Taking Plant Pharmaceuticals from Research to Production

From Farm to Pharm: Taking plant pharmaceuticals from research to production

Benjamin Doffek

BSc, MSc (Hons)

0000-0001-7583-426X

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 Institute for Molecular Bioscience Abstract

Abstract

Affordable production of pharmaceuticals with high potency and low side effects is a major challenge of the 21st century. Peptides are an emerging class of therapeutics that have the potential to marry the specificity and efficacy of protein drugs with the stability and membrane permeability of small molecule drugs. Although many peptides are amenable to chemical synthesis, their cost of production is high, as is the generation of waste products. Peptide production in plants has the potential to be a scalable, cost effective, and a less environmentally taxing alternative. Cyclotides, first discovered in Oldenlandia affinis, are a unique class of backbone cyclic peptides containing three stabilising disulfide bridges that form a knot-like structure. Their stability, and for some variants, the ability to traverse cellular membranes make them ideal candidates for pharmaceutical and agricultural applications. Even though highly constrained sterically, cyclotides are amenable to engineering by replacing native sequences with bioactive epitopes. In this thesis, cyclotide production strategies in plant cell suspensions are examined with a special focus placed on O. affinis as an archetypical cyclotide producer. Additional species investigated are Clitoria ternatea, Hybanthus enneaspermus, Nicotiana benthamiana, and Petunia hybrida. Insights into how cyclotides and their biosynthetic processing machinery are regulated in suspension plant cells are reported and provide first steps towards an affordable and environmentally friendly peptide production system in plants. Chapter 1 introduces the relevant scientific knowledge. The journey starts with a description of proteins and peptides, emphasising cyclotides and their synthesis. Then, current production strategies are compared with plant-based systems. The advantages and disadvantages of bioreactor setups suitable for medium to large scale production of cyclotides in plant cell suspensions are subsequently outlined. Finally, the importance of downstream processing is discussed. At the end of Chapter 1, the scope of this thesis is outlined to give an overview of the scientific questions addressed. Chapter 2 lays the experimental groundwork upon which the rest of this thesis is built. Protocols to establish C. ternatea, H. enneaspermus, N. benthamiana, O. affinis, and P. hybrida suspension cultures were developed and their cyclotide accumulation profiles presented. Cycloviolacin O2, a cyclotide previously reported only in the Violaceae, was discovered in O. affinis suspension cells and corroborated in the O. affinis leaf transcriptome. Two new cyclotides, kalata B22 and kalata B23, were characterized from O. affinis suspensions and were shown to have high sequence similarity to a group of cyclotides found in suspension cells of multiple plant families. Additionally, a strong time dependent effect was observed for cyclotide production in O. affinis suspension cells and was connected to reduced cyclotide mRNA expression. Cyclotide production

Abstract was tissue specific in all tested species, an outcome with potential consequences for large scale production. Chapter 3 presents the scale-up of plant cell suspension cultures for cyclotide production. C. ternatea, H. enneaspermus, and O. affinis suspensions were cultivated in a rocking motion bioreactor with culture volumes up to 5 L. Growth characteristics and biomass yields of all species were promising; however, contamination, sampling and consequential downregulation of cyclotides in suspension cultures presented both challenges and opportunities which are discussed in depth. Chapter 4 investigates several elicitors for boosting cyclotide production in plant cell suspensions. O. affinis, C. ternatea, and H. enneaspermus cultures were either immobilized or treated with methyl jasmonate or sodium chloride. Cyclotide production in C. ternatea and H. enneaspermus could not be enhanced, but their base level was already high. Cyclotide production in O. affinis suspension cells was low but was enhanced by treatment with 3 mM sodium chloride and by cell immobilization. Chapter 5 explores Agrobacterium-mediated transformation of plant suspension cells for production of natural and engineered cyclotides. The first reported transformation of O. affinis was achieved in plant cell packs, which is a stacked filter cake of suspension cells devoid of liquid medium. Several parameters necessary for transformation of plant cell packs were identified, but the observed transformation rates varied greatly. Still, the insights gained will prove useful for research of cyclotide biosynthesis and production in O. affinis. Chapter 6 presents four protocols tested to enable cell cryo-banking of O. affinis, a feat necessary for large scale production in engineered cell lines. No positive results were observed, but future attempts might find this work a useful staring point. Chapter 7 summarizes the results, focused on plant cell suspensions and cyclotide production respectively, whilst they are put in context and possible research paths going forward are discussed. In conclusion, new species were brought into suspension, new cyclotides were discovered, new cyclotide production methods were explored and the first transformation of O. affinis is reported. The application of knowledge generated in this thesis by analysing cell suspension of cyclotide producing plants could lead to affordable and flexible production of pharmaceutical peptides in plants.

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.

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Publications included in this thesis

No publications included in this thesis.

Submitted manuscripts in this thesis

No submitted manuscripts in this thesis.

Other publications during candidature

Publications Craik, D. J., Lee, M.-H., Rehm, F. B. H., Tombling, B., Doffek, B., & Peacock, H. (2017). Ribosomally-synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds. Bioorganic & Medicinal Chemistry. Conference abstracts Doffek, B., Gilding, E. K., Jackson, M. A., Huang, Y.-H., Huang, Y. and Craik, D. J. UQ-TUM Bioeconomy Symposium (2017). Poster presentation

Doffek, B., Gilding, E. K., Jackson, M. A., Huang, Y.-H., Huang, Y. and Craik, D. J. 8th International Postgraduate Symposium in Biomedical Sciences (2017). Poster presentation

Doffek, B., Gilding, E. K., Jackson, M. A., Huang, Y.-H., Huang, Y. and Craik, D. J. Chemical and Structural Biology Division Symposium (2019). Oral presentation.

Doffek, B., Gilding, E. K., Jackson, M. A., Huang, Y.-H., Huang, Y. and Craik, D. J. 21st EMBL PhD Symposium: Facing the Future: Challenges and Perspectives in Life Sciences in the 21st Century (2019). Oral presentation.

Contribution by others to the thesis

Dr. Edward Gilding and Dr. Mark Jackson supervised the experimental design, implementation and data analysis of Chapters 2 to 6. Prof. David Craik supervised the research progress and the reactor acquisition. Prof. Ben Hankamer and Dr. Jennifer Yarnold gave critical feedback as part of the thesis committee. Dr. Gilding helped with the optimisation of the RNA extraction protocols, the PCR experiments, the primer design for qPCR analysis, the transcriptome data analysis, Agrobacterium transformation, and initial suspension initiation. Dr. Jackson oversaw the qPCR experiments, helped with the Nicotiana benthamiana transient expression, designed most of the constructs used for the Agrobacterium-mediated transformation, and guided the RNA extraction. The three cryopreservation protocols were tested with the help of Dr. Elizabete de Souza Cândido. The initial condition screening of Clitoria ternatea and Hybanthus enneaspermus callus and suspension cultures and the elicitation experiments of C. ternatea and H. enneaspermus suspensions were undertaken by Yvonne Huang as iv

part of her honour’s degree under supervision of Benjamin Doffek. N-terminal labelling and imaging protocols of transmembrane proteins in plant cell suspensions were developed in collaboration with Fabian Rehm. Dr. Ian Ross contributed with discussions about cryopreservation and handling of suspended cells. LC-MS and LC-MS/MS analysis and purification was supported by Dr. Yen-Hua Huang and Qingdan Du. Qingdan Du also elucidated the sequences of the cyclotide found in H. enneaspermus. Prof. David Craik, Dr. Gilding and Dr. Jackson assisted the revision of this thesis.

Statement of parts of the thesis submitted to qualify for the award of another degree

No work submitted towards another degree have been included in this thesis.

Research involving human or animal subjects

No animal or human subjects were involved in this research.

Acknowledgments

This thesis would not have been possible without the help of countless people. My sincere apologies to those whom I forgot to mention. Prof. David Craik, thank you for taking the risk of bringing an engineer into a group of chemists and biologists, for always supporting me and always believing in me, for providing the laboratory, for trusting me with the bioreactor acquisition, and for your outstanding scientific writing coaching. Dr. Edward Gilding, thank you for all the help in the lab, for teaching me, for supporting my wacky tinkering, for many hours of brainstorming, and for your everyday upbeat attitude. Dr. Mark Jackson, thank you for all your critical feedback, for teaching me, for the countless hours proofreading my works, and for never giving up on me. Dr. Yen-Hua Huang, Dr. Aaron Poth, Alun Jones, Dr. Quentin Kaas, Haiou Qu, Yvonne Huang, Fabian Rehm and Qingdan Du, thank you for your scientific consultations and help with the experiments. To all members of the Craik and Hankamer group, thank you for your camaraderie and collegiality both inside and outside of the laboratory. Abhishek Bajpai, Alessandro Satta, Andrew White, Ben Tombling, Dake Xiong, Ferran Nadal-Bufi, Mehdi Mobli, Simon de Veer, and Xiaosa Wu, thank you for forming an awesome basketball team. Bronwyn Smithies, Gregoire Philippe, and Guillaume Petit, thank you for your friendship. You filled my time in Australia with joy and laughter. To my friends and family, thank you for supporting me, for visiting me, for chatting with me, and for gaming with me. You keep me sane and give my life direction. Last but not least, Georgianna Doffek, thank you for your trust, your support, your unending happiness. Thank you for sharing your life with me. I would not be where I am now without you.

Financial support

This thesis was supported by the University of Queensland through a Research Higher Degree Scholarship.

