Engineering an Escherichia coli Autolytic System for Overexpression of a Collagen-Mimetic Peptide with Enhanced Hemostatic Activity

We previously identified that the collagen mimetic peptide (CMP)-E3 is sufficient to induce in vitro blood clotting. In this study, we further demonstrated its ability to induce in vivo blood coagulation through a rat tail bleeding assay. However, intracellular expression of CMP-E3 negatively impacted Escherichia coli growth, causing cell density to fluctuate within an optical density at 600 nm (OD600) range of 1.8-2.2, which limited high-level expression. To address this challenge, we developed a programmed autolysis system by regulating the expression of lysis proteins. We identified Colicin M (CoIM) as the most effective cell lysis protein among the test samples. We hypothesized that dynamically controlling cell death while maintaining the cell population at an optimal level would enable continuous recombinant protein production. To achieve this, we used stationary-phase promoters to induce CoIM expression, showing that the OD600 loss within less than 14.8% using PbolA, and achieved a CMP-E3 yield of 363.9 mg/L, 91% higher than that of directly expressing CMP-E3. Fed-batch fermentation in a 5-L bioreactor was performed, where glucose supplementation was carefully controlled to create a nutrient-deficient environment. This approach resulted in dynamic changes in cell density during the stationary phase and a maximum CMP-E3 production level of 758.6 mg/L. This system may be a desired platform for the large-scale production of functional proteins or toxic natural products in E. coli.