Applied Microbiology and Biotechnology

, Volume 89, Issue 4, pp 1029–1037 | Cite as

Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants

  • Ildar NisamedtinovEmail author
  • Kaspar Kevvai
  • Kerti Orumets
  • Liisa Arike
  • Inga Sarand
  • Matti Korhola
  • Toomas Paalme
Biotechnological Products and Process Engineering


Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.


Saccharomyces cerevisiae Glutathione Cysteine GSH1 CYS3 YAP1 



The financial support for this research was provided by the Enterprise Estonia project EU22704, Estonian Ministry of Education and Research grant SF0140090s08, and by Estonian Science Foundation grant G7323. We would like to thank Dr. Chris Powell for critical revision of the manuscript.


  1. Alfafara CG, Kanda A, Shioi T, Shimizu H, Shioya S, Suga K (1992a) Effect of amino acids on glutathione production by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 36:538–540CrossRefGoogle Scholar
  2. Alfafara CG, Miura K, Shimizu H, Shioya S, Suga K (1992b) Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 37:141–146CrossRefGoogle Scholar
  3. Bachhawat AK, Ganguli D, Kaur J, Kasturia N, Thakur A, Kaur H, Kumar A, Yaday A (2009) Glutathione production in yeast. In: Satyanarayana T, Kunze G (eds) Yeast biotechnology: diversity and applications. Springer, Dordrecht, pp 259–280CrossRefGoogle Scholar
  4. Bagnyukova TV, Vasylkiv OY, Storey KB, Lushchak VI (2005) Catalase inhibition by amino triazole induces oxidative stress in goldfish brain. Brain Res 1052:180–186CrossRefGoogle Scholar
  5. Biterova EI, Barycki JJ (2010) Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase. J Biol Chem 285:14459–14466CrossRefGoogle Scholar
  6. Delaunay A, Isnard AD, Toledano MB (2000) H2O2 sensing through oxidation of the Yap1 transcription factor. EMBO J 19:5157–5166CrossRefGoogle Scholar
  7. Galant NJ, Wang H, Lee DR, Mucsi Z, Setiadi DH, Viskolcz B, Csizmadia IG (2009) Thermodynamic role of glutathione oxidation by peroxide and peroxybicarbonate in the prevention of Alzheimer’s disease and cancer. J Phys Chem A 113:9138–9149CrossRefGoogle Scholar
  8. Grant CM, MacIver FH, Dawes IW (1997) Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide γ-glutamylcysteine. Mol Biol Cell 8:1699–1707Google Scholar
  9. Gulshan K, Rovinsky SA, Coleman ST, Moye-Rowley WS (2005) Oxidant-specific folding of Yap1p regulates both transcriptional activation and nuclear localization. J Biol Chem 280:40524–40533CrossRefGoogle Scholar
  10. Hamada S, Tanaka H, Sakato K (1986) Process for producing glutathione. US Patent 4(582):801Google Scholar
  11. Hiraishi H, Miyake T, Ono B (2008) Transcriptional regulation of Saccharomyces cerevisiae CYS3 encoding cystathionine γ-lyase. Curr Genet 53:225–234CrossRefGoogle Scholar
  12. Ikeno Y, Tanno K, Omori I, Yamada R (1977) Glutathione. JP patent 52(087):296Google Scholar
  13. Kaiser P, Flick K, Wittenberg C, Reed SI (2000) Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCFMet30-mediated inactivation of the transcription factor Met4. Cell 102:303–314CrossRefGoogle Scholar
  14. Kasemets K, Nisamedtinov I, Laht T, Abner K, Paalme T (2007) Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study. Antonie Leeuwenhoek 92:109–128CrossRefGoogle Scholar
  15. Köcher T, Pichler P, Schutzbier M, Stingl C, Kaul A, Teucher N, Hasenfuss G, Penninger JM, Mechtler K (2009) High precision quantitative proteomics using iTRAQ on an LTQ Orbitrap: a new mass spectrometric method combining the benefits of all. J Proteome Res 8:4743–4752CrossRefGoogle Scholar
  16. Kono G, Harada M, Sugisaki K, Nishida M (1977) High glutathione-containing yeast. JP Patent 52(125):687Google Scholar
  17. Kuge S, Jones N, Nomoto A (1997) Regulation of yAP-1 nuclear localization in response to oxidative stress. EMBO J 16:1710–1720CrossRefGoogle Scholar