Keywords

Cyclotides, plant cell suspensions, molecular farming, elicitation, transformation, scale-up, mass- cultivation, wave-mixed bioreactor

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 100302, Bioprocessing, Bioproduction and Bioproducts, 50% ANZSRC code: 060702, Plant Cell and Molecular Biology, 25% ANZSRC code: 060101, Analytical Biochemistry, 25%

Fields of Research (FoR) Classification

FoR code: 1003, Industrial Biotechnology, 50% FoR code: 0601, Biochemistry and Cell Biology, 35% FoR code: 1004, Medical Biotechnology, 15%

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Table of contents

Abstract ...... i Table of contents ...... viii List of figures ...... xi List of tables ...... xiii List of abbreviations...... xiv 1 Introduction ...... 2 1.1 A closer look at proteins therapeutics ...... 3 1.1.1 Market analysis ...... 3 1.1.2 Protein production ...... 4 1.2 Cyclotides ...... 11 1.2.1 Discovery of cyclotides in Oldenlandia affinis ...... 12 1.2.2 Distribution and biological function of cyclotides ...... 12 1.2.3 Grafting of cyclotides ...... 14 1.2.4 Production of cyclotides ...... 15 1.3 Bioreactors for plant cell suspensions ...... 19 1.3.1 Stirred tank bioreactor (STB) ...... 19 1.3.2 Pneumatic bioreactor ...... 20 1.3.3 Rocking motion bioreactor ...... 20 1.3.4 Downstream processing ...... 21 1.4 Scope of this work ...... 22 1.4.1 Establishment and analysis of plant cell suspension cultures ...... 22 1.4.2 Scale-up of plant cell cultivations for cyclic peptide production ...... 23 1.4.3 Cyclotide elicitation in suspension cultures ...... 23 1.4.4 Transformation of plant cells in suspension ...... 24 1.4.5 Cryopreservation of O. affinis suspension cells ...... 24 1.5 Summary ...... 24 1.6 References ...... 25 2 Establishment and analysis of plant cell suspensions for cyclic peptide production ...... 34 2.1 Materials and methods ...... 35 2.1.1 Plant material and in vitro culture ...... 35 2.1.2 Callus initiation and maintenance ...... 36 2.1.3 Plant cell suspension initiation and maintenance ...... 36 2.1.4 Extraction and purification of cyclotides from plant tissue samples ...... 38 2.1.5 Reduction, alkylation and digestion of cyclotides ...... 39 2.1.6 Cyclotide detection via MALDI-TOF analysis ...... 39 2.1.7 Cyclotide sequencing via LC/MS-MS analysis ...... 41 2.1.8 In silico transcriptome analysis ...... 41 2.1.9 RNA extraction, purification and conversion ...... 42 2.1.10 Quantitative PCR analysis ...... 44 2.2 Results ...... 45 2.2.1 Establishment of in vitro cultures for cyclotide analysis and tissue culture ...... 45 2.2.2 Callus formation for suspension initiation ...... 46 2.2.3 Initiation and maintenance of suspension cultures for cyclotide analysis and production ...... 49 2.2.4 Cyclotide production in plant tissues ...... 53 2.2.5 Real-time PCR of O. affinis RNA ...... 62 viii

Table of contents

2.2.6 Suspension type cyclotides ...... 64 2.3 Discussion ...... 66 2.3.1 Establishment of plant cell suspensions ...... 66 2.3.2 Cyclotide production in plant tissues ...... 67 2.3.3 Cyclotide discovery in Oldenlandia affinis cell suspension ...... 70 2.3.4 Cyclotide and AEP mRNA translation in Oldenlandia affinis ...... 72 2.3.5 Suspension type cyclotides ...... 73 2.4 References ...... 75 3 Mass-cultivation of plant cell suspensions for cyclic peptide production ...... 79 3.1 Materials and methods ...... 80 3.1.1 Suspension culture initiation and scale-up ...... 80 3.1.2 Reactor cultivation of suspension cultures ...... 81 3.1.3 Harvesting and extraction of cyclotides ...... 82 3.1.4 MALDI-TOF analysis of cyclotide expression ...... 82 3.1.5 LC/MS analysis of suspension extracts ...... 82 3.1.6 Purification of cyclotides in plant suspension extracts ...... 83 3.2 Results ...... 83 3.2.1 Oldenlandia affinis cultivation in single-use bags – Proof of concept ...... 83 3.2.2 Optimisation and analysis of O. affinis scale-up ...... 84 3.2.3 Hybanthus enneaspermus cultivation in single-use bags – Proof of concept ...... 87 3.2.4 Clitoria ternatea cultivation in single-use bags – Proof of concept ...... 88 3.3 Discussion ...... 90 3.3.1 Limitations of the rocking motion bioreactor design ...... 90 3.3.2 Prospects of Oldenlandia affinis cultivation in single-use bags ...... 92 3.3.3 Prospects of Hybanthus enneaspermus cultivation in single-use bags ...... 94 3.3.4 Prospects of Clitoria ternatea cultivation in single-use bags ...... 95 3.4 References ...... 97 4 Cyclotide elicitation in suspension cultures ...... 100 4.1 Materials and methods ...... 101 4.1.1 Oldenlandia affinis ...... 101 4.1.2 Clitoria ternatea ...... 104 4.1.3 Hybanthus enneaspermus ...... 105 4.2 Results ...... 107 4.2.1 Elicitation of cyclotide production in Oldenlandia affinis suspensions ...... 107 4.2.2 Elicitation of cyclotide production in Clitoria ternatea suspensions ...... 112 4.2.3 Elicitation of cyclotide production in Hybanthus enneaspermus suspensions ...... 114 4.3 Discussion ...... 116 4.3.1 Enhancing cyclotide production in Oldenlandia affinis ...... 116 4.3.2 Enhancing cyclotide production in Clitoria ternatea ...... 120 4.3.3 Enhancing cyclotide production in Hybanthus enneaspermus ...... 120 4.4 References ...... 122 5 Transformation of plant suspension cells using cell pack technology ...... 125 5.1 Materials and methods ...... 127 5.1.1 Agrobacterium transformation ...... 127 5.1.2 Agrobacterium-mediated infiltration of suspension cells in plant cell packs ...... 128 5.1.3 GUS staining of transformed plant cell packs ...... 129 5.1.4 GFP detection in transformed plant cells ...... 129 5.1.5 MADLI-TOF analysis ...... 129 5.2 Results ...... 130 5.2.1 Transient transformation of Oldenlandia affinis cell packs...... 130 5.2.2 Scale-up of plant cell pack transformation ...... 133 5.2.3 Transient expression in Hybanthus enneaspermus and Clitoria ternatea PCPs ... 134 ix

Table of contents

5.2.4 Transient cyclotide expression in Nicotiana benthamiana PCPs ...... 134 5.3 Discussion ...... 136 5.3.1 Agrobacterium-based transformation of plant cell packs ...... 136 5.3.2 Cyclotide elicitation in O. affinis PCPs ...... 137 5.3.3 Plant cell packs for research and production of cyclic peptides ...... 137 5.4 References ...... 140 6 Cryopreservation of Oldenlandia affinis cell suspensions ...... 143 6.1 Materials and methods ...... 144 6.1.1 Cryopreservation ...... 144 6.1.2 Regrowth of frozen cells ...... 145 6.1.3 Cell viability assays ...... 145 6.2 Results and discussion ...... 146 6.3 References ...... 149 7 Conclusions ...... 151 7.1 Disappearing cyclotides ...... 151 7.2 Elicitation to bring back cyclotides ...... 152 7.3 Transgenic plant cell cultures to produce cyclotides ...... 152 7.4 Cryopreservation as a roadblock ...... 153 7.5 From farm to pharm ...... 153 7.6 References ...... 155

List of figures

Figure 1 | Oldenlandia affinis...... 12 Figure 2 | Anti-obesity drug ...... 14 Figure 3 | From farm to pharm. Scope of this work...... 22 Figure 4 | Settled cell volume (SCV) measurement...... 38 Figure 5 | Calculated isotopic pattern of cycloviolacin O2 with charge +1...... 40 Figure 6 | Calculated isotopic pattern of kalata B23 with charge +1...... 41 Figure 7 | In vitro germination after one week on agar medium...... 45 Figure 8 | O. affinis callus...... 46 Figure 9 | Callus formation under 35 µmol m 2 s 1 photon density at a 16:8 light-dark cycle...... 47

Figure 10 | Initiation of O. affinis suspension ⁻from⁻ fresh, friable green calli...... 50 Figure 11 | O. affinis cell suspension...... 51 Figure 12 | Screening for optimised growth conditions of C. ternatea and H. enneaspermus...... 52 Figure 13 | Cyclotide production in O. affinis plant parts...... 54 Figure 14 | Cyclotide concentration in different O. affinis tissues and the development over time in suspension...... 56 Figure 15 | Tandem MS of cycloviolacin O2 enzymatically digested by endo-GluC...... 57 Figure 16 | Transcript of cycloviolacin O2 found in O. affinis...... 57 Figure 17 | DNA and corresponding peptide sequence of kalata B22 as found in O. affinis...... 58 Figure 18 | DNA and corresponding peptide sequence of kalata B23 as found in O. affinis...... 58 Figure 19 | C. ternatea tissue dependent cyclotide concentration...... 59 Figure 20 | H. enneaspermus tissue dependent cyclotide concentration...... 60 Figure 21 | MALDI traces of cyclotides found in suspension cells, root-like structures formed via organogenesis in suspension, and in vitro roots of H. enneaspermus...... 61 Figure 22 | Alignment of Cter A sequences found in C. ternatea and H. enneaspermus...... 62 Figure 23 | Agarose gel of RNA extracted from O. affinis suspension cells...... 63 Figure 24 | Expression levels of mRNA transcripts necessary for cyclotide production ...... 64 Figure 25| Front and back surface view of the suspension type cyclotides ...... 65 Figure 26 | First cultivation of O. affinis in a rocking motion bioreactor...... 84 Figure 27 | O. affinis cultivation for cyclotide production on a BioStat® rocking motion bioreactor with 5 L culture volume...... 85 Figure 28 | Cyclotide expression of O. affinis cells ...... 86

List of figures

Figure 29 | Fed batch cultivation of 5 L H. enneaspermus suspension in a disposable bag on a rocking motion bioreactor...... 88 Figure 30 | MALDI-TOF trace of a four-week-old C. ternatea suspension culture after one day on a rocking motion bioreactor...... 89 Figure 31 | Cyclotide elicitation in O. affinis suspension cells...... 108 Figure 32 | Microscopy images of selected O. affinis suspension cultures during elicitation experiments...... 109 Figure 33 | Relative cyclotide expression in O. affinis...... 110 Figure 34 | Cyclotide profile of green O. affinis wall growth of 77 days old suspension cells...... 111 Figure 35 | Cyclotide profile of floating O. affinis suspension cells ...... 111 Figure 36 | Cyclotide profile of O. affinis callus initiated from suspension cells...... 111 Figure 37 | Cyclotide profile of O. affinis suspension after two weeks without phytohormones and one week without agitation...... 112 Figure 38 | Influence of hormone treatment and explant tissue type on cyclotide expression in C. ternatea callus and suspension cells...... 113 Figure 39 | C. ternatea suspension elicitation with 50 and 100 µM methyl jasmonate...... 114 Figure 40 | Influence of hormone treatment and explant tissue type on cyclotide expression in H. enneaspermus callus and suspension cells...... 114 Figure 41 | H. enneaspermus suspension elicitation with 50 and 100 µM methyl jasmonate...... 115 Figure 42 | Summary of cycloviolacin O2 production in O. affinis suspension after elicitation. .... 117 Figure 43 | pProGG vector map...... 127 Figure 44 | O. affinis PCP transformation...... 131 Figure 45 | Plant cell pack transformation parameter screening...... 133 Figure 46 | Cyclotide elicitation in O. affinis PCPs during Agrobacterium-mediated transformation...... 133 Figure 47 | Scale-up of O. affinis transformation...... 134 Figure 48 | Transient co-expression of butelase-1 with Oak1 or Oak1[T20K] in N. benthamiana . 135 Figure 49 | Microscopy images of O. affinis cryopreservation experiments...... 146 Figure 50 | MTT assay of O. affinis cells after cryopreservation...... 147

