Research Projects

Roseocin, the novel two-peptide lantibiotic developed in our lab from Streptomyces roseosporus, carries extensive intramolecular (methyl)lanthionine bridging in both the peptides and exhibits synergistic bactericidal activity against clinically relevant Gram-positive pathogens. Both peptides have a conserved leader but a diverse core region. The biosynthesis of roseocin involves post-translational modification of the two precursor peptides by a single promiscuous lanthipeptide synthetase, RosM, to install an indispensable disulfide bond in the Rosα core along with four and six thioether rings in Rosα and Rosβ cores, respectively. Since, roseocin has no known homologs, we searched for its synthetase (RosM) homologs in the phylum actinobacteria and found 12 other members of the roseocin family. The naturally occurring diverse congeners of roseocin peptides, identified from these 12 novel BGCs, were carefully aligned to identify the conserved sites and the substitutions in the core peptide region. Considering that a more potent roseocin homolog may exist in nature, we selected the substitutions that should not alter the Rosα peptide thioether rings and hence its structure, but could significantly influence its activity. The selected few sites in the Rosα peptide were mutated for naturally permitted substitutions. All the variant peptides were expressed heterologously and post-translationally modified in vivo by RosM in E. coli host. The semi-in vitro reconstitution (SIVR) strategy established earlier in our lab (Singh et al. 2020) was followed to obtain the bioactive core peptides for the determination of minimum inhibitory concentration (MIC) and structural characterization by mass spectrometry. Out of the 4 characterized variants, two variants, RosαL8F and RosαL8W, exhibited significantly improved inhibitory activity in a species-dependent manner, compared to the wild-type roseocin. Many more variants are being generated and characterized in our lab based upon the naturally existing repository of evolved variants of roseocin. We have demonstrated that instead of random mutagenesis or site-saturation mutagenesis, a targeted approach assisted by the phylogeny-guided genome analysis is more beneficial in terms of time and resources with a promising outcome. Using the knowledge generated on the diversity of the roseocin family lantibiotics (Chaudhary et al, 2023), we could identify novel motifs in roseocin peptides, for a similar or divergent action mechanism than that of earlier known lacticin family lantibiotics. Current study has been planned to understand roseocin peptides’ structure and the role of each of the two peptides in their mechanism of bacterial cell membrane disruption. The precise structural elucidation of roseocin peptides and validation of the bacterial cell wall components as their target will help in developing a more potent Roseocin variant against the clinically relevant bacterial strains, including Gram-negative pathogens.