Discovery, Biosynthesis, and Characterization of Rodencin, a Two-Component Lanthipeptide, Harboring <scp>d</scp>-Amino Acids Introduced by the Unusual Dehydrogenase RodJ_A

Lanthipeptides, a group of ribosomally synthesized and post-translationally modified peptides (RiPPs), exhibit diverse structures and bioactivities. Their biosynthetic enzymes serve as valuable tools for peptide bioengineering. Here, we report a class II lanthipeptide biosynthetic gene cluster in a <i>Bacillus</i> strain, driving the biosynthesis of a two-component lanthipeptide, termed rodencin, featured by the presence of two different d-amino acids, i.e., d-Ala and d-Abu. Rodencin displays synergistic antimicrobial activity against food-borne pathogens such as <i>Bacillus cereus</i>, <i>Staphylococcus aureus</i>, and <i>Listeria monocytogenes</i>. The α-peptide of rodencin contains one d-Ala and the β-peptide features both d-Ala and d-Abu. These are installed by dehydratases RodM1 and RodM2 and dehydrogenase RodJ_A, the activities of which were successfully reconstituted using a dedicated <i>E. coli</i> expression system. To illustrate the unusual d-Abu incorporation potential of the enzymes, analogous to the d-amino acid-containing β peptide of lacticin 3147, was successfully produced with the rodencin heterologous expression system, by employing RodM2 and the dehydrogenase RodJ_A.