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 KangEmail author
Research Paper


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.


glutamate decarboxylase directed evolution γ-aminobutyric acid enzyme engineering 


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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


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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
    Email author
  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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