Biosynthetic Functionalization of Nonribosomal Peptides

Area of Science:

• Biochemistry
• Synthetic Biology
• Medicinal Chemistry

Background:

• Nonribosomal peptides (NRPs) are vital therapeutic compounds produced by modular synthetases.
• Previous work demonstrated a Trp-to-Ser mutation redirects tyrocidine synthetase specificity to clickable analogues.

Purpose of the Study:

• To investigate the pathway-level functionalization of bioactive NRPs using minimally invasive mutations.
• To expand the utility of engineered synthetases for incorporating diverse functional groups into NRPs.

Main Methods:

• Utilized site-directed mutagenesis (W227S and W2742S) in tyrocidine synthetase.
• Incorporated phenylalanine analogues with alkyne, halogen, and benzoyl substituents.
• Employed Cu(I)-catalyzed alkyne-azide cycloaddition for fluorescent labeling.

Main Results:

• The W227S mutation enabled selective incorporation of functionalized Phe analogues by the initiation module.
• The W2742S mutation allowed functionalized analogue incorporation at position 4, extending to elongation modules.
• Site-selective fluorescent labeling of tyrocidine A analogues was achieved via an alkyne handle.

Conclusions:

• Engineered tyrocidine synthetases facilitate efficient, site-specific functionalization of NRPs.
• This combined synthetic biology and bioorthogonal chemistry approach accelerates NRP research.
• Potential applications include NRP isolation, target elucidation, and therapeutic optimization.