Artificial Multidomain Ribosomally Synthesized and Post-translationally Modified Peptide Enzymes for Farnesylated...

Area of Science:

• Biochemistry
• Synthetic Biology
• Medicinal Chemistry

Background:

• Cyanobactin pathways are valuable in synthetic biology for generating peptide libraries with drug-like properties, including macrocyclization and prenylation.
• Expressing multiple enzymes from RiPP (RiPP) pathways for targeted product synthesis remains a significant challenge.

Purpose of the Study:

• To design a robust and efficient method for producing and assessing multiple enzymes involved in cyanobactin pathways.
• To enable the rational production of macrocyclic peptides with selective isoprenoid appending to tyrosine residues.

Main Methods:

• Fusing multiple biosynthetic genes into a single, well-expressed, soluble construct for enzyme production.
• Developing and assaying a peptide library to identify sequence features essential for prenylation.
• Utilizing a system for selective C5, C10, or C15 isoprenoid attachment to tyrosine side chains.

Main Results:

• Achieved a >40% success rate in generating peptide derivatives with desired modifications.
• Defined critical sequence features required for successful prenylation.
• Created a library with an estimated maximum size of 2.6 million peptide derivatives.

Conclusions:

• The developed system provides a flexible and robust platform for generating novel compounds and large peptide libraries.
• This method minimizes side products, enabling efficient production of modified peptides in living organisms.
• Facilitates the discovery of new drug-like molecules through combinatorial peptide synthesis.