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Originally published In Press as doi:10.1074/jbc.M102082200 on April 4, 2001

J. Biol. Chem., Vol. 276, Issue 24, 21257-21261, June 15, 2001
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Design and Production of Active Cellulosome Chimeras
SELECTIVE INCORPORATION OF DOCKERIN-CONTAINING ENZYMES INTO DEFINED FUNCTIONAL COMPLEXES*

Henri-Pierre FierobeDagger , Adva Mechaly§, Chantal TardifDagger , Anne BelaichDagger , Raphael Lamed||, Yuval Shoham**, Jean-Pierre BelaichDagger , and Edward A. Bayer§Dagger Dagger

From the Dagger  Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, Institut de Biologie Structurale et Microbiologie-Institut Fédératif de Recherche 1, 13402 Marseille, France, the § Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel, the  Université de Provence, 13331 Marseille, France, the || Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel, and the ** Department of Food Engineering and Biotechnology and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.

* This work was supported by a contract from the European Commission (Fourth Framework, Biotechnology Programme, BIO4-97-2303) and by grants from the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem). Additional support was provided by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany), and by funds from the Technion-Niedersachsen Cooperation (Hannover, Germany).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: +972-8-934-2373; Fax: +972-8-946-8256; E-mail: bfbayer@wicc.weizmann.ac.il.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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