Abstract
The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.
Similar content being viewed by others
References
Meister, A. and M. E. Anderson (1983) Glutathione. Annu. Rev. Biochem. 52: 711–760.
Izawa, S., Y. Inoue, and A. Kimura (1995) Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae. FEBS Lett. 368: 73–76.
Penninckx, M. (2000) A short review on the role of glutathione in the response of yeast to nutritional, environmental, and oxidative stresses. Enzyme Microb. Technol. 26: 737–742.
Sies, H. (1999) Glutathione and its role in cellular functions. Free Radic. Biol. Med. 27: 916–921.
Murata, K. and A. Kimura (1990) Overproduction of glutathione and its derivatives by genetically engineered microbial cells. Biotechnol. Adv. 8: 59–96.
Penninckx, M. J. (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res. 2: 295–305.
Van Urk, H., W. S. Leopold Voll, W. Alexander Scheffers, and J. P. van Dijken (1990) Transient-state analysis of metabolic fluxes in Crabtree-positive and Crabtree-negative yeasts. Appl. Environ. Microbiol. 56: 281–287.
Rodriguez, L., F. P. Chavez, L. Basabe, T. Rivero, and J. M. Delgado (1998) Development of an integrative DNA transformation system for the yeast Candida utilis. FEMS Microbiol. Lett. 165: 335–340.
Castrillo, J. I., J. Kaliterna, R. A. Weusthuis, J. P. van Dijken, and J. T. Pronk (1996) High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures. Biotechnol. Bioeng. 49: 621–628.
Espinel, A. E., V. Gomez-Toribio, and J. M. Peinado (1996) The inactivation of hexokinase activity does not prevent glucose repression in Candida utilis. FEMS Microbiol. Lett. 135: 327–332.
Divjak, S. and J. R. Mor (1973) On the activity of carbon dioxide fixation in growing yeasts. Arch. Microbiol. 94: 191–199.
Shay, L. K. and G. H. Wegner (1985) Improved fermentation process for producing Torula yeast. Food Technol. 39: 61–66.
Watanabe, K., N. Kato, and N. Yoshida (1989) Glutathione, its manufacture with yeast, and the effects of complex amino acids in medium. JP 01,148,181.
Alfafara, C. G., K. Miura, H. Shimizu, S. Shioya, and K. Suga (1992) Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 37: 141–146.
Wen, S., T. Zhang, and T. Tan (2004) Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae. Enzyme Microb. Technol. 35: 501–507.
Wei, G. Y., Y. Li, G. C. Du, and J. Chen (2003) Application of a two-stage temperature control strategy for enhanced glutathione production in the batch fermentation by Candida utilis. Biotechnol. Lett. 25: 887–890.
Wei, G. Y., Y. Li, G. C. Du, and J. Chen (2005) Fedbatch fermentative production of glutathione by Candida utilis. Chin. J. Process Eng. 5: 327–331.
Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Anal. Biochem. 27: 502–522.
Miller, G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426–428.
Maw, G. A. (1961) Effects of cysteine and other thiols on the growth of a brewer’s yeast. J. Inst. Brew. 67: 57–63.
Alfafara, C. G., A. Kanda, T. Shioi, H. Shimizu, S. Shioya, and K. Suga (1992) Effect of amino acids on glutathione production by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 36: 538–540.
Shimizu, H., K. Araki, S. Shioya, and K. Suga (1991) Optimal production of glutathione by controlling the specific growth rate of yeast in fed-batch culture. Biotechnol. Bioeng. 38: 196–205.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wei, GY., Wang, DH. & Chen, J. Overproduction of glutathione by l-cysteine addition and a temperature-shift strategy. Biotechnol Bioproc E 13, 347–353 (2008). https://doi.org/10.1007/s12257-007-0191-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-007-0191-9