Functionalizing Nisin with a Sugar Moiety Improves Its Solubility and Results in an Altered Antibacterial Spectrum and Mode of Action

Glycosylation, a widespread post-translational modification, is present in all kingdoms of life. Despite the extensive structural diversity found in ribosomally synthesized and post-translationally modified peptides (RiPPs), only a few glycosylated bacteriocins, known as glycocins, have been identified. Notably, glycocins such as glycocin F, ASM1, and enterocin F4-9, exhibit antimicrobial properties and distinct glycoactivity, indicating that glycosylation is crucial for their bioactivity. The development of practical, and widely applicable systems for glycosylation of RiPPs is therefore highly desirable. In this study, we introduce an expression system that utilizes <i>Lactococcus lactis</i> as a host for the efficient incorporation of the noncanonical amino acid homopropargylglycine (Hpg) into the well-studied RiPP nisin, and some structurally related variants. Hpg, which has an alkyne functional group, allows for further chemical modifications with azido-sugar containing substrates through click chemistry. We reveal that glycosylated nisin at position 17 shows strong activity against <i>Enterococcus faecium</i> strains, but its activity against other pathogens such as <i>Staphylococcus aureus</i>, <i>Enterococcus faecalis</i>, and <i>Bacillus cereus</i> is reduced. Moreover, mode of action studies show that the addition of sugar diminishes its typical pore-forming ability of nisin against <i>E. faecium</i> while preserving its lipid II binding ability. Interestingly, the addition of a hydrophilic sugar significantly enhances its water solubility around 4-fold at neutral pH, indicating potential for improved drug applications. These findings highlight the potential of this methodology for glycosylation of RiPPs, leading to the creation of new antimicrobial products with varied characteristics. This also broadens the toolkit for enhancing and discovering peptide-based drugs.