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Prospecting for natural products by genome mining and microcrystal electron diffraction

Nature Chemical Biology volume 17, pages 872–877 (2021)Cite this article

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Abstract

More than 60% of pharmaceuticals are related to natural products (NPs), chemicals produced by living organisms. Despite this, the rate of NP discovery has slowed over the past few decades. In many cases the rate-limiting step in NP discovery is structural characterization. Here we report the use of microcrystal electron diffraction (MicroED), an emerging cryogenic electron microscopy (CryoEM) method, in combination with genome mining to accelerate NP discovery and structural elucidation. As proof of principle we rapidly determine the structure of a new 2-pyridone NP, Py-469, and revise the structure of fischerin, an NP isolated more than 25 years ago, with potent cytotoxicity but hitherto ambiguous structural assignment. This study serves as a powerful demonstration of the synergy of MicroED and synthetic biology in NP discovery, technologies that when taken together will ultimately accelerate the rate at which new drugs are discovered.

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Fig. 1
Fig. 2: Genome mining and MicroED determination of Py-469 (3), a new pyridone NP.
Fig. 3: Rediscovery of fischerin.
Fig. 4: Structure of fischerin (2).

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Data availability

Crystallographic information files (CIFs) for compounds 2, 3 and 8 containing atomic coordinates and structure factors have been deposited at the Cambridge Crystallographic Data Center (deposition numbers 2020516, 2038723 and 2020510, respectively). Copies of the data can be obtained free of charge at https://www.ccdc.cam.ac.uk/structures/. Source data for Extended Data Fig. 5 has been provided in Supplementary Table 6.

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Acknowledgements

The authors thank M. R. Sawaya (UCLA-DOE Institute) for assistance in crystallography in data processing and refinement. This research used resources at the X-ray Crystallography Core Facility of the UCLA-DOE Institute, which is supported by the US Department of Energy (DE-FC02-02ER63421). J.A.R. acknowledges support from STROBE, an NSF Science and Technology Center through Grant DMR-1548924, DOE Grant DE-FC02-02ER63421 and NIH-NIGMS Grant R35 GM128867. J.A.R. is supported as a Pew Scholar and a Beckman Young Investigator. Y.T. acknowledges support from the NIH (1R01AI141481). The authors also thank the David and Lucile Packard Foundation (Fellowships to H.M.N., J.A.R. and Y.T.) and Bristol Myers Squibb (Unrestricted Grant in Synthetic Organic Chemistry to H.M.N.) for generous support.

Author information

Author notes

  1. These authors contributed equally: Lee Joon Kim, Masao Ohashi, Zhuan Zhang.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Lee Joon Kim, Matthew Asay, Duilio Cascio, José A. Rodriguez, Yi Tang & Hosea M. Nelson

  2. Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA, USA

    Masao Ohashi, Zhuan Zhang, Dan Tan & Yi Tang

  3. UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, Los Angeles, CA, USA

    Duilio Cascio & José A. Rodriguez

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Contributions

H.M.N. and Y.T. supervised the project. M.O., Z.Z. and D.T. performed in vivo experiments, as well as compound isolation and characterization. L.J.K. performed crystallization experiments, collected and processed the MicroED data, and solved the structures. L.J.K. and M.A. refined the structures. D.C. assisted in structure refinement. L.J.K. and D.C. performed the atom substitution test. J.A.R. assisted in designing MicroED experiments and helped with MicroED data analysis. L.J.K. and M.O. prepared the figures. H.M.N., Y.T., L.J.K. and M.O. wrote the manuscript.

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Correspondence to Yi Tang or Hosea M. Nelson.

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Extended data

Extended Data Fig. 1 Biosynthetic gene clusters that are homologous to those of fischerin (2) and N-hydroxyapiosporamide (7); and multiple sequence alignment of SAM-binding motif of cis-MT domain in PKS-NRPSs.

Shown here are the putative biosynthetic gene clusters of 2 and 7 and their homologous biosynthetic gene clusters found in NCBI database. SAM binding motif is shown in an alignment with those from FinD and ApiD homologs. Previous study reported that active cis-MT domains contain conserved EXGXGTG sequence as a SAM binding motif23. Based on this, we hypothesized that the cis-MT domains in PKS-NRPSs which do not have this conserved this motif are inactive, and the biosynthetic gene clusters which contain the PKS-NRPSs could be responsible for formation of 2. For example, the cis-MT domains from FinD (Aspergillus carbonarius), CG_v00450 (Colletotrichum fructicola Nara gc5), and BO_621233 (Aspergillus sclerotioniger) do not contain this conserved EXGXGTG motif as the threonine residues are mutated to alanines. As shown in Fig. 3d, the biosynthetic gene cluster, which contains FinD, is indeed responsible for formation of 2.

Extended Data Fig. 2

1H NMR spectra of 2 in CDCl3, 500 MHz for 1H NMR.

Extended Data Fig. 3 Proposed biosynthetic pathway of 2.

Based on the reported proposed biosynthetic pathway of other 2-pyridone alkaloids such as leporins and ilicicolin H (3) (see Extended Data Fig. 1a), we proposed the biosynthetic pathway of 2. FinD (PKS-NRPS) and the partnering FinC (ER) form the tetramic acid intermediate. A P450 FinE catalyzes the oxidative ring-expansion reaction of the tetramic acid to the 2-pyridone compound. Then, a Diels-Alderase likely catalyze the Diels-Alder reaction to form the energetically disfavored cis-decalin ring, since the previous study24 showed that nonenzymatic Diels-Alder reaction of the analog of 2-pyridone compound in water only led to formation of the trans-decalin compound. Further redox modification by FinA, FinE, and FinH forms 2.

Extended Data Fig. 4

Atom substitution test for fischerin (2) with (top) and without (bottom) electron scattering factors.

Extended Data Fig. 5 Electron microgram of austinol crystal and its diffraction pattern from 3 ng of sample.

Holes are 1 µm wide in diameter.

Supplementary information

Supplementary Information

Supplementary Figs. 1–26, Notes and Tables 1–13.

Reporting Summary

Supplementary Data 1

Crystallographic data containing structure factors for Py-469.

Supplementary Data 2

Crystallographic data containing structure factors for fischerin.

Supplementary Data 3

Crystallographic data containing structure factors for austinol.

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Kim, L.J., Ohashi, M., Zhang, Z. et al. Prospecting for natural products by genome mining and microcrystal electron diffraction. Nat Chem Biol 17, 872–877 (2021). https://doi.org/10.1038/s41589-021-00834-2

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