Skip to main content
SpringerLink
Log in

A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

Gamma-aminobutyric acid (GABA), a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods, and the biodegradable plastic polyamide 4. Corynebacterium glutamicum shows great potential for the production of GABA from glucose. GABA added to the growth medium hardly affected growth of C. glutamicum, since a half-inhibitory concentration of 1.1 M GABA was determined. As alternative to GABA production by glutamate decarboxylation, a new route for the production of GABA via putrescine was established in C. glutamicum. A putrescine-producing recombinant C. glutamicum strain was converted into a GABA producing strain by heterologous expression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) genes from Escherichia coli. The resultant strain produced 5.3 ± 0.1 g L−1 of GABA. GABA production was improved further by adjusting the concentration of nitrogen in the culture medium, by avoiding the formation of the by-product N-acetylputrescine and by deletion of the genes for GABA catabolism and GABA re-uptake. GABA accumulation by this strain was increased by 51 % to 8.0 ± 0.3 g L−1, and the volumetric productivity was increased to 0.31 g L−1 h−1; the highest volumetric productivity reported so far for fermentative production of GABA from glucose in shake flasks was achieved.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Abe S, Takayarna K, Kinoshita S (1967) Taxonomical studies on glutamicum acid producing bacteria. J Gen Appl Microbiol 13:279–301

    Article  Google Scholar 

  • Albrecht AM, Vogel HJ (1964) Acetylornithine Delta-Transaminase. Partial purification and repression behavior. J Biol Chem 239:1872–1876

    CAS  PubMed  Google Scholar 

  • Arndt A, Eikmanns BJ (2007) The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to carbon catabolite repression. J Bacteriol 189:7408–7416

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arndt A, Auchter M, Ishige T, Wendisch VF, Eikmanns BJ (2008) Ethanol catabolism in Corynebacterium glutamicum. J Mol Microbiol Biotechnol 15:222–233

    Article  CAS  PubMed  Google Scholar 

  • Becker J, Wittmann C (2012) Systems and synthetic metabolic engineering for amino acid production—the heartbeat of industrial strain development. Curr Opin Biotechnol 23:718–726

    Article  CAS  PubMed  Google Scholar 

  • Becker J, Lange A, Fabarius J, Wittmann C (2015) Top value platform chemicals: bio-based production of organic acids. Curr Opin Biotechnol 36:168–175

    Article  CAS  PubMed  Google Scholar 

  • Blombach B, Seibold GM (2010) Carbohydrate metabolism in Corynebacterium glutamicum and applications for the metabolic engineering of l-lysine production strains. Appl Microbiol Biotechnol 86:1313–1322

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Choi JW, Yim SS, Lee SH, Kang TJ, Park SJ, Jeong KJ (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

    Article  PubMed  PubMed Central  Google Scholar 

  • Chung H, Yang JE, Ha JY, Chae TU, Shin JH, Gustavsson M, Lee SY (2015) Bio-based production of monomers and polymers by metabolically engineered microorganisms. Curr Opin Biotechnol 36:73–84

    Article  CAS  PubMed  Google Scholar 

  • Dhakal R, Bajpai VK, Baek KH (2012) Production of GABA (gamma—Aminobutyric acid) by microorganisms: a review. Braz J Microbiol 43:1230–1241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dugar D, Stephanopoulos G (2011) Relative potential of biosynthetic pathways for biofuels and bio-based products. Nat Biotechnol 29:1074–1078

    Article  CAS  PubMed  Google Scholar 

  • Eberhardt D, Jensen JV, Wendisch VF (2014) L-citrulline production by metabolically engineered Corynebacterium glutamicum from glucose and alternative carbon sources. AMB Express 4:85

    Article  PubMed  PubMed Central  Google Scholar 

  • Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. CRC Press

  • Eikmanns BJ, Kleinertz E, Liebl W, Sahm H (1991) A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene 102:93–98

    Article  CAS  PubMed  Google Scholar 

  • Follmann M, Ochrombel I, Kramer R, Trotschel C, Poetsch A, Ruckert C, Huser A, Persicke M, Seiferling D, Kalinowski J, Marin K (2009) Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis. BMC Genom 10:621

