(139c) Consolidated Bioprocessing of Lignocellulosic Biomass Components Via Synergistic Bacterial Co-Culture Consortia

Lignocellulosic biomass is the most abundant untapped renewable resource which can be utilized for the sustainable production of fuels and chemicals. Lignocellulosic biomass is composed of the complex polymers cellulose, hemicellulose and lignin which can be depolymerized to produce monomeric sugars for bacterial fermentations. 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 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. Consolidated bioprocessing overcomes this drawback by enabling microbial hosts to secrete hydrolases without the need for expensive purification. 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.

In the first part, B. subtilis was engineered to breakdown hemicellulose as hemicellulose hydrolysis requires only one enzyme, endo-β-1,4 xylanase. Different combinations of signal peptides YwmC, SacC, and AmyE with the two xylanases from Trichoderma resei (Tr) and Bacillus pumilis (Bp) were explored for the selection of an optimal design. The supernatant of the engineered strain SSL26 harbouring YwmC-XynA (Bp) displayed a 2-fold increase in endoxylanase activity. In situ depolymerization of xylan with SSL26 resulted in a maximum xylose titer of 15 g/L. As a final step, a B. subtilis: E. coli consortia was developed to break down xylan and simultaneously produce succinate from xylose in a single pot achieving a succinate yield of 3.7 g/L from 10 g/L xylan.

Secondly, for cellulose breakdown two major celluloses required are β glucosidases (Bglu) and endoglucanases (Eglc). Similar to previous work combination of signal peptides YwmC, SacC and AmyE with two β glucosidases were developed. AmyE- Bglu showed 6 folds higher activity compared to wildtype in pNPG assay. Similarly, endoglucanases constructs were developed with Bglc, YwmC, NprE signal peptide library to demonstrate complete breakdown of cellulose using β glucosidases and endoglucanases secreting strain cocultures.We envision that the two approaches together will play a major role in providing alternatives for improving the economics of the biorefinery.