Abstract：
Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains.
摘要：
提高细菌分泌蛋白质的能力对于食品酶的大规模生产至关重要。然而,由于缺乏有效的靶蛋白跟踪技术，分泌系统的优化面临许多问题。在这项研究中,我们利用分裂-GFP系统在解淀粉芽孢杆菌中实现自组装成成熟的GFP，并成功跟踪碱性蛋白酶AprE。分裂GFP系统用于评估信号肽酶，分泌系统的一个重要组成部分,信号肽酶sipA被鉴定为在AprE的分泌中起作用。与其他信号肽酶缺失菌株相比,sipA的缺失导致AprE前体蛋白的更高积累。探讨信号肽酶对信号肽的作用机制,进行分子对接和自由能计算。信号肽酶的作用强度由其与信号肽C末端的三肽的结合亲和力决定。信号肽YdbK和NucB的功能依赖于sipA,通过将sipA整合到解淀粉芽孢杆菌基因组中，使细胞外AprE的活性提高了19.9％。这些发现为提高底盘菌株的分泌效率提供了见解。
