Skip to main content

Comparative study on central metabolic fluxes of Bacillus megaterium strains in continuous culture using 13C labelled substrates

Bioprocess and Biosystems Engineering volume 30, pages 47–59 (2007)Cite this article

Abstract

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by 13C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Vary PS (1994) Prime time for Bacillus megaterium. Microbiology 140:1001–1013

    CAS  Article  Google Scholar 

  2. Rygus T, Hillen W (1991) Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl Microbiol Biotechnol 35(5):594–599

    Article  CAS  Google Scholar 

  3. Meinhardt F, Stahl U, Ebeling W (1989) Highly efficient expression of homologous and heterologous genes in Bacillus megaterium. Appl Microbiol Biotechnol 30:343–350

    Article  CAS  Google Scholar 

  4. Malten M, Hollmann R, Deckwer WD, Jahn D (2004) Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89(2):206–218. DOI 10.1002/bit.20341

  5. Yang Y, Malten M, Grote A, Jahn D, Deckwer WD (2006) Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium. Biotechnol Bioeng (in press). DOI 10.1002/bit.21167

  6. Wang W, Hollmann R, Fürch T, Nimtz M, Malten M, Jahn D, Deckwer WD (2005) Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glycosyltransferase. Proteome Sci 3:4. DOI 10.1186/1477-5956-3-4

  7. Hollmann R, Deckwer WD (2004) Pyruvate formation and suppression in recombinant Bacillus megaterium cultivation. J Biotechnol 111:89–96. DOI 10.1016/j.jbiotec.2004.03.006

  8. Lee WNP, Bergner EA (1992) Mass isotopomer pattern and precursor–product relationship. Biol Mass Spectrom 21:114–122

    Article  CAS  Google Scholar 

  9. Wittchen KD, Meinhardt F (1995) Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl Microbiol Biotechnol 42(6):871–877. DOI 10.1007/s002530050345

  10. Rygus T, Hillen W (1992) Catabolite repression of the xyl operon in Bacillus megaterium. J Bacteriol 174(9):3049–3055

    CAS  Google Scholar 

  11. Dauner M, Sauer U (2001) Stoichiometric growth model for riboflavin-producing Bacillus subtilis. Biotechnol Bioeng 76(2):132–143. DOI 10.1002/bit.1153

  12. Lee WNP, Byerley LO, Bergner EA (1991) Mass isotopomer analysis: theoretical and practical considerations. Biol Mass Spectrom 20:451–458

    Article  CAS  Google Scholar 

  13. Szyperski T (1995) Biosynthetically directed fractional 13C-labelling of proteinogenic amino acids—an efficient tool to investigate intermediary metabolism. Eur J Biochem 232:433–448

    Article  CAS  Google Scholar 

  14. Fischer E, Sauer U (2003) Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC–MS. Eur J Biochem 270:880–891

    Article  CAS  Google Scholar 

  15. Dauner M, Bailey JE, Sauer U (2001) Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. Biotechnol Bioeng 76(2):144–156. DOI 10.1002/bit.1154

  16. Fischer E, Zamboni N, Sauer U (2004) High-throughput metabolic flux analysis based on gas chromatography–mass spectrometry derived 13C constraints. Anal Biochem 325:308–316. DOI 10.1016/j.ab.2003.10.036

  17. Sauer U, Bailey JE (1999) Estimation of P-to-O ratio in Bacillus subtilis and its influence on maximum riboflavin yield. Biotechnol Bioeng 64(6):750–754. DOI 10.1002/(SICI)1097-0290(19990920)64:6 < 750::AID-BIT15 > 3.0.CO;2-S

  18. Dauner M, Tazio S, Sauer U (2001) Bacillus subtilis metabolism and energetics in carbon-limited and excess-carbon chemostat culture. J Bacteriol 183(24):7308–7317. DOI 10.1128/JB.183.24.7308-7317.2001

  19. Zeng AP, Deckwer WD (1995) A kinetic model for substrate and energy consumption of microbial growth under substrate-sufficient conditions. Biotechnol Prog 11:71–79

    Article  CAS  Google Scholar 

  20. Follstad BD, Stephanopoulos G (1998) Effect of reversible reactions on isotope label redistribution. Analysis of the pentose phosphate pathway. Eur J Biochem 252:360–371. DOI 10.1046/j.1432-1327.1998.2520360.x

  21. Christiansen T, Christensen B, Nielsen J (2002) Metabolic network analysis of Bacillus clausii on minimal and semirich medium using 13C-labelled glucose. Metab Eng 4:159–169. DOI: 10.1006/mben.2001.0219

  22. 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(17):6062–6070

    CAS  Google Scholar 

  23. Stouthamer AH, Bettenhaussen C (1973) Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. Biochim Biophys Acta 301:53–70

    CAS  Google Scholar 

  24. Neidhardt FC, Ingraham JL, Schaechter M (1990) Physiology of the bacterial cell—a molecular approach, 1st edn. Sinauer Associates Inc., Sunderland

    Google Scholar 

Download references

Acknowledgments

This work was carried out as part of the DFG-Sonderforschungsbereich 578 and the authors gratefully acknowledge financial support granted by the Deutsche Forschungsgemeinschaft. Thanks are also due to the Structure Biology Department of the Helmholtz Zentrum für Infektionsforschung (HZI), especially to Dr Manfred Nimtz and Claudia Hanko for their support in GC/MS analysis.

Author information

Affiliations

  1. Institute of Biochemical Engineering (TU-BCE), Technical University Braunschweig/HZI-Helmholtz Zentrum für Infektionsforschung, Inhoffenstrasse 7, 38124, Braunschweig, Germany

    Tobias Fürch, Rajan Hollmann, Wei Wang & Wolf-Dieter Deckwer

  2. Institute of Biochemical Engineering, Saarland University, Im Stadtwald, POB 151150, 66123, Saarbrucken, Germany

    Christoph Wittmann

Authors
  1. Tobias Fürch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rajan Hollmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christoph Wittmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolf-Dieter Deckwer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tobias Fürch.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fürch, T., Hollmann, R., Wittmann, C. et al. Comparative study on central metabolic fluxes of Bacillus megaterium strains in continuous culture using 13C labelled substrates. Bioprocess Biosyst Eng 30, 47–59 (2007). https://doi.org/10.1007/s00449-006-0095-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-006-0095-7

Keywords

  • Bacillus megaterium
  • Metabolic flux analysis
  • Central carbon pathways
  • Futile cycle