Discovery and synthesis of leaderless bacteriocins from the Actinomycetota

Leaderless bacteriocins are a unique class of bacteriocins that possess antimicrobial activity after translation and have few cases of documented resistance. Aureocin A53 and lacticin Q are considered two of the most well-studied leaderless bacteriocins. Here, we used in silico genome mining to search for novel aureocin A53-like leaderless bacteriocins in GenBank and MGnify. We identified 757 core peptides across 430 genomes with 75 species found currently without characterized leaderless bacteriocin production. These include putative novel species containing bacteriocin gene clusters (BGCs) from the genera Streptomyces (sp. NBC_00237) and Agrococcus (sp. SL85). To date, all characterized leaderless bacteriocins have been found within the phylum Bacillota, but this study identified 97 core peptides within the phylum Actinomycetota. Members of this phylum are traditionally associated with the production of antibiotics, such is the case with the genus Streptomyces. Actinomycetota is an underexplored phylum in terms of bacteriocin production with no characterized leaderless bacteriocin production to date. The two novel leaderless bacteriocins arcanocin and arachnicin from Actinomycetota members Arcanobacterium sp. and Arachnia sp., respectively, were chemically synthesized and antimicrobial activity was verified. These peptides were encoded in human gut (PRJNA485056) and oral (PRJEB43277) microbiomes, respectively. This research highlights the biosynthetic potential of Actinomycetota in terms of leaderless bacteriocin production and describes the first antimicrobial peptides encoded in the genera Arcanobacterium and Arachnia.IMPORTANCEBacteriocins are gathering attention as alternatives to current antibiotics given the increasing incidence of antimicrobial resistance. Leaderless bacteriocins are considered a commercially attractive subclass of bacteriocins due to the ability to synthesize active peptide and low levels of documented resistance. Therefore, in this work, we mined publicly available data to determine how widespread and diverse leaderless bacteriocins are within the domain of bacteria. Actinomycetota, known for its antibiotic producers but lacking described and characterized bacteriocins, proved to be a rich source of leaderless bacteriocins-97 in total. Two such peptides, arcanocin and arachnicin, were chemically synthesized and have antimicrobial activity. These bacteriocins may provide a novel source of novel antimicrobials that could aid in the development of future alternative antimicrobials and highlight that the Actinomycetota are an underexplored resource of bacteriocin peptides.