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Applied and Environmental Microbiology, December 2004, p. 6984-6991, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.6984-6991.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Design and Production in Aspergillus niger of a Chimeric Protein Associating a Fungal Feruloyl Esterase and a Clostridial Dockerin Domain

Anthony Levasseur,1* Sandrine Pagès,2,3 Henri-Pierre Fierobe,2 David Navarro,1 Peter Punt,4 Jean-Pierre Belaïch,2,3 Marcel Asther,1 and Eric Record1

UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, IFR86-BAIM, Universités de Provence et de la Méditerranée, ESIL,1 Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, IBSM,2 Université de Provence, Marseille, France,3 Department of Microbiology, TNO Nutrition and Food Research Institute, Zeist, The Netherlands4

Received 27 April 2004/ Accepted 22 July 2004

A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergillus niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from A. niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.

* Corresponding author. Mailing address: UMR 1163 INRA/Université de Provence de Biotechnologie des Champignons Filamenteux, IFR-IBAIM, Universités de Provence et de la Mediterranée, ESIL, 163 avenue de Luminy, Case Postale 925, 13288 Marseille cedex 09, France. Phone: 33 4 91 82 86 07. Fax: 33 4 91 82 86 01. E-mail: anthony.levasseur{at}esil.univ-mrs.fr.

Applied and Environmental Microbiology, December 2004, p. 6984-6991, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.6984-6991.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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  • Levasseur, A., Navarro, D., Punt, P. J., Belaich, J.-P., Asther, M., Record, E. (2005). Construction of Engineered Bifunctional Enzymes and Their Overproduction in Aspergillus niger for Improved Enzymatic Tools To Degrade Agricultural By-Products. Appl. Environ. Microbiol. 71: 8132-8140 [Abstract] [Full Text]  


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