Abstract
Noncanonical amino acids form a highly diverse pool of building blocks that can render unique physicochemical properties to peptides and proteins. Here, four methionine analogues with unsaturated and varying side chain lengths were successfully incorporated at four different positions in nisin in Lactococcus lactis through force feeding. This approach allows for residue-specific incorporation of methionine analogues into nisin to expand their structural diversity and alter their activity profiles. Moreover, the insertion of methionine analogues with biorthogonal chemical reactivity, e.g., azidohomoalanine and homopropargylglycine, provides the opportunity for chemical coupling to functional moieties and fluorescent probes as well as for intermolecular coupling of nisin variants. All resulting nisin conjugates retained antimicrobial activity, which substantiates the potential of this method as a tool to further study its localization and mode of action.