(175y) Engineering Bacillus Subtillis for the Secretion of Hydrolases Towards Consolidated Bioprocessing of Cellulosic Biomass

Increasing global concerns about limited reserves of conventional fuel sources has led to exploration of alternative fuel sources worldwide. Agricultural waste is a fascinating alternative which mainly constitutes of cellulose, hemicellulose and lignin. Agricultural waste constituting hemicellulosic and cellulosic waste is a promising renewable feedstock for increasing future demand of renewable biofuels and value-added products. Efficient and cost-effective methods are essential for breakdown of cellulosic materials into elementary components. Cost effective enzymatic hydrolysis of the cellulosic and hemicellulosic biomass can play a vital role in making economical biorefineries viable. Bacillus subtillis strains are enzyme workhorses and are widely used in biotech industries for enzyme production. Sec pathway in Bacillus subtillis secretes highest number of extracellular enzymes compared to other secretory pathway. Signal peptides play an important role in enzyme export across the bacterial cell membrane in the Sec pathway. Present work aims at development of bacterial platform for heterologous expression and extracellular export of hydrolases (cellulases and xylanases) for extracellular catalysis via the Sec pathway. Different combinations of signal peptides with genes were designed for optimal functionality. Three signal peptides namely YwmC, SacC and AmyE; two b- glucosidases from Trichoderma resei (Tr) and Neosartorya fischeri (Nf) and xylanases from Trichoderma resei and Bacillus pumilis (Bp); were selected based on compatible functionality with respect to pH temperature conditions. Three constructs were made using combinations of signal peptide-xylanase-glucosidase as; YwmC-Xyl(Tr), YwmC-Bg(Tr) and YwmC-Bg(Nf) these components. Other constructs with different combinations of these components is underway. The recombinant and wild type strains of B. subtillis WB800N were screened for extracellular hydrolysis of the substrates and comparative analysis was performed on the basis of DNSA assay. All the recombinants exhibited higher cellobiose and xylan degradation over wild type. Recombinant with Bg(Tr) displayed 4.2 fold increase in cellobiose hydrolysis, while those with Bg(Nf) and Xy (Tr) showed 2.7 and 2.4 fold increase in cellobiose and xylan hydrolysis, respectively. Hence, we, for the first time, have demonstrated one pot consolidated process for enzymatic degradation of lignocellulosic biomass components for feedstock generation. This approach will be further explored for effective degradation of complex polymers like cellulose and hemicellulose to obtain substrates for second generation biofuel production.