Biosynthesis, processing and engineering of circularin A

The global antibiotic resistance crisis demands urgent solutions. Ribosomally synthesized and post-translationally modified peptides (RiPPs) offer a diverse range of antimicrobial possibilities. Among them, circular bacteriocins are well-known for their remarkable stability, making them attractive for various applications, including food and pharmaceutical purposes. Despite their potential, the biosynthesis mechanism of circular bacteriocins remains underexplored, limiting their use in novel antibiotic development. This thesis focuses on investigating the biosynthesis, bioactivity and bioengineering potential of circularin A, a circular bacteriocin from Clostridium beijerinckii ATCC 25752. We first developed robust systems for circularin A production in Lactococcus lactis NZ9000 and established efficient methods for activity screening and peptide purification of engineered circularin A variants. These methods facilitated extensive mutagenesis studies of circularin A and functional investigations of its biosynthetic proteins. Mutagenesis studies indicated that the terminal residues near the circularization site, rather than the leader sequence, probably engage in interactions with biosynthetic proteins. The aromatic and cationic residues play important roles in circularin A biosynthesis and bioactivity. Furthermore, we have demonstrated promising prospects for engineering circular bacteriocins and recognized the critical role of structural flexibility in achieving the optimal antimicrobial activity of circularin A. Through functional investigations of biosynthetic proteins, we propose that CirC is responsible for catalyzing leader removal and possible circularization, whereas CirB and CirD form an ABC transporter for bacteriocin secretion. Overall, our work advances the understanding of the biosynthesis, bioactivity and bioengineering potential of circularin A, providing valuable insights for the development of novel antimicrobial agents with circular bacteriocins