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Reprints and permissions. Alfafara, C. et al. Effect of amino acids on glutathione production by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 36 , — Download citation. Received : 12 May Accepted : 09 September Issue Date : January Anyone you share the following link with will be able to read this content:.

Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Summary The constituent amino acids of the glutathione GSH tripeptide chain, glutamate, cysteine and glycine, were investigated for positive effects on GSH production in shake-flask cultures of Saccharomyces cerevisiae with glucose as the carbon source.

Access this article Log in via an institution. References Anderson M, Meister A Transport and direct utilization of γ-glutamylcyst e ine for glutathione synthesis. Proc Natl Acad Sci USA — Google Scholar Dennda G, Kula MR Assay of the glutathione synthesizing enzymes by high performance liquid chromatography.

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Anal Biochem Google Scholar Download references. Author information Authors and Affiliations Department of Biotechnology, Faculty of Engineering, Osaka University, , Suita, Osaka, Japan Catalino G. Alfafara View author publications. View author publications. Additional information Offprint requests to: S.

Rights and permissions Reprints and permissions. polymorpha DL-1 wild-type strain during batch cultivation in fermenter was studied depending on growth parameters, pH and aeration Figure 1. It was shown that this yeast produced more TIG during cultivation in glucose minimal medium under conditions of spontaneous pH acidification from pH 5.

It was demonstrated also that TIG was higher at low stirrer speed rpm than under intensive aeration rpm , independently of pH control regime Figure 1 - B, C, D ; Table 2.

Maximal cell biomass concentration was higher under conditions of high saturation with oxygen shake agitation at rpm as compared to low aeration rpm , independently on the mode of pH control Figure 1A ; Table 2. Growth and glutathione accumulation of H. polymorpha DL-1 at fermenter batch cultivations.

polymorpha DL-1 wild type strain was cultivated with glucose at different modes of pH control and aeration: fermentation with pH control at 5. Values shown are the means of three independent determinations.

To explain these results, one may suggest that supply cells with more oxygen at rpm provided more optimal growth conditions as we observed increase in biomass concentration.

Oxygen-limited yeast cells cultivated at low stirrer speed, rpm, most probably are suffered from metabolic stress induced by oxygen limitation. This stress could reduce growth activity and turned on cellular protection mechanisms including glutathione biosynthesis [ 29 ].

We compared biomass and glutathione producing capacities of H. We hypothesize that yeast cells supplied with enhanced oxygen suffered from excess of reactive oxygen species. It is known that the lasts could suppress growth activity and trapped accessible for measurement glutathione in the reactions of S-glutathiolation of protein and conjugation with product of lipid peroxidation [ 30 , 31 ].

polymorpha DL-1 at fermenter fed-batch cultivation. D - data of dissolved oxygen tension in culture media at different mode of aeration.

Values shown are the means of two independent determinations. In the meantime, intensification of oxygen supply led to slight increase in TEG level Table 2. We hypothesized that accumulation of elevated TIG level can be achieved in recombinant H.

polymorpha strains which overexpress this tripeptide. Consequently, we have studied TIG production in several H. polymorpha recombinant strains which harbored the additional copies of HpGSH2 gene.

As it was revealed earlier, H. cerevisiae GSH1 gene, encoding GCS, the first enzyme of GSH biosynthesis, is essential for yeast growth and stress reply [ 20 , 21 ]. One of the obtained in the present study recombinant strains designated as mcGSH2 transformant possessed copies of HpGSH2 expression cassette under native H.

polymorpha GSH2 promoter Figure 3. Another recombinant strain, named as MOXp-GSH2 transformant, acquired HpGSH2 expression cassette under control of the strong promoter of H. polymorpha alcohol oxidase MOX gene.

The MOX promoter is known to be regulated in H. polymorpha by mechanisms of glucose repression-derepression and methanol induction [ 32 ].

We also studied transformant named as mcMET4 that obtained additional copies of H. polymorpha MET4 gene Figure 3 , putative homologue of S.

cerevisiae MET4 gene involved in biosynthesis of GSH and its precursor, cysteine [ 24 , 25 ]. Southern blot analysis of copy number of HpGSH2 and HpMET4 genes in mcGSH2 and mcMET4 transformants.