    Article  Google Scholar 

  • Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345

    Article  CAS  PubMed  Google Scholar 

  • Guyer MS, Reed RR, Steitz JA, Low KB (1981) Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol 45(Pt 1):135–140

    Article  CAS  PubMed  Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Article  CAS  PubMed  Google Scholar 

  • Hashimoto K, Hamano T, Okada M (1994) Degradation of several polyamides in soils. J Appl Polym Sci 54:1579–1583

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Heider SA, Wendisch VF (2015) Engineering microbial cell factories: Metabolic engineering of Corynebacterium glutamicum with a focus on non-natural products. Biotechnol J 10:1170–1184

    Article  CAS  PubMed  Google Scholar 

  • Huhn S, Jolkver E, Kramer R, Marin K (2011) Identification of the membrane protein SucE and its role in succinate transport in Corynebacterium glutamicum. Appl Microbiol Biotechnol 89:327–335

    Article  CAS  PubMed  Google Scholar 

  • Ikeda M, Mitsuhashi S, Tanaka K, Hayashi M (2009) Reengineering of a Corynebacterium glutamicum l-arginine and l-citrulline producer. Appl Environ Microbiol 75:1635–1641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jakob K, Satorhelyi P, Lange C, Wendisch VF, Silakowski B, Scherer S, Neuhaus K (2007) Gene expression analysis of Corynebacterium glutamicum subjected to long-term lactic acid adaptation. J Bacteriol 189:5582–5590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jensen JV, Wendisch VF (2013) Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum. Microb Cell Fact 12:63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jensen JV, Eberhardt D, Wendisch VF (2015) Modular pathway engineering of Corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine. J Biotechnol 214:85–94

    Article  CAS  PubMed  Google Scholar 

  • Jeon JM, Rajesh T, Song E, Lee HW, Lee HW, Yang YH (2013) Media optimization of Corynebacterium glutamicum for succinate production under oxygen-deprived condition. J Microbiol Biotechnol 23:211–217

    Article  CAS  PubMed  Google Scholar 

  • Kawasaki N, Nakayama A, Yamano N, Takeda S, Kawata Y, Yamamoto N, Aiba S (2005) Synthesis, thermal and mechanical properties and biodegradation of branched polyamide 4. Polymer 46:9987–9993

    Article  CAS  Google Scholar 

  • Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175:5595–5603

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kelle R, Hermann T, Bathe B (2004) l-lysine production. In: Bott M, Eggeling L (eds) Handbook of C. glutamicum. CRC Press, Boca Raton

    Google Scholar 

  • Kind S, Jeong WK, Schroder H, Zelder O, Wittmann C (2010) Identification and elimination of the competing N-acetyldiaminopentane pathway for improved production of diaminopentane by Corynebacterium glutamicum. Appl Environ Microbiol 76:5175–5180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kind S, Kreye S, Wittmann C (2011) Metabolic engineering of cellular transport for overproduction of the platform chemical 1,5-diaminopentane in Corynebacterium glutamicum. Metab Eng 13:617–627

    Article  CAS  PubMed  Google Scholar 

  • Kramer R, Lambert C, Hoischen C, Ebbighausen H (1990) Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium. Eur J Biochem 194:929–935

    Article  CAS  PubMed  Google Scholar 

  • Lessmeier L, Hoefener M, Wendisch VF (2013) Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase. Microbiology 159:2651–2662

    Article  CAS  PubMed  Google Scholar 

  • Li H, Cao Y (2010) Lactic acid bacterial cell factories for gamma-aminobutyric acid. Amino Acids 39:1107–1116

    Article  CAS  PubMed  Google Scholar 

  • Li H, Qiu T, Huang G, Cao Y (2010) Production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 using fed-batch fermentation. Microb Cell Fact 9:85

    Article  PubMed  PubMed Central  Google Scholar 

  • Netzer R, Krause M, Rittmann D, Peters-Wendisch PG, Eggeling L, Wendisch VF, Sahm H (2004a) Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch Microbiol 182:354–363

    Article  CAS  PubMed  Google Scholar 

  • Netzer R, Peters-Wendisch P, Eggeling L, Sahm H (2004b) Cometabolism of a nongrowth substrate: l-serine utilization by Corynebacterium glutamicum. Appl Environ Microbiol 70:7148–7155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nguyen AQ, Schneider J, Wendisch VF (2015) Elimination of polyamine N-acetylation and regulatory engineering improved putrescine production by Corynebacterium glutamicum. J Biotechnol 201:75–85

