ADAPTOR SCAFFOLDINS: AN ORIGINAL STRATEGY FOR EXTENDED DESIGNER CELLULOSOMES, INSPIRED FROM NATURE

The designer cellulosome concept consists of chimaeric scaffoldins for controlled incorporation of recombinant enzymes [1,2]. The largest designer cellulosome that has been reported to date was designed by our group and is a 6-cohesin chimaeric scaffoldin [3]. This size raised a technical limit of sorts, since adding another cohesin to this chimaeric scaffoldin proved problematic. Nevertheless, increasing the number of enzymes that can be incorporated into designer cellulosomes is critical in order to further enhance degradation of plant cell residues.

Adaptor scaffoldins comprise an intermediate scaffoldin that can both incorporate various enzymes and attach to a primary scaffoldin. This model of intricate architecture for designer

cellulosome was inspired by naturally occurring adaptor scaffoldins. By using this simple strategy, we can both extend the number of enzymes and resolves the limited number of available cohesin-dockerin specificities.

We have constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional (primary) scaffoldin and a CBM for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by incorporation of an appropriate type II cohesin. We were thus able to incorporate 8 recombinant T. fusca enzymes. The contribution of the adaptor scaffoldin to the hexavalent scaffoldin was proved by a wide variety of controls that clearly demonstrated that the proximity between the two scaffoldins is indeed relevant for optimized degradation. The potency of our new complex designer cellulosome was evaluated compared to the native C. thermocellum cellulosome. By using the adaptor-mediated configuration, we obtained a ratio of approximately 70%.