Li Yang, Wu Yaokang, Liu Yanfeng, Li Jianghua, Du Guocheng, Lv Xueqin, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, China.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, China.
Bioresour Technol. 2022 Nov;363:127885. doi: 10.1016/j.biortech.2022.127885. Epub 2022 Sep 3.
Bacillus subtilis is a microbial cell factory widely used to produce recombinant proteins, but the expression of heterologous proteins is often severely hampered. This study constructed a genetic toolkit for improving the secretory efficiency of heterologous proteins in Bacillus subtilis. First, the protease-deficient hosts were reconstructed. Then, two endogenous constitutive promoters, P and P, were screened. Next, a method called systemic combinatorial optimization of ribosome binding site (RBS) equipped with signal peptide (SCORES) was designed for optimizing the secretion and translation of the heterologous protein. Finally, Serratia marcescens nonspecific endonuclease (SMNE), which causes cell death by degrading nucleic acids, was expressed. The enzyme activity in the shake flask reached 7.5 × 10 U/L, which was 7.5-times that of the control RBS and signal peptide combination (RS0). This study not only expanded on the synthetic biology toolbox in B. subtilis but also provided strategies to create a prokaryotic protein expression system.
枯草芽孢杆菌是一种广泛用于生产重组蛋白的微生物细胞工厂，但异源蛋白的表达常常受到严重阻碍。本研究构建了一套遗传工具包，用于提高枯草芽孢杆菌中异源蛋白的分泌效率。首先，对蛋白酶缺陷型宿主进行了改造。然后，筛选了两个内源性组成型启动子P和P。接下来，设计了一种名为配备信号肽的核糖体结合位点系统组合优化（SCORES）的方法，用于优化异源蛋白的分泌和翻译。最后，表达了通过降解核酸导致细胞死亡的粘质沙雷氏菌非特异性核酸内切酶（SMNE）。摇瓶中的酶活性达到7.5×10 U/L，是对照核糖体结合位点和信号肽组合（RS0）的7.5倍。本研究不仅扩展了枯草芽孢杆菌中的合成生物学工具包，还提供了创建原核蛋白表达系统的策略。
