The compatibility of trypsin inhibitor cyclotides with plant-based recombinant expression systems

Abstract

Plant-based production of modern pharmaceutical products could provide cheaper, greener production alternatives to laboratory-based methods. This is particularly relevant when it comes to producing peptides, where the cost of chemical synthesis can be prohibitive on a large scale and where environmentally damaging reagents are required. Cyclotides are a class of plant-derived cyclic peptides that are amenable to chemical synthesis for small-scale laboratory-based studies, but clinical trials and applications employing peptides require scale-up. Recombinant expression is an attractive alternative to chemical synthesis for scaling up cyclotide production because cyclotides are naturally ribosomally-synthesised and consist of natural amino acids. In planta expression has already been demonstrated for some cyclotides, particularly in relation to studying their biosynthesis. This thesis focuses primarily on one subclass of cyclotides, the trypsin inhibitors, whose in planta expression is yet to be fully established.The trypsin inhibitor cyclotides have been re-engineered to develop promising drug leads for chronic myeoloid leukemia, cardiovascular disease and inflammation, but the expression of these valuable lead compounds in recombinant plant systems is largely unexplored. Key challenges for plant-based production of trypsin inhibitor cyclotides include compatibility with biosynthetic enzymes and optimising the folding and accumulation conditions in suitable biofactory host plants. This poses a challenge for large scale plant-based production strategies that must consider the requirement for accessory biosynthetic elements. Ultimately, the compatibility of trypsin inhibitor cyclotides with plant-based production will rely on their ability to be cyclised by enzymes present in the plant, and their accumulation in productive plant tissues. This thesis addresses aspects of recombinant expression of trypsin inhibitor cyclotides over the course of six chapters.Firstly, this thesis details the current status of cyclotide research with a focus on re-engineered cyclotides for pharmaceutical purposes, and the current options for cyclotide synthesis or biosynthesis. Primary research findings are then presented in Chapters 2 to 5. Chapter 2 focuses on identifying promising genetic constructs for trypsin inhibitor expression in seeds and characterising the challenges for leaf-based expression. In Chapter 3, the investigation moves to the in vitro space to explore the compatibility between trypsin inhibitor cyclotides and plant-derived enzymes capable of backbone cyclisation. Modified trypsin inhibitor cyclotides are redesigned to be compatible with cyclising enzymes in vitro to enable semi-enzymatic production from either synthetic or recombinant substrates. Inthefourthchapter, thetransientleaf-basedexpressionsystemofNicotianabenthamianaisenhanced by the co-expression of a master regulator of seed development to promote a seed-like environment within the leaf tissue, resulting in greater accumulation of cyclotides. To conclude the primary research, Chapter 5 is dedicated to the documentation of a plant collection trip undertaken in the remote Kimberley region of Western Australia in May 2018. Finally, Chapter 6 presents a discussion of the results, including insights into the future directions and applications for the results presented throughout the thesis.In conclusion, the trypsin inhibitor cyclotide scaffold has been re-engineered to be compatible with both therapeutic engineering applications and enzyme-mediated cyclisation, and the foundations for in planta expression have been laid with consideration for the most widely used plant biofactory systems. An important step going forward will be the elucidation of intracellular transport pathways, specifically for trypsin inhibitor cyclotides and their cyclising enzyme partners. Additional discovery effortsareencouraged,particularlytoidentifyandcharacterisemoreplant-derivedenzymescompatible with head-to-tail cyclisation of the cyclotide backbone. Although challenging, the development of plant-based expression for trypsin inhibitor cyclotides should be pursued to provide a cheap, rapid and scalable production platform to support the development of this class of cyclotides that hold so much therapeutic potential.