Copy number of integrated into chromosomal DNA of H. polymorpha DL-1L wild type strain 1 , mcGSH2 2 and mcMET4 3 transformants pGLG HpGSH2 and pGLG HpMET4 plasmids bearing LEU2 gene. The DNA probes to LEU2 gene discovered mutated LEU2 gene a in all tested strains and introduced LEU2 gene based on indicated plasmids only in transformants b.

We explained such data discrepancy by differences in protein content of the compared strains. polymorpha DL-1 recombinant strains at glucose-feeding fermenter cultivation. TEG concentration is indicated with open symbols C.

Thus, introduction of additional copies of putative HpMET4 gene into the genome of recipient strain 1. mcGSH2 transformant displayed only 1. We have found the HpGSH2 gene overexpression in mcGSH2 transformant cultivated in medium without any carbon substrate Figure 4. Such conditions resembled fed-batch cultivation where glucose concentrations was maintained at the level lower than 0.

It seems that GSH excess restricted glutathione overproduction in our recombinant strains by mechanism of feedback inhibition of GCS activity. Nevertheless we did not confirm regulated from MOX promoter expression of HpGSH2 gene data of RT-PCR in the recombinant strain that obtained such construct.

Mode of expression of HpGSH2 gene from MOX promoter was the same as in the wild-type strain Figure 5. So, 2. Transcript level of HpGSH2. RT-PCR was performed on RNA samples using HpGSH2 primers from cultures of wild type, mcGSH2 and MOXp-GSH2 recombinant strains shifted for 6 hours from glucose medium to glucose Glc , methanol Meth or with no carbon added -C media.

As loading control transcript levels of actin ACT1 were analysed. HpGSH2 transcript analysis revealed methanol-dependent induction of this gene expression in all studied strains compared to that of glucose incubated yeast cells Figure 5.

We suggested that application of oxygen control during methanol feeding under fed-batch cultivation prevents the over-feeding of methanol and overaccumulation of toxic intermediates that could additionaly induce GSH biosynthesis [ 18 ].

Studied here MOXp-GSH2 recombinant strain was characterized by normal growth in glucose fed-batch cultures with minimal medium Figure 4A but displayed retarded growth in methanol as the sole carbon source as compared to the wild-type strain Figure 6A.

During growth in methanol fed-batch culture, MOXp-GSH2 recombinant strain accumulated about polymorpha DL-1 recombinant strain at methanol-feeding fermenter cultivation.

TEG concentration is indicated with filled symbols C. Reduced ability of MOXp-GSH2 recombinant strain to grow in methanol medium could be caused by inhibition of key enzyme of formaldehyde assimilation, dihydroxyacetone synthetase, by excess of GSH [ 35 ] or partial impairment of MOX gene expression as the result of MOXp-GSH2 expression vector integration into the chromosomal MOX locus [ 36 ].

So, high-cell-density cultivation of H. polymorpha DL-1 wild type strain led to accumulation of large amount of glutathione under dual control of substrate feeding exponential feeding rate mode combined with feedback control of substrate feed by DO set point using on-off regime.

Genetic engineering of sulfur metabolism and glutathione biosynthetic pathway improved the productivity of glutathione synthesis in H. polymorpha recombinant strains 1.

One of the best published results on GSH and biomass level for high-GSH-accumulated strain of S. Genetically modified strains of the methylotrophic yeast H.

polymorpha DL-1 studied in the present work, appeared to be competitive glutathione producer with good perspectives for further improvement. To develop the process of glutathione production based on described here H.

polymorpha producers, we plan to further optimize growth conditions to increase biomass concentration. Other approach we plan to use will be based on engineering GCS protein for impairment of feedback inhibition normally exerted by glutathione. GCS feedback inhibition engineering will be carried out on the isolated in our laboratory H.

polymorpha mutants defective both in glutathione uptake and secretory pathways which we found to possess elevated glutathione pool V. Ubiyvovk et al. It is known that S. cerevisiae mutants defective in glutathione transport and in secretory pathways overaccumulate glutathione in the medium [ 37 ].

This study has estimated biothechnological potential of the methylotrophic yeast H. polymorpha to produce antioxidant glutathione used as medicine, food additives and in cosmetic industry.

Our data showed that recombinant strains of H. polymorpha with genetically engineered pathway of global sulfur and glutathione metabolism accumulated the highest to our knowledge titre of intracellular and extracellular glutathione under optimized conditions.

We consider H. polymorpha as the yeast with good perspectives for further improvement especially for production of extracellular form of glutathione. Strains of the methylotrophic yeast H.

polymorpha used in this study are listed in Table 1. For shake-flasks and batch cultivation, strains were grown in YNB medium: 0.

