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Biosynthesis and biotechnological production of the anti-obesity agent celastrol

Nature Chemistry volume 15, pages 1236–1246 (2023)Cite this article

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Abstract

Obesity is a major health risk still lacking effective pharmacological treatment. A potent anti-obesity agent, celastrol, has been identified in the roots of Tripterygium wilfordii. However, an efficient synthetic method is required to better explore its biological utility. Here we elucidate the 11 missing steps for the celastrol biosynthetic route to enable its de novo biosynthesis in yeast. First, we reveal the cytochrome P450 enzymes that catalyse the four oxidation steps that produce the key intermediate celastrogenic acid. Subsequently, we show that non-enzymatic decarboxylation-triggered activation of celastrogenic acid leads to a cascade of tandem catechol oxidation-driven double-bond extension events that generate the characteristic quinone methide moiety of celastrol. Using this acquired knowledge, we have developed a method for producing celastrol starting from table sugar. This work highlights the effectiveness of combining plant biochemistry with metabolic engineering and chemistry for the scalable synthesis of complex specialized metabolites.

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Fig. 1: Combining metabolomics and transcriptomics to identify candidate celastrol biosynthetic genes in T. wilfordii.
Fig. 2: Functional characterization of CYP716C52 and its variants in S. cerevisiae.
Fig. 3: Functional characterization of CYP81AM1 and its variant in S. cerevisiae.
Fig. 4: Non-enzymatic oxidation and C24 decarboxylation of celastrogenic acid.
Fig. 5: A tandem catechol oxidation cascade leads to celastrol.
Fig. 6: Scalable de novo biosynthetic platform of celastrol production in yeast from sugar.

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Data availability

All data supporting the findings of this work are available within the paper and its Supplementary Information files. A reporting summary for this article is available as a Supplementary Information file. The T. wilfordii cDNA sequences identified here have been deposited in GenBank under accession numbers OP970829 to OP970833. Source data are provided with this paper.

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Acknowledgements

We thank F. Geu-Flores (University of Copenhagen), V. Roussis and E. Ioannou (National and Kapodistrian University of Athens, Greece) for critical reading of the manuscript, and H. Chen (Kunming University of Science and Technology, China) and H. Zhang (Swiss Federal Institute of Technology Lausanne, Switzerland) for discussions on the chemical mechanism. We also thank D. R. Nelson (University of Tennessee, USA) for assigning the CYP names. We thank J. Olsen, M. Ramirez, D. Pattison, I. Ovejero-Lopez and L. Kjærulff (University of Copenhagen) for their assistance in running analytical instruments. This work was financially supported by the Novo Nordisk Foundation (grants NNF17OC0027646 to S.B. and S.C.K. and NNF16OC0021760 and NNF19OC0055204 to S.C.K.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Biochemical Engineering Group, Plant Biochemistry Section, Department of Plant and Environment Sciences, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark

    Yong Zhao, Nikolaj L. Hansen, Yao-Tao Duan, Meera Prasad, Mohammed S. Motawia, Birger L. Møller, Irini Pateraki, Søren Bak, Karel Miettinen & Sotirios C. Kampranis

  2. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Dan Staerk

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Contributions

Y.Z., K.M., B.L.M., S.B. and S.C.K. conceived and designed the experiments. Y.Z., N.L.H. and M.P. conducted all cloning, the engineering of N. benthamiana and S. cerevisiae, and analysed the chromatography data. Y.Z. and Y.-T.D. isolated the products from S. cerevisiae and T. wilfordii roots. Y.Z. and D.S. analysed the NMR data. M.S.M. proposed the chemical mechanism. Y.Z., I.P., K.M. and S.C.K. wrote the paper. All authors contributed to the final version of the paper.

Corresponding authors

Correspondence to Søren Bak, Karel Miettinen or Sotirios C. Kampranis.

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Competing interests

S.C.K., Y.Z., K.M. and N.L.H. are co-inventors in a patent application (European Patent Office no. P101284EP00, 2022) describing the bioproduction of celastrol in yeast. The other authors claim no competing interests.

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Nature Chemistry thanks Jens Nielsen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Methods, Figs. 1–35 and Tables 1–7.

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Supplementary Data 1

Oligo sequences used in this study.

Supplementary Data 2

Source data for Supplementary Figs. 6 and 7.

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Statistical source data.

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Statistical source data.

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Zhao, Y., Hansen, N.L., Duan, YT. et al. Biosynthesis and biotechnological production of the anti-obesity agent celastrol. Nat. Chem. 15, 1236–1246 (2023). https://doi.org/10.1038/s41557-023-01245-7

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