Using Agrobacterium-mediated transformation, University of Queensland (UQ) researchers have developed transgenic plants capable of producing cyclic peptides.
Increased rigidity and stability are some of the numerous benefits of cyclic peptides. Cyclotides are a class of cyclic peptides that contain three disulfide bonds arranged in a cystine knot motif, which confers exceptional stability. Cyclotides have exhibited a range of bioactivities, from agricultural pests to human pathogens and diseases. The stable cyclotide framework can also be used as a pharmaceutical scaffold for the grafting of peptide sequences conferring bioactivity. Despite their attractive properties, efficient manufacture of cyclotides is a significant obstacle to their commercial development.
Cyclisation of synthetic peptides in vitro is challenging, and the limited availability of enzymes capable of this process has been a hurdle to large-scale production. Plant-host engineering allows the production of proteins with distinct and uniform post-translational modifications, allowing the development of biologics that demonstrate higher effectiveness than those produced using other protein expression systems (e.g. mammalian or yeast) eliminate the threat of animal-derived contaminants.
Using Agrobacterium-mediated transformation, University of Queensland (UQ) researchers have developed transgenic plants capable of producing cyclic peptides. The plants express the cyclising enzyme OaAEP1b ligase which efficiently cyclises both cyclotides and peptides that are naturally linear. OaAEP1 functions by coupling C-terminal cleavage of pro-peptide substrates with backbone cyclisation. The plants also express a precursor to the peptide of interest, which contains modified N- and C-termini to facilitate cyclisation and achieve OaAEP1 recognition.
Proof of concept
The UQ team has demonstrated that the cyclotide precursor (Oak1) can be effectively cyclised using OaAEP1b in three plant species:
• Tobacco relative (Nicotiana benthamiana)
• Bush bean (Phaseolus vulgaris cv. Royal Burgundy)
• Lettuce (Lactuca sativa cv. Green Cos).
Turnover in some plants (P. vugaris), from the precursor Oak1 to the cyclic product, can occur at low yield in the absence of OaAEP1 (due to naturally occurring others cyclisation plant enzymes). The introduction of an OaAEP1b enzyme significantly increases cyclisation turnover across all plant species tested. Optimal cyclisation occurs when the precursor gene Oak1 and the cyclising enzyme OaAEP1b are linked to each other via a 6-residue linker and expressed in the plant using a single gene construct.
Alternatively, purified OaAEP1b enzyme can be used to cyclise peptides in vitro. The research team is currently developing new OaAEP1b variants with optimised function for use in vitro or in planta.
• Production of difficult to manufacture cyclic peptides in a plant-based expression system
• Improved yields over synthetic or in vitro production
• Compatible with existing molecular pharming methods (including Agrobacterium-mediated transformation)
• Peptide products with exceptional stability for therapeutic and/or agricultural applications
• Ongoing development of optimised enzyme variants and expression systems.
Transgenic OaAEP1-expressing plants can be used as biofactories in molecular pharming to produce cyclic peptides based on the exceptionally stable cyclotide scaffold. The peptide(s) may be used agriculturally to protect the plant from pathogens or can be extracted from the plant for use in pharmacological applications.