Skip to main content
SpringerLink
Log in

High-level expression of Clostridium thermocellum cellulase genes in Escherichia coli

  • Applied Genetics and Regulation
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Summary

The Clostridium thermocellum cellulase genes celA and celC encoding endoglucanase A and C were subcloned in a temperature-regulated Escherichia coli expression vector containing the leftward promoterpl of bacteriophage lambda. The level of gene expression was controlled by thermal inactivation of the heat-sensitive lambda cI857 repressor. Under optimal conditions the recombinant endoglucanases A and C were expressed to a level of 10–15% of total cellular protein. Endoglucanase A was partially exported into the periplasmic space, whereas endoglucanase C was found sequestered within the cytoplasm. Overexpression of the celA gene resulted in decreased cell viability concomitant with the accumulation of endoglucanase A in the membrane fraction. In contrast, high-level synthesis of the celC gene product was well tolerated by the host cell. Overproduced endoglucanase C accumulated as a soluble enzyme without detectable formation of inactive inclusion bodies.

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.

Similar content being viewed by others

References

  • Aiba S, Kitai K, Imanaka T (1983) Cloning of a thermostable alpha-amylase gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis. Appl Environ Microbiol 46:1059–1065

    Google Scholar 

  • Beguin P, Cornet P, Aubert JP (1985) Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. J Bacteriol 162:102–105

    Google Scholar 

  • Caulcott CA, Rhodes M (1986) Temperature-induced synthesis of recombinant proteins. Trends Biotechnol 4:142–146

    Google Scholar 

  • Coughlan MP (1985) The properties of fungal and bacterial cellulases with comment on their production and application. Biotechnol Genet Eng Rev 3:39–109

    Google Scholar 

  • Hon-nami K, Coughlan MP, Hon-nami H, Ljungdahl LG (1986) Separation and characterization of the complexes constituting the cellulolytic enzyme system of Clostridium thermocellum. Arch Microbiol 145:13–19

    Google Scholar 

  • Ito K, Bassford PJ, Beckwith J (1981) Protein localization in Escherichia coli: Is there a common step in the secretion of periplasmic and outer-membrane, proteins? Cell 24:707–717

    Google Scholar 

  • Johnson EA, Demain AL (1984) Probable involvement of sulfhydryl groups and a metal as essential components of the cellulase system of Clostridium thermocellum. Arch Microbiol 137:135–138

    Google Scholar 

  • Johnson EA, Sakajoh M, Halliwell G, Madia A, Demain AL (1982) Saccharification of complex cellulosic substrates by the cellulase system from Clostridium thermocellum. Appl Environ Microbiol 43:1125–1132

    CAS  Google Scholar 

  • Johnson EA, Bouchot F, Demain AL (1985) Regulation of cellulase formation in Clostridium thermocellum. J Gen Microbiol 131:2303

    Google Scholar 

  • Joliff G, Beguin P, Juy M, Millet J, Ryter A, Poljak R, Aubert JP (1986a) Isolation, crystallization and properties of a new cellulase of Clostridium thermocellum overproduced in Escherichia coli. Bio/Technology 4:896–900

    Google Scholar 

  • Joliff G, Beguin P, Aubert JP (1986b) Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum. Nucleic Acid Res 14:8605–8613

    Google Scholar 

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

    PubMed  Google Scholar 

  • Luczak H, Schwarzmoser H, Staudenbauer WL (1985) Construction of Clostridium butyricum hybrid plasmids and transfer to Bacillus subtilis. Appl Microbiol Biotechnol 23:114–122

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Habor, N. Y.

    Google Scholar 

  • Millet J, Petre D, Beguin P, Raynaud O, Aubert JP (1985) Cloning of ten distinct DNA fragments of Clostridium thermocellum coding for cellulases. FEMS Microbiol Lett 29:145–149

    Google Scholar 

  • O'Neill GP, Kilburn DG, Warren RAJ, Miller RC (1986) Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli. Appl Environ Microbiol 52:737–743

    Google Scholar 

  • Oliver D (1985) Protein secretion in Escherichia coli. Annu Rev Microbiol 39:615

    Google Scholar 

  • Petre D, Millet J, Longin R, Beguin P, Girard H, Aubert JP (1986) Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli. Biochimie 68:687–695

    Google Scholar 

  • Pugsley AP, Schwartz M (1985) Export and secretion of proteins by bacteria. FEMS Microbiol Rev 32:3

    Google Scholar 

  • Remaut E, Stanssens P, Fiers W (1981) Plasmid vectors for high-efficiency expression controlled by thepl promoter of coliphage lambda. Gene 15:81–93

    Google Scholar 

  • Schwarz W, Bronnenmeier K, Staudenbauer WL (1985) Molecular cloning of Clostridum thermocellum genes involved in β-glucan degradation in bacteriophage lambda. Biotechnol Lett 7:859–864

    Google Scholar 

  • Schwarz WH, Gräbnitz F, Staudenbauer WL (1986) Properties of a Clostridium thermocellum endoglucanase produced in Escherichia coli. Appl Environ Microbiol 51:1293–1299

    Google Scholar 

  • Schwarz WH, Bronnenmeier K, Gräbnitz F, Staudenbauer WL (1987) Activity staining of cellulases in polyacrylamide gels containing mixing linkage β-glucans. Anal Biochem (in press)

  • Schüller A, Harkness RE, Rüther U, Lubitz, W (1985) Deletion of C-terminal amino acid codons of PhiX174 gene E: Effect on its lysis inducing properties. Nucleic Acid Res 13:4143–4152

    Google Scholar 

  • Sedmark JJ, Grossberg SE (1977) A rapid, sensitive assay for protein using Coomassie brilliant blue G250. Anal Biochem 79:544–552

    Google Scholar 

  • Siegel R, Ryu DDY (1985) Kinetic study of instability of recombinant plasmid pPLc23trpA1 in Escherichia coli using two-stage continuous culture system. Biotechnol Bioeng 27:28

    Google Scholar 

  • Soutschek-Bauer E, Staudenbauer WL (1987) Synthesis and secretion of a heat-stable carboxymethylcellulase from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus. Mol Gen Genet 208:537–541

    Google Scholar 

  • Takizawa N, Murroka Y (1985) Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes. Appl Environ Microbiol 49:294–298

    Google Scholar 

  • Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute for Microbiology, Technical University Munich, Arcisstr. 21, D-8000, München 2, Germany

    Wolfgang H. Schwarz, Silke Schimming & Walter L. Staudenbauer

Authors
  1. Wolfgang H. Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Silke Schimming
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Walter L. Staudenbauer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schwarz, W.H., Schimming, S. & Staudenbauer, W.L. High-level expression of Clostridium thermocellum cellulase genes in Escherichia coli . Appl Microbiol Biotechnol 27, 50–56 (1987). https://doi.org/10.1007/BF00257253

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00257253

Keywords

Search

Navigation