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List of tables

Table 1 | Plant-derived pharmaceuticals and their clinical trial status [31]...... 7 Table 2 | Suspension cultures in an engineering environment [57]...... 10 Table 3 | Callus induction media...... 36 Table 4 | Media compositions for C. ternatea and H. enneaspermus suspension cultures...... 37 Table 5 | Optimised suspension media...... 37 Table 6 | H. enneaspermus callus induction...... 48 Table 7 | C. ternatea callus induction...... 49 Table 8 | Most abundant cyclotides detected in H. enneaspermus tissues...... 61 Table 9 | Primer pairs used for quantitative real-time PCR of O. affinis transcripts...... 63 Table 10 | Cyclotide sequences alignment...... 65 Table 11 | BLAST alignment of the amino acid sequence of kalata B17 and kalata B22...... 71 Table 12 | BLAST alignment of the amino acid sequence of kalata B18 and kalata B23...... 72 Table 13 | Comparative cyclotide expression in O. affinis suspensions...... 85 Table 14 | Optimised O. affinis suspension medium...... 101 Table 15 | O. affinis callus induction medium...... 103 Table 16 | C. ternatea suspension initiation conditions...... 104 Table 17 | C. ternatea culture conditions...... 113 Table 18 | H. enneaspermus culture conditions...... 114 Table 19 | Vector-gene combinations used for N. benthamiana transformation...... 128 Table 20 | Screened conditions for EHA105 transformation of O. affinis...... 132

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List of abbreviations

AEP Asparaginyl endopeptidase AMP Antimicrobial peptide ACN Acetonitrile bp Base pairs CCK Cyclic cystine knot CHO Chinese hamster ovaries Da Dalton DTT Dithiothreitol FA Formic acid FDA Food and Drug Administration GFP Green fluorescent protein GMP Good manufacturing practice GUS β-glucuronidase Liquid-side mass transfer coefficient [s-1] LC/MS Liquid chromatography/mass spectrometry 𝐿𝐿 LED𝑘𝑘 𝑎𝑎 Light-emitting diode MALDI Matrix assisted laser desorption/ionization MCoTI-II Momordica cochinchinensis trypsin inhibitor-II PCP Plant cell pack PCV Packed cell volume PLCP Papain-like cysteine protease PSM Photobioreactor screening module RMB Rocking motion bioreactor RT Room temperature (24°C) SPE Solid-phase extraction SPPS Solid-phase peptide synthesis TOF Time of flight T-DNA Transfer DNA TSP Total soluble proteins Ti plasmid Tumor inducing plasmid UTR Untranslated region vvm Volume of air per unit of medium per minute

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Introduction

1 Introduction

Plants are the most successful participants of the evolutionary race as measured in information generated. It has been estimated that the amount of information stored in plant DNA exceeds the information stored in the DNA of all other organisms (i.e. prokaryotes, unicellular eukaryotes, fungi, animals, and viruses) [1]. Being robust and adaptable, plants thrive in nearly every ecological niche. In the early days of human civilization, plants were gathered in their natural habitat mainly for food and sometimes for medicine. The major achievement that enabled science, arts and literature was the domestication of plants in the Mesopotamian times that brought a change from nomadic to sedentary societies. Modern biotechnologies, as developed here, might usher in another major advancement in the history of human-plant symbiosis. The adequate supply of nutrients and pharmaceuticals is a major challenge in a world of overpopulation and economic disparity. In a now famous report to The Club of Rome titled “The Limits to Growth”, Meadows et al. presented their primary finding as “[if] the present growth trends in world population, industrialization, pollution, food production, and resource depletion continue unchanged, the limits to growth on this planet will be reached sometime within the next one hundred years. The most probable result will be a rather sudden and uncontrollable decline in both population and industrial capacity” [2]. The second finding voices the authors’ belief that it is possible to circumvent this outcome and achieve a state of ecological and economic stability. The key factors identified were population, food production, and consumption of non-renewable natural resources. Since publication of this report in 1972, the human population has grown from 3.8 billion to 7.3 billion and is projected to reach 10.2 billion in 2060 [3] and climate change has joined that rank of key influencing factors. As the human population continues to grow, it is of paramount importance to find new ways of supplying the necessary goods to sustain this population without destroying the sustainability of our planet. Plant cell cultures can help protect natural plant populations, provide flexible and cheap production capabilities for many different drugs, and consequently help support our growing human population all around the globe. The use of plants as biofactories to produce biomass, fuels, food supplements, chemicals, and pharmaceuticals has been instrumental in increasing the standard of living without exploiting natural resources. Within the last decade, the natural product industry has grown substantially and it is expected to continue to expand [4]. Considering that biopharmaceuticals occupy around 25% of the global pharmaceutical market, molecular farming is of vital importance for our society [5]. Plant molecular farming, defined as the harvesting of proteins from plants or plant cell cultures including

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Introduction native and engineered proteins, is a fast-growing economical sector. Plants have the molecular machinery to synthesize a vast array of proteins including many with the required post-translational modifications, making them ideal as recombinant protein production platforms. The development of a plant cell system to produce effective pharmaceuticals at low cost and in an environmentally friendly manner would be ground-breaking. This work strives to establish plant cell suspension cultures as production vehicles for inexpensive high-end protein pharmaceuticals at industrial scale.

1.1 A closer look at proteins therapeutics

Proteins consist of amino acids linked via peptide bonds. Their immense variability originates in their sequence and the resulting secondary, tertiary and quaternary structure. The combination of sequence and structure defines the steric, electrostatic and electrokinetic features which, in turn, defines the biological and chemical characteristics. They are an essential part of life and fulfil a plethora of functions including structural scaffolding, energy conversion, molecular transportation, immune response and many more. Industrial protein applications range from nutrition and pharmaceuticals, to bio-catalysis. The food industry utilizes proteins as nutritional components, stabilizers, emulsifiers, colouring agents and many other functional agents. The pharmaceutical industry employs their efficacy, wide range of targets and specificity to design modern drugs with less side effects compared to small molecule drugs [6]. However, downsides include high production costs, low in vivo stability and interactions with the human body that habitually necessitate administration via injection.

1.1.1 Market analysis

The pharmaceutical protein market has a value of $174.7 billion with a compound annual growth rate of 7.3%, the highest of all protein ingredient segments [7]. This trend is clearly shown by the shift from small molecule drugs to proteins in all big pharmaceutical companies. In 2001, protein drugs were barely existent as a source of therapies with only 7% of the major treatment revenue worldwide [8]. In 2012, the revenue had exploded to 71% accompanied by a tenfold increase of financing for protein research, while the overall protein product sale had quadrupled [8]. With $75 billion, monoclonal antibodies products generated nearly half of the biopharmaceutical protein

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Introduction revenue in 2013. The same year, the predicted worldwide sales of monoclonal antibody products by 2020 was approximated at $125 billion [9]. This sales volume was reached in 2017 with $123 billion total monoclonal antibody sales with eight of the top ten selling biopharmaceutical products being monoclonal antibodies[10]. This trend is likely to continue as 40% of products in clinical development are biopharmaceutical proteins [10].

1.1.2 Protein production

One major challenge of protein therapeutics is their production. It is not trivial to synthesize proteins as in vitro and in vivo production systems need to be fine-tuned for each drug specifically. Additional challenges arise from low protein stability in vivo and the obstacles that present themselves before the target site is reached (i.e. saliva, gastric acid, cell walls, aqueous solutions, time, proteases, and many more). The choice of production system for recombinant proteins influences the biological activity, immunogenicity, pharmacokinetics, the yield, and consequently the price of the product. Some authors even refer to this as “the process is the product” [11]. The main reason for this is that the production in living cells often leads to impurities and heterogeneity of the target protein. One example of impurities that can be found in the final product is the coelution of 10 host cell proteins during the production of mAbs in Chinese hamster ovary cells [12]. Post-translational modifications that vary across biological host systems include amidation, carboxylation, methylation, phosphorylation and glycosylation. As a result, many biosimilars display changed efficacies that must be assessed on a case by case basis [13]. A biosimilar is a biological product, that is highly similar to an already approved biological drug in terms of structure, function, efficacy, and safety [14]. However, they can never be identical. This highlights the importance of a suitable production system and human clinical trials. The available protein production systems include solid phase peptide synthesis (SPPS), animals, mammalian tissue culture, bacteria, yeast, plants and plant tissue culture. Currently, 84% of the recombinant protein therapeutics that were approved between 2014 and 2018 were expressed in mammalian cell lines, with the remaining 16% split between Escherichia coli and Saccharomyces cerevisiae [10]. Walsh concludes in his ‘Biopharmaceutical benchmarks 2018‘ “that there is little industrial enthusiasm for exploring new expression systems” as recent approvals show [10]. Although there have been major advances in technology behind recombinant protein therapeutic production, a major limitation still exists in the cost of production when compared to small molecule drugs. Any advances in technology to facilitate cheaper therapeutic protein production has the potential to have a huge social and financial impact. 4 | Page

Introduction

1.1.2.1 Chemical synthesis of proteins

The complexity of most proteins obstructs their chemical synthesis. Peptides, being a subgroup of proteins smaller than 50 amino acids, are the exception and their chemical synthesis is discussed in Section 1.2.4.1.

