Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

<p>Abstract</p> <p>Background</p> <p>Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast <it>Hansenula polymorpha </it>is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of <it>H. polymorpha </it>DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (<it>GSH2</it>) and its precursor cysteine (<it>MET4</it>) was studied.</p> <p>Results</p> <p>Glutathione producing capacity of <it>H. polymorpha </it>DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSG_in, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSG_ex, per liter of the culture medium.</p> <p>Conclusions</p> <p><it>H. polymorpha </it>is an competitive glutathione producer as compared to other known yeast and bacteria strains (<it>Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis </it>etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione.</p

Role of gamma-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae.

GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding gamma-glutamyltranspeptidase (gammaGT). This is confirmed by the absence of the corresponding activity of gammaGT in the mutant with disrupted GGT1 gene. It was shown that gammaGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a gammaGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe

Alcoholic fermentation by wild-type Hansenula polymorpha and Saccharomyces cerevisiae versus recombinant strains with an elevated level of intracellular glutathione.

The ability of baker's yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

Accumulation of cadmium ions in the methylotrophic yeast Hansenula polymorpha.

Intracellular cadmium (Cd(2+)) ion accumulation and the ability to produce specific Cd(2+) ion chelators was studied in the methylotrophic yeast Hansenula polymorpha. Only one type of Cd(2+) intracellular chelators, glutathione (GSH), was identified, which suggests that sequestration of this heavy metal in H. polymorpha occurs similarly to that found in Saccharomyces cerevisiae, but different to Schizosaccharomys pombe and Candida glabrata which both synthesize phytochelatins. Cd(2+) ion uptake in the H. polymorpha wild-type strains appeared to be an energy dependent process. It was found that Deltagsh2 mutants, impaired in the first step of GSH biosynthesis, are characterized by increase in net Cd(2+) ion uptake by the cells, whereas Deltagsh1/Deltamet1 and Deltaggt1 mutants impaired in sulfate assimilation and GSH catabolism, respectively, lost the ability to accumulate Cd(2+) intracellularly. Apparently H. polymorpha, similarly to S. cerevisiae, forms a Cd-GSH complex in the cytoplasm, which in turn regulates Cd(2+) uptake. Genes GSH1/MET1 and GGT1 are involved in maturation and metabolism of cellular Cd-GSH complex, respectively. Transport of [(3)H]N-ethylmaleimide-S-glutathione ([(3)H]NEM-SG) conjugate into crude membrane vesicules, purified from the wild-type cells of H. polymorpha appeared to be MgATP dependent, uncoupler insensitive and vanadate sensitive. We suggest that MgATP dependent transporter involved in Cd-GSH uptake in H. polymorpha, is similar to S. cerevisiae Ycf1-mediated vacuolar transporter responsible for accumulation of organic GS-conjugates and Cd-GSH complex.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe