Yang Jianbo, Xia Yan, Zhu Li, Wang Zhiwen, Song Fuping, Guo Shuyuan
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, Beijing, 100081, China.
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, Beijing, 100071, China.
Appl Microbiol Biotechnol. 2025 Jun 14;109(1):147. doi: 10.1007/s00253-025-13533-y.
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.
异源蛋白的高效分泌对于工业和生物医学领域的应用至关重要。选择合适的信号肽和细菌菌株对于成功的蛋白质表达和输出至关重要。苏云金芽孢杆菌以其在芽孢杆菌属内强大的分泌能力而闻名，有望成为实现此目的的理想宿主。我们对苏云金芽孢杆菌HD73进行了基于基因组的生物信息学分析。共预测有525种蛋白质含有信号肽，超过了其他芽孢杆菌属物种中的数量。通过液相色谱-串联质谱法（LC-MS/MS）分析了苏云金芽孢杆菌HD73的细胞外蛋白质组，鉴定出100种分泌蛋白。通过将基于基因组的预测与实验分泌蛋白质组数据相结合，构建了一个包含30个信号肽的文库。利用该文库，在无晶体突变菌株苏云金芽孢杆菌HD73中开发了绿色荧光蛋白分泌表达系统，携带信号肽S17的菌株显示出最高的分泌效率。此外，使用表现最佳的前10个信号肽来表达和分泌便捷酶角质酶，S20融合菌株表现出最高的角质酶活性（培养上清液中为3.65 U/mL）。本研究首次对苏云金芽孢杆菌的分泌蛋白质组进行了生物信息学和实验相结合的表征。所开发的分泌蛋白表达系统和信号肽文库证明了有效性，并为苏云金芽孢杆菌未来的异源蛋白分泌提供了潜力。要点：• 对苏云金芽孢杆菌基于基因组的分泌蛋白质组和实验分泌蛋白质组进行了表征。• 构建了一个包含30个源自苏云金芽孢杆菌信号肽的文库。• 苏云金芽孢杆菌成功分泌了绿色荧光蛋白和角质酶。
