Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Intracellular Flux Prediction of Recombinant Escherichia coli Producing Gamma-Aminobutyric Acid

  • Sung Han Bae (Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology(KAIST)) ;
  • Myung Sub Sim (Department of Biotechnology and Bioengineering, Chonnam National University) ;
  • Ki Jun Jeong (Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology(KAIST)) ;
  • Dan He (College of Life Science and Agriculture Forestry, Qiqihar University) ;
  • Inchan Kwon (School of Materials Science and Engineering, Gwangju Institute of Science and Technology) ;
  • Tae Wan Kim (Department of Biotechnology and Bioengineering, Chonnam National University) ;
  • Hyun Uk Kim (Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology(KAIST)) ;
  • Jong-il Choi (Department of Biotechnology and Bioengineering, Chonnam National University)
  • Received : 2023.12.13
  • Accepted : 2024.01.10
  • Published : 2024.04.28
Download PDF
⟨ Previous Next ⟩

Abstract

Genome-scale metabolic model (GEM) can be used to simulate cellular metabolic phenotypes under various environmental or genetic conditions. This study utilized the GEM to observe the internal metabolic fluxes of recombinant Escherichia coli producing gamma-aminobutyric acid (GABA). Recombinant E. coli was cultivated in a fermenter under three conditions: pH 7, pH 5, and additional succinic acids. External fluxes were calculated from cultivation results, and internal fluxes were calculated through flux optimization. Based on the internal flux analysis, glycolysis and pentose phosphate pathways were repressed under cultivation at pH 5, even though glutamate dehydrogenase increased GABA production. Notably, this repression was halted by adding succinic acid. Furthermore, proper sucA repression is a promising target for developing strains more capable of producing GABA.

Keywords

Acknowledgement

This work was supported by the ERC Center funded by the National Research Foundation of Korea (NRF-2022R1A5A1033719), and the 2023 Research Supporting Program (2023-1315-01) by Chonnam National University.

