Gene of the transcriptional activator MET4 is involved in regulation of glutathione biosynthesis in the methylotrophic yeast Ogataea (Hansenula) polymorpha

Gene of the transcriptional activator MET4 is involved in regulation of glutathione biosynthesis... Abstract Glutathione is the most abundant cellular thiol and the low molecular weight peptide present in cells. The methylotrophic yeast Ogataea (Hansenula) polymorpha is considered as a promising cell factory for the synthesis of glutathione. In this study, a competitive O. polymorpha glutathione producer was constructed by overexpression of the GSH2 gene, encoding γ-glutamylcysteine synthetase, the first enzyme involved in glutathione biosynthesis, and the MET4 gene coding for central regulator of sulfur metabolism. Overexpression of MET4 gene in the background of overexpressed GSH2 gene resulted in 5-fold increased glutathione production during shake flask cultivation as compared to the wild-type strain, reaching 2167 mg L−1. During bioreactor cultivation, glutathione accumulation by obtained recombinant strain was 5-fold increased relative to that by the parental strain with overexpressed only GSH2 gene, on the first 25 h of batch cultivation in mineral medium. Obtained results suggest involvement of Met4 transcriptional activator in regulation of GSH synthesis in the methylotrophic yeast O. polymorpha. glutathione, Ogataea (Hansenula) polymorpha, glutathione overproducers, MET4 gene INTRODUCTION Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is an abundant tripeptide that is present in most living organisms, predominantly, in eukaryotic cells, at concentrations between 0.2 and 10 mM. This small molecule is known as an important cellular redox buffer for maintaining the reducing thiol-disulfide balance. Concentration of the reduced form of glutathione (designated as GSH) in the cell is efficiently maintained by NADPH-dependent glutathione reductase (Spector, Labarre and Toledano 2001). Glutathione is an important detoxifier, could be used as a source of nitrogen and sulfur nutrition, cofactor of numerous enzymes and helper in protein folding (Orumets et al.2012). It is involved in DNA synthesis and amino acid transport and is therefore an important metabolite for the growth of eukaryotic cells. It is also crucial for stress responses caused by nutrient starvation, xenobiotics, heavy metals and reactive oxygen species (Grabek-Lejko et al.2011). Most of the above listed functions of glutathione are related to its antioxidative property caused by the thiol group of the cysteine moiety. More than 90% of microbial, plant and mammalian cell glutathione is present in the reduced form while the oxidized form (designated as GSSG) only accounts for 10%. Both forms are located in the cytoplasm as well as in the mitochondrial membrane. Glutathione deficiency in humans can be associated with several medical disorders caused by oxidative stress, poisoning or compromised immune system. These disorders lead to such diseases as cancer, cataracts, liver cirrhosis, neurodegenerative diseases, gastrointestinal inflammations and hemolytic anemia. Both GSH and GSSG are widely used in medical, cosmetic and food industries as an active ingredient of drugs, food and cosmetic products to alleviate dangerous oxidative processes and scavenge toxic compounds. It is also used to strengthen skin whitening and repair due to its antiaging effect and as support medication in cancer therapies (Townsend, Tew and Tapiero 2003; Bachhawat et al.2009). GSSG could be used as cryoprotector and immunomodulator (Chatterjee, Lamirande and Gagnon 2001; Kozhemyakin and Balasovski 2008). The estimated global annual production of pure crystalline glutathione and glutathione-enriched yeast extract (15% GSH) exceeds 200 and 800 tons, respectively, sold at a price of 300 and 150 USD/kg (Marz 2014). This highlights the great commercial importance of glutathione produced mainly by yeasts. In Saccharomyces cerevisiae and other glutathione-containing organisms, glutathione is synthesized in two consecutive ATP-dependent reactions, catalyzed by γ-glutamylcysteine synthetase, (EC 6.3.2.2, GCS, gene GSH1) and glutathione synthetase (EC 6.3.2.3, GS, gene GSH2), respectively. However, in O. polymorpha a homolog of the S. cerevisiae GSH1 gene coding for GCS was named GSH2 (Ubiyvovk et al.2002). The activity of the first reaction of the pathway, GCS, is feedback-inhibited by GSH (but not GSSG) to avoid overaccumulation of intracellular glutathione (Li et al.2004). Glutathione could be industrially produced enzymatically in the presence of ATP and three precursor amino acids (i.e. L-glutamic acid, L-cysteine, L-glycine). However, microbial fermentation is the most common approach to produce glutathione at industrial scale. In these processes, bacteria Escherichia coli and Lactococcus lactis or yeasts S. cerevisiae and Candida utilis are used as producing organisms (Li et al.2004). In the first attempt to improve glutathione biosynthesis in S. cerevisiae, the strains with overexpression of GSH1 and GSH2 genes were used. Both metabolic and evolutionary engineering approaches, together with medium optimization were used to increase glutathione synthesis (Lorenz, Schmacht and Senz 2016; Wang et al.2015). During the last decades, the methylotrophic yeast Ogataea (Hansenula) polymorpha has gained increasing interest both for basic research and biotechnological applications, such as characterizing the mechanisms of thermotolerance, peroxisome homeostasis, production of numerous heterologous proteins and high-temperature alcoholic fermentation (Dmytruk et al.2016, 2017; Ubiyvovk et al.2011a). The industrial significance of O. polymorpha relies on several technologically interesting features such as the ability to grow at high cell densities in bioreactors, capacity to use methanol as sole carbon source, the availability of strong both regulated and constitutive promoters allowing to reach high product yields. Similarly to S. cerevisiae, O. polymorpha is characterized by simple cultivation mode in inexpensive growth media, well-established genetic tools and experience on industrial cultivation. Additionally, availability of the complete genome sequence, established proteome and transcriptome databases render O. polymorpha as a suitable organism for metabolic engineering to modify and improve particular biosynthetic pathways (Kim et al.2013; Riley et al.2016). This yeast species is highly resistant to different stress conditions, induced by heavy metals, xenobiotics and different pollutants. The methylotrophic yeast O. polymorpha is considered to be a rich source of glutathione, due to the role of this tripeptide in detoxifications of key intermediates of methanol metabolism, formaldehyde, as well as hydrogen peroxide and alkyl hydroperoxides, accumulated during methylotrophic growth (Hartner and Glieder 2006). Design and construction of novel glutathione producers are of great importance for improvement of biotechnological production of this biotechnologically and medically important tripeptide. Here, we report on the construction of an O. polymorpha strain with improved glutathione productivity by means of constitutive overexpression of the