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Research article

Biotechnology and Bioengineering (BB)  |  Enzyme, Protein Engineering, and Metabolic Engineering

Enchancement of Gamma-Aminobutyric Acid Production by Co-Localization of Neurospora crassa OR74A Glutamate Decarboxylase with Escherichia coli GABA Transporter Via Synthetic Scaffold Complex

Sivachandiran Somasundaram 1, Murali kannan Maruthamuthu 1, Irisappan Ganesh 2, Gyeong Tae Eom 3, 4 and Soon Ho Hong 1*

1School of Chemical Engineering, University of Ulsan, Ulsan 44610, Republic of Korea, 2Bioenergy and Biochemical Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea, 3Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), Ulsan 44429, Republic of Korea, 3Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), Daejeon 34144, Republic of Korea

Received: November 11, 2016; Accepted: July 10, 2017

J. Microbiol. Biotechnol. 2017; 27(9): 1664-1669

Published September 28, 2017 https://doi.org/10.4014/jmb.1611.11041

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Gamma-aminobutyric acid is a precursor of nylon-4, which is a promising heat-resistant biopolymer. GABA can be produced from the decarboxylation of glutamate by glutamate decarboxylase. In this study, a synthetic scaffold complex strategy was employed involving the Neurospora crassa glutamate decarboxylase (GadB) and Escherichia coli GABA antiporter (GadC) to improve GABA production. To construct the complex, the SH3 domain was attached to the N. crassa GadB, and the SH3 ligand was attached to the N-terminus, middle, and C-terminus of E. coli GadC. In the C-terminus model, 5.8 g/l of GABA concentration was obtained from 10 g/l glutamate. When a competing pathway engineered strain was used, the final GABA concentration was further increased to 5.94 g/l, which corresponds to 97.5% of GABA yield. With the introduction of the scaffold complex, the GABA productivity increased by 2.9 folds during the initial culture period.

Keywords

Gamma-aminobutyric acid, glutamate decarboxylase, glutamate/GABA antiporter, Neurospora crassa, synthetic protein scaffold

References

  1. Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW. 1999. Control of acid resistance in Escherichia coli. J. Bacteriol. 181: 3525-3535.
    Pubmed PMC
  2. Kim SH, Shin BH, Kim YH, Nam SW, Jeon SY. 2007. Cloning and expression of a full-length glutamate decarboxylase gene from Lactobacillus brevis BH2. Biotechnol. Bioprocess Eng. 12:707-712.
    CrossRef
  3. Saskiawan I. 2008. Biosynthesis of polyamide 4, a biobased and biodegradable polymer. Microbiol. Indonesia 2: 119-123.
    CrossRef
  4. Capitani G, De Biase D, Aurizi C, Gut H, Bossa F, Gruetter GM. 2003. Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase. EMBO J. 22: 4027-4037.
    Pubmed PMC CrossRef
  5. Park KB, Oh SH. 2006. Enhancement of gama-aminobutyric acid production in chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis. Biotechnol. Lett. 28: 1459-1463.
    Pubmed CrossRef
  6. Park KB, Ji GE, Park MS, Oh SH. 2005. Expression of rice glutamate decarboxylase in Bifidobacterium longum enhances γ-aminobutyric acid production. Biotechnol. Lett. 27: 1681-1684.
    Pubmed CrossRef
  7. Vo TD, Kim TW, Hong SH. 2012. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli. Bioprocess Biosyst. Eng. 35: 645-650.
    Pubmed CrossRef
  8. Moon TS, Dueber JE, Shiue E, Prather KL. 2010. Use of modular, synthetic scaffold for improved production of glucaric acid in engineered E. coli. Metab. Eng. 12: 298-305.
    Pubmed CrossRef
  9. Hao R, Schmit JC. 1993. Cloning of the gene for glutamate decarboxylase and its expression during conidiation in Neurospora crassa. Biochem. J. 293: 735-738.
    Pubmed PMC CrossRef
  10. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, et al. 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422: 859-868.
    Pubmed CrossRef
  11. Vo TD, Ko JS, Lee SH, Park SJ, Hong SH. 2013. Overexpression of Neurospora crassa OR74A glutamate decarboxylase in Escherichia coli for efficient GABA production. Biotechnol. Bioprocess Eng. 18: 1062-1066.
    CrossRef
  12. Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  13. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, et al. 2009. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27: 753-759.
    Pubmed CrossRef
  14. Baek JM, Mazumdar S, Lee SW, Jung MY, Lim JH, Seo SW, et al. 2013. Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol. Bioeng. 110: 2790-2794.
    Pubmed CrossRef
  15. Tsai MF, McCarthy P, Miller C. 2013. Substrate selectivity in glutamate-dependent acid resistance in enteric bacteria. Proc. Natl. Acad. Sci. USA 110: 5898-5902.
    Pubmed PMC CrossRef
  16. M a D, L u P, Y an C , Fan C, Y in P , Wang J, et al. 2012. Structure and mechanism of a glutamate-GABA antiporter. Nature 483: 632-636.