References

  1. Hayakawa K, Kimura M, Kasaha K, Matsumoto K, Sansawa H, Yamori Y. 2004. Effect of a gamma-aminobutyric acid-enriched dairy product on the blood pressure of spontaneously hypertensive and normotensive Wistar-Kyoto rats. Br. J. Nutr. 92: 411-417.
  2. Adeghate E, Ponery AS. 2002. GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats. Tissue Cell 34: 1-6.
  3. Boonstra E, de Kleijn R, Colzato LS, Alkemade A, Forstmann BU, Nieuwenhuis S. 2015. Neurotransmitters as food supplements: the effects of GABA on brain and behavior. Front. Psychol. 6: 1520.
  4. Sarasa SB, Mahendran R, Muthusamy G, Thankappan B, Selta DRF, Angayarkanni J. 2020. A Brief review on the non-protein amino acid, gamma-amino butyric acid (GABA): its production and role in microbes. Curr. Microbiol. 77: 534-544.
  5. Park SJ, Kim EY, Noh W, Oh YH, Kim HY, Song BG, et al. 2013. Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli. Bioprocess Biosyst. Eng. 36: 885-892.
  6. Yu P, Ma J, Zhu P, Chen Q, Zhang Q. 2021. Enhancing the production of γ-aminobutyric acid in Escherichia coli BL21 by engineering the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate. World J. Microbiol. Biotechnol. 37: 130.
  7. Liu Q, Cheng H, Ma X, Xu N, Liu J, Ma Y. 2016. Expression, characterization and mutagenesis of a novel glutamate decarboxylase from Bacillus megaterium. Biotechnol. Lett. 38: 1107-1113.
  8. Masuda K, Guo X, Uryu N, Hagiwara T, Watabe S. 2008. Isolation of marine yeasts collected from the Pacific Ocean showing a high production of gamma-aminobutyric acid. Biosci. Biotechnol. Biochem. 72: 3265-3272.
  9. Wang J, Lee CL, Pan TM. Improvement of monacolin K, gamma-aminobutyric acid and citrinin production ratio as a function of environmental conditions of Monascus purpureus NTU 601. J. Ind. Microbiol. Biotechnol. 30: 669-676.
  10. Tang CD, Li X, Shi HL, Jia YY, Dong ZX, Jiao ZJ, et al. 2020. Efficient expression of novel glutamate decarboxylases and high level production of γ-aminobutyric acid catalyzed by engineered Escherichia coli. Int. J. Biol. Macromol. 160: 372-379.
  11. Luo H, Liu Z, Xie F, Bilal M, Liu L, Yang R, Wang Z. 2021. Microbial production of gamma-aminobutyric acid: applications, state-of-the-art achievements, and future perspectives. Crit. Rev. Biotechnol. 41: 491-512.
  12. Yuan H, Wang H, Fidan O, Qin Y, Xiao G, Zhan J. 2019. Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli. J. Biol. Eng. 13: 24.
  13. Le 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.
  14. Yu P, Chen K, Huang X, Wang X, Ren Q. 2018. Production of γaminobutyric acid in Escherichia coli by engineering MSG pathway. Prep. Biochem. Biotechnol. 48: 906-913.
  15. Pham VD, Lee SH, Park SJ, Hong SH. 2015. Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli. J. Biotechnol. 207: 52-57.
  16. Zhao A, Hu X, Wang X. 2017. Metabolic engineering of Escherichia coli to produce gamma-aminobutyric acid using xylose. Appl. Microbiol. Biotechnol. 101: 3587-3603.
  17. Pham VD, Somasundaram S, Lee SH, Park SJ, Hong SH. 2016. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter. J. Ind. Microbiol. Biotechnol. 43: 79-86.
  18. Tang CD, Li X, Shi HL, Jia YY, Dong ZX2, Jiao ZJ, et al. 2020. Efficient expression of novel glutamate decarboxylases and high level production of γ-aminobutyric acid catalyzed by engineered Escherichia coli. Int. J. Biol. Macromol. 160: 372-379.
  19. Park SH, Sohn YJ, Park SJ, Choi J. 2020. Effect of DR1558, a Deinococcus radiodurans response regulator, on the production of GABA in the recombinant Escherichia coli under low pH conditions. Microb. Cell Fact. 19: 64.
  20. Nielsen J. 2017. Systems biology of metabolism. Annu. Rev. Biochem. 86: 245-275.
  21. Orth JD, Thiele I, Palsson BO. 2010. What is flux balance analysis?. Nat. Biotechnol. 28: 245-248.
  22. Kang S, Choi J. 2021. Enhanced cadaverine production by recombinant Corynebacterium glutamicum with a heterologous DR1558 regulator at low pH condition. Process Biochem. 111: 63-70.
  23. Lee SM, Lee GR, Kim HU. 2022. Machine learning-guided evaluation of extraction and simulation methods for cancer patient-specific metabolic models. Comput. Struct. Biotechnol. J. 20: 3041-3052.
  24. Lewis NE, Hixson KK, Conrad TM, Lerman JA, Charusanti P, Polpitiya AD, et al. 2010. Omic data from evolved E. coli are consistent with computed optimal growth from genome-scale models. Mol. Syst. Biol. 6: 390.
  25. Lee D, Smallbone K, Dunn WB, Murabito E, Winder CL, Kell DB, et al. 2012. Improving metabolic flux predictions using absolute gene expression data. BMC Syst. Biol. 6: 73.
  26. Ebrahim A, Lerman JA, Palsson BO, Hyduke DR. 2013. COBRApy: constraints-based reconstruction and analysis for python. BMC Syst. Biol. 7: 74.
  27. Park S, Kim GB, Kim HU, Park SJ, Choi J. 2019. Enhanced production of poly-3-hydroxybutyrate (PHB) by expression of response regulator DR1558 in recombinant Escherichia coli. Int. J. Biol. Macromol. 131: 29-35.
  28. Kang SB, Choi J. 2022. Production of cadaverine in recombinant Corynebacterium glutamicum overexpressing lysine decarboxylase (ldcC) and response regulator dr1558. Appl. Biochem. Biotechnol. 194: 1013-1024.
  29. Orr JS, Christensen DG, Wolfe AJ, Rao CV. 2018. Extracellular acidic pH inhibits acetate consumption by decreasing gene transcription of the tricarboxylic acid cycle and the glyoxylate shunt. J. Bacteriol. 201: e00410-18.
  30. Stancik LM, Stancik DM, Schmidt B, Barnhart DM, Yoncheva YN, Slonczewski JL. 2002. pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli. J. Bacteriol. 184: 4246-4258.