Many natural products with antibiotic, anticancer and antifungal properties are synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), yet most microbes do not express the great majority of their NRPS or PKS enzymes. In the first granting period of this R01, we developed mass spectrometry (MS) into a facile approach to directly detect covalent intermediates in ~30 systems that have been studied in vitro. We propose to continue this activity for peptide antibiotics and iterative PKS systems (Specific Aim 1).
In Specific Aims 2 and 3, we also propose to streamline NRPS/PKS assays using high throughput LC-MS. This blends aspects of enzymology with microbial proteomics of native producers into an entirely new approach to natural product discovery and the study of NRPS and PKS systems. By virtue of their huge size (often >2000 amino acids) and unique marker ions deriving from their common cofactor (phosphopantetheine), we show proof-of-concept for mass spectrometry-based proteomics with highly selective detection of expressed NRPS/PKS gene clusters in native proteomes - without requiring genome sequence information a priori. This method (dubbed """"""""PrISM"""""""") will allow unambiguous detection of NRPS/PKS systems in Bacilli, Streptomyces, and even fungi. We therefore advocate a new """"""""proteome-first"""""""" strategy to find gene clusters that are actually expressed and efficiently link them to the natural products they produce. The new approaches proposed here are especially timely in an era when new types of mega-enzyme """"""""assembly lines"""""""" that defy classification and cloning are being discovered at an accelerating rate, but relatively few new approaches are available to study them. With emphasis on this area of natural products expanding, our activities will assist in the fundamental understanding of chemical processes involved in biogenesis of new natural products of the types that have provided so many of our societies useful drugs to fight both bacterial infection and cancer.

Public Health Relevance

The discovery of new and novel natural products is burdened by the rediscovery of common antibiotics and antiproliferative compounds. Compounds made by NRPS/PKS enzymes have proven to provide diverse modes of action toward myriad different targets. The use of proteomics technology to discover natural products is a novel approach to this difficult problem.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067725-11
Application #
8306194
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Edmonds, Charles G
Project Start
2003-05-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
11
Fiscal Year
2012
Total Cost
$292,220
Indirect Cost
$93,595
Name
Northwestern University at Chicago
Department
Pharmacology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Chen, Yunqiu; McClure, Ryan A; Kelleher, Neil L (2016) Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics. Methods Mol Biol 1401:135-47
Henke, Matthew T; Kelleher, Neil L (2016) Modern mass spectrometry for synthetic biology and structure-based discovery of natural products. Nat Prod Rep 33:942-50
Goering, Anthony W; McClure, Ryan A; Doroghazi, James R et al. (2016) Metabologenomics: Correlation of Microbial Gene Clusters with Metabolites Drives Discovery of a Nonribosomal Peptide with an Unusual Amino Acid Monomer. ACS Cent Sci 2:99-108
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Le, Hoang V; Hawker, Dustin D; Wu, Rui et al. (2015) Design and mechanism of tetrahydrothiophene-based γ-aminobutyric acid aminotransferase inactivators. J Am Chem Soc 137:4525-33
Albright, Jessica C; Henke, Matthew T; Soukup, Alexandra A et al. (2015) Large-scale metabolomics reveals a complex response of Aspergillus nidulans to epigenetic perturbation. ACS Chem Biol 10:1535-41
Bok, Jin Woo; Ye, Rosa; Clevenger, Kenneth D et al. (2015) Fungal artificial chromosomes for mining of the fungal secondary metabolome. BMC Genomics 16:343
Doroghazi, James R; Albright, Jessica C; Goering, Anthony W et al. (2014) A roadmap for natural product discovery based on large-scale genomics and metabolomics. Nat Chem Biol 10:963-8

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