细胞焦亡法破碎微生物细胞在合成生物学与代谢工程的应用

Application of pyroptosis in the disruption of microbial cells in synthetic biology and metabolic engineering

摘要:

【背景】细胞焦亡是一种细胞程序性死亡。在古菌和细菌中，gasdermin同源蛋白（GSDM）能够被特定的活化caspase （protease）酶切，从而激活类似于细胞焦亡的效应，产生细胞破碎效果。【目的】合成生物学、代谢工程和生物制造等应用过程中，细胞破碎是不可或缺的一步。利用细胞焦亡法破碎细胞取代传统的破碎方法，可以简化操作、提高生产效益。【方法】在大肠杆菌（Escherichia coli） BW25113中共表达protease和不同来源的GSDM，选择有明显细胞焦亡效应即来源Runella sp.的GSDM进行蛋白截短改造，使其在诱导表达蛋白截短体GSDMJD后能直接激活细胞焦亡效应。对GSDMJD进行过表达优化，获得可控大肠杆菌细胞焦亡菌株。进一步以重组表达蔗糖磷酸化酶为研究模型，验证本系统应用于细胞破碎释放蛋白的效果。【结果】实现了大肠杆菌中细胞焦亡的人为可控。焦亡菌株在诱导表达焦亡相关蛋白2 h后大肠杆菌细胞破碎死亡，内容物释放。将上述系统和超声法应用于制备蔗糖磷酸化酶粗酶液，细胞焦亡法制备的粗酶液的相对酶活显著高于超声法制备的粗酶液。在制备粗酶液的菌液OD₆₀₀值为2.0时，细胞焦亡法制备的粗酶液相对酶活最高并且相较于超声法制备粗酶液，提高了60%的相对酶活。【结论】细胞焦亡提供了一种更加简单快捷、绿色环保的微生物细胞破碎方式，为合成生物学与代谢工程的发展奠定了基础。

关键词:大肠杆菌;细胞焦亡;微生物细胞的破碎;蔗糖磷酸化酶

Abstract:

[Background] Pyroptosis is a form of programmed cell death. Gasdermin (GSDM) can be cleaved by cysteinyl aspartate-specific caspase (protease) in archaea and bacteria, which results in cell disruption, similar to the effects of pyroptosis in eukaryotes.[Objective] Cell disruption is a crucial step in synthetic biology, metabolic engineering, and biomanufacturing. Utilizing pyroptosis as a cell disruption method can simplify the operation and improve the production efficiency compared with the conventional methods.[Methods] Protease and GSDM from different sources were co-expressed in Escherichia coli BW25113. The GSDM with obvious pyroptosis effect from Runella sp. was selected for protein truncation, so that it could directly activate pyroptosis effect after inducing the expression of truncated protein (GSDMJD). After overexpression of GSDMJD and optimization, an E. coli strain with controllable pyroptosis effect was obtained. Furthermore, sucrose phosphorylase was used to verify the effects of this system and ultrasonic disruption on enzyme activity. [Results] The regulation of pyroptosis was successfully implemented in E. coli. After the strain was induced to express the pyroptosis-related protein for 2 h, the cells were disrupted and released the contents. The above system and ultrasonic method were applied to the preparation of crude liquid of sucrose phosphorylase. The relative activity of crude enzyme liquid prepared by the pyroptosis method was significantly higher than that of crude enzyme liquid prepared by the ultrasonic method. In the case of OD₆₀₀ 2.0, the crude enzyme liquid prepared by the pyroptosis method had the highest enzyme activity, which increased by 60% compared with that of the crude enzyme liquid prepared by the ultrasonic method. [Conclusion] Pyroptosis serves as a simple, fast, and environmentally friendly method for microbial cell disruption, laying a foundation for the development of synthetic biology and metabolic engineering.

Key words:Escherichia coli;pyroptosis;disruption of microbial cells;sucrose phosphorylase