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

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Chemistry

Background:

• Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with significant potential for drug discovery.
• Their biosynthesis involves complex enzymatic processing guided by leader peptides that recruit specific enzymes.

Purpose of the Study:

• To identify sequence determinants governing leader peptide-enzyme interactions in thiopeptide biosynthesis.
• To understand how leader peptide mutations affect the recruitment and function of modifying enzymes.

Main Methods:

• Utilized the flexible *in vitro* (FIT)-Laz translation platform for studying thiopeptide biosynthesis.
• Employed single amino acid saturation mutagenesis via mRNA display to probe leader peptide sequence space.
• Validated enzyme recognition modulation by correlating enrichment scores with observed modifications.

Main Results:

• Identified specific leader peptide mutations that significantly alter enzyme recognition and modification efficiency.
• Demonstrated a direct link between leader peptide sequence and the recruitment of biosynthetic enzymes.
• Provided experimental evidence for the role of leader peptides in directing post-translational modifications.

Conclusions:

• Leader peptides play a critical, yet underexplored, role in regulating RiPP biosynthesis.
• Insights gained can inform the rational design of modified RiPPs and pseudonatural product libraries for drug discovery.
• This work advances the understanding of RiPP biosynthetic pathways and their engineering potential.