Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides

Area of Science:

• Natural Product Biosynthesis
• Synthetic Biology
• Medicinal Chemistry

Background:

• Thiopeptides are a class of natural antibiotics derived from short peptides through extensive post-translational modifications.
• Investigating thiopeptide biosynthesis, especially the role of pyridine synthases in forming the macrocyclic core, is challenging due to difficulties in reconstituting multi-enzyme pathways.
• Existing methods for studying thiopeptide enzymes are often complex and resource-intensive.

Purpose of the Study:

• To develop an accessible benchtop biosynthesis strategy for thiopeptide scaffolds.
• To overcome challenges associated with reconstituting multiple enzymes in thiopeptide pathways.
• To enable the study and validation of novel pyridine synthases and the production of new thiopeptide analogs.

Main Methods:

• Combined select RiPP (Ribosomally synthesized and Post-translationally modified Peptides) enzymes with cell-free expression systems.
• Utilized flexizyme-based codon reprogramming to facilitate the biosynthesis.
• Developed a simplified system for thiopeptide scaffold production and enzyme activity validation.

Main Results:

• Successfully established a benchtop biosynthesis method for producing thiopeptide scaffolds.
• Gained access to analytical quantities of new thiopeptide analogs.
• Demonstrated the utility of the strategy for validating new pyridine synthases without prior pathway reconstitution.

Conclusions:

• The developed cell-free biosynthesis strategy simplifies the investigation of thiopeptide enzymes.
• This approach provides a powerful tool for discovering and characterizing new thiopeptide natural products and their biosynthetic machinery.
• The method facilitates the validation of enzyme function, particularly pyridine synthases, in a streamlined manner.