Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont

Journal name:
Nature Chemical Biology
Year published:
Published online


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.

At a glance


  1. Calyculin A from the marine sponge Discodermia calyx.
    Figure 1: Calyculin A from the marine sponge Discodermia calyx.

    (a) The marine sponge Discodermia calyx. Scale bar, 5 cm. (b) Structure of ​calyculin A. The β-branches at the C3 and C7 positions are highlighted.

  2. The biosynthetic gene cluster and proposed pathway of calyculin A.
    Figure 2: The biosynthetic gene cluster and proposed pathway of ​calyculin A.

    (a) ORFs encoded in the putative biosynthetic gene cluster. The ORFs related to NRPS and PKS are highlighted in red, and the putative phosphotransferases are in blue. The other ORFs include tailoring enzymes. (b) The domain organization and biosynthetic model for calyculin assembly. The KS clade and the putative substrate of the A domain are indicated above each domain. Domains: C, condensation; ER, enoylreductase; HC, heterocyclization; Ox, oxidation; PCP, peptidyl-carrier protein. KS clades: KS0, nonelongating ketosynthase; DB, double bond; SB, single bond; Oxz, oxazole; AA, amino acid.

  3. Monitoring of the conversion of phosphocalyculin A to calyculin A.
    Figure 3: Monitoring of the conversion of phosphocalyculin A to ​calyculin A.

    ODS HPLC profiles for (i) the product of CalQ-catalyzed reaction with ​calyculin A as the substrate, (ii) the ​methanol extract of the frozen sponges, (iii) the fresh sponges soaked in ​ethanol, (iv) the ​methanol extract of the sponges lyophilized after being flash-frozen in liquid nitrogen, (v) the authentic reference of phosphocalyculin A and (vi) the authentic reference of ​calyculin A. All of the chromatograms were detected by UV at 340 nm. The 2JP,P value was measured on the proton-decoupled 31P NMR spectrum.

  4. Symbiont bacteria bearing calyculin NRPS-PKS genes.
    Figure 4: Symbiont bacteria bearing calyculin NRPS-PKS genes.

    (a) Phase contrast image of D. calyx homogenate. A filamentous bacterium is designated as 'F'. A small filamentous bacterium appearing in bright color is designated as 'S'. (b) Differential interference contrast image of the filamentous bacteria. (c) CARD-FISH imaging with the calyculin NRPS-PKS gene as a probe. (d) PCR products with the calyculin NRPS-PKS–specific primer pair using dissected cells (F or S) as templates. F1–4, single filament (F); F5–7, four filaments (F); S1-4, four filaments (S); S4–7, eight filaments (S); P, D. calyx metagenomic DNA; N, negative control. In a, scale bars are 20 μm; in b and c, scale bars are 10 μm.

  5. Conversion of phosphocalyculin A to calyculin A after wounding the sponge tissue.
    Figure 5: Conversion of phosphocalyculin A to ​calyculin A after wounding the sponge tissue.

    The ratio of each compound in the analyzed samples was calculated as the percentage of the peak area of that component divided by the sum of the peak area of phosphocalyculin A and ​calyculin A (n = 3; error bars represent s.d.). No conversion was observed in unwounded sponge tissue (control).

  6. Phylogenetic tree of phosphotransferases involved in antibiotic resistance systems.
    Figure 6: Phylogenetic tree of phosphotransferases involved in antibiotic resistance systems.

    The neighbor-joining method (CLC sequence viewer and Geneious Tree Builder) was used to generate the phylogenetic tree with a bootstrap test of 100 replicates. Bootstrap values given in percentages are shown at the nodes. Clade names are provided on the basis of the phosphorylation site of aminoglycoside antibiotics.


1 compounds View all compounds
  1. Phosphocalyculin A
    Compound 1 Phosphocalyculin A

Accession codes

Primary accessions

NCBI Reference Sequence

Referenced accessions

NCBI Reference Sequence


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Author information

  1. These authors contributed equally to this work.

    • Toshiyuki Wakimoto &
    • Yoko Egami


  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


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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