    Article  CAS  PubMed  Google Scholar 

  • Okai N, Takahashi C, Hatada K, Ogino C, Kondo A (2014) Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase. AMB Express 4:20

    Article  PubMed  PubMed Central  Google Scholar 

  • Park SJ, Kim EY, Noh W, Oh YH, Kim HY, Song BK, Cho KM, Hong SH, Lee SH, Jegal J (2013) Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli. Bioprocess Biosyst Eng 36:885–892

    Article  CAS  PubMed  Google Scholar 

  • Park SH, Kim HU, Kim TY, Park JS, Kim SS, Lee SY (2014) Metabolic engineering of Corynebacterium glutamicum for l-arginine production. Nat Commun 5:4618

    CAS  PubMed  Google Scholar 

  • Polen T, Schluesener D, Poetsch A, Bott M, Wendisch VF (2007) Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol Lett 273:109–119

    Article  CAS  PubMed  Google Scholar 

  • Prieto-Santos MI, Martin-Checa J, Balana-Fouce R, Garrido-Pertierra A (1986) A pathway for putrescine catabolism in Escherichia coli. Biochim Biophys Acta 880:242–244

    Article  CAS  PubMed  Google Scholar 

  • Qian ZG, Xia XX, Lee SY (2009) Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine. Biotechnol Bioeng 104:651–662

    CAS  PubMed  Google Scholar 

  • Rodriguez A, Martinez JA, Flores N, Escalante A, Gosset G, Bolivar F (2014) Engineering Escherichia coli to overproduce aromatic amino acids and derived compounds. Microb Cell Fact 13:126

    PubMed  PubMed Central  Google Scholar 

  • Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  • Samsonova NN, Smirnov SV, Altman IB, Ptitsyn LR (2003) Molecular cloning and characterization of Escherichia coli K12 ygjG gene. BMC Microbiol 3:2

    Article  PubMed  PubMed Central  Google Scholar 

  • Samsonova NN, Smirnov SV, Novikova AE, Ptitsyn LR (2005) Identification of Escherichia coli K12 YdcW protein as a gamma-aminobutyraldehyde dehydrogenase. FEBS Lett 579:4107–4112

    Article  CAS  PubMed  Google Scholar 

  • Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73

    Article  CAS  PubMed  Google Scholar 

  • Schneider BL, Reitzer L (2012) Pathway and enzyme redundancy in putrescine catabolism in Escherichia coli. J Bacteriol 194:4080–4088

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schneider J, Wendisch VF (2010) Putrescine production by engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol 88:859–868

    Article  CAS  PubMed  Google Scholar 

  • Schneider J, Niermann K, Wendisch VF (2011) Production of the amino acids l-glutamate, l-lysine, l-ornithine and l-arginine from arabinose by recombinant Corynebacterium glutamicum. J Biotechnol 154:191–198. doi:10.1016/j.jbiotec.2010.07.009

    Article  CAS  PubMed  Google Scholar 

  • Schneider J, Eberhardt D, Wendisch VF (2012) Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system. Appl Microbiol Biotechnol 95:169–178

    Article  CAS  PubMed  Google Scholar 

  • Schneider BL, Hernandez VJ, Reitzer L (2013) Putrescine catabolism is a metabolic response to several stresses in Escherichia coli. Mol Microbiol 88:537–550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi F, Jiang J, Li Y, Li Y, Xie Y (2013) Enhancement of gamma-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis. J Ind Microbiol Biotechnol 40:1285–1296

    Article  CAS  PubMed  Google Scholar 

  • Siebert D, Wendisch VF (2015) Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum. Biotechnol Biofuels 8:91

    Article  PubMed  PubMed Central  Google Scholar 

  • Stansen KC (2005) Charakterisierung der Ausscheidung von L-Glutamat bei Corynebacterium glutamicum, Heinrich-Heine-Universität Düsseldorf

  • Stansen C, Uy D, Delaunay S, Eggeling L, Goergen JL, Wendisch VF (2005) Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production. Appl Environ Microbiol 71:5920–5928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takahashi C, Shirakawa J, Tsuchidate T, Okai N, Hatada K, Nakayama H, Tateno T, Ogino C, Kondo A (2012) Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate decarboxylase from Escherichia coli. Enzyme Microb Technol 51:171–176

