Stationary-Phase Antimicrobial Peptide Production in Escherichia coli

Antimicrobial peptides (AMPs) are a class of naturally occurring molecules found in animals, plants, and microorganisms. Ω76 is an AMP specifically designed for treating Carbapenem-resistant Enterobacteriaceae (CRE). Escherichia coli, engineered to produce Ω76, may serve as a potential whole-cell drug delivery system for treating CRE, yet Ω76 exhibited potent antimicrobial activity against the production host. In this study, P_AtxA, a tightly controlled, high-level, modulable, and stationary-phase-specific promoter, was employed for the functional expression of Ω76 in E. coli. P_AtxA facilitates the decoupling of bacterial growth from Ω76 production, enabling efficient Ω76 expression. The results show that P_AtxA-governed Ω76 expression exhibited a relatively high OD₆₀₀ and biomass concentration. In contrast, P_T7-goverend Ω76 expression, which occurred during the log phase, showed a lower OD₆₀₀ and dormancy with cell size reducing to less than 1 μm. The expression of Ω76 was confirmed in SDS-PAGE. The extraction of Ω76 was demonstrated using methanol and ethanol as solvents, while acetate and acetonitrile were found to be ineffective. The bacteria extract containing Ω76 exhibited antimicrobial activity against both Gram-negative E. coli and Gram-positive B. subtilis. Moreover, pTOL03F-Ω76 had a Ω76 concentration of 25 ± 3 mg/L compared to 21 ± 3 mg/L for pT7-Ω76. The solid antibacterial testing suggests the formation of Ω76 tertiary structures, indicating that conventional assays designed for small-molecule antibiotics may not be suitable for AMP assays. This study first reveals the phenotypic differences between log-phase and stationary-phase Ω76 expression, suggesting two potential applications for the stationary-phase promoter: (1) the development of a whole-cell drug delivery system, where the recombinant E. coli could grow along with CRE and produce Ω76 during the stationary phase to suppress CRE, and (2) its use in the production of secondary metabolites.