  18. Lai J, Lee S, Hsieh C, Hwang C, Liao C (2008) Saccharomyces cerevisiae strains for hyper-producing glutathione and γ-glutamylcysteine and process of use. US Patent 7(371):557Google Scholar
  19. Lee J, Godon C, Lagniel G, Spector D, Garin J, Labarre J, Toledano MB (1999) Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J Biol Chem 274:16040–16046CrossRefGoogle Scholar
  20. Li Y, Wei G, Chen J (2004) Glutathione: a review on biotechnological production. Appl Microbiol Biotechnol 66:233–242CrossRefGoogle Scholar
  21. Liang G, Du G, Chen J (2008) Enhanced glutathione production by using low-pH stress coupled with cysteine addition in the treatment of high cell density culture of Candida utilis. Lett Appl Microbiol 46:507–512CrossRefGoogle Scholar
  22. Martin HL, Teismann P (2009) Glutathione–a review on its role and significance in Parkinson’s disease. FASEB J 23:3263–3272CrossRefGoogle Scholar
  23. Menant A, Baudouin-Cornu P, Peyraud C, Tyers M, Thomas D (2006) Determinants of the ubiquitin-mediated degradation of the Met4 transcription factor. J Biol Chem 281:11744–11754CrossRefGoogle Scholar
  24. Nisamedtinov I, Kevvai K, Orumets K, Rautio JJ, Paalme T (2010) Glutathione accumulation in ethanol-stat fed-batch culture of Saccharomyces cerevisiae with a switch to cysteine feeding. Appl Microbiol Biotechnol 87:175–183CrossRefGoogle Scholar
  25. Pócsi I, Prade R, Penninckx M (2004) Glutathione, altruistic metabolite in fungi. Adv Microb Physiol 49:1–76CrossRefGoogle Scholar
  26. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906CrossRefGoogle Scholar
  27. Rouillon A, Barbey R, Patton EE, Tyers M, Thomas D (2000) Feedback-regulated degradation of the transcriptional activator Met4 is triggered by the SCFMet30 complex. EMBO J 19:282–294CrossRefGoogle Scholar
  28. Soltaninassab SR, Sekhar KR, Meredith MJ, Freeman ML (2000) Multi-faceted regulation of γ-glutamylcysteine synthetase. J Cell Physiol 182:163–170CrossRefGoogle Scholar
  29. Sugiyama K, Izawa S, Inoue Y (2000) The Yap1p-dependent induction of glutathione synthesis in heat shock response of Saccharomyces cerevisiae. J Biol Chem 275:15535–15540CrossRefGoogle Scholar
  30. Thomas D, Surdin-Kerjan Y (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol R 61:503–532Google Scholar
  31. Trotter EW, Grant CM (2003) Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems. EMBO Rep 4:184–188CrossRefGoogle Scholar
  32. Wang Z, Tan T, Song J (2007) Effect of amino acids addition and feedback control strategies on the high-cell-density cultivation of Saccharomyces cerevisiae for glutathione production. Process Biochem 42:108–111CrossRefGoogle Scholar
  33. Wen S, Zhang T, Tan T (2006) Maximizing production of glutathione by amino acid modulation and high-cell-density fed-batch culture of Saccharomyces cerevisiae. Process Biochem 41:2424–2428CrossRefGoogle Scholar
  34. Wheeler GL, Trotter EW, Dawes IW, Grant CM (2003) 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 278:49920–49928CrossRefGoogle Scholar
  35. Xu H, Freitas MA (2009) MassMatrix: a database search program for rapid characterization of proteins and peptides from tandem mass spectrometry data. Proteomics 9:1548–1555CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Ildar Nisamedtinov
    • 1
    • 2
    • 4
    Email author
  • Kaspar Kevvai
    • 1
  • Kerti Orumets
    • 1
    • 2
  • Liisa Arike
    • 1
    • 2
  • Inga Sarand
    • 1
    • 2
  • Matti Korhola
    • 3
  • Toomas Paalme
    • 2
  1. 1.Competence Centre of Food and Fermentation TechnologiesTallinnEstonia
  2. 2.Department of Food ProcessingTallinn University of TechnologyTallinnEstonia
  3. 3.Department of BiosciencesUniversity of HelsinkiHelsinkiFinland
  4. 4.Lallemand, Inc.MontréalCanada

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