High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.