Recently, the food industry and the animal farming field have been working on different strategies to reduce the use of antibiotics in animal production. The use of probiotic producers of antimicrobial peptides (bacteriocins) is considered to be a potential solution to control bacterial infections and to reduce the use of antibiotics in animal production. In this study, Ligilactobacillus salivarius P1CEA3, isolated from the gastrointestinal tract (GIT) of pigs, was selected for its antagonistic activity against Gram-positive pathogens of relevance in swine production. Whole genome sequencing (WGS) of L. salivarius P1ACE3 revealed the existence of two gene clusters involved in bacteriocin production, one with genes encoding the class II bacteriocins salivaricin B (SalB) and Abp118, and a second cluster encoding a putative nisin variant. Colony MALDI-TOF MS determinations and a targeted proteomics combined with massive peptide analysis (LC-MS/MS) of the antimicrobial peptides encoded by L. salivarius P1CEA3 confirmed the production of a 3347 Da novel nisin variant, termed nisin S, but not the production of the bacteriocins SalB and Abp118, in the supernatants of the producer strain. This is the first report of a nisin variant encoded and produced by L. salivarius, a bacterial species specially recognized for its safety and probiotic potential.

Circular bacteriocins are antimicrobial peptides produced by bacteria that after synthesis undergo a head-to-tail circularization. Compared to their linear counterparts, circular bacteriocins are, in general, very stable to temperature and pH changes and more resistant to proteolytic enzymes, being considered as one of the most promising groups of antimicrobial peptides for their potential biotechnological applications. Up to now, only a reduced number of circular bacteriocins have been identified and fully characterized, although many operons potentially coding for new circular bacteriocins have been recently found in the genomes of different bacterial species. The production of these peptides is very complex and depends on the expression of different genes involved in their synthesis, circularization, and secretion. This complexity has greatly limited the identification and characterization of these bacteriocins, as well as their production in heterologous microbial hosts. In this work, we have evaluated a synthetic biology approach for the in vitro and in vivo production combined with a split-intein mediated ligation (SIML) of the circular bacteriocin garvicin ML (GarML). The expression of one single gene is enough to produce a protein that after intein splicing, circularizes in an active peptide with the exact molecular mass and amino acid sequence as native GarML. In vitro production coupled with SIML has been validated with other, well described and not yet characterized, circular bacteriocins. The results obtained suggest that this synthetic biology tool holds great potential for production, engineering, improving and testing the antimicrobial activity of circular bacteriocins.