Applied Microbiology and Biotechnology

, Volume 91, Issue 4, pp 1001–1006 | Cite as

Enzymatic glutathione production using metabolically engineered Saccharomyces cerevisiae as a whole-cell biocatalyst

  • Hideyo Yoshida
  • Kiyotaka Y. Hara
  • Kentaro Kiriyama
  • Hideki Nakayama
  • Fumiyoshi Okazaki
  • Fumio Matsuda
  • Chiaki Ogino
  • Hideki Fukuda
  • Akihiko Kondo
Biotechnological Products and Process Engineering

Abstract

We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.

Keywords

Glutathione ATP Yeast Permeated cell Biocatalyst 

Notes

Acknowledgments

We are grateful to Dr. J. Ishii (Organization of Advanced Science and Technology, Kobe University) for providing us with pGK402 and pGK405 plasmids. We thank Dr. Takashi Kondo, Dr. Naoko Okai, and Dr. Kazunori Nakashima (Organization of Advanced Science and Technology, Kobe University) for their helpful discussion. This study was supported by the Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan. Hara KY was supported by Grant-in-Aid for Young Scientists (B) (18769004, 22760608).

References

  1. Chen DC, Yang BC, Kuo TT (1992) One-step transformation of yeast in stationary phase. Curr Genet 21:83–84CrossRefGoogle Scholar
  2. Chow CKC, Palecek SP (2004) Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells. Biotechnol Prog 20:449–456CrossRefGoogle Scholar
  3. Dröge W, Breitkreutz R (2000) Glutathione and immune function. Proc Nutr Soc 59:595–600CrossRefGoogle Scholar
  4. Eydallin G, Viale AM, Morán-Zorzano MT, Muñoz FJ, Montero M, Baroja-Fernández E, Pozueta-Romero J (2007) Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12. FEBS Lett 581:2947–2953CrossRefGoogle Scholar
  5. Eydallin G, Montero M, Almagro G, Sesma MT, Viale AM, Muñoz FJ, Rahimpour M, Baroja-Fernández E, Pozueta-Romero J (2010) Genome-wide screening of genes whose enhanced expression affects glycogen accumulation in Escherichia coli. DNA Res 17:61–71CrossRefGoogle Scholar
  6. Flohé L (1985) The glutathione peroxidase reaction: molecular basis of the antioxidant function of selenium in mammals. Curr Top Cell Regul 27:473–478Google Scholar
  7. Hara KY, Mori H (2006) An efficient method for quantitative determination of cellular ATP synthetic activity. J Biomol Screen 11:310–317CrossRefGoogle Scholar
  8. Hara KY (2009a) Permeable cell assay: a method for high-throughput measurement of cellular ATP synthetic activity. Methods Mol Biol. 577:251–258CrossRefGoogle Scholar
  9. Hara KY, Shimodate N, Hirokawa Y, Ito M, Baba T, Mori H, Mori H (2009b) Glutathione production by efficient ATP-regenerating Escherichia coli mutants. FEMS Microbiol Lett 297:217–224CrossRefGoogle Scholar
  10. Ishii J, Izawa K, Matsumura S, Wakamura K, Tanino T, Tanaka T, Ogino C, Fukuda H, Kondo A (2009) A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast. J Biochem 145:701–708CrossRefGoogle Scholar
  11. Li Y, Wei G, Chen J (2004) Glutathione: a review on biotechnological production. Appl Microbiol Biotechnol 66:233–242CrossRefGoogle Scholar
  12. Li W, Li Z, Ye Q (2010) Enzymatic synthesis of glutathione using yeast cells in two-stage reaction. Bioprocess Biosyst Eng 33:675–682CrossRefGoogle Scholar
  13. Liao X, Deng T, Zhu Y, Du G, Chen J (2007) Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae. J Appl Microbiol 104:345–352CrossRefGoogle Scholar
  14. Meister A, Andersen ME (1983) Glutathione. Annu Rev Biochem 52:711–760CrossRefGoogle Scholar
  15. Murata K, Tani K, Kato J, Chibata I (1981) Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP. Enzyme Microb Technol 3:233–242CrossRefGoogle Scholar
  16. Penninckx MJ (2000) A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses. Enzyme Microb Technol 26:737–742CrossRefGoogle Scholar
  17. Penninckx MJ (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res 2:295–305Google Scholar
  18. Ray S, Watkins DN, Misso NL, Thompson PJ (2002) Oxidant stress induces gamma-glutamylcysteine synthetase and glutathione synthesis in human bronchial epithelial NCI-H292 cells. Clin Exp Allergy 32:571–577CrossRefGoogle Scholar
  19. Rolseth V, Djurhuus R, Svardal AM (2002) Additive toxicity of limonene and 50% oxygen and the role of glutathione in detoxification in human lung cells. Toxicology 170:75–88CrossRefGoogle Scholar
  20. Singh RJ (2002) Glutathione: a marker and antioxidant for aging. J Lab Clin Med 140:380–381CrossRefGoogle Scholar
  21. Tate S, Meister A (1981) γ-Glutamyltranspeptidase: catalytical, structural and functional aspects. Mol Cell Biochem 39:357–368CrossRefGoogle Scholar
  22. Thon VJ, Vigneron-Lesens C, Marianne-Pepin T, Montreuil J, Decq A, Rachez C, Ball SG, Cannon JF (1992) Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme. J Biol Chem 267:15224–15228Google Scholar
  23. Vartanyan LS, Gurevich S, Kozachenko AI, Nagler LG, Lozovskaya EL, Burlakova EB (2000) Changes in superoxide production rate and in superoxide dismutase and glutathione peroxidase activities in subcellular organelles in mouse liver under exposure to low doses of low-intensity radiation. Biochem (Mosc) 65:442–446Google Scholar
  24. Wei G, Li Y, Du G, Chen J (2003) Effect of surfactants on extracellular accumulation of glutathione by Saccharomyces cerevisiae. Process Biochem 38:1133–1138CrossRefGoogle Scholar
  25. Wilson WA, Hughes WE, Tomamichel W, Roach PJ (2004) Increased glycogen storage in yeast results in less branched glycogen. Biochem Biophys Res Commun 320:416–423CrossRefGoogle Scholar
  26. Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A (2010) Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized. Microb Cell Fact 9:32–39CrossRefGoogle Scholar
  27. Yoshida K, Hariki T, Inoue H, Nakamura T (2002) External skin preparation for whitening. JP Patent 2, 002, 284, 664Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hideyo Yoshida
    • 1
  • Kiyotaka Y. Hara
    • 2
  • Kentaro Kiriyama
    • 1
  • Hideki Nakayama
    • 2
  • Fumiyoshi Okazaki
    • 2
  • Fumio Matsuda
    • 2
  • Chiaki Ogino
    • 1
  • Hideki Fukuda
    • 1
  • Akihiko Kondo
    • 1
  1. 1.Department of Chemical Science and Engineering, Graduate School of EngineeringKobe UniversityKobeJapan
  2. 2.Organization of Advanced Science and TechnologyKobe UniversityKobeJapan

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