Nisin O is an antimicrobial peptide encoded by the human gut bacterium Blautia obeum A2-162 which has antimicrobial activity against clinically relevant organisms. The nisin O biosynthetic gene cluster (BGC) varies from other nisin BGCs as it lacks a leader-peptide cleaving protease and contains two bacterial two-component response regulator–histidine kinase (RK) systems. The dissemination of the nisin O cluster, the final proteolytic biosynthesis step and the regulation of nisin O are currently unknown and are the foci of this study. We identified six nisin O-like BGCs across Blautia, Dorea and Ruminococcus species using comparative genomics. These BGCs show evidence of genetic transfer between genera, with genes involved in transposition discovered up- and downstream of the BGCs. All nisin O-like BGCs contained two RK systems but no protease. Mining the B. obeum A2-162 genome identified candidate proteases that were cloned and used in pre-nisin O leader peptide cleavage assays. None of the candidate proteases removed the leader; however, cleavage was achieved using trypsin. To maximize the expression of the nsoA1-4 peptides, the interactions of the two RK systems with predicted promoters in the nisin O cluster were assessed using a PepI reporter assay. We observed that the PnsoR2K2 promoter was constitutively expressed, with NsoR1K1 increasing its activity, and that there was increased nsoA1-4 expression when the nisin A RK system and nisin A were present. Long-read cDNA sequencing confirmed nso gene transcription in the heterologous expression system and identified a novel, highly expressed gene. This study provides evidence that the nisin O BGC has been transferred between different gut-associated genera, with all clusters lacking a protease and containing two RK systems. We hypothesize that this BGC has lost its protease due to negative selection as a result of high trypsin concentrations in the gut. Further work is required to maximize nisin O expression for it to be used as a potential antimicrobial therapy.