Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes.
Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.
Mingardon
Florence
F
Department of Bioénergétique et Ingénierie de Protéines, UPR9036, BIP-CNRS, IBSM, 31, chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Chanal
Angélique
A
López-Contreras
Ana M
AM
Dray
Cyril
C
Bayer
Edward A
EA
Fierobe
Henri-Pierre
HP
D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D044602:Cellulases / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D044603:Cellulosomes / Q000201:enzymology*; D002850:Chromatography, Gel; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology*; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009097:Multienzyme Complexes / Q000096:biosynthesis*; D020088:Neocallimastix / Q000201:enzymology*; D015202:Protein Engineering / Q000379:methods; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D009097:Multienzyme Complexes; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D044602:Cellulases