Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes.
Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.
Fierobe
H P
HP
Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, Institut de Biologie Structurale et Microbiologie-Institut Fédératif de Recherche 1, 13402 Marseille, France.
Mechaly
A
A
Tardif
C
C
Belaich
A
A
Lamed
R
R
Shoham
Y
Y
Belaich
J P
JP
Bayer
E A
EA
D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C074775:CelA endoglucanase; D002480:Cellulase