then pelleted by centrifugation (16,000×g, 10 min) and resuspended in 20 mM potassium phosphate buffer (pH 7.0) containing 500 mM NaCl. The cell suspension was sonicated and centrifuged (16,000×g, 10 min) to remove cell debris. The His-tagged Erv1 protein in the supernatant was purified by TALON® Metal Affinity Resin (Takara Bio). The Erv1 variants were also prepared by the same method.
The activities of recombinant Erv1 and its variants were determined by measuring the initial velocity of product formation. The assay mixture containing 50 mM potassium phosphate buffer (pH 7.0), 10 mM GSH or γ-GC, and the purified recombinant protein was incubated at 30 °C for 10 min. The reaction was then stopped by adding an equal volume of 20% (w/v) trichloroacetic acid. The formed products were measured using high performance liquid chromatography (HPLC) as mentioned below. The protein concentrations were assayed by the Bradford method. All assays were separately performed three times.
The S. cerevisiae GCIΔGLR1/ERV1 strain was streaked and grown on YPD solid media with Aba at 30 °C for 72 h. A single grown colony was inoculated into 5 ml of YPD liquid medium with Aba and aerobically grown at 30 °C for 18 h. The grown cells were inoculated into 20 ml of the same medium. The initial cell density (OD600) was 0.03, and cells were grown in a 200 ml baffled Erlenmeyer flask at 30 °C with agitation at 150 rpm for up to 48 h. The other mutant strains were grown by the same method. Intracellular GSH and GSSG were analyzed by HPLC as mentioned below. All fermentations for glutathione production were separately performed three times.
To determine the cell concentration, the OD600 of the culture sample was measured using an UVmini-1240 Spectrometer (Shimadzu, Kyoto, Japan). Samples were prepared according to a previous report . GSH and GSSG concentrations were determined by HPLC (Shimadzu) equipped with the YMC-Pack ODS-A column (YMC, Kyoto, Japan). The operating condition was 30 °C, with 50 mM potassium dihydrogen phosphate buffer (pH 2.8) and 10 mM sodium 1-heptanesulfonate as the mobile phase at a flow rate of 1.0 ml min−1, and detection was performed with a UV detector SPD-20A (Shimadzu) at 210 nm. Intracellular (reduced, oxidized, and total) glutathione content was calculated using the volumetric glutathione concentration (g l−1) divided by cell concentration (OD600 × 0.3) (g l−1), represented as % values (w/w).
Protein structures and intramolecular interactions were simulated using PyMol software (https://www.pymol.org/). The structural data of the Erv1 variant protein of S. cerevisiae (PDB ID: 4E0I) was retrieved from RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) and used for constructions of Erv1 and its variants structures.
Catalytic activity of Erv1 and its variants
To obtain effective Erv1 variants, we further prepared Erv1S32A/N34A and Erv1S32T/N34A, and assayed their specific activities. Erv1S32A/N34A showed the highest relative activity for GSH (240%) among all variants, and showed comparable activity for γ-GC (96%). On the other hand, Erv1S32T/N34A showed almost the same activity for GSH (192%) compared to Erv1S32A, Erv1S32T, and Erv1N34A, and high activity for γ-GC (161%).
Thermal and kinetic profiles of Erv1 and its variants
Kinetic and thermal profiles of Erv1 and its variants
Maximum temperature (°C)a
T 1/2 (°C)b
K m (mM)c
V max (mU)c
K cat (min−1)c
The kinetic profiles of Erv1 and its variants were also analyzed. All variants exhibited higher K m , V max , and K cat values than the parental enzyme (Table 1). The Erv1S32A and Erv1S32T containing only S32 replacement showed about 2.7- to 3.0- and 2.1- to 2.6-fold higher K m and V max values than those of the parental Erv1, respectively (Table 1). Furthermore, Erv1N34A, Erv1S32A/N34A and Erv1S32T/N34A containing N34A replacement showed about 7.6- to 9.4- and 4.3- to 7.6-fold higher K m and V max values than those of the parental enzyme, respectively (Table 1). These differences in thermal and kinetic profiles between variants containing S32 and/or N34 replacements suggest that effect of the N34A replacement to the Erv1 profile was greater than the S32 replacements.
Glutathione production by recombinant S. cerevisiae strains
Growth and volumetric glutathione production of ERV1- and mutant-expressing strains
Cell growth (g-cell l−1)
GSH (mg l−1)a
GSSG (mg l−1)a
Total glutathione (mg l−1)a
1.87 ± 0.07 (100)
38.7 ± 4.0 (100)
47.1 ± 1.0 (100)
85.8 ± 4.7 (100)
1.77 ± 0.03 (95)
32.3 ± 0.6 (83)
63.4 ± 2.7 (135)**
95.8 ± 3.3 (112)*
1.61 ± 0.04 (86)*
20.3 ± 1.1 (52)**
85.2 ± 2.7 (181)**
105.5 ± 5.9 (123)**
1.70 ± 0.11 (91)
28.3 ± 2.6 (73)*
70.5 ± 8.4 (150)**
98.8 ± 10.9 (115)
1.64 ± 0.14 (88)
26.3 ± 2.8 (68)*
72.3 ± 7.1 (154)**
98.7 ± 9.9 (115)
1.83 ± 0.13 (98)
35.5 ± 3.8 (92)
60.6 ± 1.3 (129)**
96.2 ± 4.5 (112)*
1.71 ± 0.05 (91)*
29.3 ± 2.6 (76)*
76.4 ± 2.9 (162)**
105.7 ± 5.2 (123)**
The kinetic profiles of Erv1 and its variants provided interesting insights into mechanisms of GSH oxidation by Erv1 variants (Table 1). Generally, increasing of K m value indicates decreasing of substrate binding affinities. However, V max values of Erv1 variants were also increased, and the over-expressions of mutant genes coding these Erv1 variants practically increased GSSG production in S. cerevisiae cells. These facts imply that Erv1 variants became hard to be binding to GSH compared with parental Erv1, due to instabilizations around the catalytic center by amino acid replacements. However, rates of GSH oxidations and/or GSSG dissociations from Erv1 variants became fast by decreasing of inhibitory effects as mentioned above.
