CRISPR/Cas9-engineered Bacillus subtilis chassis for tailored chitooligosaccharide production from marine waste chitosan

This study establishes a sustainable bioprocess for converting chitosan from marine waste into high-value chitooligosaccharides (COSs), offering an eco-friendly alternative to conventional methods that often generate chemical waste. We achieved heterologous production of chitosanase in an engineered Bacillus subtilis chassis by knocking out its endogenous chitosanase, leveraging the dual advantages of this bacterium as a robust synthetic biology platform and an industrial microorganism. The endogenous chitosanase gene (BsCsn) in Bacillus subtilis WB800N was deleted via CRISPR/Cas9-mediated editing, generating the chassis strain B. subtilis WB800N ΔBsCsn. A codon-optimized GH46 chitosanase (CsnA) from Streptomyces coelicolor, fused to the AprE signal peptide, was then expressed in this host. Response surface methodology optimized the fermentation process, enabling a high extracellular CsnA activity of 540.08 ± 6.20 U/mL, in a 5-L bioreactor under DO-stat-controlled fed-batch conditions. This process achieved a productivity of 11.25 U/(mL·h) and a carbon conversion efficiency of 1682.86 U/g glycerol. Furthermore, MALDI-TOF MS analysis confirmed that CsnA produces COSs with defined degrees of polymerization (DP2-DP4). This integrated platform enables the upcycling of marine waste into high-value COSs, establishing B. subtilis as an eco-efficient cell factory and providing a valuable framework for the heterologous expression of other chitosanases in this host.