Optimizing the Addition of Amino Acids in the Production of Glutathione by Recombinant Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2015.09.028

Abstract

Abstract: The condition of amino acids was optimized in the production of glutathione(GSH)by recombinant Escherichia coli expressing bifunctional glutathione synthetase. Only 0.81 g/L of GSH was produced without adding any amino acid, whereas 1.16 g/L of GSH was formed when 17 mmol/L of cysteine was fed into the medium after 2 h induction, increasing 43% compared to no amino acid added. With the feeding 17 mmol/L of 3 precusor amino acids, the production was improved significantly and achieved at 3.86 g/L, which was 2.33 times of single cysteine addition. By increasing the amount of 3 amino acids to 25 mmol/L, GSH was 4.64 g/L, 4.73 times as no amino acid added, and the overall productivity was 317.8 mg/(L·h), the conversion rate of cysteine to glutathione was 0.60 mol/mol. While concerning the addition mode, the production rate of GSH was 3.09 g/(L·h) when 25 mmol/L of amino acids was once added to the fermenter, only 2.38 g/(L·h) when 25 mmol/L of amino acids added at constant speed and continuously, so the productivity increased 29.8% by adding once. The process was successfully scaled up in the 50 L fermenter and 4.31 g/L of GSH was produced with an overall productivity of 310.1 mg/(L·h), which laid a foundation for the industrial production of GSH.

Key words: glutathione, amino acid, cysteine, recombinant Escherichia coli

Wang Dezheng, Wu Hui, Li Zhimin, Ye Qin. Optimizing the Addition of Amino Acids in the Production of Glutathione by Recombinant Escherichia coli[J]. Biotechnology Bulletin, 2015, 31(9): 197-203.

References

[1]Meister A, Anderson M. Glutathione[J]. Annual Review of Biochemistry, 1983, 52(1)：711-760.
[2]Pastore A, Federici G, Bertini E, et al. Analysis of glutathione：implication in redox and detoxification[J]. Clinica Chimica Acta, 2003, 333(1)：19-39.
[3]Sies H. Glutathione and its role in cellular functions[J]. Free Radical Biology and Medicine, 1999, 27(9)：916-921.
[4]Liao X, Shen W, Chen J, et al. Improved glutathione production by gene expression in Escherichia coli[J]. Letters in Applied Microbiology, 2006, 43(2)：211-214.
[5]沈蓓英, 江志炜. 具有生物活性新型功能性食品添加剂谷胱甘肽[J]. 粮食与油脂, 1993, 6(2)：27-32.
[6]袁尔东, 郑建仙. 功能性食品基料—谷胱甘肽的研究进展[J]. 食品与发酵工业, 1999, 25(5)：52-57.
[7]Li W, Li Z, Yang J, et al. Production of glutathione using a bifunctio-nal enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli[J]. Journal of Biotechnology, 2011, 154(4)：261-268.
[8]Janowiak B, Griffith O. Glutathione synthesis in Streptococcus agalactiae：one protein accounts for γ-glutamylcysteine synthetase and glutathione synthetase activities[J]. Journal of Biological Chemistry, 2005, 280(12)：11829-11839.
[9]Gopal S, Borovok I, Ofer A, et al. A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis[J]. Journal of Bacteriology, 2005, 187(11)：3839-3847.
[10]Alfafara C, Kanda A, Shioi T, et al. Effect of amino acids on glutathione production by Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology, 1992, 36(4)：538-540.
[11]Wen S, Zhang T, Tan T. Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae[J]. Enzyme and Microbial Technology, 2004, 35(6)：501-507.
[12]Li Y, Chen J, Mao Y, et al. Effect of additives and fed-batch culture strategies on the production of glutathione by recombinant Escherichia coli[J]. Process Biochemistry, 1998, 33(7)：709-714.
[13]Wang Z, Tan T, Song J. Effect of amino acids addition and feedback control strategies on the high-cell-density cultivation of Saccharomyces cerevisiae for glutathione production[J]. Process Biochemistry, 2007, 42(1)：108-111.
[14]Alfafara C, Miura K, Shimizu H, et al. Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology, 1992, 37(2)：141-146.
[15]Liang G, Liao X, Du G, et al. Optimization of amino acids addition for efficient production of glutathione in Candida utilis[J]. Biochemical Engineering Journal, 2008, 41(3)：234-240.
[16]Wen S, Zhang T, Tan T. Maximizing production of glutathione by amino acid modulation and high-cell-density fed-batch culture of Saccharomyces cerevisiae[J]. Process Biochemistry, 2006, 41(12)：2424-2428.
[17]Liang G, Liao X, Du G, et al. Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis[J]. Journal of Applied Microbiology, 2008, 105(5)：1432-1440.
[18]Fei L, Wang Y, Chen S. Improved glutathione production by gene expression in Pichia pastoris[J]. Bioprocess and Biosystems Engineering, 2009, 32(6)：729-735.