Skip to main content
SpringerLink
Log in

Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction. However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated. We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source. Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium. A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated. DNA microarray experiments revealed increased expression of the malic enzyme gene malE. The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis. Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate. The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate. Whereas during growth of C. glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.

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

Similar content being viewed by others

References

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

    Google Scholar 

  • Al Zaid Siddiquee K, Arauzo-Bravo MJ, Shimizu K (2003) Metabolic flux analysis of pykF gene knockout Escherichia coli based on (13)C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations. Appl Microbiol Biotechnol 63:407–417

    Article  PubMed  Google Scholar 

  • Bendt AK, Burkovski A, Schaffer S, Bott M, Farwick M, Hermann T (2003) Towards a phosphoproteome map of Corynebacterium glutamicum. Proteomics 3:1637–1646

    Article  CAS  PubMed  Google Scholar 

  • Chao YP, Liao JC (1994) Metabolic responses to substrate futile cycling in Escherichia coli. J Biol Chem 269:5122–5126

    CAS  PubMed  Google Scholar 

  • Chao YP, Patnaik R, Roof WD, Young RF, Liao JC (1993) Control of gluconeogenic growth by pps and pck in Escherichia coli. J Bacteriol 175:6939–6944

    CAS  PubMed  Google Scholar 

  • Diesterhaft MD, Freese E (1973) Role of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme during growth and sporulation of Bacillus subtilis. J Biol Chem 248:6062–6070

    CAS  PubMed  Google Scholar 

  • Dominguez H, Rollin C, Guyonvarch A, Guerquin-Kern JL, Cocaign-Bousquet M, Lindley ND (1998) Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose. Eur J Biochem 254:96–102

    Article  CAS  PubMed  Google Scholar 

  • Eikmanns BJ, Thum-Schmitz N, Eggeling L, Lüdtke K, Sahm H (1994) Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology 140:1817–1828

    CAS  PubMed  Google Scholar 

  • Eikmanns BJ, Rittmann D, Sahm H (1995) Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol 177:774–782

    CAS  PubMed  Google Scholar 

  • Emmerling M, Dauner M, Ponti A, Fiaux J, Hochuli M, Szyperski T, Wuthrich K, Bailey JE, Sauer U (2002) Metabolic flux responses to pyruvate kinase knockout in Escherichia coli. J Bacteriol 184:152–164

    Article  CAS  PubMed  Google Scholar 

  • Flores S, Gosset G, Flores N, de Graaf AA, Bolivar F (2002) Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy. Metab Eng 4:124–137

    Article  CAS  PubMed  Google Scholar 

  • Fry B, Zhu T, Domach MM, Koepsel RR, Phalakornkule C, Ataai MM (2000) Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant. Appl Environ Microbiol 66:4045–4049

    Article  CAS  PubMed  Google Scholar 

  • Gerstmeir R, Wendisch VF, Schnicke S, Ruan H, Farwick M, Reinscheid D, Eikmanns BJ (2003) Acetate metabolism and its regulation in Corynebacterium glutamicum. J Biotechnol 104:99–122

    Article  CAS  PubMed  Google Scholar 

  • Gerstmeir R, Cramer A, Dangel P, Schaffer S, Eikmanns BJ (2004) RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J Bacteriol 186:2798–2809

    Article  CAS  PubMed  Google Scholar 

  • Gourdon P, Baucher MF, Lindley ND, Guyonvarch A (2000) Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism. Appl Environ Microbiol 66:2981–2987

    Article  CAS  PubMed  Google Scholar 

  • Grant SGN, Jessee J, Bloom FR, Hanahan D (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87:4645–4649

    CAS  PubMed  Google Scholar 

  • Gubler M, Jetten M, Lee SH, Sinskey AJ (1994) Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain. Appl Environ Microbiol 60:2494–2500

    CAS  PubMed  Google Scholar 

  • Hanahan D (1985) Techniques for transformation of Escherichia coli. In: Glover DM (ed) DNA cloning, vol 1. IRL, Oxford, pp 109–136

    Google Scholar 

  • Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172

    Article  CAS  PubMed  Google Scholar 

  • Ishige T, Krause M, Bott M, Wendisch VF, Sahm H (2003) The phosphate starvation stimulon of Corynebacterium glutamicum determined by DNA microarray analyses. J Bacteriol 185:4519–4529

