Abstract Description

Programmed cell autolytic Escherichia coli strains have emerged as promising hosts for releasing and recovering recombinant proteins inexpensively and less tediously. However, reported lysis efficiencies cannot be reliably used as the basis for choosing a lytic gene for cell autolysis due to discrepancies with the criteria used for calculating lysis efficiency, and different culture conditions, among other factors. In this study, two cell autolytic plasmid vectors based on phage lambda SRRz, and the engineered chimeolytic fusion gene, N-terminally fused T4 lysozyme with two repeating units of amphipathic cell-penetrating peptides (2PaT4L), were constructed and transformed into E. coli T7 Express lysY expression host cells for comparative analysis and investigation of their effect on cell lysis under the same culture conditions. Cell autolysis was triggered by the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). A time-dependent release of proteins was observed, with the released proteins increasing from 0.076 (2 hours post-induction) to 1.349 mg/mL (24 hours post-induction) for SRRz and from 1.367 (2 hours post-induction) to 1.622 mg/mL (24 hours post-induction) for 2PaT4L. Lysis efficiency of 2PaT4L and SRRz–expressing cells reached 99.99 % and 71.1 %, respectively. Furthermore, the 2PaT4L was transformed into a dye-decolorising peroxidase-expressing E. coli host. The induction of autolysis resulted in the recovery of 99.99 % of dye decolorizing peroxidase. Techno-economic analysis in enzyme production, using SuperPro Designer v13, showed wide cost variations in enzyme production, depending on the cell lysis method used, with cell autolysis offering the most cost-effective strategy in releasing intracellularly accumulated proteins. The engineered lytic protein 2PaT4L showed higher lysis efficiency using multiple criteria to determine cell autolysis, making it a preferable lytic gene for various cell autolysis applications including industrial applications.