Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain
article
A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergilhis 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.
Topics
BiotechnologyAmino acidsBacteriaBioassayElectrophoresisEnzymesFungiFeruloyl esterase A (FAEA)Fungal enzymesIntracellular productionWestern blottingProteinsAntibodyBacterial proteinCell proteinChimeric proteinCohesinEsteraseFungal enzymeGlucan 1,4 alpha glucosidaseHybrid proteinProtein dockerinProtein feruloyl esterase AUnclassified drugEnzymeFungusMicrobiologyProteinAmino acid sequenceAspergillus nigerCellular distributionClostridium thermocellumComplex formationEnzyme activityEnzyme bindingEnzyme substrate complexMolecular weightNonhumanNorthern blottingPolyacrylamide gel electrophoresisPosttranscriptional gene silencingProtein degradationProtein domainProtein processingProtein protein interactionProtein secretionTranscription initiationWestern blottingAspergillus nigerBacterial ProteinsBiotechnologyCarboxylic Ester HydrolasesCarrier ProteinsCell Cycle ProteinsChromosomal Proteins, Non-HistoneClostridium thermocellumFungal ProteinsNuclear ProteinsRecombinant Fusion ProteinsTranscription, GeneticTransformation, GeneticActinobacteria (class)AspergillusAspergillus nigerBacteria (microorganisms)ClostridiumClostridium thermocellumFungiPosibacteriauncultured actinomycete
TNO Identifier
238234
ISSN
00992240
Source
Applied and Environmental Microbiology, 70(12), pp. 6984-6991.
Pages
6984-6991