Accurate Broadcasting of Substrate Fitness for Lactazole Biosynthetic Pathway from Reactivity-Profiling mRNA Display

We
report a method for the high-throughput reactivity profiling
of genetically encoded libraries as a tool to study substrate fitness
landscapes for RiPP (ribosomally synthesized and post-translationally
modified peptide) biosynthetic enzymes. This method allowed us to
rapidly analyze the substrate preferences of the lactazole biosynthetic
pathway using a saturation mutagenesis mRNA display library of lactazole
precursor peptides. We demonstrate that the assay produces accurate
and reproducible in vitro data, enabling the quantification of reaction
yields with temporal resolution. Our results recapitulate the previously
established knowledge on lactazole biosynthesis and expand it by identifying
the extent of substrate promiscuity exhibited by the enzymes. This
work lays a foundation for the construction and screening of mRNA
display-based combinatorial thiopeptide libraries for the discovery
of lactazole-inspired thiopeptides with de novo designed biological
activities.