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 mol ecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum a nd Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporate d into the complexes by means of high-affinity species-specific cohesin-doc kerin interactions. To construct the desired complexes, a series of chimeri c scaffoldins was prepared by recombinant means. The scaffoldin chimeras we re designed to include two cohesin modules from the different species, opti onally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that leach recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropria te dockerin-containing enzymes could be assembled precisely and by design i nto a desired complex. Compared with the mixture of free cellulases, the re sultant cellulosome chimeras exhibited enhanced synergistic action on cryst alline cellulose.