Skip to main content
SpringerLink
Log in

Heterologous expression of pyruvate decarboxylase in Geobacillus thermoglucosidasius

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Expression of a pyruvate decarboxylase (Pdc) pathway in metabolically versatile thermophilic bacteria could create novel ethanologenic organisms, but no suitable thermostable Pdc is available. We have demonstrated that Pdc from Zymomonas mobilis can be expressed in an active form in Geobacillus thermoglucosidasius at up to 52°C, while expression of Pdc polypeptides up to 54°C was evident from Western blotting. By using an unstable lactate dehydrogenase (ldh) mutant of G. thermoglucosidasius, indirect evidence of Pdc activity in vivo was also obtained.

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

  • Alterthum F, Ingram LO (1989) Efficient ethanol production from glucose, lactose and xylose by recombinant Escherichia coli. Appl Environ Microbiol 55:1943–1948

    PubMed  CAS  Google Scholar 

  • Barstow DA, Clarke AR, Chia WN et al (1986) Cloning, expression and complete nucleotide sequence of Bacillus stearothermophilus L-lactate dehydrogenase gene. Gene 46:47–55

    Article  PubMed  CAS  Google Scholar 

  • Chen ZF, Wojcik SF, Welker NE (1986) Genetic analysis of Bacillus stearothermophilus by protoplast fusion. J Bacteriol 165:994–1001

    PubMed  CAS  Google Scholar 

  • Cohn NS (1961) Production of chromatid aberrations by diepoxybutane and an iron chelator. Nature 192:1093–1094

    Article  PubMed  CAS  Google Scholar 

  • Cutting SM, van der Horn PB (1990) Genetic analysis. In: Harwood CR, Cutting SM (eds) Molecular biology methods for bacillus. Wiley, Chichester, UK, pp 27–74

    Google Scholar 

  • Dien BS, Hespell RB, Wyckoff HA et al (1998) Fermentation of hexose and pentose sugars using a novel ethanologenic Escherichia coli strain. Enz Microb Technol 23:366–371

    Article  CAS  Google Scholar 

  • Green E, Cusdin, FS, Baghaei-Yazdi N et al (2002) Modification of bacteria. US Patent No 20020042134

  • Hild HM, Stuckey DC, Leak DJ (2003) Effect of nutrient limitation on product formation during continuous fermentation of xylose with Thermoanaerobacter ethanolicus JW200 Fe(7). Appl Microbiol Biotechnol 60:679–686

    PubMed  CAS  Google Scholar 

  • Ingram LO, Conway T (1988) Expression of different levels of ethanologenic enzymes from Zymomonas mobilis in recombinant strains of Escherichia coli. Appl Environ Microbiol 54:397–404

    PubMed  CAS  Google Scholar 

  • Ingram LO, Conway T, Clark DP et al (1987) Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol 53:2420–2425

    PubMed  CAS  Google Scholar 

  • Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Liao H, McKenzie T, Hageman R (1986) Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. Proc Natl Acad Sci USA 83:576–580

    Article  PubMed  CAS  Google Scholar 

  • Lynd LR (1989) Production of ethanol from lignocellulosic materials using thermophilic bacteria: criteria evaluation of potential and review. Adv Biochem Eng Biotech 38:1–52

    CAS  Google Scholar 

  • Pohl M, Grotzinger J, Wollmer A et al (1994) Reversible dissociation and unfolding of pyruvate decarboxylase from Zymomonas mobilis. Eur J Biochem 224:651–661

    Article  PubMed  CAS  Google Scholar 

  • Pohl M, Mesch K, Rodenbrock A et al (1995) Stability investigations on the pyruvate decarboxylase from Zymomonas mobilis. Biotech Appl Biochem 22:95–105

    CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis TS (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY

    Google Scholar 

  • San Martìn R, Bushell D, Leak DJ et al (1992) Development of a synthetic medium for continuous growth and ethanol production with a lactate dehydrogenase mutant of Bacillus stearothermophilus. J Gen Microbiol 138:987–996

    PubMed  Google Scholar 

  • San Martìn R, Bushell D, Leak DJ et al (1993) Pathways of ethanol production from sucrose by a mutant thermophilic Bacillus in continuous culture. J Gen Microbiol 139:1033–1040

    Google Scholar 

  • Sommer P, Georgieva T, Ahring BK (2004) Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicellulose. Biochem Soc Trans 32:283–289

    Article  PubMed  CAS  Google Scholar 

  • Talarico LA, Gil MA, Yomano LP, Ingram LO, Maupin-Furlow JA (2005) Construction and expression of an ethanol operon in Gram positive bacteria. Microbiology 151:4023–4031

    Article  PubMed  CAS  Google Scholar 

  • Wittung-Stafshede P (2002) Role of cofactors in protein folding. Acc Chem Res 35:201–208

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Lonnie Ingram for providing pLOI275 and the anti-Z. mobilis Pdc antibody, to H. Liao for pBST22 and R. Gemmell for help with SDS-PAGE and Western Blotting. AHT was supported by a BBSRC-CASE studentship with Agrol Ltd and DJS was supported by a BBSRC studentship.

Author information

Author notes

  1. David J. Studholme

    Present address: Sainsbury Laboratory, John Innes Centre, Norwich, NR4 7UH, UK

  2. Edward M. Green

    Present address: Green Biologics Ltd, 45A, Milton Park, Abingdon, Oxfordshire, OX14 4RU, UK

Authors and Affiliations

  1. Division of Biology, Faculty of Natural Sciences, Imperial College London, South Kensington, London, SW7 2AZ, UK

    Ann H. Thompson, David J. Studholme & David J. Leak

  2. Agrol Ltd, The White House, 2 Meadrow, Godalming, Surrey, GU7 3HN, UK

    Edward M. Green

Authors
  1. Ann H. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David J. Studholme
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Edward M. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David J. Leak
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David J. Leak.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thompson, A.H., Studholme, D.J., Green, E.M. et al. Heterologous expression of pyruvate decarboxylase in Geobacillus thermoglucosidasius . Biotechnol Lett 30, 1359–1365 (2008). https://doi.org/10.1007/s10529-008-9698-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-008-9698-1

Keywords

Search

Navigation