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Engineering the amoeba Dictyostelium discoideum for biosynthesis of a cannabinoid precursor and other polyketides

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Abstract

Aromatic polyketides are natural polyphenolic compounds with a broad spectrum of pharmacological activities. Production of those metabolites in the model organisms Escherichia coli and Saccharomyces cerevisiae has been limited by the extensive cellular engineering needed for the coordinated biosynthesis of polyketides and their precursors. In contrast, the amoeba Dictyostelium discoideum is a native producer of secondary metabolites and harbors a wide, but largely unexplored, repertoire of genes for the biosynthesis of polyketides and terpenoids. Here we present D. discoideum as an advantageous chassis for the production of aromatic polyketides. By expressing its native and cognate plant polyketide synthase genes in D. discoideum, we demonstrate production of phlorocaprophenone, methyl-olivetol, resveratrol and olivetolic acid (OA), which is the central intermediate in the biosynthesis of cannabinoids. To facilitate OA synthesis, we further engineered an amoeba/plant inter-kingdom hybrid enzyme that produced OA from primary metabolites in two enzymatic steps, providing a shortcut in a synthetic cannabinoid pathway using the D. discoideum host system.

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Fig. 1: PCP production by overexpression of stlB in D. discoideum.
Fig. 2: Biosynthesis of methyl-olivetol and olivetol by StlA in D. discoideum.
Fig. 3: Resveratrol production by expressing a plant type III PKS gene in D. discoideum.
Fig. 4: Heterologous production of olivetolic acid in D. discoideum.
Fig. 5: OA biosynthesis by a functional inter-kingdom PKS hybrid.

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Data availability

Nucleotide sequence data were obtained from the GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The Codon Usage for organisms was obtained from the Kazusa Codon Usage Database (https://www.kazusa.or.jp/codon/). The minimal datasets generated during the study are available as Supplementary Information or as source data. Biological and genetic materials are available from V.V. and F.H. upon reasonable request and completion of a material transfer agreement. Source data are provided with this paper.

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Acknowledgements

We thank H. Heinecke for conducting NMR experiments and L. Reimer for technical support. This work was supported by two grants of the European Social Fund ESF ‘Europe for Thuringia’ projects MiQWi (2015FGR0097, to F.H.) and SphinX (2017FGR0073, to V.V.), the Leibniz Research Cluster in the frame of the BMBF Strategic Process Biotechnology 2020+ (031A360A, to V.V.), the BMBF funding program ‘GO-Bio initial’ (FKZ161B097, to F.H.) and the BMBF-funded InfectControl consortium (03ZZ0813A, to L.R.).

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Author notes

  1. These authors contributed equally: Christin Reimer, Johann E. Kufs.

Authors and Affiliations

  1. Evolution of Microbial Interactions, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (Leibniz-HKI), Jena, Germany

    Christin Reimer & Falk Hillmann

  2. Faculty of Biological Sciences, Friedrich Schiller University Jena, Jena, Germany

    Christin Reimer & Johann E. Kufs

  3. Biobricks of Microbial Natural Product Syntheses, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (Leibniz-HKI), Jena, Germany

    Johann E. Kufs, Julia Rautschek & Vito Valiante

  4. Bio Pilot Plant, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (Leibniz-HKI), Jena, Germany

    Johann E. Kufs & Lars Regestein

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Contributions

C.R., J.E.K., V.V. and F.H. designed the research. C.R. and J.E.K. performed experiments and analyzed the data. L.R. supervised the respiration activity measurements. J.R. analyzed the NMR data. C.R., J.E.K., V.V. and F.H. wrote the manuscript.

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Correspondence to Vito Valiante or Falk Hillmann.

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Competing interests

C.R., J.E.K., V.V. and F.H. declare the following competing financial interest: part of this work was used to file patent application PCT/EP2021/068240.

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Peer review information Nature Biotechnology would like to thank Rasmus Frandsen, Rob Kay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Reimer, C., Kufs, J.E., Rautschek, J. et al. Engineering the amoeba Dictyostelium discoideum for biosynthesis of a cannabinoid precursor and other polyketides. Nat Biotechnol 40, 751–758 (2022). https://doi.org/10.1038/s41587-021-01143-8

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