    Article  CAS  PubMed  Google Scholar 

  • Jacoby M, Ngouoto-Nkili CE, Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors. Biotechnol Tech 13:437–441

    Article  Google Scholar 

  • Jetten MS, Sinskey AJ (1995) Purification and properties of oxaloacetate decarboxylase from Corynebacterium glutamicum. Antonie Van Leeuwenhoek 67:221–227

    CAS  PubMed  Google Scholar 

  • Jetten MS, Gubler ME, Lee SH, Sinskey AJ (1994) Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum. Appl Environ Microbiol 60:2501–2507

    CAS  PubMed  Google Scholar 

  • Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Krämer R, Linke B, McHardy AC, Meyer F, Möckel B, Pfefferle W, Pühler A, Rey DA, Rückert C, Rupp O, Sahm H, Wendisch VF, Wiegräbe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104:5–25

    Article  CAS  PubMed  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  Google Scholar 

  • Khodursky AB, Bernstein JA, Peter BJ, Rhodius V, Wendisch VF, Zimmer DP (2003) Escherichia coli spotted double-strand DNA microarrays: RNA extraction, labeling, hybridization, quality control, and data management. Methods Mol Biol 224:61–78

    Article  CAS  PubMed  Google Scholar 

  • Kiefer P, Heinzle E, Wittmann C (2002) Influence of glucose, fructose and sucrose as carbon sources on kinetics and stoichiometry of lysine production by Corynebacterium glutamicum. J Ind Microbiol Biotechnol 28:338–343

    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

    CAS  PubMed  Google Scholar 

  • Lange C, Rittmann D, Wendisch VF, Bott M, Sahm H (2003) Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of l-valine. Appl Environ Microbiol 69:2521–2531

    Article  CAS  PubMed  Google Scholar 

  • Lehnen D, Blumer C, Polen T, Wackwitz B, Wendisch VF, Unden G (2002) LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli. Mol Microbiol 45:521–532

    Article  CAS  PubMed  Google Scholar 

  • Marx A, deGraaf AA, Wiechert W, Eggeling L, Sahm H (1996) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng 49:111–129

    Article  Google Scholar 

  • Medina V, Pontarollo R, Glaeske D, Tabel H, Goldie H (1990) Sequence of the pckA gene of Escherichia coli K-12: relevance to genetic and allosteric regulation and homology of E. coli phosphoenolpyruvate carboxykinase with the enzymes from Trypanosoma brucei and Saccharomyces cerevisiae. J Bacteriol 172:7151–7156

    CAS  PubMed  Google Scholar 

  • Murai T, Tokushige M, Nagai J, Katsuki H (1971) Physiological functions of NAD- and NADP-linked malic enzymes in Escherichia coli. Biochem Biophys Res Commun 43:875–881

    CAS  PubMed  Google Scholar 

  • Niersbach M, Kreuzaler F, Geerse RH, Postma PW, Hirsch HJ (1992) Cloning and nucleotide sequence of the Escherichia coli K-12 ppsA gene, encoding PEP synthase. Mol Gen Genet 231:332–336

    CAS  PubMed  Google Scholar 

  • Norrander JM, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligdeoxynucleotide-directed mutagenesis. Gene 26:101–106

    Article  CAS  PubMed  Google Scholar 

  • Oh MK, Rohlin L, Kao KC, Liao JC (2002) Global expression profiling of acetate-grown Escherichia coli. J Biol Chem 277:13175–13183

    Article  CAS  PubMed  Google Scholar 

  • Petersen S, de Graaf AA, Eggeling L, Mollney M, Wiechert W, Sahm H (2000) In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum. J Biol Chem 275:35932–35941

    Article  CAS  PubMed  Google Scholar 

  • Peters-Wendisch P, Eikmanns BJ, Thierbach G, Bachmann B, Sahm H (1993) Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is dispensable for growth and lysine production. FEMS Microbiol Lett 112:269–274

    Article  CAS  Google Scholar 

  • Peters-Wendisch P, Wendisch VF, Paul S, Eikmanns BJ, Sahm H (1997) Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum. Microbiology 143:1095–1103

    CAS  Google Scholar 

  • Peters-Wendisch PG, Kreutzer C, Kalinowski J, Patek M, Sahm H, Eikmanns BJ (1998) Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene. Microbiology 144:915–927

