Engineering Escherichia coli for controlled autolytic release of intracellular proteins: bacteriophage lambda SRRz versus the 2PaT4L chimeolysin

Abstract

Programmed cell autolysis can simplify the recovery of intracellular recombinant proteins, but differences in experimental conditions and evaluation criteria often limit direct comparisons of lysis systems. This study presents a multicriteria comparison of the two distinct lysis genes, the phage lambda SRRz cassette and the engineered 2PaT4L chimeolysin under identical IPTG-inducible conditions in Escherichia coli. The 2PaT4L outperformed the SRRz across multiple metrics, including cell viability, turbidity, protein release, and escape frequency. Using the inverse of escape frequency as a quantitative measure of lysis efficiency, 2PaT4L and SRRz reached (99.99 ± 0.012)% and (71.10 ± 7.370)% lysis, respectively. Protein release into the culture medium was also higher for 2PaT4L, reaching 1.62 mg/mL after 24 h compared with 1.35 mg/mL for SRRz. These results identify 2PaT4L as the more effective autolysis system under the conditions tested and highlight its potential for intracellular protein recovery. Techno-economic analysis and sensitivity analyses of lysis efficiency, autolysis duration, and inducer concentration indicated that optimizing these conditions can significantly reduce enzyme production costs.

Keywords

Cell autolysis / SRRz / 2PaT4L / Lysis efficiency / Techno-economic analysis / Process simulation

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Funding

Durban University of Technology

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