(166i) Development of an Engineered Co-Culture Consortium for in Situ Depolymerization of Cellulose

Lignocellulosic biomass is the most abundant untapped renewable resource which can be utilized for the sustainable production of fuels and chemicals. Cellulose is the principal constituent of lignocellulosic biomass and is the most abundant biopolymer on earth. Currently, biorefineries utilize purified β-glucosidase (Bglu) and endoglucanase (Eglc) for breaking down cellulose into its sugars, which is not economical due to the prohibitive cost of purified enzymes. Therefore, efficient and cost-effective methods for hydrolysis of cellulose can play a vital role in making biorefineries economically viable. One pot bioprocess overcomes this drawback by enabling multiple microbial hosts to secrete hydrolases without expensive purification. Bacillus subtilis strains are widely used in biotechnology industries for enzyme production and therefore, has been used as a host in this work for developing a one pot process for cellulose. The present work aims to engineer Bacillus subtilis for heterologous expression and extracellular export of cellulases to reduce the cost incurred by protein purification.

First, a combination of signal peptides YwmC, SacC, and AmyE with two β glucosidases was explored to identify the optimal pair. AmyE- Bglu showed six-fold higher activity than wildtype in the para-Nitrophenyl β-D-glucopyranoside (pNPG) assay. Similarly, endoglucanases constructs were developed with Bglc, YwmC, and NprE signal peptide library to demonstrate the complete breakdown of cellulose. Furthermore, we cultured the best performing strains for Bglu secretion and Eglc secretion and isolated the supernatant for further studies. This enzymatic supernatant was further combined to see the synergistic effect of these enzymes on carboxymethylcellulose (CMC). The two enzymes' synergistic effect showed successful cellulose deconstruction into reducing sugars as quantified by dinitro salicylic acid (DNSA) assay. The highest titers obtained were 3.7 g/L of reducing sugar from 10 g/L of CMC. We plan to demonstrate in situ depolymerization by using the two screened strains for simultaneous deconstruction, assimilation, and metabolic processing of cellulose into fuels and value-added products. We envision that this approach will play a significant role in providing alternatives for improving the economics of the biorefinery.