    Article  CAS  PubMed  Google Scholar 

  • Teramoto H, Shirai T, Inui M, Yukawa H (2008) Identification of a gene encoding a transporter essential for utilization of C4 dicarboxylates in Corynebacterium glutamicum. Appl Environ Microbiol 74:5290–5296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • van der Rest ME, Lange C, Molenaar D (1999) A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl Microbiol Biotechnol 52:541–545

    Article  PubMed  Google Scholar 

  • Wang N, Ni Y, Shi F (2015) Deletion of odhA or pyc improves production of gamma-aminobutyric acid and its precursor L-glutamate in recombinant Corynebacterium glutamicum. Biotechnol Lett 37(7):1473–1481

    Article  CAS  PubMed  Google Scholar 

  • Wendisch VF (2003) Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. J Biotechnol 104:273–285

    Article  CAS  PubMed  Google Scholar 

  • Wendisch VF (2007) (ed) Amino acid biosynthesis—pathways, regulation and metabolic engineering, microbiology monographs. Springer Berlin Heidelberg, Berlin, Heidelberg

  • Witthoff S, Muhlroth A, Marienhagen J, Bott M (2013) C1 metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide. Appl Environ Microbiol 79:6974–6983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wittmann C, Kiefer P, Zelder O (2004) Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source. Appl Environ Microbiol 70:7277–7287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yamada H (1971) Putrescine oxidase (Micrococcus rubens). In: Tabor H, Tabor CW (eds) Methods in enzymology XVIIB: metabolism of amino acids and amines, New York, Academic Press, pp 726–730

  • Yamano N, Nakayama A, Kawasaki N, Yamamoto N, Aiba S (2008) Mechanism and Characterization of Polyamide 4 Degradation by Pseudomonas sp. J Polym Environ 16:141–146

    Article  CAS  Google Scholar 

  • Youn JW, Jolkver E, Kramer R, Marin K, Wendisch VF (2008) Identification and characterization of the dicarboxylate uptake system DccT in Corynebacterium glutamicum. J Bacteriol 190:6458–6466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Youn JW, Jolkver E, Kramer R, Marin K, Wendisch VF (2009) Characterization of the dicarboxylate transporter DctA in Corynebacterium glutamicum. J Bacteriol 191:5480–5488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yukawa H, Inui M (2013) (eds) Corynebacterium glutamicum—biology and biotechnology. microbiology monographs. Springer, Berlin Heidelberg, Berlin, Heidelberg

  • Zahoor A, Otten A, Wendisch VF (2015) Metabolic engineering of Corynebacterium glutamicum for glycolate production. J Biotechnol 192:366–375

    Article  Google Scholar 

  • Zhao Z, Ding JY, Ma WH, Zhou NY, Liu SJ (2012) Identification and characterization of gamma-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum. Appl Environ Microbiol 78:2596–2601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu N, Xia H, Yang J, Zhao X, Chen T (2014) Improved succinate production in Corynebacterium glutamicum by engineering glyoxylate pathway and succinate export system. Biotechnol Lett 36:553–560

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We wish to thank Dr. Anh Q. D. Nguyen for providing the strain PU21ΔcgmA.

Author information

Authors and Affiliations

  1. Chair of Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany

    João M. P. Jorge & Volker F. Wendisch

  2. evocatal GmbH, Alfred-Nobel-Str. 10, 40789, Monheim am Rhein, Germany

    Christian Leggewie

Authors
  1. João M. P. Jorge
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian Leggewie
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Volker F. Wendisch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Volker F. Wendisch.

Ethics declarations

Conflict of interest

This work is patented by evocatal GmbH under the patent number WO2015132213 A1.

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

João Jorge is a fellow of the CLIB2021 graduate cluster at Bielefeld University. This work was supported in part by evocatal GmbH.

Additional information

Handling Editor: C.-A. A. Hu.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jorge, J.M.P., Leggewie, C. & Wendisch, V.F. A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose. Amino Acids 48, 2519–2531 (2016). https://doi.org/10.1007/s00726-016-2272-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-016-2272-6

Keywords

Search

Navigation