Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Prospecting for natural products by genome mining and microcrystal electron diffraction


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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).

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 Source data for Extended Data Fig. 5 has been provided in Supplementary Table 6.


  1. 1.

    Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J. Nat. Prod. 83, 770–803 (2020).

    CAS  Article  Google Scholar 

  2. 2.

    Perfect, J. R. The antifungal pipeline: a reality check. Nat. Rev. Drug Discov. 16, 603–616 (2017).

    CAS  Article  Google Scholar 

  3. 3.

    Fair, R. J. & Tor, Y. Antibiotics and Bacterial Resistance in the 21st Century. Perspect. Medicin. Chem. 6, 25–64 (2014).

  4. 4.

    Pye, C. R., Bertin, M. J., Lokey, R. S., Gerwick, W. H. & Linington, R. G. Retrospective analysis of natural products provides insights for future discovery trends. Proc. Natl Acad. Sci. USA 114, 5601–5606 (2017).

    CAS  Article  Google Scholar 

  5. 5.

    Fisch, K. M. et al. Rational domain swaps decipher programming in fungal highly reducing polyketide synthases and resurrect an extinct metabolite. J. Am. Chem. Soc. 133, 16635–16641 (2011).

    CAS  Article  Google Scholar 

  6. 6.

    Nicolaou, K. C. & Snyder, S. A. Chasing molecules that were never there: misassigned natural products and the role of chemical synthesis in modern structure elucidation. Angew. Chem. Int. Ed. 44, 1012–1044 (2005).

    CAS  Article  Google Scholar 

  7. 7.

    Maier, M. E. Structural revisions of natural products by total synthesis. Nat. Prod. Rep. 26, 1105–1124 (2009).

    CAS  Article  Google Scholar 

  8. 8.

    Bifulco, G., Dambruoso, P., Gomez-Paloma, L. & Riccio, R. Determination of relative configuration in organic compounds by NMR spectroscopy and computational methods. Chem. Rev. 107, 3744–3779 (2007).

    CAS  Article  Google Scholar 

  9. 9.

    Jones, C. G. et al. The CryoEM method MicroED as a powerful tool for small molecule structure determination. ACS Cent. Sci. 4, 1587–1592 (2018).

    CAS  Article  Google Scholar 

  10. 10.

    Gruene, T. et al. Rapid structure determination of microcrystalline molecular compounds using electron diffraction. Angew. Chem. Int. Ed. 57, 16313–16317 (2018).

    CAS  Article  Google Scholar 

  11. 11.

    Rodriguez, J. A. et al. Structure of the toxic core of α-synuclein from invisible crystals. Nature 525, 486–490 (2015).

    CAS  Article  Google Scholar 

  12. 12.

    Jones, C. J. et al. Characterization of reactive organometallic species via MicroED. ACS Cent. Sci. 5, 1507–1513 (2019).

    CAS  Article  Google Scholar 

  13. 13.

    Hayakawa, S., Minato, H. & Katagiri, K. The ilicicolins, antibiotics from Cylindrocladium ilicicola. J. Antibiot. 24, 653–654 (1971).

    CAS  Article  Google Scholar 

  14. 14.

    Du, L. et al. Crowdsourcing natural products discovery to access uncharted dimensions of fungal metabolite diversity. Angew. Chem. Int. Ed. 53, 804–809 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Miyadera, H. et al. Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase). Proc. Natl Acad. Sci. USA 100, 473–477 (2003).

    CAS  Article  Google Scholar 

  16. 16.

    Ziemert, N., Alanjary, M. & Weber, T. The evolution of genome mining in microbes – a review. Nat. Prod. Rep. 33, 988–1005 (2016).

    CAS  Article  Google Scholar 

  17. 17.

    Singh, S. B. et al. Antifungal spectrum, in vivo efficacy, and structure–activity relationship of ilicicolin H. ACS Med. Chem. Lett. 3, 814–817 (2012).

    Article  Google Scholar 

  18. 18.

    Zhang, Z. et al. Enzyme-catalyzed inverse-electron demand Diels–Alder reaction in the biosynthesis of antifungal ilicicolin H. J. Am. Chem. Soc. 141, 5659–5663 (2019).

    CAS  Article  Google Scholar 

  19. 19.

    Liu, N. et al. Identification and Heterologous production of a benzoyl-primed tricarboxylic acid polyketide intermediate from the zaragozic acid A biosynthetic pathway. Org. Lett. 19, 3560–3563 (2017).

