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A proteomics approach to discovering natural products and their biosynthetic pathways

Abstract

Many natural products with antibiotic, anticancer and antifungal properties are synthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs)1. Although genome sequencing has revealed the diversity of these enzymes, identifying new products and their biosynthetic pathways remains challenging2. By taking advantage of the size of these enzymes (often >2,000 amino acids) and unique marker ions derived from their common phosphopantetheinyl cofactor, we adapted mass spectrometry–based proteomics to selectively detect NRPS and PKS gene clusters in microbial proteomes without requiring genome sequence information. We detected known NRPS systems in members of the genera Bacillus and Streptomyces, and screened 22 environmental isolates to uncover production of unknown natural products from the hybrid NRPS-PKS zwittermicin A biosynthetic gene cluster3. We also discovered an NRPS cluster that generates a seven-residue lipopeptide. This 'protein-first' strategy complements bioassay- and sequence-based approaches by finding expressed gene clusters that produce new natural products.

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Figure 1: The workflow for PrISM.
Figure 2: Identification of a phosphopantetheinylated peptide in the NK2018 proteome, with subsequent analyses by tandem mass spectrometry and targeted PCR using genomic DNA.
Figure 3: Identification of new lipoheptapeptides in NK2018.

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Acknowledgements

We thank W. Metcalf for providing the S. viridochromogenes DSM 40736, B. subtilis ATCC 6633 strains and Taq polymerase, and for, along with W. van der Donk and P. Yau, technical assistance. We would also like to thank D. Dlott and the following members of the Kelleher Research Group for their assistance in this work: L. Zamdborg, J. Osuji, J. Norris, J. Anderson and H. Hannon. This work was supported in part by National Institutes of Health (NIH) grants (N.L.K.; R01 GM 067725-07, P01 GM 077596-03), NIH Chemistry Biology Interface Training grant (P.M.T.), NIH Molecular Biophysics Training Grant (B.S.E.) and NIH Cell & Molecular Biology Training Grant (S.B.B.). P.M.T. was also supported by an Analytical Chemical Society (ACS)-Division of Analytical Chemistry Fellowship sponsored by Eli Lilly and Co. S.B.B. is currently supported by an ACS-Division of Analytical Chemistry Fellowship sponsored by the Society for Analytical Chemists of Pittsburgh.

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Authors and Affiliations

Authors

Contributions

S.B.B. designed and performed proteomic analyses, performed gel-based analyses, identified the natural products discussed herein, conducted LC-MS analyses and wrote the paper. B.S.E. isolated and characterized strains, performed gel-based analyses, performed LC-MS analyses, designed and executed genomic analyses of NK2018 and wrote the paper. P.M.T. assisted in experimental design and performed gel-based analyses and wrote the paper. I.N. assisted in experimental design and conducted LC-MS analyses. N.L.K. designed experiments and wrote the paper. S.B.B. and B.S.E. contributed equally to this study. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Neil L Kelleher.

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Supplementary Figs. 1–18, Supplementary Tables 1–10 and Supplementary Discussion (PDF 1683 kb)

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Bumpus, S., Evans, B., Thomas, P. et al. A proteomics approach to discovering natural products and their biosynthetic pathways. Nat Biotechnol 27, 951–956 (2009). https://doi.org/10.1038/nbt.1565

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