Assembly of Minicellulosomes on the Surface of Bacillus subtilis

To cost-efficiently produce biofuels new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface displayed multi-cellulase containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of B. subtilis that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by co-expressing them with the B. anthracis sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from C. thermocellum to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of B. subtilis via non-covalent interactions with a cell wall attached cohesin module. We also demonstrate that it is possible to assemble multi-enzyme complexes on the cell surface. A three enzyme containing minicellulosome was displayed on the cell surface that consists of a cell wall attached scaffoldin protein non-covalently bound to three cellulase-dockerin fusion proteins that were produced in E. coli. B. subtilis has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of B. subtilis may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.