Glutathione Production by Yarrowia lipolytica Showing Both Methyglyoxal Resistance and a High Activity of Glutathione Synthetase

Naofumi Shiomi, Emi Harada, Risako Nabeshima

Graduate School of Human Sciences, Kobe College, Kobe, Japan.

DOI: 10.4236/aim.2012.22022                

Abstract

Although Yarrowia lipolytica is an important host strain, there have so far been few studies on the production of glutathione by the strain. We therefore performed a study to obtain an improved strain of Y. lipolytica ATCC20688, which could produce a high yield of glutathione. First, the capability of glutathione production in the ATCC20688 strain was estimated. In comparison with other yeasts, the yield of this strain was higher than those in Pichia strains. Furthermore, this strain could produce glutathione by assimilating sodium oleate. We next performed mutation and gene cloning to improve the yield. After the yield of glutathione was improved in the isolated methylglyoxal-resistant mutant (MGR3), the glutathione synthetase gene was cloned into the MGR3 strain. By using this recombinant strain, we could reach the maximum yield and intracellular content of glutathione of 54 mg/L-medium and 30 mg/g-dry cell weight, respectively.

Keywords

Glutathione; Yarrowia lipolytica; Glutathione Synthetase; GSH2; Methylglyoxal

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	M. E. Anderson, “Glutathione: An Overview of Biosynthesis and Modulation,” Chemico-Bioligal Interactions, Vol. 111-112, 1998, pp. 1-14. doi:10.1016/S0009-2797(97)00146-4
[2]	Y. Li, G. Wei and J. Chen, “Glutathione: A Review on Biotechnological Production,” Applied Microbiology and Biotechnology, Vol. 66, No. 3, 2004, pp. 233-242. doi:10.1007/s00253-004-1751-y
[3]	E. Beutler, “Nutritional and Metabolic Aspects of Glutathione,” Annual Review of Nutrition, Vol. 9, 1989, pp. 287-302. doi:10.1146/annurev.nu.09.070189.001443
[4]	B. M. Lomaestro and M. Malone, “Glutathione in Health and Disease: Pharmacotherapeutic Issues,” The Annals of Pharmacotherapy, Vol. 29, No. 12, 1995, pp. 1263-1273.
[5]	C. Alfafara, K. Miura, H. Shimizu, S. Shioya and K. Suga, “Cysteine Addition Strategy for Maximum Glutathione Production in Fed-Batch Culture of Saccharomyces cerevisiae,” Applied Microbiology and Biotechnology, Vol. 37, No. 2, 1992, pp. 141-146. doi:10.1007/BF00178160
[6]	K. O. Udeh and B. Achremowicz, “High-Glutathione Containing Yeast Sacchromyces cerevisiae: Optimization and Production,” Acta Microbiologica Polonica, Vol. 46, No. 1, 1997, pp. 105-114.
[7]	J. Y. Cha, J. C. Park, B. S. Jeon, Y. C. Lee and Y. S. Cho, “Optimal Fermentation Conditions for Enhanced Glutathione Production by Saccharomyces cerevisiae FF-8,” Journal of Microbiology, Vol. 42, No. 1, 2004, pp. 51-55.
[8]	K. Sakato and H. Tanaka, “Advanced Control of Glutathione Fermentation Process,” Biotechnology and Bioengineering, Vol. 40, No. 8, 1992, pp. 904-912. doi:10.1002/bit.260400806
[9]	W. Nie, G. Wei, G. Du and Y. Li, J. Chen, “Enhanced Intracellular Glutathione Synthesis and Excretion Capability of Candida utilis by Using a Low pH-Stress Strategy,” Letters in Applied Microbiology, Vol. 40, No. 5, 2005, pp. 378-384. doi:10.1111/j.1472-765X.2005.01687.x
[10]	Y. Ohtake and S. Yabuuchi,“Molecular Cloning of the Gamma-Glytamylcysteine Synthetase Gene of Sacchromyces cerevisiae,” Yeast, Vol. 7, No. 9, 1991, pp. 953-961. doi:10.1002/yea.320070907
[11]	Y. Inoue, K. Sugiyama, S. Izawa and A. Kimura, “Molecular Identification of Glutathione Synthetase (GSH2) Gene from Saccharomyces cerevisiae,” Biochimica Biophysoca Acta, Vol. 1395, No. 3, 1998, pp. 315-320.
[12]	V. M. Ubiyvovk, T. Y. Nazarko, O. G. Stasyk, M. J. Sohn, H. A. Kang and A. A. Sibirny, “GSH2, a Gene Encoding Gamma-Glutamylcysteine Synthetase in the Methyltrophic Yeast,” FEMS Yeast Research, Vol. 2, No. 3, 2002, pp. 327-332.
[13]	L. Yin, J. Chen and L. Shiyi,“Production of Glutathione by High Cell Density Culture of Recombinant Escherichia coli,” Chinese Journal of Pharmaceticls, Vol. 30, No. 1, 1999, pp. 1-4.
[14]	M. Rollini, H. Pagani, S. Riboldi and M. Manzoni, “Influence of Carbon Source on Glutathione Accumulation in Methyltrophic Yeasts,” Annals of Microbiology, Vol. 55, No. 3, 2005, pp. 199-203.
[15]	H. P. Fickers, P. H. Benetti, Y. Waché, A. Marty, S. Mauersberger, M. S. Smit and J. M. Nicaud, “Hydrophobic Substrate Utilization by the Yeast Yarrowia lipolytica, and Its Potential Applications,” FEMS Yeast Research, Vol. 5, No. 607, 2005, pp. 527-543. doi:10.1016/j.femsyr.2004.09.004
[16]	N. Shiomi, Y. Inoue, K. Tomioka and T. Yasuda, “Immobilization of Candida Cells Showing Mycelium-Like Shapes in Porous Polyvinyl Formal Resin and Their Applications,” Journal of Chemical Engineering of Japan, Vol. 36, No. 2, 2003, pp.161-165. doi:10.1252/jcej.36.161
[17]	T. H. Kim, J. H. Lee, Y. S. Bae and S. J. Kim, “Identification and Characterization of an Oil-Degrading Yeast, Yarrowia lipolytica 180,” Journal of Microbiology, Vol. 37, No. 3, 1999, pp. 128-135.
[18]	S. Mauersberger, H. J. Wang, C. Gaillardin, G. Barth and J. M. Nicaud, “Insertional Mutagenesis in then-Alkane-Assimilating Yeast Yarrowia lipolytica: Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization,” Journal of Bacteriology, Vol. 183, No. 17, 2001, pp. 5102-5109. doi:10.1128/JB.183.17.5102-5109.2001
[19]	P. Founier, A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J. W. Xuan, A. Peito, A. M. Ribet, C. Feyrol, F. He and C. Gillardin, “Colocalization of Centromeric and Replicative Functions on Autonomously Replicating Sequences Isolated from the Yeast Yarrowia lipolytica,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 11, 1993, pp. 4912-4916. doi:10.1073/pnas.90.11.4912
[20]	C. A. Strick, L. C. James, M. M. O’Donnell, M. G. Gollaher and A. E. Franke, “The Isolation and Characterization of the Pyruvate Kinase-Encoding Gene from the Yeast Yarrowia lipolytica,” Gene, Vol. 118, No. 1, 1992, pp. 65-72. doi:10.1016/0378-1119(92)90249-O
[21]	G. Pignede, H. Wang, F. Fudalej, C. Gaillardin, M. Seman and J. M. Nicaud, “Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica,” Journal of Bacteriology, Vol. 182, No. 10, 2000, pp. 2802-2810. doi:10.1128/JB.182.10.2802-2810.2000
[22]	C. W. Owens and R. V. Belcher, “A Colorimetric Micro-Method for the Determination of Glutathione,” The Biochemical Journal, Vol. 94, No. 3, 1965, pp. 705-711.
[23]	Y. Inoue, H. Rhee, K. Watanabe, K. Murata and A. Kimura, “Metabolism of 2-Ketoaldehydes in Mold: Purification and Characterization of Glyoxalase I from Aspergillusniger,” The Journal of Biochemistry, Vol. 102, No. 3, 1987, pp. 583-589.
[24]	R. E. Allen, T. W. C. Lo and P. J. Thornalley, “Purification and Characterization of Glyoxalase II from Human Red Blood Cells,” European Journal of Biochemistry, Vol. 213, No. 3, 1993, pp.1261-1267. doi:10.1111/j.1432-1033.1993.tb17877.x
[25]	C. C. White, H. Viernes, C. M. Krejsa, D. Botta and T. J. Kavanagh, “Fluorescence-Based Microtiter Plate Assay for Glutamate-Cysteine Ligase Activity,” Analytical Biochemistry, Vol. 318, No. 2, 2003, pp. 175-180. doi:10.1016/S0003-2697(03)00143-X
[26]	N. Shiomi, H. Fukuda, F. Yasuki, K. Murata and A. Kimura, “Expression and Stability of the Plasmid Carring Ethionine-Resistant Gene in Saccharomyces cerevisiae”, Kagakukougakuronbunsyu, Vol. 17, No. 3, 1991, pp. 674-678.
[27]	Y. Inoue and A. Kimura, “Methylglyoxal and Regulation of Its Metabolism in Microorganisms,” Advances in Microbial Physiology, Vol. 37, 1995, pp. 177-227. doi:10.1016/S0065-2911(08)60146-0
[28]	C. S. Newlon, “Yeast Chromosome Replication and Segregation,” Microbiological Reviews, Vol. 52, No. 4, 1988, pp. 568-601.
[29]	A. B. Fucher and B. S. Cox, “Copy Number and the Stability of 2-Micron Circle-Based Artificial Plasmids of Saccharomyces cerevisiae,” Journal of Bacteriology, Vol. 157, No. 1, 1984, pp. 283-290.
[30]	T. Juretzek, M. T. Le Dall, S. Mauersberger and C. Gaillardin, “Vectors for Gene Expression and Amplification in the Yeast Yarrowia lipolytica,” Yeast, Vol. 18, No. 2, 2001, pp. 97-113. doi:10.1002/1097-0061(20010130)18:2<97::AID-YEA652>3.0.CO;2-U
[31]	M. T. Le Dall and J. M. Nicaud, “Multiple-Copy Integration in the Yeast Yarrowia lipolytica,” Current Genetics, Vol. 26, No. 1, 1994, pp. 38-44. doi:10.1007/BF00326302