State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Mol Biol Rep. 2024 Aug 28;51(1):939. doi: 10.1007/s11033-024-09881-z.
Plasmids are the most commonly used vectors for heterologous protein expression in Escherichia coli. However, the plasmid copy number decreases with the segregational instability, which inevitably leads to a decrease in the yield of heterologous protein.
In this study, plasmid stabilization systems were used to enhance the expression level of heterologous proteins in E. coli. With the investigation of protein expression level, biomass and plasmid retention rate in different plasmid stabilization systems, the hok/sok system had the greatest potential on plasmid stabilization. In order to further investigate the molecular mechanism of hok/sok system, the structure of the binding region of hok mRNA and sok antisense RNA was modified based on the minimum free energy of mRNA, which resulted in the reduction of the binding efficiency of hok mRNA and sok asRNA, and then the toxicity of the Hok protein led to the decreased viability of the host cells. Finally, the hok/sok plasmid stabilization system was testified in 5 L fermenter, and the plasmid retention rate and protein expression level were significantly increased without the addition of antibiotics.
This study lays a solid foundation for a deeper understanding of the mechanism of the hok/sok plasmid stabilization system and improving the productivity of heterologous protein in E. coli.
质粒是大肠杆菌中用于异源蛋白表达最常用的载体。然而，质粒拷贝数会因分离不稳定而减少，这不可避免地导致异源蛋白产量下降。
在本研究中，使用质粒稳定系统来提高大肠杆菌中异源蛋白的表达水平。通过研究不同质粒稳定系统中的蛋白表达水平、生物量和质粒保留率，hok/sok 系统在质粒稳定化方面具有最大的潜力。为了进一步研究 hok/sok 系统的分子机制，基于 mRNA 的最小自由能对 hok mRNA 和 sok 反义 RNA 的结合区域结构进行了修饰，从而降低了 hok mRNA 和 sok asRNA 的结合效率，进而导致 Hok 蛋白的毒性降低了宿主细胞的活力。最后，在 5L 发酵罐中验证了 hok/sok 质粒稳定系统，无需添加抗生素即可显著提高质粒保留率和蛋白表达水平。
本研究为深入了解 hok/sok 质粒稳定系统的机制以及提高大肠杆菌中异源蛋白的生产效率奠定了基础。
