Abstract Description

The critical need for the discovery of new and more effective antimicrobial agents against antibiotic resistant pathogenic microbes is highlighted by the global increase in microbial antibiotic resistance. Epidermin is a potent lantibiotic peptide naturally produced by Staphylococcus epidermidis Tü329, with demonstrated antimicrobial activity against pathogens such as Propionibacterium acnes and Staphylococcus aureus. Its targeted efficacy highlights its therapeutic potential for the topical treatment of acne and other skin infections. Lantibiotics are ribosomal peptides that undergo extensive post-translational modifications (PTMs) catalysed by specific enzymes, yielding compounds with unique modes of action. However, natural producers often yield low quantities, hindering their application on a large scale. This study aimed to construct a heterologous bacterial expression system to produce epidermin and two genome mined epidermin-like derivatives using the biosynthetic pathway of the prototypical nisinA lantibiotic and the Nisin-Inducible Expression System. This system makes use of a plasmid-free Lactococcus lactis derivative and the nisin-inducible promoter in a two-plasmid system. The pNZ8048 expression plasmid carries a chloramphenicol resistance marker and a 6x histidine tag while the pMSP plasmid carries an erythromycin resistance marker for selection of recombinant cells. The nisinA PTM genes were PCR amplified using genomic DNA isolated from the L. lactis F10 nisinA producer and cloned into the pMSP plasmid. Precursor peptide genes were synthesised by Twist Bioscience followed by insertion into the pNZ plasmid. Each recombinant pNZ and the pMSP plasmid were successfully co-transformed into electrocompetent L. lactis cells. Recombinant single colonies were selected on M17 media containing both antibiotic markers. This was followed by protein expression trials, IMAC purification of the cell-free culture supernatants and SDS-Page gel electrophoresis. Only one of the lantibiotics demonstrated antimicrobial activity as determined using agar spot plate assays. Future experiments will use LC-MS to confirm molecular weights and predicted PTMs, followed by bioactivity screening against clinically relevant acne-associated bacteria.