Structural requirements for the biosynthesis of backbone cyclic peptide libraries

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Combinatorial methods are crucial for generating molecular libraries and ligand diversity in chemical biology.
• Synthetic methods produce small molecules requiring cell membrane traversal, while genetic methods yield large intracellular molecules.
• Existing methods have limitations in producing diverse, small, intracellular molecules.

Purpose of the Study:

• To develop a versatile method for producing intracellular libraries of small, stable cyclic peptides.
• To combine the strengths of synthetic and genetic methods for ligand diversity.
• To enable facile manipulation of vast numbers of compounds for pharmacophore identification.

Main Methods:

• Utilized an intein-based approach for the biosynthesis of backbone cyclic peptide libraries.
• Employed site-directed mutagenesis of the DnaE intein from Synechocystis sp. PCC6803.
• Generated libraries with varying peptide lengths (4-9 amino acids) and compositions (5 variable, 1 or 4 fixed residues).

Main Results:

• Demonstrated the promiscuity of the DnaE intein regarding peptide substrate composition.
• Successfully generated cyclic peptide products ranging from four to nine amino acids.
• Produced libraries yielding 10^7 to 10^8 transformants, with most clones yielding cyclic products.

Conclusions:

• Developed a versatile method for intracellular production of small, stable cyclic peptide libraries.
• Genetic encoding allows for easy manipulation of large compound libraries.
• The method's flexibility in peptide length and composition enhances ligand diversity generation.