1.1.2.2 Protein production in bacteria, yeast and mammalian cell cultures

Since the first expression of a mammalian peptide hormone, somatostatin, in Escherichia coli in 1977 [15], this microorganism has been used extensively for the production of biopharmaceuticals. Only five years after the transgenic expression of somatostatin, regular insulin became the first genetically engineered protein produced in bacteria approved by the Food and Drug Administration (FDA) [16]. Combining bacterial expression with recombinant DNA techniques enabled the production of proteins with large molecular weight and/or unusual sequences [17]. The limited folding and posttranslational machinery present within bacteria is often overcome by using yeast cells, which offer an advanced eukaryotic machinery while retaining the benefits of easy cultivation and high growth rates in suspension culture. Yeast was unknowingly used for fermentations since ancient Egyptian times and is still popular today. Saccharomyces cerevisiae, for example, is a well-studied model organism and regularly deployed in modern bioprocesses. S. cerevisiae is the third largest production host for biopharmaceuticals, after E. coli and mammalian cells [18]. The first FDA approved biopharmaceutical produced in yeast, in this case Pichia pastoris, was recombinant ecallantide [19]. Ecallantide is a protein of 7053.83 g mol-1 that is applied to treat hereditary angioedema and blood loss during cardiothoracic surgery under the trade name Kalbitor®. Mammalian cell cultures are nowadays the gold standard for pharmaceutical protein production [10]. With the rise of monoclonal antibodies as therapeutic agents, mammalian cell cultures, especially CHO (Chinese Hamster Ovary) cells, became increasingly important for industrial production. As a consequence, over 70% of all recombinant biopharmaceutical proteins are produced in CHO cells [20]. CHO cell suspensions are suitable for large scale production systems with high production rates in serum-free medium [20]. Having been extensively optimised over decades, multiple gene amplification systems are available for CHO cells enabling high titre yields (1-5 g L-1) and specific productivity of recombinant glycoproteins with human-like glycans [21]. Disadvantages include high production and purification costs as well as possible contamination with mammalian pathogens [18]. Any new production system will have to compete with mammalian cells.

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Introduction

1.1.2.3 Protein production in plants

In 1986, four years after insulin produced in E. coli was approved by the FDA, and ten years after Agrobacterium tumefaciens mediated plasmid DNA transfer was achieved [22], Barta et al. reported the first successful expression of a mammalian gene, human growth hormone, in plant cells [23]. The first described production of a functional recombinant antibody in tobacco plants regenerated from transformed leaf segments was reported in 1989 and founded the field of molecular farming [24]. In the following years, Agrobacterium-mediated plant transformation became the gold standard for recombinant gene expression in plants [25]. This technique utilizes the natural ability of Agrobacterium to transfer DNA into plant cells. The transferred DNA (T-DNA) is located on tumour- inducing (Ti) or rhizogenic (Ri) plasmids, that are typically 10 to 30 kbp in size. These plasmids also harbor required virulence genes for T-DNA processing and transfer. Many additional chromosomal located genes also play a key role in this process [26]. The DNA regions to be transferred are bookended by 25 bp T-DNA border sequences. This allows the insertion of engineered sequences and ensure their export into the plant cell. Today, many of the economically important plant species have been transformed and the number of transformable species continues to grow constantly [27]. Nevertheless, it is still impossible to predict if a species will be transformable and, if so, what the required parameters will be. It took a further eight years after the successful production of insulin in E. coli for the first genetically engineered food to be approved by the FDA. Hitting the shelves in 1994, the FLAVR SAVR tomato was engineered with traits for prolonged shelf life. Initially, the FLAVR SAVR tomato experienced high interest, but was never accepted by the broader market. Only five years after its introduction the FLAVR SAVR tomato vanished from stores [28]. This product is not directly related to recombinant drug production, but highlights the importance of market analysis, marketing and the right timing in the field of genetic engineering. In 2012, the first FDA approved drug produced in plant cell culture entered the market [29]. Taliglucerase alfa is a recombinant glucocerebrosidase produced in carrot cell culture. Protalix distributes the purified product as treatment for Gaucher disease. Another successful example of plant based pharmaceutical production is the Ebola drug ZMapp, a cocktail of highly purified monoclonal antibodies that was able to rescue rhesus macaques 5 days post-challenge. Mapp Biopharmaceutical Incorporation achieved ZMapp production in Nicotiana benthamiana leaves in 2014. Field tests of the drug in Africa and randomized control trials showed promising results for the standard of care plus ZMapp treatment of Ebola [30]. The plant-based system was chosen for its fast response time and massive scale-up potential. Today many products expressed in plants are in phase 1, phase 2 or

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Introduction in the case of Medicago’s quadrivalent VLP vaccine phase 3 clinical trial and are anticipated to enter the market soon as shown in Table 1.

Table 1 | Plant-derived pharmaceuticals and their clinical trial status [31]. Product Host Application Status Phase 3 completed (2012); Taliglucerase alfa Carrot cell culture Gaucher disease FDA approved (2012) ZMapp Tobacco Ebola virus Phase 1 and 2 (2015) PRX-102 Tobacco cell culture Fabry disease Phase 1 and 2 (2014) Vaccine Pfs25 VLP Tobacco Malaria Phase 1 (2015) Vaccine recombinant Tobacco Anthrax Phase 1 (2014) protective antigen HAI-05 Tobacco H5N1 vaccine Phase 1 (2011) Hepatitis B surface Potato Hepatitis B vaccine Phase 1 [32] antigen Recombinant Vitamin B12 Phase 2 completed human intrinsic Arabidopsis thaliana deficiency (2006) factor H5-VLP + GLA-AF Influenza A subtype Phase 1 completed Tobacco Vaccine H5N1 infection (2014) Phase 1 completed P2G12 antibody Tobacco HIV (2011) Moss-aGal Moss Fabry disease Phase 1 (2015) [33] Nicotiana Phase 2 completed QVLP vaccine Influenza A benthamiana (2019) [34]

Plant growth is only limited by available fertile soil, they are easy to cultivate, relatively cheap to grow and the risk of introducing an infectious agent is low because plants are unable to harbor mammalian pathogens. Open fields of close cropped tobacco can achieve a wet biomass production of up to 10,000 t km-2 y-1 [35] and specialized methods like vertical farming units achieve around 91,000 t km-2 y-1 [36]. The yield of recombinant protein per fresh biomass in plants is in the order of 1 g kg-1 for human antibodies in N. benthamiana leaves [37]. Obviously, these yields differ between species and target product, but the values given here allow a rough comparison with other systems. Open systems are subject to seasonal changes, prone to anomalies such as droughts, floods, tornadoes, etc., and do not fulfil GMP requirements. Specialized systems, like greenhouses or vertical farming units, entail larger initial investments [38], but can easily satisfy GMP requirements with lower running costs than mammalian systems [39]. From an engineering perspective, downstream processing is similar in plant-based and mammalian production systems [40]. However, clever design can eliminate the need for downstream processing in plant-based systems as discussed in Section 1.3.4. Disadvantages of plants include the 7 | Page

Introduction generally low target protein yield, the often-necessary cell disruption for product recovery, and differences in the glycosylation machinery compared to mammalian cells. Subtle differences in glycosylation of proteins can affect stability, efficacy and inducing unwanted immune responses. Interestingly, these changes are not always detrimental. Reski et al., for example, reported a 40-fold increase in efficacy of a moss produced glycol-optimised monoclonal antibody compared to the same antibody produced in CHO cells [33]. In cases of disadvantageous plant glycosylation, it is possible to knock out plant transferases using CRISPR/Cas9. Jansing et al. reported increased binding affinity of antibodies produced in xylosyl- and fucosyl-transferase N. benthamiana knock out lines compared to wild-type plants with an affinity similar to the same antibodies produced in CHO cells [41]. The proper maturation of viral proteins and low contamination risks in plants present advantages for vaccine production as well [42]. Low production costs, easy scale-up, omission of purification before administration and possible bioencapsulation led to the production of immunogenic antigens in many plant species, some edible, with transgene expression of up to 70% of total soluble protein for engineered chloroplast genomes [43]. Haemagglutinin, the only plant- based immunogenic antigen tested in humans, yielded ~50 mg per kg fresh weight in N. benthamiana and is produced by Medicago Inc. [44, 45]. Finally, plant-based production systems can easily adhere to GMP guidelines that stipulate self–contained traceable batch production in a defined environment. In summary, after decades of development and initial setbacks plants are viable and economically competitive protein production systems that can offer advantages over traditional systems in terms of safety and scalability [46, 47]. Another important aspect of plant-based protein production, aside from their recombinant production, is that most medicinal and aromatic plants are collected from the wild to harvest their vast spectrum of bioactive compounds. This practice endangers the natural populations and often leads to extinction [4]. To be affordable and conserve the natural biodiversity, industrial production of plant-based bioactive compounds is becoming imperative.

1.1.2.4 Protein production in plant cell suspension cultures deals with secondary metabolites and proteins

Plant cell suspension cultures have been instrumental for the study of cellular differentiation, regeneration, elucidating biosynthetic pathways and membrane interactions [48]. They have several unique advantages over plants. Cell suspensions are homogeneous and isotropic. Therefore, it is possible to set up a plethora of tests including multiple parameters using identical biological systems. This is impossible for plants, as each cell, leaf, or plant experiences a diverse environment.