MET4 gene encoding the central regulator of sulfur metabolism in yeasts (Thomas, Jacquemin and Surdin-Kerjan 1992; Ubiyvovk et al.2011a) using recipient strain with overexpressed gene GSH2 coding GCS, the first reaction of glutathione synthesis. Production of glutathione in the resulting strain was 5-fold increased relative to that of the parental strain after first 25 h of batch bioreactor cultivation. MATERIALS AND METHODS Microorganisms and cultivation conditions In this study, the following O. polymorpha strains were used: the DL-1 leu2—wild-type strain (WT); the mcGSH2—strain, overexpressing GSH2 gene under control of native promoter due to multicopy integration (Ubiyvovk et al.2011b); the mcGSH2/MET4(pGAP)—strains, overexpressing, respectively, the GSH2 gene under control of native promoter due to multicopy integration and the MET4 gene under control of strong constitutive promoter of GAP1 gene coding for glyceraldehyde-3-phosphate dehydrogenase (pGAP). The E. coli DH5α strain (Φ80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(rK−, mK+), supE44, relA1, deoR, Δ(lacZYA-argF)U169) was used as a host for plasmid propagation as previously described (Sambrook et al.1989). Transformed E. coli cells were maintained on LB medium supplemented with 0.1 g L−1 of ampicillin. Yeast cells were grown on YPD (10 g L−1 yeast extract, 10 g L−1 peptone, 20 g L−1 glucose, 20 g L−1 agar) supplemented with 0.1 g L−1 of nourseotricin for selection of yeast transformants. For shake flask cultivation on glucose, strains were grown in YNB medium (6.7 g L−1 YNB without amino acids, 20 g L−1 glucose) at 37°C under aerobic condition for 5 days. For shake flask cultivation on methanol, yeast cells were grown in mineral medium supplemented with 10 g L−1 glucose. Glucose was completely utilized within 24 h and after that methanol was added to the culture medium at concentration 10 g L−1. For cultivation in bioreactors, yeast cells were grown in mineral medium: (NH4)2SO4—5 g L−1, KH2PO4—3 g L−1, MgSO4*7H2O—0.5 g L−1, glucose—20 g L−1; trace elements solution (×100): EDTA—15 mg; ZnSO4*7H2O—4.5 mg; CoCl2*6H2O—0.3 mg; MnCl2*4H2O—1 mg, CuSO4*5H2O—4.5 mg; NaMoO4*2H2O—0.4 mg; H3BO3—1 mg; CaCl2*2H2O—4.5 mg; FeSO4*7H2O—3 mg; KI—0.1 mg. Moreover, vitamins solution (×1000): biotin—0.5 mg; calcium panthotenate—10 mg; nicotinic acid—10 mg; inositol—250 mg; thiamine—10; pyridoxine—10 mg; para-aminobenzoic acid—2 mg was filter sterilized and added to the autoclaved medium. Bioreactor cultures were performed in a 2-L Biostat B-Twin fermentor (Sartorius) containing 1 L of medium and kept at 37°C without pH control. Stirrer speed was automatically adjusted in the range of 140–1000 rpm to maintain a dissolved oxygen value of 30% of saturation. All cultures were started at an initial optical density at 600 nm of 1. Precultures were performed for 24 h in shake flask in mineral medium. Samples for cell growth and glutathione quantification were taken daily. Molecular-biology techniques Standard cloning techniques were used as described (Sambrook et al.1989). Genomic DNA of O. polymorpha was isolated using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases and DNA ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer specifications. Plasmid isolation from E. coli was performed with the Wizard®Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). DNA fragments were separated on a 0.8% agarose (Fisher Scientific, Fair Lawn, NJ, USA) gel. Isolation of DNA fragments from the gel was carried out with a DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). PCR amplifications were performed using the Platinum®Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) according to the manufacturer specification. PCRs were performed in GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Transformation of the yeast O. polymorpha by electroporation was carried out as described previously (Faber et al.1994). Plasmid construction The sequence of MET4 gene was obtained from the O. polymorpha genome database (http://genome.jgi-psf.org/Hanpo2/Hanpo2.home.html). Genomic DNA of O. polymorpha was used as a template for MET4 gene PCR amplification. Native promoter of this gene was substituted by strong constitutive GAP promoter. First, GAP1 promoter and terminator were amplified from O. polymorpha genomic DNA using primers pairs Кo644/Кo645 and Кo646/Кo647, respectively (Table 1). Then two fragments were fused by overlap PCR using primers Кo644 and Кo647. The resulting 0.8-kb fragment was digested with BamHI and SalI endonucleases and ligated with BamHI/SalI-linearized vector pUC19 to yield pUC19/pGAP plasmid. Beside this, the natNT2 gene conferring resistance to nourseothricin was amplified from plasmid pRS41N (Taxis and Knop 2006) using primers OK42/OK43 (Table 1). The resulting 1.3-kb fragment was digested with NdeI endonuclease and ligated with the NdeI-linearized pUC19/pGAP plasmid, to yield pUC19/pGAP/NTC plasmid. Finally, gene MET4 was amplified from O. polymorpha genomic DNA using primers Кo655/Кo656 and cloned into XbaI/XhoI-linearized plasmid pUC19/pGAP/NTC. The final constructed plasmid was named pUC19/pGAP_MET4/NTC (Fig. 1). Figure 1. View largeDownload slide Linear scheme of plasmid pUC19/pGAP_MET4/NTC. The plasmid is derived from the plasmid pUC19 (explanation in the text). Expression cassette pGAP-MET4-GAPt is shown as gray box; natNT2 gene conferring resistance to nourseothricin is designated with the hatched lines; ampicillin resistance gene (bla) is shown as black box. Restriction sites are designated as follows B, BamHI; Xb, XbaI; Xh, XhoI; Sl, SalI; Nd, NdeI; NotI; Ad, AdhI. Figure 1. View largeDownload slide Linear scheme of plasmid pUC19/pGAP_MET4/NTC. The plasmid is derived from the plasmid pUC19 (explanation in the text). Expression cassette pGAP-MET4-GAPt is shown as gray box; natNT2 gene conferring resistance to nourseothricin is designated with the hatched lines; ampicillin resistance gene (bla) is shown as black box. Restriction sites are designated as follows B, BamHI; Xb, XbaI; Xh, XhoI; Sl, SalI; Nd, NdeI; NotI; Ad, AdhI. Table 1. Primers used in this study. Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  View Large Table 1. Primers used in this study. Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  View Large Quantitative real-time PCR Quantification of MET4 gene expression was performed by quantitative real-time PCR (qRT-PCR). Total RNA was extracted using the GeneMATRIX Universal RNA Purification Kit with DNAse I (EURx Ltd, Gdansk, Poland). RNA was quantified using Picodrop Microliter UV/Vis Spectrophotometer and diluted in RNAse free water. The qRT-PCR was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems) using the SG OneStep qRT-PCR kit, gene-specific primer pairs, extracted RNA as a template and ROX reference passive dye according to the manufacturer's instructions (EURx Ltd, Gdansk, Poland). The following primer pairs were used: OK63/OK64 for the 3΄ fragment of O. polymorpha MET4 gene and OK61/OK62 for the 3΄ fragment of O. polymorpha ORF of the ACT1 gene used as a reference. In brief, normalized amount of RNA (100 ng) and 0.4 μmol of each of the two primers were used in a total reaction volume of 20 μL. The amplification was performed with the following cycling profile: reverse transcription step at 50°C for 30 min; initial denaturation at 95°C for 3 min at preparation step; followed by 40 cycles of 15 s at 94°C and 30 s at 60°C. Melting curve analysis was performed to verify the specificity and identity of PCR products from 65°C to 95°C using the software of real-time cycler. The amplification for over 40 cycles gave abundance of PCR product indicating saturation phase. The fold change of each amplicon in each sample relative to the control sample was normalized to the internal control gene ACT1 and calculated according to the comparative Ct (ΔΔCt) method. All data points were analyzed in triplicate. Glutathione assay Total glutathione (GSH and GSSG) concentration was measured in cell-free extracts by means of the standard recycling assay based on the colorimetric method with Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic) acid) in the presence of glutathione reductase and NADPH as described earlier (Grabek-Lejko et al.2011). Glutathione extraction was done by glass beads disruption in buffer (0.1 M Tris-HCl pH 7.5/0.2 mM EDTA). Protein precipitation was done with 4% TCA with subsequent neutralization of cell extracts with 2N NaOH. For dry cell weight determination, the cell pellet was collected by centrifugation, washed twice with distilled water and dried at 95°C until constant weight. Experiments were performed at least in triplicates. Additionally, a glutathione standard curve was obtained with standard solution of GSH in concentration ranging from 1 to 100 μmol L−1. Intracellular GSH production was calculated in nmol mg−1 protein, mg L−1 and μmol L−1. Protein concentration was determined with Folin reagent (Lowry et al.1951). RESULTS AND DISCUSSION In our previous study, the physiological role of O. polymorpha genes involved in glutathione homeostasis, namely GSH2, a homolog of S. cerevisiae GSH1 gene coding for GCS, and MET4, similar to S. cerevisiae MET4 gene involved in global sulfur regulation, was characterized (Ubiyvovk et al.2002). We demonstrated the positive effect of the separate multicopy overexpression of GSH2 and MET4 genes under control of their native promoters on glutathione production titer. The recombinant strains mcGSH2 overexpressing GSH2 gene accumulated, during cultures in bioreactor, 15% more intracellular glutathione as compared to the best known S. cerevisiae glutathione overproducer (Ubiyvovk et al.2011a). Therefore, we hypothesized that overexpression of MET4 gene under control of the strong GAP promoter instead of the native one in a mcGSH2 strain would further increase glutathione production titer. At the first step, a strain which expresses MET4 gene under control of the strong constitutive GAP1 promoter was constructed. For this purpose, O. polymorpha strain overexpressing GSH2 gene under control of native promoter was used as a recipient strain. The constructed plasmid pUC19/pGAP_MET4/NTC was HindIII-linearized and introduced into genome of O. polymorpha mcGSH2 strain. The transformants were selected on the solid YPD medium supplemented with 0.1 g L−1 of nourseothricin and subsequently were stabilized by alternating cultivation in nonselective and selective media. The genotype of the resulting strains was verified by analytical PCR using the forward primer Ko644, specific to the GAP1 promoter and the reverse one Ko647, specific to the MET4 gene. Finally, the overexpression of MET4 gene was confirmed by qRT-PCR. The transcribed level of MET4 gene was more than 200 times higher in mcGSH2/MET4(pGAP) strain as compared to the mcGSH2 strain (data not shown). As shown in Fig. 2, co-overexpression of GSH2 and MET4 genes led to a significant increase of glutathione level during shake flask cultivation in YNB medium. The dynamics of glutathione accumulation in the WT strain and recombinant strains mcGSH2 and mcGSH2/MET4(pGAP) is quite different. No significant change in glutathione production by the WT strain was noted during the entire course of cultivation. The maximal glutathione amount was observed in mcGSH2 strain after 48 h of cultivation probably as a result of stimulation of glutathione biosynthetic pathway. The glutathione amount after 48 h was significantly higher (2.5-fold increase) for mcGSH2 strain as compared to the WT strain (Fig. 2). The maximal glutathione accumulation of mcGSH2/MET4(pGAP) was reached after 72 h, probably due to the impact of Met4 transcriptional factor for activation of reactions involved in cysteine and glutathione synthesis. One of the possible explanations also can be the shifted balance between the reactions involved in glutathione synthesis and degradation in obtained recombinant strains overexpressing GSH2 and MET4 genes. Figure 2. View largeDownload slide Intracellular GSH accumulation in O. polymorpha recombinant strains co-overexpressing MET4 and GSH2 genes (mcGSH2/MET4(pGAP)) as compared to the parental strain (mcGSH2) during cultivation in YNB medium with glucose as carbon source under aerobic conditions at 37°C. Figure 2. View largeDownload slide Intracellular GSH accumulation in O. polymorpha recombinant strains co-overexpressing MET4 and GSH2 genes (mcGSH2/MET4(pGAP)) as compared to the parental strain (mcGSH2) during cultivation in YNB medium with glucose as carbon source under aerobic conditions at 37°C. The glutathione level of the mcGSH2/MET4(pGAP) strain reached 837 nmol mg−1 protein, which is 1.8- and 4.4-fold higher than that of the parental (mcGSH2) and the WT strain, respectively. The recombinant strain mcGSH2/MET4(pGAP) accumulated 5- and 1.8-fold higher concentration of glutathione as compared to the WT and the parental strains, reaching 2167 mg L−1. The glutathione level in the WT strain, mcGSH2 and mcGSH2/MET4(pGAP) reached 39, 85 and 134 mg g−1 cell dry weight, respectively (Table 2). Moreover, it was found that the mcGSH2/MET4(pGAP) mutant produced 187 μmol L−1 of extracellular glutathione on fifth day of cultivation which represents a 2.3-fold increase as compared to the WT strain (Fig. 3). Figure 3. View largeDownload slide Extracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during cultivation in YNB medium under aerobic conditions at 37°C. Figure 3. View largeDownload slide Extracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during cultivation in YNB medium under aerobic conditions at 37°C. Table 2. Maximal intracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during shake flask cultivation at 37°C. Intracellular GSH production was calculated in nmol mg−1 protein and in mg L−1 (μmol L−1). Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  a 72h of cultivation b 48 h of cultivation at 37°C c96 h of cultivation at 37°C View Large Table 2. Maximal intracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during shake flask cultivation at 37°C. Intracellular GSH production was calculated in nmol mg−1 protein and in mg L−1 (μmol L−1). Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  a 72h of cultivation b 48 h of cultivation at 37°C c96 h of cultivation at 37°C View Large We additionally tested glutathione accumulation of constructed strains during shake flask cultivation using methanol as a carbon source. The intracellular level of glutathione in the mcGSH2/MET4(pGAP) strain was 1.9- and 2-fold increased after 48 h and 72 h of shake flask cultivation in methanol containing medium, as compared to the WT strain, reaching 289 and 228 nmol mg−1 protein, respectively. No significant difference was observed in glutathione production between mcGSH2 and mcGSH2/MET4(pGAP) strains under these growth conditions. The maximal amount of glutathione was 3-fold higher in glucose-containing medium during shake flask cultivation as compared to that with methanol. As a next step, bioreactors batch cultivations of 75 h were performed with strains mcGSH2 and mcGSH2/MET4(pGAP) to assess glutathione production in these conditions. No significant differences in cell growth (Fig. 4) and in glucose consumption rate (Fig. 5) were observed between the two strains. By contrast, glutathione accumulation by mcGSH2/MET4(pGAP) strain was 5-fold increased as compared to that of mcGSH2 strain, on first 25 h of batch cultivation, reaching 249 nmol mg−1 of protein. The glutathione production by mcGSH2/MET4(pGAP) remains higher during the 71 h of the batch cultivation. The final glutathione production titer reached 420 nmol mg−1 of protein for both strain (Fig. 6). The quite low glutathione production could be attributed to the composition of the defined mineral medium used during batch culture as compared to the laboratory YNB medium. However, the rate of glutathione synthesis during the first 24 h of cultivation was significantly higher for strain mcGSH2/MET4(pGAP) as compared to the parental strain (249 and 55 nmol mg−1 protein, respectively) while rate of synthesis were quite similar during shake flask experiment 179 and 274 nmol mg−1 protein, respectively. Figure 4. View largeDownload slide Biomass accumulation during batch cultivation in bioreactors of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Figure 4. View largeDownload slide Biomass accumulation during batch cultivation in bioreactors of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Figure 5. View largeDownload slide Glucose consumption during batch cultivation of O. polymorpha recombinant strains mcGSH2 and mcGSH2/MET4(pGAP) strain. Figure 5. View largeDownload slide Glucose consumption during batch cultivation of O. polymorpha recombinant strains mcGSH2 and mcGSH2/MET4(pGAP) strain. Figure 6. View largeDownload slide GSH production during batch cultivation of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Figure 6. View largeDownload slide GSH production during batch cultivation of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Our results clearly demonstrated that the glutathione production by the constructed mcGSH2/MET4(pGAP) strain is one of the highest ever reported for yeast glutathione producers; however, it is still lower than the one reported in E. coli (Table 3) (Lorenz, Schmacht and Senz 2016; Wang et al.2015). Table 3. Overview of current research on GSH production. Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  View Large Table 3. Overview of current research on GSH production. Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  View Large Thus, data of this study show importance of MET4 gene coding for transcription activator involved in sulfur metabolism in yeasts for regulation of glutathione biosynthesis in O. polymorpha. The transcription of the genes encoding proteins involved in the synthesis of sulfur-containing amino acids, known as the MET regulon, is rapidly induced if methionine and cysteine are limiting for growth. This induction is mediated by the transcription factor Met4, whose function requires different combinations of the cofactors Cbf1, Met28, Met31 and Met32 (Lee et al.2010; Sadhu et al.2014). The sites bound by Cbf1, Met31 and Met32 have been mapped genome wide in S. cerevisiae. Both Cbf1 and Met31/Met32 sites were found in promoter of S. cerevisiae GSH1 gene, encoding γ-glutamylcysteine synthetase (Lee et al.2010). Previously, it was also shown that Yap1 and Met4 transcription factors regulate the expression of S. cerevisiae GSH1 gene (Weeler et al.2003). Met4 is also known to regulate the response of S. cerevisiae GSH1 to cadmium stress, which additionally requires Yap1 (Dormer et al.2000). In O. polymorpha homolog of S. cerevisiae Gsh1, we revealed potential CDEI motifs similar to Cbf1-binding sites involved in expression of S. cerevisiae GSH1 gene under cadmium stress. Therefore, molecular mechanisms of regulatory network mediated by described O. polymorpha Met4 may be an intriguing subject for the following study to provide further insight into how this transcriptional factor is involved in regulation of glutathione biosynthesis. Used here approach (co-overexpression of the first gene involved in glutathione synthesis (GSH2 in O. polymorpha) along with MET4), was found to be a novel successful strategy to increase glutathione biosynthesis in yeast. It was found that simultaneous overexpression of GSH2 and MET4 genes led to 5-fold increased glutathione production after first 25 h of batch bioreactor cultivation and 1.8-fold during flask cultivation as compared to the parental strain with overexpressed only GSH2 gene. FUNDING This work was partly supported by bilateral Polish-Wallonian grant DS.183.132.2013/MBA ‘Metabolic engineering of the yeast Hansenula polymorpha for increase the level of glutathione’ awarded to AS and PF, by the Wallonie-Bruxelles International grant N°2015/252690 and by FEMS Fellowship grant 2016-1 awarded to MY. Conflict of interest. None declared. REFERENCES Bachhawat A, Gangul D, Kaur J et al.   Glutathione production in yeast. In: Satyanarayana T, Kunze G (ed). Yeast Biotechnology: Diversity and Applications . Dordrecht, The Netherlands: Springer, 2009, 259– 80. Google Scholar CrossRef Search ADS   Chatterjee S, Lamirande E, Gagnon C. Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione. Mol Reprod Dev  2001; 60: 498– 506. Google Scholar CrossRef Search ADS PubMed  Dmytruk K, Kurylenko O, Ruchala J et al.   Development of the thermotolerant methylotrophic yeast Hansenula polymorpha as efficient ethanol producer. 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Effect of amino acids addition and feedback control strategies on the high-cell-density cultivation of Saccharomyces cerevisiae for glutathione production. Process Biochem  2007; 42: 108– 11. Google Scholar CrossRef Search ADS   Wheeler GL, Trotter EW, Dawes IW et al.   Coupling of the transcriptional regulation of glutathione biosynthesis to the availability of glutathione and methionine via the Met4 and Yap1 transcription factors. J Biol Chem  2003; 278: 49920– 8. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Yeast Research Oxford University Press

Gene of the transcriptional activator MET4 is involved in regulation of glutathione biosynthesis in the methylotrophic yeast Ogataea (Hansenula) polymorpha

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Abstract

