Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Discovery

Background:

• Liquid chromatography-mass spectrometry (LC-MS)-based proteomics enables high-throughput protein expression profiling.
• Identifying enzymes involved in natural product biosynthesis is crucial for discovering new compounds.

Purpose of the Study:

• To develop and report a protocol using LC-MS/MS proteomics for screening bacterial enzymes.
• To identify nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) involved in natural product biosynthesis.

Main Methods:

• Utilized LC-MS/MS proteomics for screening bacterial strains.
• Employed size-based separation (SDS-PAGE) for large modular enzymes (>200 kDa) prior to LC-MS/MS analysis.
• Protein identification through software search to pinpoint expressed gene clusters.

Main Results:

• Successfully identified expressed NRPS and/or PKS gene clusters from bacterial strains.
• Demonstrated the ability to pinpoint specific gene clusters based on protein identification.

Conclusions:

• The developed proteomics screening method accurately identifies key biosynthetic enzymes.
• This approach guides the discovery of novel nonribosomal peptide and polyketide natural products.