Intein-mediated assembly of tunable scaffoldins for facile synthesis of designer cellulosomes

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coh(t)), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Coh(c)) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (A(t)) and C. cellulolyticum exoglucanase (E-c), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both E-c and A(t) on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Coh(c)/Coh(t) ratio (i.e., increased E-c/A(t) ratio). Furthermore, among scaffoldins having the same Coh(c)/Coh(t) ratio, highest rates of Avicel hydrolysis are noted when Coh(t), and hence an endoglucanase, is situated next to the CBM and not flanked by Coh(c). These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.