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Biotechnology and Bioprocess Engineering

, Volume 23, Issue 4, pp 473–479 | Cite as

Selection of Escherichia coli Glutamate Decarboxylase Active at Neutral pH from a Focused Library

  • Chen Yuan Hou
  • Cheeyoon Ahn
  • Byung-Kwan Cho
  • Taek Jin Kang
Research Paper

Abstract

Bacterial glutamate decarboxylase (GAD) converts glutamate (Glu) into γ-aminobutyric acid (GABA) at acidic conditions. Since the reaction consumes a proton per GABA synthesis, cells use this reaction to survive in the acidic environments. Characteristically, the enzyme displays a sigmoidal decrease in its activity as pH rises becoming completely inactive at or above pH 6. This cooperative activity loss is accompanied by several distinct structural changes. Previously, by examining structures at acidic and neutral pH, two key regions had been chosen and mutated to break the cooperativity; Glu89 and C-terminal 15 residues. In this study, we included Asp86 in candidate key residues for mutation to break the cooperativity of GAD. We devised a selection strategy according to which only Escherichia coli cells expressing a variant GAD that was active at neutral pH could survive. In this scheme, an alanine (Ala) auxotroph was rescued by the intracellular synthesis of GABA that was subsequently converted into Ala by heterologously expressed GABA-pyruvate transaminase. New GAD variants were readily selected using this strategy and the most of them indeed had a mutation at residue 86. The results suggest that the role of Asp86 in the wild-type enzyme might be the same as Glu89; to make GAD keep its activity only at acidic environments. Characterization of representative variants are also presented.

Keywords

glutamate decarboxylase directed evolution γ-aminobutyric acid enzyme engineering 

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Notes

Acknowledgements

This work was supported by National Research Foundation of Korea (NRF-2013R1A1A2006018 and 2016M1A5A1027458 to T.J.K and B.-K.C., respectively).

Supplementary material

12257_2018_258_MOESM1_ESM.pdf (961 kb)
Selection of Escherichia coli Glutamate Decarboxylase Active at Neutral pH from a Focused Library

