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Journal of Bacteriology, October 2004, p. 6544-6552, Vol. 186, No. 19
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.19.6544-6552.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Towards Designer Cellulosomes in Clostridia: Mannanase Enrichment of the Cellulosomes Produced by Clostridium cellulolyticum

Stéphanie Perret,1,2,{dagger} Anne Bélaich,1 Henri-Pierre Fierobe,1 Jean-Pierre Bélaich,1,2 and Chantal Tardif1,2*

Laboratoire de Bioénergétique et Ingénierie des Protéines, UPR 9036-CNRS,1 Université de Provence, Marseille, France2

Received 1 March 2004/ Accepted 29 June 2004

The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

* Corresponding author. Mailing address: Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Phone: 33 (0)491-16-43-40. Fax: 33 (0)491-71-33-21. E-mail: tardif{at}ibsm.cnrs-mrs.fr.

{dagger} Present address: Centre d'Etudes des Macromolécules Végétales, CNRS, Domaine Universitaire, Grenoble Cedex 9, France.

Journal of Bacteriology, October 2004, p. 6544-6552, Vol. 186, No. 19
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.19.6544-6552.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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