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Introduction

Additionally, supplied chemicals are immediately available for all cells in suspension, whereas plants rely on transportation systems. However, disadvantages of suspension cultures include the necessity of a sterile environment, specific growth medium, and agitation. These factors result in higher investment and maintenance costs of suspensions compared to traditionally grown plants. As plant suspension cultures exhibit high growth rates under defined conditions, they have been utilized as production systems for secondary metabolites for four decades. In 1983 shikonin, a dye and pharmaceutical produced in Lithospermum erythrorhizon, became the first industrial plant cell suspension product [49]. Under certain circumstances, plant cell suspensions offer unique production avenues. One such example is the production of paclitaxel, one of the most common natural-source cancer drugs for the treatment of breast, lung and ovarian cancer [50]. It is isolated from the bark of the Pacific yew, a slow growing tree. One kilogram of paclitaxel requires the bark of over 1000 trees that need 100 years to grow [50]. With its chemical synthesis not economically feasible and natural harvesting limited, plant cell suspensions have become the solution [51]. The advantages of plant cell suspensions include sterility in a contained and controlled environment, higher growth rates than plants, cheap medium, no mammalian pathogens, no limitation by climatic, environmental and ecological factors, and the ability to seamlessly implement pharmaceutical GMP [52]. The latter is especially important for adherence to the rulebook of the International Conference on Harmonization [53]. The faster turnover rate is a consequence of shorter production cycles of a few weeks in suspension cultures compared to several months in plants. The shorter production cycles combined with controlled environmental factors lead to consistent high product quality between batches [54]. Whereas plants generally do not harbor human pathogens, the safety of closed and sterile bioreactors is much higher compared to field grown plants in terms of product contamination with endotoxins and mycotoxins as well as possible release of genetically modified material into the wild. Serendipitously, plant viruses typically cannot infect suspension cultures, as their means of transportation, the plasmodesmata, is not available [55]. One major problem is the general low yield of plant cell based systems (ranging from 0.5 µg L-1 to 200 mg L-1 [52]) compared to bacteria (100 mg L-1 to 200 mg L-1 [56]) and mammalian cells (1-5 g L-1). To overcome this hurdle, strategies tried and tested in established bacterial cultures are applied in plant cell cultures (e.g. high throughput screening, process optimisation, culture optimisation and genetic engineering). Minor problems of plant cell cultures include the absence of gene amplification systems (e.g. the CHO mutant cell line DG44 allows tandem transfection that results in greatly enhanced gene amplification), difficult cell banking, agglomeration, somaclonal variation and non-mammalian glycosylation [57]. Table 2 compares mammalian cells with various plant-based production systems and outlines the opportunities provided by plant cell suspensions.

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Introduction

Table 2 | Suspension cultures in an engineering environment [57]. N-glycosylation Contamination Time to Overall System Scalability ability risk production cost* Plant cell Yes Very low Medium High Medium suspensions Whole plant No terminal Low High Very high Low systems galactose or sialic acid; Core-xylose; Plant different fucose transient linkage Low High High Low expression Yes, but different potential sialic Mammalian acid (NGNA) and High Medium Medium High Cells alpha-Gal epitope both potential immunogenic * Low $20-100/g; Medium $50-1000/g; High $1000-10,000/g.

Transient expression of recombinant proteins in plants can be achieved through microprojectile bombardment or by Agrobacterium-mediated transfection. The latter is commonly referred to as agroinfiltration and has been applied to study plant-pathogen interactions, abiotic stresses and for recombinant protein production [58]. Both are standard methods of the plant molecular biology tool set. However, DNA transfer using the natural machinery of Agrobacterium is advantageous when it comes to production, as the resulting transient gene expression is generally very high and rearrangements of transgenes low. Whereas scalable protocols for agroinfiltration of leaves have been developed [59], transient expression in suspension cultures is still in its infancy. A very interesting protocol to achieve Agrobacterium-mediated transient expression in suspension cultures forming cell packs was published in 2019 [60, 61]. Whereas bombardment works with every species, specific protocols are required for Agrobacterium-mediated DNA transfer and many species do not have optimised protocols for transformation in this way, or are at present, recalcitrant to transformation. Cells in suspension consist of dedifferentiated single cells and are potentially more amenable to agroinfiltration. Specifically, the method for the generation and cultivation of a plant cell pack patented by Thomas Rademacher is a promising platform to transform new species [61]. It utilizes a loose pack of cells taken out of the exponential growth phase, to create the ideal parameters for agroinfiltration. The successful transformation of a new species enables a whole set of experiments to answer many questions regarding gene knockdowns, overexpression, gene transfer, pathways, and

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Introduction regeneration. Additionally, transgenic cells could be used to produce recombinant pharmaceutical proteins in a plant-based system with good scalability much cheaper than mammalian cell cultures.

1.2 Cyclotides

Cyclotides are small backbone cyclised peptides with a characteristic cyclic cystine knot (CCK) motif consisting of six cysteine residues forming three disulfide bridges that together knot the cyclic peptide into a stable and compact conformation [62]. Although the CCK is conserved in all cyclotides, the remaining amino acids vary and can readily be substituted with different amino acids. The three dimensional structure of cyclotides does not change from solution, to crystal to membrane bound [63], highlighting not only their resistance to degradation but also to conformational changes, retaining their biological activity. Aside from plants, the cystine knot motif is found in many animals, bacteria, fungi and viruses [64]; however, these non-plant peptides do not belong to the cyclotide group. The peptides containing the CCK motif are often connected to a host defence role. Linear, functional peptides containing the CCK are also present in many living organisms, indicating that the CCK formed first before the cyclization took place on the evolutionary timeline [65]. Cyclotides are divided into three subgroups. The interested reader is referred to a representative list of cyclotides for each group published in ‘Plant Toxins’ [66] and the latest review on cyclotides published in ‘Phytochemistry Reviews’ [67]. The bracelet and Möbius group are named after their three-dimensional structure, whereas the trypsin inhibitor group is named after its inhibitory properties. This trypsin inhibitor group, sometimes referred to as cyclic knottins, is found only in the Momordica genus of the Cucurbitaceae family and is more closely related to squash trypsin inhibitors than it is to the other cyclotides [68]. Möbius cyclotides contain a twist in their backbone introduced by a cis-Pro residue in loop 5. This twist is not present in the bracelet group leading to a smooth backbone. Even though being the most abundant group in nature attributing for around two thirds of all known cyclotide sequences, bracelet cyclotides are notoriously hard to fold during chemical synthesis [69].

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Introduction

1.2.1 Discovery of cyclotides in Oldenlandia affinis

Cyclotides were first discovered in the 1960s by the Norwegian doctor Lorents Gran as the active component of an herbal tea in Africa [70]. Local tribes’ women used the indigenous herb Oldenlandia affinis (Figure 1) to brew a medicine they named “Kalata-kalata” to facilitate labour. The structure of the uterotonic compound, a macrocyclic cystine-knot peptide, was not solved until 1995 [71]. The following years brought the discovery of a large group of plant peptides containing the characteristic cyclic cystine knot (CCK) motif.

Figure 1 | Oldenlandia affinis. A) Plant pot-grown in Queensland (Australia). B) Suspension cells (200x, autofluorescence, pseudo- colour image).

The genus name Oldenlandia of the Rubiaceae family was established in 1703 by Plumier. O. affinis (R.&S.) DC. is comprehensively described by Bremekamp [72]. The distribution of O. affinis includes tropical Africa, Madagascar and western Asia up to Malaysia. In these regions, it grows among the grasses from sea level to around 1,500 meters above sea level. The flowers are blue-violet and are located at the end of stipules on the filiform, divaricating branches. The stem of adult plants is typically decumbent, branched, and around 40 cm in length. The lanceolate leaves sit on opposite sides of the green, shiny stem [73].

1.2.2 Distribution and biological function of cyclotides

Vastly abundant in nature, cyclotides have been found in plants of the Rubiaceae [74], Violaceae [75], Cucurbitaceae [76], Fabaceae [77], Apocynaceae [74], Solanaceae [78] families, and cyclotide-like sequences in some Poaceae [79] plants. The up to date online database CyBase (http://www.cybase.org.au) lists over 1000 discovered cyclotides including their origin. Interestingly, every species of Violaceae analysed thus far contained cyclotides [75], whereas only 5% of the analysed Rubiaceae species showed evidence of cyclotides [74]. These findings indicate that the 12 | Page

Introduction evolutionary history of cyclotides in different plant families is unique and the result of convergent evolution [74]. The biological role of cyclotides is ascribed to host defence by protecting plants from pests and pathogens [80]. Several studies observed the effect of a cyclotide rich diet on larvae and ubiquitously found increased mortality rates as well as significantly reduced growth rates [81-84]. Similar setups for molluscs [85] severely hindered growth and development, further supporting the host defence hypothesis. Interestingly, cyclotides display biological activity against several organisms that do not have plants on their menu. These include bacteria, fungi, nematodes, trematodes, insects and other plants [86]. Additionally, cyclotides have displayed anti-microbial [87], anthelmintic [88-90], anti-HIV [91], anti-fouling [92], cytotoxic [93], haemolytic and uterotonic properties [94]. Plant antimicrobial peptides (AMPs) have long been considered as very promising next generation antibiotic drug leads [95, 96]. However, not a single AMP has made it into clinical trials after several decades of research as high dose requirements and post-translational modification continue to remain challenging [97], highlighting the complexity of the development of peptide-based pharmaceuticals. Host defence studies have led to several patents issued for the use of cyclotides as insecticides. The first cyclotide product hit the market in 2017, when Innovate Ag launched their “bee-friendly bio insecticide” called Sero-XTM [98] in Australia. The active ingredient of this product is a cyclotide extract of Clitoria ternatea (Butterfly Pea) a plant known to contain many different cyclotides. Curiously, cyclotides affect not only plant insect pathogens, but also sheep, cow and human parasites [99]. The proposed mode of action in lepidopterans is a midgut membrane disruption of the larvae [100]. Consequently, the chance of widespread immunity to cyclotides developing is low, as it would require major membrane restructuring, making cyclotides prime targets for crop protection. However, recent studies on Bacillus thuringiensis toxin resistance mechanisms in target pests have shown that nature is adept at overcoming biotechnological controls [101]. The membrane interactions of cyclotides have been extensively studied and linked to specific lipid-binding domains in Möbius and bracelet type cyclotides [99] Their cyclic backbone in concert with the cystine knot fosters extraordinary chemical, biological and thermal stability. Exopeptidases, a group of enzymes found in the intestine that catalyzes the cleavage of a single amino acid from the amino terminus (aminopeptidase) or carboxy terminus (carboxypeptidase) of a peptide, need N- or C-terminal amino acids to cleave peptides. The lack thereof, enhances the resistance to digestion and enables oral bioavailability of cyclic peptides. In vivo this stability is essential to withstand the acidic environment in the plant vacuole and the digestive environment of pest guts.

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Introduction

1.2.3 Grafting of cyclotides

Peptides combine the specificity of larger proteins (> 5000 Da) with the oral delivery of classical small organic molecules (< 500 Da). Cyclotides, a group of peptides, display resistance to pH, heat, enzymes, denaturation and organic solvents making them ideal pharmaceutical scaffolds. Nevertheless, cyclotides are naturally occurring peptides and as such degrade over time. This enables the application of cyclotides in open environments. Many studies have shown their tolerance to insertions of different peptide epitopes in their amino acid sequence, a process called grafting [102, 103]. One example of a pharmaceutically active epitope grafted into kalata B1 to address obesity is shown in Figure 2 [104].

Figure 2 | Anti-obesity drug (MC4R antagonist, black) grafted into the kalata B1 scaffold (pink, disulfide bridges in yellow).

