(424f) One-Pot Hydrolysis of Lignocellulosic Biomass Components Via Engineered Bacillus Subtillis.

Advent of 21^st century has seen surge in demand of energy, the depletion of fossil fuel reserves and detrimental effect of fossil fuels on environment. Therefore, alternative energy sources have been extensively explored to reduce our dependence on non-renewable fuel sources. Agricultural waste is fascinating alternative which is mainly composed of cellulose, hemicellulose and lignin. This untapped resource can be utilised by breaking down complex biopolymers in agricultural waste into fermentable sugars by process called hydrolysis. Both chemicals and enzymes can be used as catalyst in hydrolysis of biomass subunits but chemical hydrolysis produces furfural, hydroxymethylfurfural and other byproducts which can be toxic for microorganisms. Hence, Enzymes are safer and efficient alternative for biomass hydrolysis. Currently, biorefineries utilize purified hydrolases (class of enzyme) for breaking down cellulose and hemicellulose into its sugars, which is not cost effective. Therefore, efficient and cost-effective methods for hydrolysis of the cellulosic and hemicellulosic biomass can play vital role in making biorefineries economically viable. Bacillus subtillis strains are enzyme workhorses and are widely used in biotechnology industries for enzyme production. The present work aims at the engineering of Bacillus subtillis for heterologous expression and extracellular export of hydrolases (cellulases and xylanases) to reduce the cost incurred by protein purification. Extracellular enzyme export can be facilitated with the help of signal peptide (a short chain of amino acids which help in export of enzymes outside a bacterial cell). Exhaustive study on signal peptides of B. Subtilis has been reported by Brockmeier et al. 2006a, 2006b which shows that signal peptides can increase the extracellular enzyme export in B subtilis. Different combinations of signal peptides and genes were explored to identify the optimal secretion system. Three signal peptides namely YwmC, SacC and AmyE; two b- glucosidases (Bg) from Trichoderma resei (Tr) and Neosartorya fischeri (Nf) and two xylanases from Trichoderma resei and Bacillus pumilis (Bp) were selected. The selection was based upon their activity at the pH and temperature conditions in which the microbials host is still viable. 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 and p-NPG assay. Recombinant strains with AmyE- Bg (Tr) and YwmC- Xy (Tr) displayed 4.1 fold increase in cellobiose hydrolysis and 2.2 fold increase in xylan hydrolysis compared to wild type strain, respectively. Insitu breakdown of xylan was studied for screened strain consisting YwmC- Xy (Tr) which demonstrated production of 7.1 g/L of xylose from 1% xylan after 72 hours of fermentation. Hence, we, have demonstrated one pot process for enzymatic degradation of lignocellulosic biomass components for feedstock generation.Further work is underway to breakdown the biomass components and produce high value products simultaneously.