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The Compatibility of Trypsin Inhibitor Cyclotides with Plant-Based Recombinant Expression Systems

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The Compatibility of Trypsin Inhibitor Cyclotides with Plant-Based Recombinant Expression Systems The compatibility of trypsin inhibitor cyclotides with plant-based recombinant expression systems Bronwyn Smithies B. BiotechMedRes (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 The Institute for Molecular Bioscience Abstract The compatibility of trypsin inhibitor cyclotides with plant-based recombinant expression systems Bronwyn Smithies, The University of Queensland, 2019 Plant-based production of modern pharmaceutical products could provide cheaper, greener pro- duction 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. In the fourth chapter, the transient leaf-based expression system of Nicotiana benthamiana is enhanced 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 ii 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 efforts are encouraged, particularly to identify and characterise more plant-derived enzymes compatible 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. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. iv Publications included in this thesis 1. Smithies, B. J., Huang Y-H., Jackson M. A., Yap K., Gilding E. K., Harris K. S., Anderson M. A. and Craik D. J. (2020) Circular permutation of the native enzyme-mediated cyclization position in cyclotides, ACS Chemical Biology 15, pp. 962-969. This work is reported in Chapter 3 of this thesis. Submitted manuscripts included in this thesis No manuscripts submitted for publication. Other publications during candidature Peer-reviewed articles 1. Qu H., Smithies B. J., Durek T., and Craik D. J. (2017). Synthesis and protein engineering applications of cyclotides, Australian Journal of Chemistry 70, p. 152. Conference abstracts 1. Smithies B. J., Huang Y-H., Gilding E., Jackson M. and Craik D. J. Using plants as biofactories for therapeutic peptides. ComBio, October 2016, Brisbane, Australia. Poster ‘teaser’ oral presentation and poster presentation. 2. Smithies B. J., Jackson M., Huang Y-H., Gilding E., and Craik D. J. Using plants as biofactories for therapeutic cyclic peptides. EMBL Australia Postgraduate Symposium, November 2016. Adelaide, Australia. Poster presentation. 3. Smithies B. J., Jackson M., Huang Y-H., Gilding E., and Craik D. J. Closing the ring: Reor- ganising cyclic peptide structure to enable expression of promising peptide pharmaceuticals in plants. 3rd Congress of the International Society for Plant Molecular Farming, June 2018. Helsinki, Finland. Poster presentation. 4. Smithies B. J., Jackson M., Huang Y-H., Gilding E., and Craik D. J. Closing the ring: a new cyclisation position for the cyclic peptide MCoTI-II. 4th International Conference on Circular Peptides and Proteins, November 2018. Kawasaki, Japan. ‘Hotspot’ talk and poster presentation. v Contributions by others to the thesis Dr Mark Jackson and Dr Edward Gilding helped conceptualise, design and analyse the results of experiments in Chapters 2 to 5. Dr Jackson designed some of the expression constructs, provided guidance and suggestions for further construct design, and gave instruction on molecular cloning and plant expression techniques used in Chapters 2 and 4. Dr Gilding designed and provided the pDONR221 expression vector used in Chapters 2 and 4, gave instruction on molecular cloning and plant expression techniques for Chapters 2 and 4, and developed the CRISPR-Cas9 edited plants used in Chapter 4. Dr Jackson and Dr Yen-Hua Huang provided the original concept for the alternative cyclisation experiments in Chapters 2, 3 and 4. Dr Huang gave instruction on peptide purification and NMR techniques and analysed NMR data for Chapter 3. Kuok Yap conducted E. coli expression, provided purified AEPs, and assisted with co-elution studies for Chapter 3. Dr Gilding organised the itinerary, car hire, collection permits and compiled the target taxa list for the field trip to the Kimberleys (Chapter 5). Dr Gilding, Dr Jackson, Dr Quentin Kaas and Prof. David Craik assisted in collecting plant specimens and providing photographic records for Chapter 5. Dr Jackson, Dr Gilding, Professor Marilyn Anderson and Prof. Craik provided critical revision during
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