T20K another kalata B1 variant has been shown to silence T-cell proliferation and the authors of a recent study surmised that cyclotides are promising for the treatment of diseases linked to a hypersensitive immune system [105]. T20K is still in preclinical trials, however phase 1 and 2 clinical studies are planned and a large scale synthesis method is available [105]. The patent introducing T20K as an immunosuppressive agent for the treatment of autoimmune disorders, hypersensitivity disorders, and lymphocyte-mediated inflammation was granted in February 2019 [106]. Also in 2019, the first in vivo dose-exposure response of cyclotides and cyclotide grafts was reported by Poth et al. [107]. They demonstrate the viability of oral and intravenous administration, while maintaining high efficiency and resistance to in vivo degradation [107]. After many years of research, two cyclic peptides are in phase 3 and one is close to phase 3 clinical trials, with many more in the pipeline [108]. Cyclotides are easy to produce in small quantities, possibly produced cost efficiently in plant based systems on a large scale, very stable and can carry cargo through membranes [109]. Thus, they can introduce significant improvements to the modern, protein-based drug market. These reports highlight the immense potential of cyclotides in a pharmaceutical context.

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Introduction

In summary, cyclotides and their engineered analogues represent promising drug candidates that retain structural stability opening opportunities for oral delivery and extended half-life in vivo. Furthermore, plant-based cyclotide production is promising as the biosynthetic machinery is already in place, which is lacking in bacterial, yeast and mammalian systems. Current production of cyclic peptide therapeutics focusses mainly on costly and chemically intensive synthesis. Plant based systems can make a difference in terms of affordability and availability of peptide therapeutics.

1.2.4 Production of cyclotides

1.2.4.1 Biosynthesis of cyclotides

Elucidating the cyclotide biosynthesis pathway is a field of active research and any insights discovered will no doubt enhance the prospects of developing an economically competitive plant- based cyclic peptide production platform. All cyclotide genes characterized so far contain an endoplasmic reticulum (ER) signal sequence that enables co-translational translocation across the ER membrane. Once within the ER, the cyclotide precursor protein folds and disulfide bonds are formed [110]. From the ER the cyclotide precursor is thought to continue to traffic towards the vacuole, where cyclization is predicted to occur via the action of resident asparaginyl endopeptidases [111]. The first cyclotide precursors characterized from O. affinis contain an endoplasmic reticulum signal sequence followed by an N-terminal pro-peptide, an N-terminal repeat sequence, the core peptide sequence and a C-terminal pro-peptide. The order and presence of these elements vary greatly in the cyclotide precursors found in other plant families, underlining the high diversity of the precursor protein arrangements for cyclotides[112]. Irrespective of the arrangement, a pre-requisite for cyclotide backbone cyclisation is N-terminal processing. For the prototypic cyclotide kalata B1, N-terminal processing was recently linked to specific members of the papain-like cysteine protease (PLCP) family [113]. PLCPs require a leucine residue in the P2 position, which is a common residue present in O. affinis cyclotide precursors [113]. This requirement must be kept in mind when expressing cyclotide grafts in plants. Once the cyclotide’s N-terminus is liberated, asparaginyl endopeptidases (AEPs) initiate the backbone cyclisation of cyclotides. Whereas their usual role is the activation of cellular pathways and target specific proteolysis, some can act as competent peptide ligases [114]. Butelase 1, for example, an AEP found in Clitoria ternatea (butterfly pea) is an especially efficient peptide ligase when tested in vitro on a range of peptide substrates [115]. All native cyclotide producing plants have their own versions of ligating AEPs, for example O. affinis has OaAEP1b [114] and garden petunia (Petunia x

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Introduction hybrida E.Vilm.) has PxAEP3b [116]. All discovered cyclotides contain an asparagine or aspartic acid residue at the precursor C-terminus, which is necessary for AEP mediated cyclisation [113]. AEPs have great potential for targeted ligation of proteins and peptides. Even large intrinsically disordered proteins can be cyclized and therefore stabilized as was recently demonstrated [117]. PLCPs and AEPs autocatalytically mature in low pH, suggesting the plant cell vacuole is a likely final processing compartment for cyclotide biosynthesis.

1.2.4.2 Chemical and chemoenzymatic synthesis of cyclotides

The short chain length of around 30 amino acids enables cyclotides to be synthesized by solid- phase peptide synthesis [109]. The two most common strategies today use 9H-fluoren-9-ylmethoxycarbonyl (Fmoc) or tert-butoxy carbonyl (Boc) as protective groups [118, 119]. Native chemical ligation allows folding in aqueous buffer at pH 7. Another approach successfully utilized reduced glutathione in a single pot reaction for cyclization and folding [120]. Most chemical grafting studies have focused on cyclotides of the Möbius or trypsin inhibitor group, as in vitro folding of bracelet cyclotides is very difficult [102]. Mimicking nature, chemoenzymatic synthesis of MCoTI-I and MCoTI-II of the trypsin inhibitor group was achieved using an immobilised protease [121]. Production of macrocyclised peptides in vitro utilizing natural enzymes has shown some potential, even for designed sequences and unnatural amino acids [122]. The reliability and high product purity make chemical synthesis a relevant competitor for lab-scale production of peptides; however, the low yield and large amount of chemical waste makes chemical synthesis unfavourable for industrial production of most peptides. Peptides synthesized by SPPS cost around 100 USD to 600 USD per gram [123].

1.2.4.3 Cyclotide production in bacteria

Production of the cyclic sunflower trypsin inhibitor 1 (SFTI-1) peptide in vivo has been reported to produce a yield of 180 µg L-1 in E. coli utilizing an intein-based cyclisation approach that circumvents the inherent inability of bacteria to cyclise peptides [124]. This opens the possibility of cyclotide and cyclotide graft production in large scale bacterial culture [125]. Another application of cyclotide production in bacteria is the screening of large combinatorial libraries of biological active compounds stabilized by the SFTI-1 scaffold using high throughput fluorescence activated cell sorting [124]. However, the intein fusion step requires specific N- and C-termini limiting the possible sequences.

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Introduction

1.2.4.4 Cyclotide production in plants

Plants produce cyclotides in different concentrations and compositions in different tissues. The reported concentration of kalata B1 in O. affinis range from 0.36 mg g-1 DW in stems to 1.82 mg g-1 DW in leaves [126]. Another cyclotide producing species, Clitoria ternatea, contains around 30 mg kg-1 FW Cter A in leaves and 7.6 mg kg-1 FW Cter M in roots [78]. Consequently, cyclotide production in planta is feasible; however, higher concentrations are desirable and achievable via bioengineering [127]. The role of cyclotides is linked to the innate plant immune system and it is possible that their production could be enhanced by external stress signals [128, 129]. Ultimately, under controlled growth conditions free of any pathogens this could lead to low cyclotide yields. However, if the stress signals are identified, growth and elicited production could be decoupled under controlled growth conditions. Applying stress signals, like light, chemicals, hormones, temperature and physical stress, has been shown to up-regulate the expression of secondary metabolites and defensive proteins [86]. A case study of O. affinis found that cyclotides play important roles in the constitutive immune system and are subject to seasonal changes [130]. Cyclotide synthesis is regulated both at the transcriptional and post translational level, with regulation potentially differing across tissue types [130], opening the avenue of genetically engineered upregulation. The high stability of cyclotides combined with advanced knowledge of their precursors and in planta processing might enable biopharmaceutical expression in orally administered edible seeds [131]. Each cyclotide producing plant encodes multiple cyclotide genes, with some genes encoding multiple cyclotide domains. Consequently, one plant species can harbor many cyclotides possibly complicating product purification because most cyclotides have similar molecular masses and retention times. A conceivable solution is the production in a plant free of non-target cyclotides. Cyclotides have many interesting properties, but most drug candidates require grafting of an active sequence into a cyclotide scaffold. For heterologous proteins expression in planta, the introduction of the engineered gene into the host’s genome is necessary. Agrobacterium-mediated transient expression can produce high yields of target proteins [132] and enables pathway studies in vivo [133]. Cyclotides have been produced in N. benthamiana leaves, by simultaneous introduction of a gene for C-terminal processing and cyclisation (OaAEP1) as well as the gene encoding kalata B1 (Oak1) via Agrobacterium infiltration [116]. These results were a milestone for cyclotide production in planta; however, the yield of cyclic product was only 0.2 mg g-1 DW [125]. Co-transfer of a cyclotide gene with a ligase-type AEP has also led to cyclotide production in tobacco, bush bean, lettuce and canola [125], all species not natively producing cyclotides.

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Introduction

1.2.4.5 Cyclotide production in plant cell suspensions

Compared to whole plants, plant cell suspensions have some distinct advantages. Their production cycles are much shorter, product yield and quality are more consistent, and the closed reactor design enables easy compliance with GMP (good manufacturing practices) without contaminations or unintended gene flow in the environment [134]. As cyclotide production is connected to specific tissue types, cell suspensions produce novel cyclotides, while losing other cyclotides normally found in the species [135]. This poses challenges and opportunities to produce cyclotides in cell suspension cultures. This work will investigate the development of the cyclotide profile of several species in suspension. Plant hormones (i.e. phytohormones) regulate the life cycle of every plant cell [136]. Every species requires a specific cocktail of administered phytohormones for dedifferentiated growth in suspension. These unfamiliar conditions often lead to a very different peptide and protein profile compared to cells in the whole plant. In theory, the tailor-made cocktail allows a fine-tuning of the desired peptide levels, in reality the interplay of different hormones and stress conditions is most of the time too complex to be predictable and trial and error is necessary [136, 137]. The only reported quantified cyclotide production in suspension achieved an accumulation of 0.1 g kalata B1 per 1 kg dry weight of Oldenlandia affinis culture with an estimated productivity of 1 mg L-1 d-1[138]. Building on these results, the establishment of cyclotide producing plant cell suspension cultures is described in Chapter 2 and the scale up in Chapter 3. In Chapter 4, elicitors are tested to enhance the yield of cyclotide production in planta. Finally, the feasibility of a scalable transformation system to enable the production of engineered cyclotides in plant cell suspension is investigated in Chapter 5.