    CAS  Article  Google Scholar 

  20. 20.

    Alfatafta, A. A., Gloer, J. B., Scott, J. A. & Malloch, D. Apiosporamide, a new antifungal agent from the coprophilous fungus Apiospora montagnei. J. Nat. Prod. 57, 1696–1702 (1994).

    CAS  Article  Google Scholar 

  21. 21.

    Williams, D. R., Kammler, D. C., Donnell, A. F. & Goundry, W. R. F. Total synthesis of (+)-apiosporamide: assignment of relative and absolute configuration. Angew. Chem. Int. Ed. 44, 6715–6718 (2005).

    CAS  Article  Google Scholar 

  22. 22.

    Fujimoto, H., Ikeda, M., Yamamoto, K. & Yamazaki, M. Structure of fischerin, a new toxic metabolite from an ascomycete, Neosartorya fischeri var. fischeri. J. Nat. Prod. 56, 1268–1275 (1993).

    CAS  Article  Google Scholar 

  23. 23.

    Amini, S. K. Assignment of the absolute configuration of fischerin by computed nmr chemical shifts. J. Struct. Chem. 56, 1334–1341 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Ugai, T., Minami, A., Gomi, K. & Oikawa, H. Genome mining approach for harnessing the cryptic gene cluster in Alternaria solani: production of PKS–NRPS hybrid metabolite, didymellamide B. Tetrahedron Lett. 57, 2793–2796 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Skiba, M. A. et al. Domain organization and active site architecture of a polyketide synthase C-methyltransferase. ACS Chem. Biol. 11, 3319–3327 (2016).

    CAS  Article  Google Scholar 

  26. 26.

    Nannenga, B. L. MicroED methodology and development. Struct. Dyn. 7, 014304, (2020).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    de la Cruz, M. J. et al. Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED. Nat. Methods 14, 399–402 (2017).

    Article  Google Scholar 

  28. 28.

    Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228 (1988).

    CAS  Article  Google Scholar 

  29. 29.

    Natesh, R. in Structural Bioinformatics: Applications in Preclinical Drug Discovery Process, Edition 1, Vol. 27 (ed. Mohan, C. G.) 375–400 (Springer Nature, 2019).

  30. 30.

    Kato, K. et al. A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry. Acta Cryst. D64, 525–531 (2008).

    Google Scholar 

  31. 31.

    Matsuda, Y. & Abe, Ikuro Biosynthesis of fungal meroterpenoids. Nat. Prod. Rep. 33, 26–53 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Ohashi, M. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502 (2017).

    Article  Google Scholar 

  33. 33.

    Liu, N. et al. Identification and Heterologous production of a benzoyl-primed tricarboxylic acid polyketide intermediate from the zaragozic acid A biosynthetic pathway. Org. Lett. 19, 3560–3563 (2017).

    CAS  Article  Google Scholar 

  34. 34.

    Nannenga, B. L., Shi, D., Leslie, A. G. W. & Gonen, T. High-resolution structure determination by continuous-rotation data collection in MicroED. Nat. Methods 11, 927–930 (2014).

    CAS  Article  Google Scholar 

  35. 35.

    Hattne, J. et al. MicroED data collection and processing. Acta Cryst. A71, 353–360 (2015).

    Google Scholar 

  36. 36.

    Kabsch, W. XDS. Acta Cryst. D66, 125–132 (2010).

    Google Scholar 

  37. 37.

    Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Cryst. D66, 133–144 (2010).

    Google Scholar 

  38. 38.

    Sheldrick, G. M. A short history of SHELX. Acta Cryst. A64, 112–122 (2008).

    Article  Google Scholar 

  39. 39.

    Sheldrick, G. M. SHELXT – Integrated space-group and crystal-structure determination. Acta Cryst. A71, 3–8 (2015).

    Google Scholar 

  40. 40.

    Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Cryst. C71, 3–8 (2015).

    Google Scholar 

  41. 41.

    Hübschle, C. B., Sheldrick, G. M. & Dittrich, B. ShelXle: a Qt graphical user interface for SHELXL. J. Appl. Cryst. 44, 1281–1284 (2011).

    Article  Google Scholar 

  42. 42.

    Delano, W. The PyMOL Molecular Graphics System version 2.3.3 (Schrödinger LLC, 2019);

  43. 43.

    Van Rossum, G. & Drake, F. L. Python 3 Reference Manual (CreateSpace, 2009).

Download references


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




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.

Corresponding authors

Correspondence to Yi Tang or Hosea M. Nelson.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing