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Punt, Peter
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article
Design and Production in <i>Aspergillus niger</i> of a Chimeric Protein Associating a Fungal Feruloyl Esterase and a Clostridial Dockerin Domain
Abstract
<jats:title>ABSTRACT</jats:title><jats:p>A chimeric enzyme associating feruloyl esterase A (FAEA) from<jats:italic>Aspergillus niger</jats:italic>and dockerin from<jats:italic>Clostridium thermocellum</jats:italic>was produced in<jats:italic>A. niger</jats:italic>. 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<jats:italic>A. niger</jats:italic>glucoamylase and a dibasic proteolytic processing site (<jats:italic>kex</jats:italic>-<jats:italic>2</jats:italic>) 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.</jats:p>