Engineering of multiple modules to enhance lignocellulose degradation ability in Bacillus subtilis using CRISPR/Cas9 system

Bacillus subtilis is widely employed for lignocellulose degradation. However, wild-type strains typically exhibit low and incomplete cellulase activities. This study aimed to engineer recombinant B. subtilis strains harboring complete, high-efficiency cellulase systems using CRISPR/Cas9-mediated genome editing. After optimization of signal peptides, transcriptional terminators, and chromosomal integration sites, two endoglucanase expression cassettes were integrated into B. subtilis 168, yielding a strain with a 16.29-fold increase in endoglucanase activity relative to the parental strain. In parallel, a bifunctional cellulase was successfully expressed and optimized, achieving exoglucanase (549.77 U/mL) and β-glucosidase (349.26 U/mL) activities. Moreover, the strain BSKI3Cel, containing three optimized expression cassettes in its genome, exhibited high endoglucanase (129.59 U/mL), exoglucanase (596.75 U/mL), and β-glucosidase (447.42 U/mL) activities. Subsequent transformation of BSKI3Cel with plasmid pJOE2006Bf, carrying genes encoding a composite cellulase system, yielded BSK3P2C, which achieved peak cellulase activities of 533.16, 2959.83, and 2829.61 U/mL on day 8. As a proof of concept, fermentation of wheat straw using BSK3P2C was found to significantly reduce hemicellulose (16.70 %), neutral detergent fiber (7.46 %) and acid detergent fiber (9.93 %) contents. Microscopic analyses confirmed extensive lignocellulose degradation. Overall, this study establishes a high-performance B. subtilis platform with complete cellulase systems for efficient cellulosic biomass conversion.