For shake-flasks cultivation it was used shaking diameter of 15 cm. For fed-batch fermentations it was used synthetic salt medium per litre : NH 4 2 SO 4 - Standard DNA manipulations were performed as described in Sambrook et al.

Shuttle vector pGLG61, which bears LEU2 selectable marker and gives tandem integrated and mitotically stable copies near the end of the chromosome, was used [ 39 ]. pGLG HpGSH2 plasmid bearing H. polymorpha GSH2 gene, homologue of S. cerevisiae GSH1 gene, was constructed by subcloning of Sma I- Hpa I fragment of pG2 plasmid [ 21 ] bp upstream and bp downstream regions of ATG codon of H.

polymorpha CBS chromosomal HpGSH2 gene into Not I digested and blunted pGLG61 vector. Plasmid with HpGSH2 driven by strong promoter of H. Yeast cells of H. polymorpha DL-1 leu2 were transformed with appropriate plasmids by electroporation as described earlier [ 21 ]. Copy number of integrated into chromosomal DNA plasmids was estimated with Southern blot analysis.

For that total chromosomal DNA was digested with Cla I endonuclease, fractionated on 0. Schull GmbH, Dassel, Germany. The DNA probes for Southern blot analysis were labelled with digoxigenin DIG , using DIG-labelling kit Roche Diagnostics, Mannheim, Germany.

Pelleted cells of each studied strain were once washed with water, resuspended in YNB medium to OD Total RNA was extracted from yeast cells using the Trizol method Invitrogen Corporation, Carlsbad, California, USA following the manufacturer's protocol. RNA was quantified using UV spectrophotometry and diluted in RNAse-free water.

Single-stranded cDNA was synthesized using MuLV reverse transcriptase First Strand cDNA Synthesis Kit, Fermentas, Vilnius, Lithuania. Quantitative RT-PCR analysis was carried out using gene-specific primer pairs and cDNA as a template.

polymorpha ORF of the ACT1 gene. Sequence of the gene ACT1 was taken from the H. polymorpha database [ 14 ]. Inoculum culture of H. polymorpha was prepared by 24 h cultivation in shake-flasks in YNB medium supplemented with glucose.

All kinds of fermentations were carried out in BioFlo III bench-top fermenter New Brunswick Scientific Co. The fermentations were computer controlled and monitored. High cell density cultivation experiments were carried out as fed-batch fermentations using dual control algorithms of carbon source feeding.

Dual control algorithms were programmed using AFS-BioCommand control diagrams, a pictorial function-block language.

This control mode was combined with feedback control of substrate feed by dissolved oxygen tension so called DO-stats using on-off regime. In other words, current value of substrate feed rate was equal to exponentially increasing set point of substrate feed rate till the moment when current value of controlling parameter DO CV was passed through the DO set point DO SP.

After return DO CV to the DO SP predetermined exponential feeding control continued operation. Finally fed-batch processes were represented as pulsing feeding with exponentially increased magnitude of pulsation [ 34 , 40 ].

Glucose feeding was started after stoppage of batch culture growth and followed with gradual increase of stirrer speeds: for fed-batch fermentation of mode I - from to rpm during 24 h and for mode II - from to rpm during 12 h.

Mode I of fed-batch was applied for cultivation of all tested strains. Mode II of process was used for cultivation of wild type only. Methanol feeding was begun only after complete consuming of glycerol which indicated by sharp increasing of DO level.

Next 24 h of cultivation were carried out at gradual increase of stirrer speed from to rpm to avoid strong oxygen limitation.

At the time when no changes of biomass concentration observed process was terminated. Dry cells were prepared by centrifuging 5 ml of the culture medium at × g for 5 min, followed by drying at °C for 4 h.

The concentration of glucose in cultivation medium was measured using glucose oxidase method with a glucose assay kit Sigma-Aldrich Corporation, St.

Louis, Missouri, USA. The lasts were prepared by yeast cells vortexing in Eppendorf microtubes at 4°C for 20 min with 0. Protein was determined using Bio Rad DC protein assay in microplate reader at nm.

Procedure was adapted to well plates: each well contained μl of 0. Reaction was started with 20 μl of 3 mM DTNB promptly added by multipipetman. Microplate was incubated at room temperature for min and the absorbance of each well was read at nm using microplate reader.

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