Abstract
The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of calyculin A by a metagenome mining approach. Single-cell analysis revealed that the gene cluster originates in the symbiont bacterium 'Candidatus Entotheonella' sp. A phosphotransferase encoded in the gene cluster deactivated calyculin A to produce a newly discovered diphosphate, which was actually the biosynthetic end product. The diphosphate had been previously overlooked because of the enzymatic dephosphorylation that occurred in response to sponge tissue disruption. Our work presents what is to our knowledge the first evidence for the biosynthetic process of calyculin A along with a notable phosphorylation-dephosphorylation mechanism to regulate toxicity, suggesting activated chemical defense in the most primitive of all multicellular animals.
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Acknowledgements
We thank H. Morita (University of Toyama) and M.F. Freeman (ETH Zurich) for technical assistance; K. Takada (University of Tokyo) for assistance with the sponge collection; Y. Ise (University of Tokyo) for species identification; and M. Kamio (Tokyo University of Marine Science and Technology), N. Fusetani (Hokkaido University) and S. Matsunaga (University of Tokyo) for fruitful discussions. This work was carried out in part as joint research with the Japanese Association for Marine Biology (JAMBIO) and was partly supported by the Mitsubishi Foundation (I.A.), the Nagase Science Technology Foundation (T.W.), the Astellas Foundation for Research on Metabolic Disorders (T.W.), the CREST program from the Japan Science and Technology Agency (I.A.), the Bilateral Program between Japan and Switzerland from the Japan Society for the Promotion of Science (JSPS) (I.A.) and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (T.W. and I.A.).
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T.W. and I.A. designed the research. T.W. collected sponge specimens. Y.E. constructed libraries and isolated cal genes. T.W., Y.E., T.I., H.K., Y.A., J.P. and I.A. sequenced and analyzed cal genes. T.W. and Y.E. performed the cell separation and elucidation of chemical structures as well as bioconversion experiments. Y.E. and Y.N. conducted the single-cell studies. Y.E., Y.N., Y.W., T.M. and T.A. characterized the enzymes. T.W., Y.E. and I.A. wrote the paper.
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Wakimoto, T., Egami, Y., Nakashima, Y. et al. Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont. Nat Chem Biol 10, 648–655 (2014). https://doi.org/10.1038/nchembio.1573
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DOI: https://doi.org/10.1038/nchembio.1573