Nonclassical Secretion and Translational Optimization for Enhancing Alginate Lyase Expression in Bacillus subtilis

Alginate lyase is a crucial enzyme for the production of alginate oligosaccharides, a versatile functional sugar widely utilized in the pharmaceutical, agricultural, and food industries. However, achieving high-level expression of alginate lyase in food-grade Bacillus subtilis remains a significant challenge. This study revealed that the alginate lyase from Paenibacillus sp. YN15 (PyAly) is secreted via a signal peptide-independent, nonclassical pathway in B. subtilis, while its native signal peptide is essential for translational initiation. To enhance PyAly translation, various N-terminal coding sequences (NCSs) and 5' untranslated region (5'-UTR) elements were employed to replace the native signal peptide of PyAly and the original 5'-UTR of the pP43NMK vector, respectively. The optimal combination of superior NCSs (ydbp-up, MLD62, and MLD42) and 5'-UTR elements (UTR4, UTR7, and UTR8) identified the UTR4-MLD62 pairing, which effectively reduced nontarget protein content in the secreted fraction and maximized extracellular PyAly activity, reaching 171.3 U/mL. This enhancement demonstrated the synergistic effect of NCS and 5'-UTR optimizations. This study establishes a platform for fine-tuning the translation and secretion of alginate lyase in B. subtilis, with promising potential for industrial enzyme production.