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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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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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).
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Supplementary Figs. 1–65, Tables 1–12 and synthetic procedures.
Primary data underlying Supplementary Figs. 42, 43, 45, 46 and 47
Growth of 3Y1 cancer cells treated with DMSO. Growth of cells is not affected.
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.
Growth of 3Y1 cancer cells treated with 0.34 μM doxorubicin. Cells die on the first day of treatment.
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.
Growth of HeLa cancer cells treated with DMSO. Growth of cells is not affected.
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.
Growth of 3Y1 cancer cells treated with 3.7 nM janustatin B (14). Cells grow normally for the first two days.
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