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Introduction

1.3 Bioreactors for plant cell suspensions

Lab-scale plant suspension cultures have been grown in Erlenmeyer flasks up to 1 litre culture volume for over 60 years [139]. The first industrially produced plant secondary metabolite was introduced in 1983 [49]. Nowadays, there are many different reactor concepts available for the transfer to the industrial scale, however there is no standard formula for scale-up and every system must be optimised and tested individually. Successful examples of plant cell and plant tissue cultivations in bioreactors include the production of taliglucerase alfa in multiuse disposable bag reactors by Protalix Biopharmaceutics [140], the production of a full-size human anti-HIV antibody in stirred-tank reactors [141], and the production of cyclotides in a photobioreactor [138]. The largest operational plant cell cultivation encompass a volume of two times 75,000 litres and produce taxanes for Phyton Biotech [142]. Taxanes are a class of notoriously difficult to synthesize diterpenes. Many members of the group are chemotherapeutic agents (e.g. paclitaxel, docetaxel or carbazitaxel). The following subsections outline the most important reactor candidates for cyclotide production in plant cell suspensions. A distinction must be made between classical (generally made of glass and steel) bioreactors and single use bioreactors (generally made of plastic). Traditionally, bioreactors have been built from glass and stainless steel to enable autoclaving at 121°C, 2 bar under superheated steam atmosphere. The consequence is a robust, expensive reactor that requires downtime for sterilization. The single use bioreactor is a different concept with smaller investment costs that enables production without downtimes but requires a new reactor vessel for every batch. Although stirred tank reactors are still the model reactor system, pneumatic bioreactors and rocking motion bioreactors are the two most relevant reactor types for this work. Plant cell suspensions can grow in the dark under chemotrophic conditions [57]; however, light can sometimes be used to activate pathways of secondary metabolite production [143].

1.3.1 Stirred tank bioreactor (STB)

The stirred tank reactor is the gold standard for a large variety of processes. As such, it is well studied and tested. Although several bioprocesses like yeast fermentation or bacterial cultivation can be carried out in STBs, many biological systems require a different reactor. Plant cell suspensions, for example, are susceptible to shear stress, which can lead to problems especially at the impeller tips [57]. Algae require light, but high-density cultures only allow for a light transfer of a few centimetres and consequently most of the volume of a STB stays in the dark. Differentiated plant systems (e.g.

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Introduction hairy roots or shoots) need a retaining support system in STBs or a special reactor setups [134]. Even though STBs come with many problems, they are very well researched and documented and can be adapted to a variety of situations. Greenovation Biotech GmbH, a small company producing pharmaceutical proteins in moss suspension, recently switched their largest production vessels to STBs, whereas their original medium sized production vessels were rocking motion bioreactors.

1.3.2 Pneumatic bioreactor

Pneumatic bioreactors are suitable for animal and plant cell cultures. They provide medium mass transfer rates (component mixing inside the reactor) and can be utilized as photobioreactors [144]. One major drawback of pneumatic bioreactors is the high shear stress present at the liquid-gas surface of a bursting bubble, damaging cells attached to the bubble and in close vicinity [145]. Even if the cells are sturdy enough to withstand the shear stress, the rising bubbles lead to a concentration of cells in the upper layers of the reactor. This in turn favours aggregation and diminished nutrients and gas transfer to the cells within the aggregate. This problem is aggravated in high density cultures and accompanied by reduced fluid mixing resulting in unfavourable pH, nutrient and biomass gradients throughout the culture [146].

1.3.3 Rocking motion bioreactor

Rocking motion bioreactors inherit the advantages of disposable bioreactors and qualify as a low shear stress production system that is easily scalable. The low costs and high flexibility combined with conditions suited for high cell density cultures (i.e. low shear stress and high oxygen transfer rate) make rocking motion bioreactors the ideal candidates for the interface between research and production [146]. Disposable reactors require disposable sensors or a sterilizable sensor-reactor interface. Although this imposes new challenges to miniaturization and production costs, it simultaneously circumvents problems of classical sensors such as their limited lifetime, resistance to steam, resistance to heat and degradation even outside of the reactor. New sensors for every batch increase the accuracy of all monitored parameters and prevent shifts that can occur over longer uptimes. This is very advantageous for reliable secondary metabolite production, which often can be influenced by minimal fluctuations in pH or concentration of chemicals.

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Recent studies have shown that rocking motion bioreactors can be used as temporary immersion systems for the cultivation of differentiated in vitro plant cultures (e.g. hairy roots) [147]. The combination of cheap disposable culture bags with cheap hormone-free hairy root medium could lead to cost-competitive plant based secondary metabolite production. Sufficient oxygen supply to the cells is crucial for all aerobic bioreactor setups. The defining parameter for oxygen transfer from the gas phase to the cells is the volumetric liquid-side mass transfer coefficient ( ). In rocking motion bioreactors the oxygen transfer can be adjusted by the rocking speed, rocking𝑘𝑘𝐿𝐿 𝑎𝑎angle, and volumetric gas flow [148].

1.3.4 Downstream processing

Although not part of this work, a short word on downstream processing is warranted here. This process step requires up to 50% of the energy and 80% of the capital necessary to produce a pharmaceutical in cell culture [18]. Consequently, early thought must be put into downstream processing. For plant cell suspensions, this normally requires filtration or centrifugation. Interestingly, one major challenge is the down-scaling of the established industrial processes to be usable in a lab environment [149], contrasting the up-scaling challenges of bringing the culture from the flask to the reactor. In general, the aim is to accumulate the target product in the liquid phase. As the cells are separated from the supernatant during cell harvesting, there is no major difference in the setup if the supernatant or the cells containing the product. However, if the cells contain the product, an additional step for extracting the product from the cells is required. Typically, this step includes cell lysis or non-destructive permeabilization [50]. Parallel to the trend of producing pharmaceuticals in smaller single use reactors, centrifuges are also shifting to smaller single use equipment. This allows greater flexibility (shorter installation time, lower investment costs, no cleaning validation), cheaper maintenance (time and resources), and easier GMP compliance. The downsides are the smaller g-force and the lower maximal volume [149]. Utilizing natural production systems opens another avenue possibly bypassing downstream processing completely. Namely the “eat the reactor” approach. If the pharmaceutical target product is expressed in an edible scaffold, oral delivery with no or minimal downstream processing is achievable. The product needs to retain its bioactivity when exposed to the digestive system for this method to work. Plant cells as production and storage systems can function as bio-capsules as their cell walls can protect the target product on its journey through the stomach [43]. These are further considerations that may enhance molecular farming capabilities.

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1.4 Scope of this work

Production of cyclotides and engineered cyclotide grafts in plant cell suspensions requires a holistic view encompassing plant biology, molecular biosynthesis, small scale cultivation, large scale production, downstream processing, mode of action, and target market. Each part influences the others and may be instrumental for success or failure. The focus was on the first four subjects as there was no cyclotide drug developed for production in plant cell suspensions at available the time of writing. However, this work achieved a proof of concept presented in Chapter 3 and included the three latter subjects as part of the introduction in Chapter 1. All other subjects are presented in detail. An overview of the scope of this work is given in Figure 3.

Figure 3 | From farm to pharm. Scope of this work.

1.4.1 Establishment and analysis of plant cell suspension cultures

Several species for cyclotide production in cell suspension were investigated as part of this thesis as presented in Chapter 2. Special focus was placed on Oldenlandia affinis as first plant to discover cyclotides in and first cell suspension to produce cyclotides. Stable cultures of O. affinis and Petunia hybrida have been achieved and their cyclotide profile characterized. Reliable protocols to 22 | Page

Introduction achieve Clitoria ternatea and Hybanthus enneaspermus suspension cultures were established and analysed. Nicotiana benthamiana cell suspensions were maintained as comparative model species. Cycloviolacin O2 was detected in O. affinis suspensions, verified via LC/MS-MS and found in the transcriptome. Additionally, new cyclotide transcriptome sequences, named kalata B22 and kalata B23 were discovered. Part of the mystery of the vanishing cyclotides in O. affinis suspensions was unravelled.

1.4.2 Scale-up of plant cell cultivations for cyclic peptide production

There are many choices when it comes to the symbiotic design of bioreactor and biological host. The considerations that led to the bioreactor scale-up are presented in Chapter 3. The available literature represented a good starting point for the biological host. The first cell suspension reported to produce cyclotides was O. affinis with a yield of 0.09 g kalata B1 per 1 kg dried cells. These cell lines have been scaled up from the lab bench to 25 L culture volume in an Medusa type photobioreactor [138, 150]. Illumination was reported as necessary elicitation for cyclotide production. Following the result presented in Chapter 2, O. affinis, H. enneaspermus and C. ternatea were investigated for their potential to produce cyclic peptides in a rocking motion bioreactor (Chapter 3). Higher plant suspension cultures grown in a rocking motion bioreactor (RMB) proved to be scalable and aseptic, with every parameter controllable. These features make this system a prime target for GMP compliant pharmaceutical production. This work characterized plant cell suspensions on a laboratory scale and small industrial scale to establish a scalable, flexible, and economical production platform for cyclic peptides.

1.4.3 Cyclotide elicitation in suspension cultures

Initial experiments showed a significant drop of cyclotide numbers and concentrations in O. affinis suspension. Therefore, elicitation to improve cyclotide production in plant suspensions was investigated. Methyl jasmonate, salicylic acid, sodium chloride, nutrient starvation, hormone starvation and cell immobilization were tested for their effect on cyclotide production in C. ternatea, H. enneaspermus, and O. affinis suspension cultures as presented in Chapter 4. 3 mM sodium chloride was found to enhance production of cycloviolacin O2, kalata B22, and kalata B23 in O. affinis suspension.

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1.4.4 Transformation of plant cells in suspension

The potential of transient expression in different species was evaluated in Chapter 5. Cyclic peptide drug targets are predominantly designed. This necessitates the introduction of foreign DNA into the plant-based production system. Furthermore, the assembly of cyclic peptides requires a specific enzymatic machinery already present in native cyclotide producers. Consequently, agroinfiltration of native cyclotide producers is preferable. Cell suspensions are more amendable to agroinfiltration than plants. Additionally, the observed loss of endogenous cyclotides in suspension cells over time might simplify the purification during downstream processing. In this work, infiltration of O. affinis suspension cell packs with EHA105 led to transformed cells, but reproducibility remained challenging. The same method did not result in transformed cells for C. ternatea and H. enneaspermus. Transformation of cell packs could enable a process with decoupled growth and production for grafted cyclic peptides.

