Construction and evaluation of an inside-out lysis system based on T4 lysozyme.

Lysozyme can effectively lyse Escherichia coli (E. coli), providing a method for the release of intracellular substances. In this study, an inside-out lysis system based on T4 lysozyme was constructed. Three secretion signal peptides (PelB, NapA, and HybA) were fused to T4 lysozyme to create fusion proteins, among which, NapA-T4 lysozyme (N-T4L) exhibited the highest level of intracellular protein leakage. Microscopic analysis revealed significant cell wall damage in N-T4L expressing cells, confirming the initiation of cell lysis. The influence of chemical factors on cell lysis was also evaluated. Expression of N-T4L reduced cell viability by 2.09 log units following Triton X-100 treatment and pH adjustment. Antibacterial assays demonstrated that N-T4L in the fermentation supernatant maintained substantial inhibitory activity against bacteria. These findings provide a strong foundation for synthetic biology applications involving programmed cell destruction in E. coli.