High-efficiency heterologous expression of functional RVG29-BoNT/A light chain fusion protein in Bacillus subtilis based on a combinatorial strategy

Botulinum neurotoxin type A (BoNT/A) has been used as an injectable therapeutic agent in medical aesthetics, neuroscience research, and other medical fields. In this study, the non-endotoxin-producing and food-safe bacterium Bacillus subtilis WB800N was selected as the host strain to express functional RVG29-BoNT/A light chain (Lc) fusion proteins through a combinatorial strategy involving promoter engineering, signal peptide optimization, and overexpression of transport-related proteins and molecular chaperones. The RVG29 peptide, known for its neuronal targeting ability, was fused to the Lc of BoNT/A to enhance its transmembrane delivery and substrate cleavage activity. Furthermore, a series of promoter screening, tandem promoter construction, and site-specific mutations were conducted to maximize transcriptional activity of the target protein. Moreover, a library of 244 native signal peptides was constructed, and high-throughput fluorescence screening identified Sec pathway-related signal peptides that significantly improved secretion efficiency. Meanwhile, transport-related proteins, particularly FtsY, and molecular chaperones like GroEL/DnaJ, were overexpressed to further enhance the secretion and soluble expression of the RVG29-Lc. Additionally, knockdown of the hrcA gene, a repressor of heat-shock genes, improved the expression of molecular chaperones, as well as the RVG29-Lc. The combinatorial strategy achieved a final intracellular expression level of 500.33 +/- 5.00 mg/L and the secretion level of 75.15 +/- 4.33 mg/L under the same experimental conditions. This work presents a robust framework for the high-efficiency production of functional RVG29-Lc fusion proteins in B. subtilis, offering valuable insights into the industrial-scale production of therapeutic BoNT-derived proteins.