Screening signal peptides from Bacillus thuringiensis secretome for heterologous protein secretion.

Efficient secretion of heterologous proteins is crucial for applications in industrial and biomedical fields. Selecting appropriate signal peptides and bacterial strains is critical for successful protein expression and export. Bacillus thuringiensis, known for its robust secretion capabilities within the Bacillus genus, shows promise as an ideal host for this purpose. We performed genome-based bioinformatic analysis of B. thuringiensis HD73. A total of 525 proteins were predicted to contain signal peptides, exceeding those in other Bacillus species. The extracellular proteome of B. thuringiensis HD73 was analyzed via LC-MS/MS, identifying 100 secreted proteins. A library of 30 signal peptides was constructed by integrating genome-based predictions with experimental secretome data. Using this library, green fluorescent protein secretory expression systems were developed in the acrystalliferous mutant strain B. thuringiensis HD73, and the strain carrying signal peptide S17 showed the highest secretion efficiency. Additionally, the top 10 performing signal peptides were used to express and secrete the convenient enzyme cutinase, with the S20 fusion strain exhibiting the highest cutinase activity (3.65 U/mL in the culture supernatant). This study provides the first combined bioinformatic and experimental characterization of the B. thuringiensis secretome. The developed secreted protein expression system and signal peptide library demonstrate effectiveness and offer potential for future heterologous protein secretion in B. thuringiensis. KEY POINTS: • Genome-based secretome and experimental secretome of B. thuringiensis were characterized. • A SP library comprising 30 SPs derived from B. thuringiensis was constructed. • GFP and cutinase were successfully secreted by B. thuringiensis.