Abstract
Glutathione (GSH) is an important reducing agent in the living cells. It is synthesized by a two-step reaction and requires two molecules of adenosine triphosphate (ATP) for one molecule GSH. The enzymatic cascade reaction in vitro is a promising approach to achieve a high titer and limit side reactions; although, a cost-effective phosphate donor for ATP regeneration is required. Triphosphate (PolyP(3)), tetraphosphate (PolyP(4)), and hexametaphosphate (PolyP(6)) were investigated in this study. Triphosphate inhibited the bifunctional GSH synthetase (GshF) from Streptococcus agalactiae, while no significant inhibition was observed by adding hexametaphosphate. The polyphosphate kinase from Corynebacterium glutamicum was hence investigated to use hexametaphosphate for regeneration of ATP. Further, the orthogonal experiment, which includes seven factors (buffer concentration, pH value, ADP concentration, GshF dosage, polyphosphate kinase (PPK) dosage, reaction temperature, substrate ratio of amino acid, and reaction times), indicated that the capacity of buffer is the most significant factor of the reaction conditions for enzymatic production of glutathione coupling with a PPK-based ATP regeneration system. After optimizing the Mg2+ concentration, the reaction was scaled up to 250 mL in a stirred reactor with pH feedback control to stabilize the pH value of reaction system and nitrogen protection to avoid the oxidation of product. A yield of 12.32 g/L was achieved. This work provided a potential GshF-based enzymatic way coupling the PPK-based ATP regeneration to product GSH in the optimal conditions towards cost-effectiveness at the industrial scale.
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References
Meister, A., & Anderson, M. E. (1983). Glutathione. Annual Review of Biochemistry, 52(1), 711–760. https://doi.org/10.1146/annurev.bi.52.070183.003431.
Li, Y., Wei, G., & Chen, J. (2004). Glutathione: a review on biotechnological production. Applied Microbiology and Biotechnology, 66(3), 233–242. https://doi.org/10.1007/s00253-004-1751-y.
Penninckx, M. J., & Elskens, M. T. (1993). Metabolism and functions of glutathione in micro-organisms. Advan. Microb Physiol., 34, 239–301.
Wang, J., Gong, S., Xu, D., Sui, J., & Mu, J. (2005). A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis. Journal of Bacteriology, 187, 3839–3847.
Janowiak, B. E., & Griffith, O. W. (2005). Glutathione synthesis in Streptococcus agalactiae one protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities. The Journal of Biological Chemistry, 280(12), 11829–11839. https://doi.org/10.1074/jbc.M414326200.
Iwamoto, S., Motomura, K., Shinoda, Y., Urata, M., Kato, J., Takiguchi, N., Ohtake, H., Hirota, R., Kuroda and Akio. (2007) Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate. Applied and Environmental Microbiology 73, 5676, 17, 5678, DOI: https://doi.org/10.1128/AEM.00278-07.
Andexer, J. N., & Richter, M. (2015). Emerging enzymes for ATP regeneration in biocatalytic processes. Chem. Bio. Chem., 16(3), 380–386. https://doi.org/10.1002/cbic.201402550.
Caschera, F., & Noireaux, V. (2015). A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. Metabolic Engineering, 27, 29–37. https://doi.org/10.1016/j.ymben.2014.10.007.
Del Campo, J. S. M., Chun, Y., Kim, J. E., Patiño, R., & Zhang, Y. H. (2013). Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima. Journal of Industrial Microbiology & Biotechnology, 40(7), 661–669. https://doi.org/10.1007/s10295-013-1265-7.
Resnick, S. M., & Zehnder, A. J. (2000). In vitro ATP regeneration from polyphosphate and AMP by polyphosphate: AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A. Applied and Environmental Microbiology, 66(5), 2045–2051. https://doi.org/10.1128/AEM.66.5.2045-2051.2000.
Li, W., Li, Z., Yang, J., & Ye, Q. (2011). Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. Journal of Biotechnology, 54, 261–268.
Zhang, X., Wu, H., Huang, B., Li, Z., & Ye, Q. (2017). One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system. Journal of Biotechnology, 241, 163–169. https://doi.org/10.1016/j.jbiotec.2016.11.034.
Jiang, Y., Tao, R., Shen, Z., Sun, L., Zhu, F., & Yang, S. (2016). Enzymatic production of glutathione by bifunctional γ-glutamylcysteine synthetase/glutathione synthetase coupled with in vitro acetate kinase-based ATP generation. Appl. Biochem Biotech, 180, 1446–1455.
Vergauwen, B., De Vos, D., & Van Beeumen, J. J. (2006). Characterization of the bifunctional γ-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida. The Journal of Biological Chemistry, 281(7), 4380–4394. https://doi.org/10.1074/jbc.M509517200.
Lindner, S. N., Vidaurre, D., Willbold, S., Schoberth, S. M., & Wendisch, V. F. (2007). NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum. Applied and Environmental Microbiology, 73(15), 5026–5033. https://doi.org/10.1128/AEM.00600-07.
Nocek, B., Kochinyan, S., Proudfoot, M., Brown, G., Evdokimova, E., Osipiuk, J., Edwards, A. M., Savchenko, A., Joachimiak, A., & Yakunin, A. F. (2008). Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria. Proc. Nat Academy of Sciences, 105(46), 17730–17735. https://doi.org/10.1073/pnas.0807563105.
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This study was funded by the National 973 Basic Research Program of China (grant number 2014CB745100) and National Natural Science Foundation of China (grant number 21676016).
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Cao, H., Li, C., Zhao, J. et al. Enzymatic Production of Glutathione Coupling with an ATP Regeneration System Based on Polyphosphate Kinase. Appl Biochem Biotechnol 185, 385–395 (2018). https://doi.org/10.1007/s12010-017-2664-4
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DOI: https://doi.org/10.1007/s12010-017-2664-4