Abstract Glutathione is the most abundant cellular thiol and the low molecular weight peptide present in cells. The methylotrophic yeast Ogataea (Hansenula) polymorpha is considered as a promising cell factory for the synthesis of glutathione. In this study, a competitive O. polymorpha glutathione producer was constructed by overexpression of the GSH2 gene, encoding γ-glutamylcysteine synthetase, the first enzyme involved in glutathione biosynthesis, and the MET4 gene coding for central regulator of sulfur metabolism. Overexpression of MET4 gene in the background of overexpressed GSH2 gene resulted in 5-fold increased glutathione production during shake flask cultivation as compared to the wild-type strain, reaching 2167 mg L−1. During bioreactor cultivation, glutathione accumulation by obtained recombinant strain was 5-fold increased relative to that by the parental strain with overexpressed only GSH2 gene, on the first 25 h of batch cultivation in mineral medium. Obtained results suggest involvement of Met4 transcriptional activator in regulation of GSH synthesis in the methylotrophic yeast O. polymorpha. glutathione, Ogataea (Hansenula) polymorpha, glutathione overproducers, MET4 gene INTRODUCTION Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is an abundant tripeptide that is present in most living organisms, predominantly, in eukaryotic cells, at concentrations between 0.2 and 10 mM. This small molecule is known as an important cellular redox buffer for maintaining the reducing thiol-disulfide balance. Concentration of the reduced form of glutathione (designated as GSH) in the cell is efficiently maintained by NADPH-dependent glutathione reductase (Spector, Labarre and Toledano 2001). Glutathione is an important detoxifier, could be used as a source of nitrogen and sulfur nutrition, cofactor of numerous enzymes and helper in protein folding (Orumets et al.2012). It is involved in DNA synthesis and amino acid transport and is therefore an important metabolite for the growth of eukaryotic cells. It is also crucial for stress responses caused by nutrient starvation, xenobiotics, heavy metals and reactive oxygen species (Grabek-Lejko et al.2011). Most of the above listed functions of glutathione are related to its antioxidative property caused by the thiol group of the cysteine moiety. More than 90% of microbial, plant and mammalian cell glutathione is present in the reduced form while the oxidized form (designated as GSSG) only accounts for 10%. Both forms are located in the cytoplasm as well as in the mitochondrial membrane. Glutathione deficiency in humans can be associated with several medical disorders caused by oxidative stress, poisoning or compromised immune system. These disorders lead to such diseases as cancer, cataracts, liver cirrhosis, neurodegenerative diseases, gastrointestinal inflammations and hemolytic anemia. Both GSH and GSSG are widely used in medical, cosmetic and food industries as an active ingredient of drugs, food and cosmetic products to alleviate dangerous oxidative processes and scavenge toxic compounds. It is also used to strengthen skin whitening and repair due to its antiaging effect and as support medication in cancer therapies (Townsend, Tew and Tapiero 2003; Bachhawat et al.2009). GSSG could be used as cryoprotector and immunomodulator (Chatterjee, Lamirande and Gagnon 2001; Kozhemyakin and Balasovski 2008). The estimated global annual production of pure crystalline glutathione and glutathione-enriched yeast extract (15% GSH) exceeds 200 and 800 tons, respectively, sold at a price of 300 and 150 USD/kg (Marz 2014). This highlights the great commercial importance of glutathione produced mainly by yeasts. In Saccharomyces cerevisiae and other glutathione-containing organisms, glutathione is synthesized in two consecutive ATP-dependent reactions, catalyzed by γ-glutamylcysteine synthetase, (EC 6.3.2.2, GCS, gene GSH1) and glutathione synthetase (EC 6.3.2.3, GS, gene GSH2), respectively. However, in O. polymorpha a homolog of the S. cerevisiae GSH1 gene coding for GCS was named GSH2 (Ubiyvovk et al.2002). The activity of the first reaction of the pathway, GCS, is feedback-inhibited by GSH (but not GSSG) to avoid overaccumulation of intracellular glutathione (Li et al.2004). Glutathione could be industrially produced enzymatically in the presence of ATP and three precursor amino acids (i.e. L-glutamic acid, L-cysteine, L-glycine). However, microbial fermentation is the most common approach to produce glutathione at industrial scale. In these processes, bacteria Escherichia coli and Lactococcus lactis or yeasts S. cerevisiae and Candida utilis are used as producing organisms (Li et al.2004). In the first attempt to improve glutathione biosynthesis in S. cerevisiae, the strains with overexpression of GSH1 and GSH2 genes were used. Both metabolic and evolutionary engineering approaches, together with medium optimization were used to increase glutathione synthesis (Lorenz, Schmacht and Senz 2016; Wang et al.2015). During the last decades, the methylotrophic yeast Ogataea (Hansenula) polymorpha has gained increasing interest both for basic research and biotechnological applications, such as characterizing the mechanisms of thermotolerance, peroxisome homeostasis, production of numerous heterologous proteins and high-temperature alcoholic fermentation (Dmytruk et al.2016, 2017; Ubiyvovk et al.2011a). The industrial significance of O. polymorpha relies on several technologically interesting features such as the ability to grow at high cell densities in bioreactors, capacity to use methanol as sole carbon source, the availability of strong both regulated and constitutive promoters allowing to reach high product yields. Similarly to S. cerevisiae, O. polymorpha is characterized by simple cultivation mode in inexpensive growth media, well-established genetic tools and experience on industrial cultivation. Additionally, availability of the complete genome sequence, established proteome and transcriptome databases render O. polymorpha as a suitable organism for metabolic engineering to modify and improve particular biosynthetic pathways (Kim et al.2013; Riley et al.2016). This yeast species is highly resistant to different stress conditions, induced by heavy metals, xenobiotics and different pollutants. The methylotrophic yeast O. polymorpha is considered to be a rich source of glutathione, due to the role of this tripeptide in detoxifications of key intermediates of methanol metabolism, formaldehyde, as well as hydrogen peroxide and alkyl hydroperoxides, accumulated during methylotrophic growth (Hartner and Glieder 2006). Design and construction of novel glutathione producers are of great importance for improvement of biotechnological production of this biotechnologically and medically important tripeptide. Here, we report on the construction of an O. polymorpha strain with improved glutathione productivity by means of constitutive overexpression of the MET4 gene encoding the central regulator of sulfur metabolism in yeasts (Thomas, Jacquemin and Surdin-Kerjan 1992; Ubiyvovk et al.2011a) using recipient strain with overexpressed gene GSH2 coding GCS, the first reaction of glutathione synthesis. Production of glutathione in the resulting strain was 5-fold increased relative to that of the parental strain after first 25 h of batch bioreactor cultivation. MATERIALS AND METHODS Microorganisms and cultivation conditions In this study, the following O. polymorpha strains were used: the DL-1 leu2—wild-type strain (WT); the mcGSH2—strain, overexpressing GSH2 gene under control of native promoter due to multicopy integration (Ubiyvovk et al.2011b); the mcGSH2/MET4(pGAP)—strains, overexpressing, respectively, the GSH2 gene under control of native promoter due to multicopy integration and the MET4 gene under control of strong constitutive promoter of GAP1 gene coding for glyceraldehyde-3-phosphate dehydrogenase (pGAP). The E. coli DH5α strain (Φ80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(rK−, mK+), supE44, relA1, deoR, Δ(lacZYA-argF)U169) was used as a host for plasmid propagation as previously described (Sambrook et al.1989). Transformed E. coli cells were maintained on LB medium supplemented with 0.1 g L−1 of ampicillin. Yeast cells were grown on YPD (10 g L−1 yeast extract, 10 g L−1 peptone, 20 g L−1 glucose, 20 g L−1 agar) supplemented with 0.1 g L−1 of nourseotricin for selection of yeast transformants. For shake flask cultivation on glucose, strains were grown in YNB medium (6.7 g L−1 YNB without amino acids, 20 g L−1 glucose) at 37°C under aerobic condition for 5 days. For shake flask cultivation on methanol, yeast cells were grown in mineral medium supplemented with 10 g L−1 glucose. Glucose was completely utilized within 24 h and after that methanol was added to the culture medium at concentration 10 g L−1. For cultivation in bioreactors, yeast cells were grown in mineral medium: (NH4)2SO4—5 g L−1, KH2PO4—3 g L−1, MgSO4*7H2O—0.5 g L−1, glucose—20 g L−1; trace elements solution (×100): EDTA—15 mg; ZnSO4*7H2O—4.5 mg; CoCl2*6H2O—0.3 mg; MnCl2*4H2O—1 mg, CuSO4*5H2O—4.5 mg; NaMoO4*2H2O—0.4 mg; H3BO3—1 mg; CaCl2*2H2O—4.5 mg; FeSO4*7H2O—3 mg; KI—0.1 mg. Moreover, vitamins solution (×1000): biotin—0.5 mg; calcium panthotenate—10 mg; nicotinic acid—10 mg; inositol—250 mg; thiamine—10; pyridoxine—10 mg; para-aminobenzoic acid—2 mg was filter sterilized and added to the autoclaved medium. Bioreactor cultures were performed in a 2-L Biostat B-Twin fermentor (Sartorius) containing 1 L of medium and kept at 37°C without pH control. Stirrer speed was automatically adjusted in the range of 140–1000 rpm to maintain a dissolved oxygen value of 30% of saturation. All cultures were started at an initial optical density at 600 nm of 1. Precultures were performed for 24 h in shake flask in mineral medium. Samples for cell growth and glutathione quantification were taken daily. Molecular-biology techniques Standard cloning techniques were used as described (Sambrook et al.1989). Genomic DNA of O. polymorpha was isolated using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases and DNA ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer specifications. Plasmid isolation from E. coli was performed with the Wizard®Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). DNA fragments were separated on a 0.8% agarose (Fisher Scientific, Fair Lawn, NJ, USA) gel. Isolation of DNA fragments from the gel was carried out with a DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). PCR amplifications were performed using the Platinum®Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) according to the manufacturer specification. PCRs were performed in GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Transformation of the yeast O. polymorpha by electroporation was carried out as described previously (Faber et al.1994). Plasmid construction The sequence of MET4 gene was obtained from the O. polymorpha genome database (http://genome.jgi-psf.org/Hanpo2/Hanpo2.home.html). Genomic DNA of O. polymorpha was used as a template for MET4 gene PCR amplification. Native promoter of this gene was substituted by strong constitutive GAP promoter. First, GAP1 promoter and terminator were amplified from O. polymorpha genomic DNA using primers pairs Кo644/Кo645 and Кo646/Кo647, respectively (Table 1). Then two fragments were fused by overlap PCR using primers Кo644 and Кo647. The resulting 0.8-kb fragment was digested with BamHI and SalI endonucleases and ligated with BamHI/SalI-linearized vector pUC19 to yield pUC19/pGAP plasmid. Beside this, the natNT2 gene conferring resistance to nourseothricin was amplified from plasmid pRS41N (Taxis and Knop 2006) using primers OK42/OK43 (Table 1). The resulting 1.3-kb fragment was digested with NdeI endonuclease and ligated with the NdeI-linearized pUC19/pGAP plasmid, to yield pUC19/pGAP/NTC plasmid. Finally, gene MET4 was amplified from O. polymorpha genomic DNA using primers Кo655/Кo656 and cloned into XbaI/XhoI-linearized plasmid pUC19/pGAP/NTC. The final constructed plasmid was named pUC19/pGAP_MET4/NTC (Fig. 1). Figure 1. View largeDownload slide Linear scheme of plasmid pUC19/pGAP_MET4/NTC. The plasmid is derived from the plasmid pUC19 (explanation in the text). Expression cassette pGAP-MET4-GAPt is shown as gray box; natNT2 gene conferring resistance to nourseothricin is designated with the hatched lines; ampicillin resistance gene (bla) is shown as black box. Restriction sites are designated as follows B, BamHI; Xb, XbaI; Xh, XhoI; Sl, SalI; Nd, NdeI; NotI; Ad, AdhI. Figure 1. View largeDownload slide Linear scheme of plasmid pUC19/pGAP_MET4/NTC. The plasmid is derived from the plasmid pUC19 (explanation in the text). Expression cassette pGAP-MET4-GAPt is shown as gray box; natNT2 gene conferring resistance to nourseothricin is designated with the hatched lines; ampicillin resistance gene (bla) is shown as black box. Restriction sites are designated as follows B, BamHI; Xb, XbaI; Xh, XhoI; Sl, SalI; Nd, NdeI; NotI; Ad, AdhI. Table 1. Primers used in this study. Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  View Large Table 1. Primers used in this study. Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  Primers  Sequence 5΄–3΄  Кo644  CGC GGATCC TAG ACC ACA TCC GTG CAC CAG  Кo645  GTA AAT ATG TAG ATG GAG CCG AGC CTCGAG CCCGGG GCGGCCGC TCTAGA TTT GT T TCT ATA TTA TCT TTG TAC TAA AG  Кo646  CTT TAG TAC AAA GAT AAT ATA GAA ACA AA TCTAGA GCGGCCGC CCCGGG CTCGAG GCT CGG CTC CAT CTA CAT ATT TAC  Кo647  CGC GTCGAC CTG CCA CGA GGT ACC ACA AAG  OK42  CGC CATATG ATA ACT TCG TAT AGC ATA CAT TAT ACG    AAG TTA T CT TAA CTA TGC GGC ATC AGA G  OK43  CGC CATATG ATA ACT TCG TAT AAT GTA TGC TAT ACG    AAG TTA TCC GAG ATT CAT CAA CTC ATT GC  Кo655  TAG TCTAGA ATG TGT GGC GCA GTA TGG C  Кo656  CCG CTCGAG CTA GTT TGG CTT CGG GAA AC  View Large Quantitative real-time PCR Quantification of MET4 gene expression was performed by quantitative real-time PCR (qRT-PCR). Total RNA was extracted using the GeneMATRIX Universal RNA Purification Kit with DNAse I (EURx Ltd, Gdansk, Poland). RNA was quantified using Picodrop Microliter UV/Vis Spectrophotometer and diluted in RNAse free water. The qRT-PCR was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems) using the SG OneStep qRT-PCR kit, gene-specific primer pairs, extracted RNA as a template and ROX reference passive dye according to the manufacturer's instructions (EURx Ltd, Gdansk, Poland). The following primer pairs were used: OK63/OK64 for the 3΄ fragment of O. polymorpha MET4 gene and OK61/OK62 for the 3΄ fragment of O. polymorpha ORF of the ACT1 gene used as a reference. In brief, normalized amount of RNA (100 ng) and 0.4 μmol of each of the two primers were used in a