Poster Presentation International Plant Molecular Biology Conference 2024

Optimising peptide cyclisation pathways in Nicotiana benthamiana enables plant molecular farming of therapeutic peptides. (#51)

Mark A Jackson 1 , Edward Gilding 1 , Max Harding 1 , David Craik 1
  1. Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, St Lucia, Queensland, Australia

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species are known to harbour enzymatic capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. We recently demonstrated that by co-expressing a specific class of plant asparaginyl endopeptidases (AEPs) that cyclic peptide yields in planta can be dramatically improved, opening opportunities for large scale production in plant hosts (1,2). However, for some peptides we find that premature proteolysis occurs before the peptide can be stabilised by backbone cyclisation. Here we address this limitation by re-engineering subcellular targeting of peptide precursors and AEPs to more peptide friendly organelles. Furthermore, we use a gene editing strategy to develop Nicotiana benthamiana transgenics with reduced interfering protease activity, thus endowing this plant molecular farming host with an expanded repertoire of peptide sequence space (3).  Here, we describe these approaches that together enable the in planta production of a range of peptide therapeutic candidates to treat multiple sclerosis, prostate cancer, Netherton syndrome, and neuropathic pain.

  1. Jackson MA, Gilding EK, Shafee T, Harris KS, Kaas Q, Poon S, Yap K, Jia H, Guarino R, Chan LY, Durek T, Anderson MA, Craik DJ (2018) Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases. Nature Communications 9: 2411
  2. Jackson MA, Xie J, Nguyen LTT, Wang X, Yap K, Harvey PJ, Gilding EK, Craik DJ. Plant-based production of an orally active cyclotide for the treatment of multiple sclerosis. Transgenic Res. 2023 Apr 32(1-2):121-133.
  3. Mark A Jackson, Lai Yue Chan, Maxim D Harding, David J Craik, Edward K Gilding, Rational domestication of a plant-based recombinant expression system expands its biosynthetic range, Journal of Experimental Botany, Volume 73, Issue 18, 18 October 2022, Pages 6103–6114.