Nonribosomal biosynthesis of backbone-modified peptides

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzyme Engineering

Background:

• Nonribosomal peptides are a crucial class of therapeutics.
• Modifying their backbones offers a route to new drug structures.
• Nonribosomal peptide synthetases (NRPSs) are large enzymes responsible for their assembly.

Purpose of the Study:

• To engineer an L-phenylalanine (L-Phe)-specific NRPS module to accept and process (S)-β-phenylalanine ((S)-β-Phe).
• To understand the structural basis for altered substrate specificity in NRPS modules.
• To demonstrate the utility of engineered NRPS modules for producing novel peptide frameworks.

Main Methods:

• High-throughput screening assay for NRPS catalytic activity.
• Site-directed mutagenesis and protein engineering of an NRPS module.
• X-ray crystallography to determine the co-crystal structure of the engineered NRPS module with an aminoacyl-AMP analogue.
• In vitro and in vivo production of (S)-β-Phe-containing peptides.

Main Results:

• The engineered NRPS module accepted (S)-β-Phe with high specificity and efficiency, achieving a 40,000-fold α/β-specificity switch.
• Structural analysis revealed precise active site remodelling responsible for the specificity change.
• (S)-β-Phe-containing peptides were produced at preparative scale in vitro and high titres in vivo.

Conclusions:

• Biosynthetic pathway engineering of NRPSs is a viable strategy for creating novel peptide therapeutics.
• Enzyme reprogramming can overcome natural substrate limitations in peptide biosynthesis.
• This work opens avenues for generating diverse nonribosomal peptide backbones with unique properties.