Synthetic Biology‐Based Heterologous Expression and Purification of Enterocin A: Advancing Antimicrobial Peptide Applications

ABSTRACT Antibiotic resistance is a global health challenge, requiring the development of alternative antimicrobial strategies where pathogens can be inhibited by safe antimicrobials with different modes of action and less possibilities for developing resistance. Bacteriocins, ribosomally synthesized antimicrobial peptides produced by lactic acid bacteria, have emerged as promising natural agents for food preservation and pathogen control. This study reports the design and synthesis of a complete genetic cassette encoding the mature form of enterocin A, a bacteriocin derived from Enterococcus faecium . The vector (pJ404‐pEntApep), containing the synthetic entA gene, was entirely constructed de novo and included as functional regulatory elements along with an N‐terminal His 6 ‐tag for efficient purification. The genetic construct was validated through PCR and followed a sequencing of the generated amplicon. Expression in Escherichia coli BL21 (DE3) under optimized conditions (28°C, 0.5 mM IPTG) resulted in production of recombinant leaderless enterocin A (EntA), primarily in the form of inclusion bodies. Solubilization using 6 M guanidine‐HCl, followed by protein refolding, enabled efficient recovery. The refolded peptide was purified via Ni‐NTA affinity chromatography, with recorded elution at 400 mM imidazole. The purified enterocin A demonstrated potent antimicrobial activity against multidrug‐resistant pathogens, including Staphylococcus aureus subsp. aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Acinetobacter lwoffii GPE 3002, Micrococcus luteus GPE 3001, and Bacillus cereus GPE 3003, with minimum inhibitory concentration (MIC) values ranging from 27 to 109 µg/mL. This study establishes a robust and cost‐effective method for producing bioactive bacteriocins from inclusion bodies, underscoring their potential as sustainable alternatives to conventional antibiotics and chemical preservatives.