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Article

Research article

J. Microbiol. Biotechnol. 2017; 27(9): 1664-1669

Published online September 28, 2017 https://doi.org/10.4014/jmb.1611.11041

Copyright © The Korean Society for Microbiology and Biotechnology.

Enchancement of Gamma-Aminobutyric Acid Production by Co-Localization of Neurospora crassa OR74A Glutamate Decarboxylase with Escherichia coli GABA Transporter Via Synthetic Scaffold Complex

Sivachandiran Somasundaram 1, Murali kannan Maruthamuthu 1, Irisappan Ganesh 2, Gyeong Tae Eom 3, 4 and Soon Ho Hong 1*

1School of Chemical Engineering, University of Ulsan, Ulsan 44610, Republic of Korea, 2Bioenergy and Biochemical Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea, 3Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), Ulsan 44429, Republic of Korea, 3Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), Daejeon 34144, Republic of Korea

Received: November 11, 2016; Accepted: July 10, 2017

Abstract

Gamma-aminobutyric acid is a precursor of nylon-4, which is a promising heat-resistant
biopolymer. GABA can be produced from the decarboxylation of glutamate by glutamate
decarboxylase. In this study, a synthetic scaffold complex strategy was employed involving
the Neurospora crassa glutamate decarboxylase (GadB) and Escherichia coli GABA antiporter
(GadC) to improve GABA production. To construct the complex, the SH3 domain was
attached to the N. crassa GadB, and the SH3 ligand was attached to the N-terminus, middle,
and C-terminus of E. coli GadC. In the C-terminus model, 5.8 g/l of GABA concentration was
obtained from 10 g/l glutamate. When a competing pathway engineered strain was used, the
final GABA concentration was further increased to 5.94 g/l, which corresponds to 97.5% of
GABA yield. With the introduction of the scaffold complex, the GABA productivity increased
by 2.9 folds during the initial culture period.

Keywords: Gamma-aminobutyric acid, glutamate decarboxylase, glutamate/GABA antiporter, Neurospora crassa, synthetic protein scaffold

References

  1. Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW. 1999. Control of acid resistance in Escherichia coli. J. Bacteriol. 181: 3525-3535.
    Pubmed KoreaMed
  2. Kim SH, Shin BH, Kim YH, Nam SW, Jeon SY. 2007. Cloning and expression of a full-length glutamate decarboxylase gene from Lactobacillus brevis BH2. Biotechnol. Bioprocess Eng. 12:707-712.
    CrossRef
  3. Saskiawan I. 2008. Biosynthesis of polyamide 4, a biobased and biodegradable polymer. Microbiol. Indonesia 2: 119-123.
    CrossRef
  4. Capitani G, De Biase D, Aurizi C, Gut H, Bossa F, Gruetter GM. 2003. Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase. EMBO J. 22: 4027-4037.
    Pubmed KoreaMed CrossRef
  5. Park KB, Oh SH. 2006. Enhancement of gama-aminobutyric acid production in chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis. Biotechnol. Lett. 28: 1459-1463.
    Pubmed CrossRef
  6. Park KB, Ji GE, Park MS, Oh SH. 2005. Expression of rice glutamate decarboxylase in Bifidobacterium longum enhances γ-aminobutyric acid production. Biotechnol. Lett. 27: 1681-1684.
    Pubmed CrossRef
  7. Vo TD, Kim TW, Hong SH. 2012. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli. Bioprocess Biosyst. Eng. 35: 645-650.
    Pubmed CrossRef
  8. Moon TS, Dueber JE, Shiue E, Prather KL. 2010. Use of modular, synthetic scaffold for improved production of glucaric acid in engineered E. coli. Metab. Eng. 12: 298-305.
    Pubmed CrossRef
  9. Hao R, Schmit JC. 1993. Cloning of the gene for glutamate decarboxylase and its expression during conidiation in Neurospora crassa. Biochem. J. 293: 735-738.
    Pubmed KoreaMed CrossRef
  10. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, et al. 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422: 859-868.
    Pubmed CrossRef
  11. Vo TD, Ko JS, Lee SH, Park SJ, Hong SH. 2013. Overexpression of Neurospora crassa OR74A glutamate decarboxylase in Escherichia coli for efficient GABA production. Biotechnol. Bioprocess Eng. 18: 1062-1066.
    CrossRef
  12. Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  13. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, et al. 2009. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27: 753-759.
    Pubmed CrossRef
  14. Baek JM, Mazumdar S, Lee SW, Jung MY, Lim JH, Seo SW, et al. 2013. Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol. Bioeng. 110: 2790-2794.
    Pubmed CrossRef
  15. Tsai MF, McCarthy P, Miller C. 2013. Substrate selectivity in glutamate-dependent acid resistance in enteric bacteria. Proc. Natl. Acad. Sci. USA 110: 5898-5902.
    Pubmed KoreaMed CrossRef
  16. M a D, L u P, Y an C , Fan C, Y in P , Wang J, et al. 2012. Structure and mechanism of a glutamate-GABA antiporter. Nature 483: 632-636.