total reaction volume of 20 μL. The amplification was performed with the following cycling profile: reverse transcription step at 50°C for 30 min; initial denaturation at 95°C for 3 min at preparation step; followed by 40 cycles of 15 s at 94°C and 30 s at 60°C. Melting curve analysis was performed to verify the specificity and identity of PCR products from 65°C to 95°C using the software of real-time cycler. The amplification for over 40 cycles gave abundance of PCR product indicating saturation phase. The fold change of each amplicon in each sample relative to the control sample was normalized to the internal control gene ACT1 and calculated according to the comparative Ct (ΔΔCt) method. All data points were analyzed in triplicate. Glutathione assay Total glutathione (GSH and GSSG) concentration was measured in cell-free extracts by means of the standard recycling assay based on the colorimetric method with Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic) acid) in the presence of glutathione reductase and NADPH as described earlier (Grabek-Lejko et al.2011). Glutathione extraction was done by glass beads disruption in buffer (0.1 M Tris-HCl pH 7.5/0.2 mM EDTA). Protein precipitation was done with 4% TCA with subsequent neutralization of cell extracts with 2N NaOH. For dry cell weight determination, the cell pellet was collected by centrifugation, washed twice with distilled water and dried at 95°C until constant weight. Experiments were performed at least in triplicates. Additionally, a glutathione standard curve was obtained with standard solution of GSH in concentration ranging from 1 to 100 μmol L−1. Intracellular GSH production was calculated in nmol mg−1 protein, mg L−1 and μmol L−1. Protein concentration was determined with Folin reagent (Lowry et al.1951). RESULTS AND DISCUSSION In our previous study, the physiological role of O. polymorpha genes involved in glutathione homeostasis, namely GSH2, a homolog of S. cerevisiae GSH1 gene coding for GCS, and MET4, similar to S. cerevisiae MET4 gene involved in global sulfur regulation, was characterized (Ubiyvovk et al.2002). We demonstrated the positive effect of the separate multicopy overexpression of GSH2 and MET4 genes under control of their native promoters on glutathione production titer. The recombinant strains mcGSH2 overexpressing GSH2 gene accumulated, during cultures in bioreactor, 15% more intracellular glutathione as compared to the best known S. cerevisiae glutathione overproducer (Ubiyvovk et al.2011a). Therefore, we hypothesized that overexpression of MET4 gene under control of the strong GAP promoter instead of the native one in a mcGSH2 strain would further increase glutathione production titer. At the first step, a strain which expresses MET4 gene under control of the strong constitutive GAP1 promoter was constructed. For this purpose, O. polymorpha strain overexpressing GSH2 gene under control of native promoter was used as a recipient strain. The constructed plasmid pUC19/pGAP_MET4/NTC was HindIII-linearized and introduced into genome of O. polymorpha mcGSH2 strain. The transformants were selected on the solid YPD medium supplemented with 0.1 g L−1 of nourseothricin and subsequently were stabilized by alternating cultivation in nonselective and selective media. The genotype of the resulting strains was verified by analytical PCR using the forward primer Ko644, specific to the GAP1 promoter and the reverse one Ko647, specific to the MET4 gene. Finally, the overexpression of MET4 gene was confirmed by qRT-PCR. The transcribed level of MET4 gene was more than 200 times higher in mcGSH2/MET4(pGAP) strain as compared to the mcGSH2 strain (data not shown). As shown in Fig. 2, co-overexpression of GSH2 and MET4 genes led to a significant increase of glutathione level during shake flask cultivation in YNB medium. The dynamics of glutathione accumulation in the WT strain and recombinant strains mcGSH2 and mcGSH2/MET4(pGAP) is quite different. No significant change in glutathione production by the WT strain was noted during the entire course of cultivation. The maximal glutathione amount was observed in mcGSH2 strain after 48 h of cultivation probably as a result of stimulation of glutathione biosynthetic pathway. The glutathione amount after 48 h was significantly higher (2.5-fold increase) for mcGSH2 strain as compared to the WT strain (Fig. 2). The maximal glutathione accumulation of mcGSH2/MET4(pGAP) was reached after 72 h, probably due to the impact of Met4 transcriptional factor for activation of reactions involved in cysteine and glutathione synthesis. One of the possible explanations also can be the shifted balance between the reactions involved in glutathione synthesis and degradation in obtained recombinant strains overexpressing GSH2 and MET4 genes. Figure 2. View largeDownload slide Intracellular GSH accumulation in O. polymorpha recombinant strains co-overexpressing MET4 and GSH2 genes (mcGSH2/MET4(pGAP)) as compared to the parental strain (mcGSH2) during cultivation in YNB medium with glucose as carbon source under aerobic conditions at 37°C. Figure 2. View largeDownload slide Intracellular GSH accumulation in O. polymorpha recombinant strains co-overexpressing MET4 and GSH2 genes (mcGSH2/MET4(pGAP)) as compared to the parental strain (mcGSH2) during cultivation in YNB medium with glucose as carbon source under aerobic conditions at 37°C. The glutathione level of the mcGSH2/MET4(pGAP) strain reached 837 nmol mg−1 protein, which is 1.8- and 4.4-fold higher than that of the parental (mcGSH2) and the WT strain, respectively. The recombinant strain mcGSH2/MET4(pGAP) accumulated 5- and 1.8-fold higher concentration of glutathione as compared to the WT and the parental strains, reaching 2167 mg L−1. The glutathione level in the WT strain, mcGSH2 and mcGSH2/MET4(pGAP) reached 39, 85 and 134 mg g−1 cell dry weight, respectively (Table 2). Moreover, it was found that the mcGSH2/MET4(pGAP) mutant produced 187 μmol L−1 of extracellular glutathione on fifth day of cultivation which represents a 2.3-fold increase as compared to the WT strain (Fig. 3). Figure 3. View largeDownload slide Extracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during cultivation in YNB medium under aerobic conditions at 37°C. Figure 3. View largeDownload slide Extracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during cultivation in YNB medium under aerobic conditions at 37°C. Table 2. Maximal intracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during shake flask cultivation at 37°C. Intracellular GSH production was calculated in nmol mg−1 protein and in mg L−1 (μmol L−1). Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  a 72h of cultivation b 48 h of cultivation at 37°C c96 h of cultivation at 37°C View Large Table 2. Maximal intracellular GSH production of O. polymorpha WT, parental mcGSH2 and constructed mcGSH2/MET4(pGAP) strains during shake flask cultivation at 37°C. Intracellular GSH production was calculated in nmol mg−1 protein and in mg L−1 (μmol L−1). Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  Strains  GSH, nmol mg−1 protein  GSH, mg L−1 (μmol L−1)  GSH, mg g−1 of dry cells  WTa  191.9 ± 19.2  418.1 ± 37.6 (1393 ± 125.37)  39 ± 3.9  mcGSH2b  475.5 ± 38  1223 ± 61.1 (4076.7 ± 203.8)  85 ± 6.6  mcGSH2/MET4 (pGAP)c  837 ± 58.5  2167 ± 65.3 (7223.3 ± 216.7)  134 ± 9.4  a 72h of cultivation b 48 h of cultivation at 37°C c96 h of cultivation at 37°C View Large We additionally tested glutathione accumulation of constructed strains during shake flask cultivation using methanol as a carbon source. The intracellular level of glutathione in the mcGSH2/MET4(pGAP) strain was 1.9- and 2-fold increased after 48 h and 72 h of shake flask cultivation in methanol containing medium, as compared to the WT strain, reaching 289 and 228 nmol mg−1 protein, respectively. No significant difference was observed in glutathione production between mcGSH2 and mcGSH2/MET4(pGAP) strains under these growth conditions. The maximal amount of glutathione was 3-fold higher in glucose-containing medium during shake flask cultivation as compared to that with methanol. As a next step, bioreactors batch cultivations of 75 h were performed with strains mcGSH2 and mcGSH2/MET4(pGAP) to assess glutathione production in these conditions. No significant differences in cell growth (Fig. 4) and in glucose consumption rate (Fig. 5) were observed between the two strains. By contrast, glutathione accumulation by mcGSH2/MET4(pGAP) strain was 5-fold increased as compared to that of mcGSH2 strain, on first 25 h of batch cultivation, reaching 249 nmol mg−1 of protein. The glutathione production by mcGSH2/MET4(pGAP) remains higher during the 71 h of the batch cultivation. The final glutathione production titer reached 420 nmol mg−1 of protein for both strain (Fig. 6). The quite low glutathione production could be attributed to the composition of the defined mineral medium used during batch culture as compared to the laboratory YNB medium. However, the rate of glutathione synthesis during the first 24 h of cultivation was significantly higher for strain mcGSH2/MET4(pGAP) as compared to the parental strain (249 and 55 nmol mg−1 protein, respectively) while rate of synthesis were quite similar during shake flask experiment 179 and 274 nmol mg−1 protein, respectively. Figure 4. View largeDownload slide Biomass accumulation during batch cultivation in bioreactors of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Figure 4. View largeDownload slide Biomass accumulation during batch cultivation in bioreactors of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Figure 5. View largeDownload slide Glucose consumption during batch cultivation of O. polymorpha recombinant strains mcGSH2 and mcGSH2/MET4(pGAP) strain. Figure 5. View largeDownload slide Glucose consumption during batch cultivation of O. polymorpha recombinant strains mcGSH2 and mcGSH2/MET4(pGAP) strain. Figure 6. View largeDownload slide GSH production during batch cultivation of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Figure 6. View largeDownload slide GSH production during batch cultivation of O. polymorpha mcGSH2 and mcGSH2/MET4(pGAP) recombinant strains at 37°C. Our results clearly demonstrated that the glutathione production by the constructed mcGSH2/MET4(pGAP) strain is one of the highest ever reported for yeast glutathione producers; however, it is still lower than the one reported in E. coli (Table 3) (Lorenz, Schmacht and Senz 2016; Wang et al.2015). Table 3. Overview of current research on GSH production. Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  View Large Table 3. Overview of current research on GSH production. Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  Process  Strain  Max. (GSH) (mg L−1)  Reference  Fed-batch   O. polymorpha MOXp-GSH2a  2257.0  (Ubiyvovk et al.2011b)  Fed-batch   C. utilis WSH02-08  2448.0  (Wang et al.2010)  Fed-batch   S. cerevisiae G-14  2020.0  (Wang, Tan and Song 2007)  Batch  S. bayanus Sa-00645  852.7  (Lorenz, Schmacht and Senz 2016)  Batch  S. boulardii Sa-07145  629.3  (Lorenz et al.2016)  Fed-batch   E. coli BL21(pUC18-gshF)a  15 210.0  (Wang et al.2015)  Flask cultivation  O. polymorpha mcGSH2  1223  This study  Flask cultivation  O. polymorpha mcGSH2/MET4(pGAP)  2167  This study  View Large Thus, data of this study show importance of MET4 gene coding for transcription activator involved in sulfur metabolism in yeasts for regulation of glutathione biosynthesis in O. polymorpha. The transcription of the genes encoding proteins involved in the synthesis of sulfur-containing amino acids, known as the MET regulon, is rapidly induced if methionine and cysteine are limiting for growth. This induction is mediated by the transcription factor Met4, whose function requires different combinations of the cofactors Cbf1, Met28, Met31 and Met32 (Lee et al.2010; Sadhu et al.2014). The sites bound by Cbf1, Met31 and Met32 have been mapped genome wide in S. cerevisiae. Both Cbf1 and Met31/Met32 sites were found in promoter of S. cerevisiae GSH1 gene, encoding γ-glutamylcysteine synthetase (Lee et al.2010). Previously, it was also shown that Yap1 and Met4 transcription factors regulate the expression of S. cerevisiae GSH1 gene (Weeler et al.2003). Met4 is also known to regulate the response of S. cerevisiae GSH1 to cadmium stress, which additionally requires Yap1 (Dormer et al.2000). In O. polymorpha homolog of S. cerevisiae Gsh1, we revealed potential CDEI motifs similar to Cbf1-binding sites involved in expression of S. cerevisiae GSH1 gene under cadmium stress. Therefore, molecular mechanisms of regulatory network mediated by described O. polymorpha Met4 may be an intriguing subject for the following study to provide further insight into how this transcriptional factor is involved in regulation of glutathione biosynthesis. Used here approach (co-overexpression of the first gene involved in glutathione synthesis (GSH2 in O. polymorpha) along with MET4), was found to be a novel successful strategy to increase glutathione biosynthesis in yeast. It was found that simultaneous overexpression of GSH2 and MET4 genes led to 5-fold increased glutathione production after first 25 h of batch bioreactor cultivation and 1.8-fold during flask cultivation as compared to the parental strain with overexpressed only GSH2 gene. FUNDING This work was partly supported by bilateral Polish-Wallonian grant DS.183.132.2013/MBA ‘Metabolic engineering of the yeast Hansenula polymorpha for increase the level of glutathione’ awarded to AS and PF, by the Wallonie-Bruxelles International grant N°2015/252690 and by FEMS Fellowship grant 2016-1 awarded to MY. Conflict of interest. None declared. REFERENCES Bachhawat A, Gangul D, Kaur J et al.   Glutathione production in yeast. In: Satyanarayana T, Kunze G (ed). Yeast Biotechnology: Diversity and Applications . Dordrecht, The Netherlands: Springer, 2009, 259– 80. Google Scholar CrossRef Search ADS   Chatterjee S, Lamirande E, Gagnon C. Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione. Mol Reprod Dev  2001; 60: 498– 506. Google Scholar CrossRef Search ADS PubMed  Dmytruk K, Kurylenko O, Ruchala J et al.   Development of the thermotolerant methylotrophic yeast Hansenula polymorpha as efficient ethanol producer. 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FEMS Yeast ResearchOxford University Press

Published: Mar 1, 2018

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