The over-expression of mutant ERV1 genes also increased GSSG and total glutathione production in S. cerevisiae cells as expected (Fig. 4b, c). These strains also showed higher volumetric GSSG and total glutathione production, whereas decreases in growth were observed in these recombinant strains (Table 2). In Table 2, the relationship between cell growth and GSH production is seemed to be proportional. This relationship is probably caused by reduced redox potentials of GSH in engineered strains. Generally, GSH works as a redox and antidotal agent in cells and therefore is an essential for various biological activities in all organisms . Indeed, GSH1 or GSH2 deleted mutant S. cerevisiae strains lost their growth abilities in growth media without GSH , and genetically engineered S. cerevisiae strains that produce high concentration of GSH exhibited significant tolerance against diverse stresses such as high temperatures and presence of toxic agents [30, 31].
The in vivo GSSG production of the recombinant strains expressing ERV1 and its mutant genes was not necessarily reflected by in vitro enzyme activities (Figs. 1a, 4b). Generally, thiol oxidase oxidizes various substrates that are not only small molecules [27, 28], but also macromolecules such as mitochondrial intermembrane space (MIMS) proteins including Mia40, Cox19, and so on [24, 25]. Therefore, the Erv1 variants, especially Erv1S32A/N34A, may oxidize unexpected substrates and form disulfide bonds between GSH and thiols in other substrates in S. cerevisiae cells, due to a change in substrate specificity by the amino acid replacements. Indeed, the existence of unproductive oxidized MIMS proteins  and a wide variety of protein-glutathione adducts in yeast cells  have been reported in previous studies. Generally, Mia40 oxidizes thiols in proteins, and is consequently oxidized by Erv1 in the MIMS . In this study, Erv1 protein was successfully improved for oxidation of GSH, and over-expression of its coding genes meaningfully increased GSSG production in S. cerevisiae cells. These results suggest that the Erv1 protein directly oxidized GSH in S. cerevisiae cells. The suppression of GSSG production by the simultaneous over-expression of ERV1 and MIA40 genes in our previous study  and the much lower oxidation activity of Mia40 for GSH  also support this idea.
GSSG and total glutathione production of S. cerevisiae were increased by over-expressing improved ERV1 genes. However, glutathione has been produced by industrial fermentation methods using high glutathione producing strains such as Candida utilis and S. cerevisiae, and their glutathione productions have been improved by screening from randomly mutated strains and metabolic engineering. These improved strains produce much higher amount of glutathione (e.g., S. cerevisiae K-2 strain produces 2700 mg l−1 glutathione for 24 h) than the host strain used in this study . However, in many cases for improving glutathione production by metabolic engineering, genes involved in synthesis of GSH and its precursors, such as GSH1, PRO1, and CYS3 are frequently focused [19, 30, 33]. On contrast, ERV1 has never been applied for industrial glutathione production, and therefore Erv1 has even greater potential for industrial GSSG production when ERV1 and its mutant genes were overexpressed in the industrial glutathione producing S. cerevisiae host strain.
In this study, mitochondrial thiol oxidase Erv1 was applied for improvement of GSSG production by S. cerevisiae, and its enzyme activity for GSH oxidation was improved for the first time by site-directed mutations. The critical roles of S32 and N34 residues for GSH oxidation and protein stability were revealed with possible reasons. Five engineered Erv1 variants containing S32 and/or N34 replacements exhibited about 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of the parental Erv1. The over-expression of mutant ERV1 genes coding these variants also demonstrated in vivo validity, showing 1.5-fold higher GSSG production than that of the strain over-expressing ERV1 gene. This study indicates potential of Erv1 for high GSSG production by S. cerevisiae.
- Aba A:
complementary deoxyribonucleic acid
high performance liquid chromatography
mitochondrial intermembrane space
polymerase chain reaction
JK performed all experiments and wrote the manuscript. KYH planned the study. DS and KYH revised the manuscript. DS, KYH, TH and AK supervised the study. All authors read and approved the final manuscript.
We are grateful to Dr. J. Ishii (Organization of Technology and Innovation, Kobe University) for providing us with the pGK plasmid series.
The authors declare that they have no competing interests.
Availability of data and materials
The figures and tables supporting the conclusions of this study are included in this article. The primer sequences are listed in the Additional file 1.
Consent for publication
All authors consented to publication this paper.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
This work was supported by a Special Coordination Fund for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction, Kobe) from the Ministry of Education, Culture, Sports, and Science and Technology (MEXT), Japan.
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