Enhanced lignocellulose degradation in Bacillus subtilis RLI2019 through CRISPR/Cas9-mediated chromosomal integration...

Bacillus subtilis (B. subtilis) is a crucial industrial microorganism for lignocellulose biomass degradation. However, wild-type strains from natural environments have inherent deficiencies in the composition of cellulase genes, so constructing recombinant strains through genome engineering is a generalizable strategy to overcome these shortcomings. Herein, eglS, cel48S, and bglS were integrated into the aprE, epr, and amyE loci of the B. subtilis RLI2019 chromosome, respectively, through CRISPR/Cas9-mediated genome editing, deriving the engineered strain B. subtilis AEA3. The activities of endoglucanase, exoglucanase, β-glucosidase, xylanase, and total cellulase in B. subtilis AEA3 were enhanced by 3.1-fold, 6.6-fold, 3.0-fold, 1.2-fold, and 1.8-fold, respectively, reaching 26.31 U/mL, 9.77 U/mL, 3.91 U/mL, 19.63 U/mL, and 2.42 U/mL. Notably, the engineered strain improved the saccharification efficiency of crop straws, effectively disrupting fiber structure, and significantly reducing the content of neutral and acid detergent fibers, lignocellulose and hemicellulose. In summary, this study provides a general strategy for enhancing the cellulose degradation capabilities of B. subtilis through comprehensive and systematic multi-module genetic engineering, broadening its potential application in lignocellulose biomass conversion.