Evolution-inspired engineering of nonribosomal peptide synthetases

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Many pharmaceuticals originate from bacterial natural products synthesized by nonribosomal peptide synthetases (NRPSs).
• NRPSs are large enzyme complexes that assemble amino acids sequentially.
• Engineering NRPSs is crucial for discovering new drugs.

Purpose of the Study:

• To identify novel recombination sites within NRPS thiolation (T) domains for improved engineering.
• To develop a new method for assembling NRPS fragments with diverse properties.
• To demonstrate the utility of this method by producing a specific proteasome inhibitor.

Main Methods:

• Phylogenetic analysis of bacterial NRPS sequences to find recombination sites.
• Development of the "eXchange Unit between T domains" (XUT) approach for NRPS engineering.
• Assembly and production of a designed proteasome inhibitor using five distinct NRPS fragments.

Main Results:

• Identification of previously undescribed recombination sites in NRPS T domains.
• Successful development and application of the XUT approach.
• Demonstrated ability to assemble complex NRPS constructs from diverse fragments, yielding a functional proteasome inhibitor.

Conclusions:

• The XUT approach provides a versatile platform for NRPS engineering.
• This method facilitates the modular construction of novel NRPS-derived compounds.
• The identified recombination sites offer new avenues for NRPS-based drug discovery.