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

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.