Plant derived cyclic peptides: from discovery to biotechnological applications

Abstract

Plant-derived cysteine-rich cyclic peptides are a class of small peptides that range from 14-37 amino acids in size and are characterised by a head-to-tail cyclized backbone. Their unique cyclized and disulfide‑constrained structures make them exceptionally stable molecules. Members include the six‑cysteine containing cyclotides, which have been detected in species spanning five plant families, and the two-cysteine PawS-derived cyclic peptides found in sunflower. The sequences between cysteines residues are tolerant to residue substitutions in both of these examples, thus making them promising scaffolds for grafting and stabilizing bioactive epitopes. Indeed, a number of pharmaceutical protein‑engineering applications based on cyclic peptides have been demonstrated in recent years. Chemical synthesis has been the dominant approach in the past for producing cysteine-rich cyclic peptides, with native chemical ligation being the most commonly used to generate the cyclic backbone. However, production using this approach can be expensive, especially upon scale-up production, and not all cyclic peptides are amenable to synthesis and correct folding. Furthermore, the large amounts of chemical reagents required for synthesis has impacts for the environment. To overcome this issue, an alternative strategy is to produce cyclic peptides in plants, as some plant species are naturally efficient at cyclic peptide production. Other advantages of developing plant-based production systems include reduced production costs, reduced chemical waste, and the possibility of developing innovative oral delivery drugs by the packaging of peptides in edible plant products. The overall goal of this thesis is to explore the potential of plant-based production of cyclic peptides. Chapter 1 provides a comprehensive background of plant-derived cysteine‑rich cyclic peptides and the current synthesis approaches.The first aim was to develop rice as a production system to produce cyclic peptides (Chapter 2). Rice was selected as a candidate biofactory host as it has been proven to be efficient for the recombinant production of complex proteins, especially for those with disulfide bonds. Moreover, rice does not naturally produce cyclic peptides, which eliminates the possible interference of native cyclic peptides during separation and purification. A stable transformation platform was set up to produce cyclic peptides in rice suspension cells and seeds. Transgenes encoding prototypical cyclic peptides and engineered analogues were co-expressed with asparaginyl endopeptidases (AEPs) which are enzymes required for peptide backbone cyclization. The yields and structures of rice-derived cyclic peptides and transcript expression levels of AEPs were characterized. The second aim was to investigate the diversity and cyclization capability of cyclotide-like peptides in monocots (Chapter 3). To date, no native cyclic peptides have been identified in any monocot species, and only genes encoding linear cyclotide-like peptides have been identified. The essential residues known to be required for backbone cyclization are missing in these precursors. To investigate this further, monocot lineages were explored for cyclotide-like gene sequences using transcriptome analysis of monocots spanning the breadth of the taxonomic group. When expressing cyclotide-like genes in planta, a pyroGlu modification at the N-terminus was observed. Additionally, it was observed that some monocot cyclotide‑like genes could be engineered with minimal residue changes to allow backbone cyclization both in vitroand in planta. These results will aid efficient cyclic peptide production in monocot cereal plants (e.g. rice, maize).The third aim was to define the plasticity of seeds for the production of diverse cyclic peptides (Chapter 4). Some cyclic peptides are naturally produced in seeds, which suggests that plant seeds may provide a beneficial environment for the production of heterologous cyclic peptides. To investigate this hypothesis, a number of cyclic peptide genes were expressed in Arabidopsis seeds. Furthermore, the cyclization efficiencies of three different sunflower AEPs were determined. To circumvent the low efficiency of co‑transformation of cyclic peptide precursors and AEP genes, a homozygous AEP transgenic line exhibiting high expression was created. This stable AEP expressing line with cyclic peptide gene stacking experiments would shed light on the production of cyclic peptides in seeds.Throughout my PhD, a range of plants, tissues and cell types has been investigated for their plasticity to produce cyclic peptides. Rice suspension cells and seeds were developed to produce cyclic peptides in continuous production or stable long term storage respectively. Arabidopsis seeds were developed as a simple platform for the seed production of cyclic peptides. Furthermore, the diversity of monocot cyclotide‑like genes was investigated, as well as their capability to be engineered for backbone cyclization using a transient leaf expression system. All in all, these studies provide valuable information on the selection of biofactory hosts, tissue specificity and the genetic modifications required to produce cyclic peptides efficiently in plants.