    CAS  PubMed  Google Scholar 

  • Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Möckel B, Sahm H, Eikmanns BJ (2001) Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol 3:295–300

    CAS  PubMed  Google Scholar 

  • Polen T, Wendisch VF (2004) Genome-wide expression analysis in amino acid producing bacteria using DNA microarrays. Appl Biochem Biotechnol 118:215–232

    Article  CAS  PubMed  Google Scholar 

  • Polen T, Rittmann D, Wendisch VF, Sahm H (2003) DNA microarray analyses of the long-term adaptive response of Escherichia coli to acetate and propionate. Appl Environ Microbiol 69:1759–1774

    Article  CAS  PubMed  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 

  • van der Rest ME, Frank C, Molenaar D (2000) Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Escherichia coli. J Bacteriol 182:6892–6899

    Article  PubMed  Google Scholar 

  • Rhodius V, Van Dyk TK, Gross C, LaRossa RA (2002) Impact of genomic technologies on studies of bacterial gene expression. Annu Rev Microbiol 56:599–624

    Article  CAS  PubMed  Google Scholar 

  • Riedel C, Rittmann D, Dangel P, Mockel B, Petersen S, Sahm H, Eikmanns BJ (2001) Characterization of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production. J Mol Microbiol Biotechnol 3:573–583

    CAS  PubMed  Google Scholar 

  • Rittmann D, Schaffer S, Wendisch VF, Sahm H (2003) Fructose-1,6-bisphosphatase from Corynebacterium glutamicum: expression and deletion of the fbp gene and biochemical characterization of the enzyme. Arch Microbiol 180:285–292

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratoy, Cold Spring Harbor

    Google Scholar 

  • Schäfer A, Kalinowski J, Pühler A (1994) Increased fertility of Corynebacterium glutamicum recipients in intergeneric matings with Escherichia coli after stress exposure. Appl Environ Microbiol 60:756–759

    PubMed  Google Scholar 

  • Shaw WV (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol 43:737–755

    Article  CAS  PubMed  Google Scholar 

  • Vasicova P, Abrhamova Z, Nesvera J, Patek M, Sahm H, Eikmanns B (1998) Integrative and autonomously replicating vectors for analysis of promoters in Corynebacterium glutamicum. Biotechnol Tech 12:743–746

    Article  CAS  Google Scholar 

  • Velayudhan J, Kelly DJ (2002) Analysis of gluconeogenic and anaplerotic enzymes in Campylobacter jejuni: an essential role for phosphoenolpyruvate carboxykinase. Microbiology 148:685–694

    CAS  PubMed  Google Scholar 

  • Wagner A (2000) Robustness against mutations in genetic networks of yeast. Nat Genet 24:355–361

    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, de Graaf AA, Sahm H, Eikmanns BJ (2000) Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J Bacteriol 182:3088–3096

    Article  CAS  PubMed  Google Scholar 

  • Wendisch VF, Zimmer DP, Khodursky A, Peter B, Cozzarelli N, Kustu S (2001) Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays. Anal Biochem 290:205–213

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Christian Lange and Tino Polen for help with DNA microarray experiments. Malgorzata Krause received an ERASMUS fellowship from the European Union. Roman Netzer and Petra Peters-Wendisch acknowledge support from the Deutsche Bundesstiftung Umwelt (grant AZ13037) and Hermann Sahm acknowledges support by the Fonds der Chemischen Industrie. Roman Netzer and Malgorzata Krause contributed equally to this study.

Author information

Authors and Affiliations

  1. Institute of Biotechnology 1, Research Centre Jülich, 52425, Juelich, Germany

    Roman Netzer, Malgorzata Krause, Doris Rittmann, Petra G. Peters-Wendisch, Lothar Eggeling, Volker F. Wendisch & Hermann Sahm

Authors
  1. Roman Netzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Malgorzata Krause
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Doris Rittmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Petra G. Peters-Wendisch
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lothar Eggeling
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Volker F. Wendisch
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hermann Sahm
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Volker F. Wendisch.

Additional information

First two authors contributed equally.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Netzer, R., Krause, M., Rittmann, D. et al. Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch Microbiol 182, 354–363 (2004). https://doi.org/10.1007/s00203-004-0710-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-004-0710-4

Keywords

Search

Navigation