Secretion of heterologous thermostable cellulases in Bacillus subtilis

Abstract

Bacillus subtilis is used industrially for the production of secreted enzymes. The most characteristic feature of the secreted enzymes is variation in the N-terminal signal peptides that is recognized by secretion machinery, which is one of the determinants of efficiency and must be customized in each case. Culturing cellulolytic B. subtilis to secrete heterologous cellulases combined with customized signal peptides would be beneficial for producing biocommodities from cellulosic biomass. Four Clostridium thermocellum genes, encoding endoglucanases (celA and celB) and exoglucanases (celK and celS) were cloned to construct random libraries of combinations with 173 different signal peptides originating from the B. subtilis genome. The libraries were successfully screened to identify the signal peptides most efficient in secretion of each of the four cellulases, which were theoretically unpredictable. The secreted cellulases were assayed on carboxymethyl cellulose, phosphoric acid swollen cellulose, and microcrystalline cellulose to determine the possible effects of the signal peptides on substrate specificity. The customized signal peptides for CelA, CelB, and CelS did not affect enzyme performance but those for CelK might influence its substrate specificity.