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Applied and Environmental Microbiology, June 2007, p. 3822-3832, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00398-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes{triangledown}

Florence Mingardon,1 Angélique Chanal,1 Ana M. López-Contreras,2 Cyril Dray,1,{dagger} Edward A. Bayer,3 and Henri-Pierre Fierobe1*

Department of Bioénergétique et Ingénierie des Protéines, CNRS, IBSM, 13402 Marseille, France,1 Bioconversion Group, Agrotechnology and Food Sciences Group, WUR, P.O. Box 17, 6700 AA Wageningen, The Netherlands,2 Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel3

Received 20 February 2007/ Accepted 18 April 2007

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.

* Corresponding author. Mailing address: UPR9036, BIP-CNRS, 31, chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Phone: 33-491-16-42-99. Fax: 33-491-71-33-21. E-mail: hpfierob{at}ibsm.cnrs-mrs.fr

{triangledown} Published ahead of print on 27 April 2007.

{dagger} Present address: IBDML, UMR 6216, 13288 Marseille, France.

Applied and Environmental Microbiology, June 2007, p. 3822-3832, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00398-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Caspi, J., Barak, Y., Haimovitz, R., Irwin, D., Lamed, R., Wilson, D. B., Bayer, E. A. (2009). Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode. Appl. Environ. Microbiol. 75: 7335-7342 [Abstract] [Full Text]  
  • Mingardon, F., Chanal, A., Tardif, C., Bayer, E. A., Fierobe, H.-P. (2007). Exploration of New Geometries in Cellulosome-Like Chimeras. Appl. Environ. Microbiol. 73: 7138-7149 [Abstract] [Full Text]  


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