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Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont

Nature Chemical Biology volume 10, pages 648–655 (2014)Cite this article

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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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Figure 1: Calyculin A from the marine sponge Discodermia calyx.
Figure 2: The biosynthetic gene cluster and proposed pathway of calyculin A.
Figure 3: Monitoring of the conversion of phosphocalyculin A to calyculin A.
Figure 4: Symbiont bacteria bearing calyculin NRPS-PKS genes.
Figure 5: Conversion of phosphocalyculin A to calyculin A after wounding the sponge tissue.
Figure 6: Phylogenetic tree of phosphotransferases involved in antibiotic resistance systems.

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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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  1. Toshiyuki Wakimoto and Yoko Egami: These authors contributed equally to this work.

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Toshiyuki Wakimoto, Yoko Egami, Yu Nakashima, Yukihiko Wakimoto, Takahiro Mori, Takayoshi Awakawa & Ikuro Abe

  2. Japan Science and Technology Agency, CREST, Tokyo, Japan

    Toshiyuki Wakimoto, Yoko Egami, Takahiro Mori, Takayoshi Awakawa & Ikuro Abe

  3. Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan

    Takuya Ito, Hiromichi Kenmoku & Yoshinori Asakawa

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

    Jörn Piel

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Contributions

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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Correspondence to Toshiyuki Wakimoto or Ikuro Abe.

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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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