References

  1. 1.
    Ueno, H. (2000) Enzymatic and structural aspects on glutamate decarboxylase. J. Mol. Catal. B Enzym. 10: 67–79.CrossRefGoogle Scholar
  2. 2.
    Diana, M., J. Quilez, and M. Rafecas (2014) Gamma–aminobutyric acid as a bioactive compound in foods: a review. J. Funct. Food. 10: 407–420.CrossRefGoogle Scholar
  3. 3.
    Komatsuzaki, N., J. Shima, S. Kawamoto, H. Momose, and T. Kimura (2005) Production of gamma–aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods. Food Microbiol. 22: 497–504.CrossRefGoogle Scholar
  4. 4.
    Yamano, N., N. Kawasaki, S. Takeda, and A. Nakayama (2013) Production of 2–pyrrolidone from biobased glutamate by using Escherichia coli. J. Polym. Environ. 21: 528–533.CrossRefGoogle Scholar
  5. 5.
    _Lammens, T. M., M. C. R. Franssen, E. L. Scott, and J. P. M. Sanders (2010) Synthesis of biobased N–methylpyrrolidone by one–pot cyclization and methylation of gamma–aminobutyric acid. Green Chem. 12: 1430–1436.CrossRefGoogle Scholar
  6. 6.
    Kawasaki, N., A. Nakayama, N. Yamano, S. Takeda, Y. Kawata, N. Yamamoto, and S. Aiba (2005) Synthesis, thermal and mechanical properties and biodegradation of branched polyamide 4. Polyme. 46: 9987–9993.CrossRefGoogle Scholar
  7. 7.
    Park, S. J., E. Y. Kim, W. Noh, Y. H. Oh, H. Y. Kim, B. K. Song, K. M. Cho, S. H. Hong, S. H. Lee, and J. Jegal (2012) Synthesis of nylon 4 from gamma–aminobutyrate (GABA) produced by recombinant Escherichia coli. Bioprocess Biosyst. Eng. 36: 885–892.CrossRefGoogle Scholar
  8. 8.
    Pham, V. D., S. Somasundaram, S. H. Lee, S. J. Park, and S. H. Hong (2016) Engineering the intracellular metabolism of Escherichia coli to produce gamma–aminobutyric acid by colocalization of GABA shunt enzymes. Biotechnol. Lett. 38: 321–327.CrossRefGoogle Scholar
  9. 9.
    Pham, V. D., S. Somasundaram, S. H. Lee, S. J. Park, and S. H. Hong (2016) Gamma–aminobutyric acid production through GABA shunt by synthetic scaffolds introduction in recombinant Escherichia coli. Biotechnol. Bioprocess Eng. 21: 261–267.CrossRefGoogle Scholar
  10. 10.
    Lammens, T. M., D. De Biase, M. C. R. Franssen, E. L. Scott, and J. P. M. Sanders (2009) The application of glutamic acid a–decarboxylase for the valorization of glutamic acid. Green Chem. 11: 1562–1567.CrossRefGoogle Scholar
  11. 11.
    De Biase, D., A. Tramonti, R. A. John, and F. Bossa (1996) Isolation, overexpression, and biochemical characterization of the two isoforms of glutamic acid decarboxylase from Escherichia coli. Protein Expr. Purif. 8: 430–438.CrossRefGoogle Scholar
  12. 12.
    Capitani, G., D. De Biase, C. Aurizi, H. Gut, F. Bossa, and M. G. Grutter (2003) Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase. EMBO J. 22: 4027–4037.CrossRefGoogle Scholar
  13. 13.
    Pennacchietti, E., T. M. Lammens, G. Capitani, M. C. R. Franssen, R. A. John, F. Bossa, and D. De Biase (2009) Mutation of His465 alters the pH–dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity towards more alkaline pH. J. Biol. Chem. 284: 31287–31596.CrossRefGoogle Scholar
  14. 14.
    Thu Ho, N. A., C. Y. Hou, W. H. Kim, and T. J. Kang (2013) Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness. J. Biosci. Bioeng. 115: 154–158.CrossRefGoogle Scholar
  15. 15.
    Choi, J. W., S. S. Yim, S. H. Lee, T. J. Kang, S. J. Park, and K. J. Jeong (2015) Enhanced production of gamma–aminobutyrate (GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range. Microb. Cell Fact. 14: 21.CrossRefGoogle Scholar
  16. 16.
    Chae, T. U., Y. S. Ko, K. S. Hwang, and S. Y. Lee (2017) Metabolic engineering of Escherichia coli for the production of four–, five–and six–carbon lactams. Metab. Eng. 41: 82–91.CrossRefGoogle Scholar
  17. 17.
    Hou, C. Y. and T. J. Kang (2018) Production of gammaaminobutyric acid by Escherichia coli using glycerol as a sole carbon source. J. Chem. Technol. Biotechnol. 93: 184–190.CrossRefGoogle Scholar
  18. 18.
    Shi, F., Y. L. Xie, J. J. Jiang, N. N. Wang, Y. F. Li, and X. Y. Wang (2014) Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near–neutral pH. Enzyme Microb. Technol. 61–62: 35–43.CrossRefGoogle Scholar
  19. 19.
    Van Cauwenberghe, O. R., A. Makhmoudova, M. D. McLean, S. M. Clark, and B. J. Shelp (2002) Plant pyruvate–dependent gamma–aminobutyrate transaminase: identification of an Arabidopsis cDNA and its expression in Escherichia coli. Can. J. Bot. 80: 933–941.CrossRefGoogle Scholar
  20. 20.
    Yoneyama, H., H. Hori, S. J. Lim, T. Murata, T. Ando, E. Isogai, and R. Katsumata (2011) Isolation of a mutant auxotrophic for L–alanine and identification of three major aminotransferases that synthesize L–alanine in Escherichia coli. Biosci. Biotechnol. Biochem. 75: 930–938.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Chen Yuan Hou
    • 1
  • Cheeyoon Ahn
    • 1
  • Byung-Kwan Cho
    • 2
  • Taek Jin Kang
    • 1
  1. 1.Department of Chemical and Biochemical EngineeringDongguk University-SeoulSeoulKorea
  2. 2.Department of Biological Sciences and KI for the BioCenturyKorea Advanced Institute of Science and TechnologyDaejeonKorea

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