Natural Products Discovery

Our Natural Products subgroup is focused on the discovery and biosynthesis of novel natural products.  Developments from this subgroup include the introduction of the PrISM platform, geared towards the identification of natural products synthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) without prior knowledge of a gene sequence. This is made possible by our ability to detect a phosphopantetheinyl (Ppant) ejection marker ion for NRPS/PKS thiolation domains.  We also work in collaboration with groups from other universities to provide mass spectrometry analysis of novel biochemical systems. Overall the goal is to develop the largest, functional library of natural products (through the most robust discovery platform) in order to facilitate a new pipeline of chemical matter into early stage pharmaceutical testing. Natural products have been honed for thousands of years to interact with real biological systems which makes them very valuable lead compounds for drug development, but their discovery has been a bottleneck in the past. The Kelleher group hopes to alleviate this bottleneck to deliver much-needed drug research to the world.

 

The Workflow for PrISM (read more here)

a, Microbial strains are grown in liquid culture. b, The proteome of the strain is subjected to proteomics or in-gel digestion of high molecular weight bands. c, LC-FTMSn is conducted on the resulting peptide mixture, with expressed T domain active site peptides identified by the Ppant ejection assay. d, Peptide sequences are used to generate primers to amplify DNA sequence portions of the expressed gene cluster. e, The gene cluster is identified and sequenced, which informs targeted detection of the natural product produced as depicted in panel f.

 

NRPS Biosynthesis (read more here)

Non-ribosomal Peptide Synthetases (NRPS) are comprised of a series of domains with specific functions. The adenylation (A) domain is responsible for the selection/activation of amino acid monomers, the thiolation (T) domain holds the intermediate structure, passing it to the next catalytic site, the condensation (C) domain, which is responsible for the formation of the peptide bond with the upstream thiolation domain. Among other tailoring domains in NRPS synthesis are epimerization (E) domains, responsible for converting the loaded amino acid to its enantiomer and thioesterase (TE) domains, responsible for the release of the NRPS. The Kelleher group analyzes these biosynthetic processes and the kinetics of this biosynthesis, along with discovering new pathways and natural products and their biosynthetic pathways using microbial proteomics and the PrISM workflow described.

Natural Product Detection by Phosphopantetheinyl Ejection Assay (read more here)

The sequence of amino acids added to an NRPS can be determined by analysis of the T domain. Fragmentation of an aminoacylated form of the T domain yields both the pantetheinyl and the phosphopantetheinyl fragment with amino acids attached.