Assembly of Minicellulosomes on the Surface of Bacillus subtilis
ABSTRACTTo 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 multicellulase-containing complexes present in cellulolyticClostridiumandRuminococcusspecies. In this study we created cellulolytic strains ofBacillus subtilisthat display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with theBacillus anthracissortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase fromClostridium thermocellumto the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface ofB. subtilisvia noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced inEscherichia coli.B. subtilishas a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface ofB. subtilismay yield cellulolytic bacteria with increased potency that can be used to degrade biomass.