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

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.

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