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Production of a structurally validated cyclotide in rice suspension cells is enabled by a supporting biosynthetic enzyme

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Abstract

Main conclusion

We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides.

Abstract

Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1b from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC–MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.

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Acknowledgements

We thank Dr. Deyun Qiu from the Australian National University for the assistance in rice transformation.

Funding

We acknowledge funding from the Australian Research Council (ARC Laureate Fellowship FL150100146 to DC and ARC Grants DP150100443 and DP200101299 to DC, EG, and TD, CE200100012). This research was also supported by a 2015 Ramaciotti Biomedical Research Award and the Simon Axelsen Memorial Fund and by the facilities of the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science.

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Authors and Affiliations

  1. Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD, 4072, Australia

    Haiou Qu, Mark A. Jackson, Kuok Yap, Peta J. Harvey, Edward K. Gilding & David J. Craik

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  1. Haiou Qu
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  2. Mark A. Jackson
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Correspondence to David J. Craik.

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Qu, H., Jackson, M.A., Yap, K. et al. Production of a structurally validated cyclotide in rice suspension cells is enabled by a supporting biosynthetic enzyme. Planta 252, 97 (2020). https://doi.org/10.1007/s00425-020-03505-z

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