Elucidating Leader Peptide–Enzyme Dynamics in Lactazole Biosynthesis Using mRNA Display

Thiopeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that show promise for drug discovery. Their biosynthesis depends on leader peptide recognition, whereby multiple enzymes are recruited to process a core region into the mature natural product. Here, we identify sequence determinants of the leader peptide-enzyme dynamics in a thiopeptide biosynthesis. Using the flexible <i>in vitro</i> (FIT)-Laz translation platform, we examined how leader mutations influence modifications performed by the enzymes that together complete lactazole biosynthesis. Initial DNA template translations informed single amino acid saturation mutagenesis using mRNA display. This revealed leader mutations that modulate enzyme recognition, as validated by aligning enrichment scores with observed modification. This study provides important insights into the underexplored role of RiPP leader peptides and informs the design of improved pseudonatural product libraries.