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Genome-based discovery and total synthesis of janustatins, potent cytotoxins from a plant-associated bacterium

Nature Chemistry volume 14, pages 1193–1201 (2022)Cite this article

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Abstract

Host-associated bacteria are increasingly being recognized as underexplored sources of bioactive natural products with unprecedented chemical scaffolds. A recently identified example is the plant-root-associated marine bacterium Gynuella sunshinyii of the chemically underexplored order Oceanospirillales. Its genome contains at least 22 biosynthetic gene clusters, suggesting a rich and mostly uncharacterized specialized metabolism. Here, in silico chemical prediction of a non-canonical polyketide synthase cluster has led to the discovery of janustatins, structurally unprecedented polyketide alkaloids with potent cytotoxicity that are produced in minute quantities. A combination of MS and two-dimensional NMR experiments, density functional theory calculations of 13C chemical shifts and semiquantitative interpretation of transverse rotating-frame Overhauser effect spectroscopy data were conducted to determine the relative configuration, which enabled the total synthesis of both enantiomers and assignment of the absolute configuration. Janustatins feature a previously unknown pyridodihydropyranone heterocycle and an unusual biological activity consisting of delayed, synchronized cell death at subnanomolar concentrations.

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Fig. 1: Biosynthetic loci identified in the genome of G. sunshinyii YC6258.
Fig. 2: The jan gene cluster in G. sunshinyii YC6258.
Fig. 3: Structure elucidation of janustatins.
Fig. 4: Total synthesis of janustatin A.
Fig. 5: Proposed biosynthesis of janustatins.
Fig. 6: Growth of 3Y1 cancer cells treated with 1.8 nM janustatin A (13).

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Data availability

The data supporting the findings of this study are available in this Article and the Supplementary Information. The genome sequence of G. sunshinyii YC6258 is accessible in GenBank under accession no. NZ_CP007142.1, the janustatin gene cluster is located at locus YC6258_05439 (AJQ97469.1) to YC6258_05446 (AJQ97476.1). The closest homologues to these proteins can be found under accession nos. WP_158657926.1, WP_086931657.1, WP_038924948.1, RKZ46405.1, WP_087684108.1 and WP_086931660.1. Candidate proteins for the free-standing KR are available under accession numbers: WP_044620326.1, WP_044617647.1, WP_044617493.1, WP_044616081.1, WP_044617507.1, WP_044618569.1, WP_044619377.1, WP_044617759.1, WP_144407613.1 and WP_052830250.1. The janustatin BGC has been deposited in MIBiG under accession no. BGC0002136.

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Acknowledgements

We thank Y. R. Chung and D. Mavrodi for insightful discussions and D. Mazel for sharing plasmid pSW8197 with us. J.P. acknowledges funding by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 742739), the Gordon and Betty Moore Foundation (#9204, https://doi.org/10.37807/GBMF9204) and the Swiss National Science Foundation (NRP72 ‘Antimicrobial Resistance’, 407240_167051). Y.Y. is supported by a Grant-in-Aid for Scientific Research on Innovative Areas (17H06411), the Japan Society for the Promotion of Science (JSPS). M.Y. and S.N. are supported in part by a JSPS Grant-in-Aid for Scientific Research (S) (19H05640).

Author information

Author notes

  1. Reiko Ueoka

    Present address: School of Marine Biosciences, Kitasato University, Sagamihara, Kanagawa, Japan

  2. Philipp Sondermann

    Present address: Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

  3. Yizhou Liu

    Present address: Analytical Research & Development, Pfizer Worldwide Research and Development, Groton, CT, USA

  4. Rei Suo

    Present address: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan

  5. R. Thomas Williamson

    Present address: Department of Chemistry & Biochemistry, University of North Carolina Wilmington, Wilmington, NC, USA

  6. These authors contributed equally: Reiko Ueoka, Philipp Sondermann, Stefan Leopold-Messer.

Authors and Affiliations

  1. Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland

    Reiko Ueoka, Stefan Leopold-Messer, Agneya Bhushan, Lida Vadakumchery, Ute Greczmiel, Annette Oxenius & Jörn Piel

  2. Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland

    Philipp Sondermann & Erick M. Carreira

  3. NMR Structure Elucidation, Process & Analytical Chemistry, Merck & Co. Inc., Rahway, NJ, USA

    Yizhou Liu & R. Thomas Williamson

  4. Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

    Rei Suo & Shigeki Matsunaga

  5. Molecular Ligand Target Research Team, RIKEN Center for Sustainable Resource Science, Wako, Saitama, Japan

    Yoko Yashiroda, Hiromi Kimura, Shinichi Nishimura & Minoru Yoshida

  6. Department of Biotechnology, The University of Tokyo, Tokyo, Japan

    Shinichi Nishimura, Yojiro Hoshikawa & Minoru Yoshida

  7. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan

    Shinichi Nishimura & Minoru Yoshida

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Contributions

All authors devised the experiments. R.U. and S.L.-M. isolated and characterized natural products. P.S. prepared and characterized synthetic compounds. Y.L. and R.T.W. analysed NMR data and conducted calculations. S.L.-M., R.S., Y.Y., H.K., A.B., U.G., S.N., Y.H., M.Y., A.O. and S.M. performed biological assays. S.L.-M., A.B. and J.P. performed bioinformatic analysis. S.L.-M. and L.V. generated the mutant strain. R.U., S.L.-M., P.S., E.M.C. and J.P. wrote the paper, with contributions from all authors.

Corresponding authors

Correspondence to Erick M. Carreira or Jörn Piel.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–65, Tables 1–12 and synthetic procedures.

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Supplementary Data 1

Primary data underlying Supplementary Figs. 42, 43, 45, 46 and 47

Supplementary Video 1

Growth of 3Y1 cancer cells treated with DMSO. Growth of cells is not affected.

Supplementary Video 2

Growth of 3Y1 cancer cells treated with 1.8 nM janustatin A (13). Cells grow normally for the first two days. After three days, janustatin-treated cells stop dividing and die in a synchronized fashion.

Supplementary Video 3

Growth of 3Y1 cancer cells treated with 0.34 μM doxorubicin. Cells die on the first day of treatment.

Supplementary Video 4

Growth of HeLa cancer cells treated with 1.8 nM janustatin A (13). Cells grow normally for the first two days. After three days, janustatin-treated cells stop dividing and die in a synchronized fashion.

Supplementary Video 5

Growth of HeLa cancer cells treated with DMSO. Growth of cells is not affected.

Supplementary Video 6

Growth of 3Y1 cancer cells treated with 3.6 nM janustatin A′ (13′). Cells grow normally for the first two days. After three days, janustatin-treated cells stop dividing and die in a synchronized fashion.

Supplementary Video 7

Growth of 3Y1 cancer cells treated with 3.7 nM janustatin B (14). Cells grow normally for the first two days.

Supplementary Video 8

Growth of 3Y1 cancer cells treated with 1.7 μM doxorubicin. Cells die on the first day of treatment.

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Ueoka, R., Sondermann, P., Leopold-Messer, S. et al. Genome-based discovery and total synthesis of janustatins, potent cytotoxins from a plant-associated bacterium. Nat. Chem. 14, 1193–1201 (2022). https://doi.org/10.1038/s41557-022-01020-0

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