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 in vitro (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.