1.4.5 Cryopreservation of O. affinis suspension cells

Cell based production requires a cell banking system to ensure the survival and availability of the high producing strains. Constant maintenance of living cells bears the risk of contamination and genetic shifts. Additionally, it is resource, time and labour intensive. Therefore, an attempt was made to develop a suitable cryopreservation protocol for O. affinis, as every plant species requires specific conditions. However, all protocols tested did not lead to any cell viability after thawing. The results were presented in Chapter 6 nevertheless, to supply information for possible future attempts.

1.5 Summary

In conclusion, this chapter has provided the vital background for cyclotide and plant cell suspension research. The available production systems for proteins have been presented and evaluated for cyclotide production. A special focus was placed on plant cell suspensions and their cultivation in bioreactors. Finally, the scope of this work was outlined. In the following chapter, suspensions of several plant species are established and analysed for their cyclotide production.

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1.6 References

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100. Barbeta, B.L., A.T. Marshall, A.D. Gillon, D.J. Craik, and M.A. Anderson, Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proceedings of the National Academy of Sciences, 2008. 105: p. 1221. 101. Xiao, Y. and K. Wu, Recent progress on the interaction between insects and Bacillus thuringiensis crops. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 2019. 374: p. 20180316. 102. Craik, D.J., M.H. Lee, F.B.H. Rehm, B. Tombling, B. Doffek, and H. Peacock, Ribosomally- synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds. Bioorg Med Chem, 2018. 26: p. 2727. 103. Wang, C.K., C.W. Gruber, M. Cemazar, C. Siatskas, P. Tagore, N. Payne, G. Sun, S. Wang, C.C. Bernard, and D.J. Craik, Molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis. ACS Chem Biol, 2014. 9: p. 156. 104. Eliasen, R., N.L. Daly, B.S. Wulff, T.L. Andresen, K.W. Conde-Frieboes, and D.J. Craik, Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1. Journal of Biological Chemistry, 2012. 287: p. 40493. 105. Gründemann, C., K.G. Stenberg, and C.W. Gruber, T20K: An Immunomodulatory Cyclotide on Its Way to the Clinic. International Journal of Peptide Research and Therapeutics, 2018. 106. Gruber, C.W. and C. Gruendemann, Cyclotides as immunosuppressive agents, U. Freiburg and M.U. Wien. 2019, US 10,159,710 B2. 107. Poth, A.G., Y.H. Huang, T.T. Le, M.W. Kan, and D.J. Craik, Pharmacokinetic characterization of kalata B1 and related therapeutics built on the cyclotide scaffold. Int J Pharm, 2019. 565: p. 437. 108. Morrison, C., Constrained peptides' time to shine? Nature Reviews Drug Discovery, 2018. 17: p. 531. 109. Wang, C.K. and D.J. Craik, Designing macrocyclic disulfide-rich peptides for biotechnological applications. Nat Chem Biol, 2018. 14: p. 417. 110. Gruber, C.W., M. Cemazar, R.J. Clark, T. Horibe, R.F. Renda, M.A. Anderson, and D.J. Craik, A novel plant protein-disulfide isomerase involved in the oxidative folding of cystine knot defense proteins. J Biol Chem, 2007. 282: p. 20435. 111. Conlan, B.F., A.D. Gillon, B.L. Barbeta, and M.A. Anderson, Subcellular targeting and biosynthesis of cyclotides in plant cells. American journal of botany, 2011. 98: p. 2018. 112. Gould, A. and J.A. Camarero, Cyclotides: Overview and Biotechnological Applications. Chembiochem, 2017. 18: p. 1350. 113. Rehm, F.B.H., M.A. Jackson, E. De Geyter, K. Yap, E.K. Gilding, T. Durek, and D.J. Craik, Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization. Proc Natl Acad Sci U S A, 2019. 116: p. 7831. 114. Harris, K.S., T. Durek, Q. Kaas, A.G. Poth, E.K. Gilding, B.F. Conlan, I. Saska, N.L. Daly, N.L. van der Weerden, D.J. Craik, and M.A. Anderson, Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat Commun, 2015. 6: p. 10199. 115. Nguyen, G.K., S. Wang, Y. Qiu, X. Hemu, Y. Lian, and J.P. Tam, Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nature chemical biology, 2014. 10: p. 732. 116. Jackson, M.A., E.K. Gilding, T. Shafee, K.S. Harris, Q. Kaas, S. Poon, K. Yap, H. Jia, R. Guarino, L.Y. Chan, T. Durek, M.A. Anderson, and D.J. Craik, Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases. Nat Commun, 2018. 9: p. 2411. 117. Harris, K.S., R.F. Guarino, R.S. Dissanayake, P. Quimbar, O.C. McCorkelle, S. Poon, Q. Kaas, T. Durek, E.K. Gilding, M.A. Jackson, D.J. Craik, N.L. van der Weerden, R.F. Anders, and M.A. Anderson, A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein. Sci Rep, 2019. 9: p. 10820.

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118. Fields, G.B. and R.L. Noble, Solid phase peptide synthesis utilizing 9‐ fluorenylmethoxycarbonyl amino acids. International journal of peptide and protein research, 1990. 35: p. 161. 119. Schnölzer, M., P. Alewood, A. Jones, D. Alewood, and S.B.H. Kent, In situ neutralization in Boc‐chemistry solid phase peptide synthesis: rapid, high yield assembly of difficult sequences. International journal of peptide and protein research, 1992. 40: p. 180. 120. Aboye, T., Y. Kuang, N. Neamati, and J.A. Camarero, Rapid parallel synthesis of bioactive folded cyclotides by using a tea-bag approach. Chembiochem, 2015. 16: p. 827. 121. Thongyoo, P., N. Roqué-rosell, R.J. Leatherbarrow, and E.W. Tate, Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides. Organic & Biomolecular Chemistry, 2008. 6: p. 1462. 122. Ludewig, H., C.M. Czekster, E. Oueis, E.S. Munday, M. Arshad, S.A. Synowsky, A.F. Bent, and J.H. Naismith, Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates. ACS Chem Biol, 2018. 13: p. 801. 123. Hancock, R.E. and H.G. Sahl, Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol, 2006. 24: p. 1551. 124. Li, Y., T. Aboye, L. Breindel, A. Shekhtman, and J.A. Camarero, Efficient recombinant expression of SFTI-1 in bacterial cells using intein-mediated protein trans-splicing. Biopolymers, 2016. 106: p. 818. 125. Poon, S., K.S. Harris, M.A. Jackson, O.C. McCorkelle, E.K. Gilding, T. Durek, N.L. van der Weerden, D.J. Craik, and M.A. Anderson, Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. J Exp Bot, 2018. 69: p. 633. 126. Dörnenburg, H., Cyclotide synthesis and supply: from plant to bioprocess. Biopolymers, 2010. 94: p. 602. 127. Dörnenburg, H., Progress in kalata peptide production via plant cell bioprocessing. Biotechnol J, 2009. 4: p. 632. 128. Jones, J.D. and J.L. Dangl, The plant immune system. Nature, 2006. 444: p. 323. 129. Pieterse, C.M.J., A. Leon-Reyes, S.V.d. Ent, and S.C.M.V. Wees, Networking by small- molecule hormones in plant immunity. Nature Chemical Biology, 2009. 5: p. nchembio.164. 130. Mylne, J.S., C.K. Wang, N.L. van der Weerden, and D.J. Craik, Cyclotides are a component of the innate defense of Oldenlandia affinis. Peptide Science, 2010. 94: p. 635. 131. Mahatmanto, T., Review seed biopharmaceutical cyclic peptides: From discovery to applications. Biopolymers, 2015. 104: p. 804. 132. Jin, T., J. Wang, X. Zhu, Y. Xu, X. Zhou, and L. Yang, A new transient expression system for large-scale production of recombinant proteins in plants based on air-brushing an Agrobacterium suspension. Biotechnol Rep (Amst), 2015. 6: p. 36. 133. Yang, Y., R. Li, and M. Qi, In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. The Plant Journal, 2000. 22: p. 543. 134. Srikantan, C. and S. Srivastava, Bioreactor Design and Analysis for Large-Scale Plant Cell and Hairy Root Cultivation, in Hairy Roots, V. Srivastava and S. Mehrotra, Editors. 2018, Springer, Singapore. p. 147. 135. Narayani, M., A. Chadha, and S. Srivastava, Callus and cell suspension culture of Viola odorata as in vitro production platforms of known and novel cyclotides. Plant Cell, Tissue and Organ Culture (PCTOC), 2017. 130: p. 289. 136. Wang, Y.H. and H.R. Irving, Developing a model of plant hormone interactions. Plant Signaling & Behavior, 2014. 6: p. 494. 137. Suzuki, N., Hormone signaling pathways under stress combinations. Plant Signal Behav, 2016. 11: p. e1247139. 138. Seydel, P., C. Walter, and H. Dörnenburg, Scale-up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production. Engineering in Life Sciences, 2009. 9: p. 219.

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139. Yesil-Celiktas, O., A. Gurel, and F. Vardar-Sukan, Large scale cultivation of plant cell and tissue culture in bioreactors. Transworld Research Network, 2010. 1: p. 54. 140. Tekoah, Y., A. Shulman, T. Kizhner, I. Ruderfer, L. Fux, Y. Nataf, D. Bartfeld, T. Ariel, S. Gingis-Velitski, U. Hanania, and Y. Shaaltiel, Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience. Plant Biotechnol J, 2015. 13: p. 1199. 141. Holland, T., M. Sack, T. Rademacher, K. Schmale, F. Altmann, J. Stadlmann, R. Fischer, and S. Hellwig, Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full‐size antibody in BY‐2 suspension culture. Biotechnology and bioengineering, 2010. 107: p. 278. 142. Pantchev, I., G. Rakleova, A. Pavlov, and A. Atanassov, History of Plant Biotechnology Development. 2018. 143. Dörnenburg, H. and P. Seydel, Effect of irradiation intensity on cell growth and kalata B1 accumulation in Oldenlandia affinis cultures. Plant Cell, Tissue and Organ Culture, 2007. 92: p. 93. 144. Schwerna, P., H. Hübner, and R. Buchholz, Quantification of oxygen production and respiration rates in mixotrophic cultivation of microalgae in nonstirred photobioreactors. Engineering in Life Sciences, 2017. 17: p. 140. 145. Walls, P.L., O. McRae, V. Natarajan, C. Johnson, C. Antoniou, and J.C. Bird, Quantifying the potential for bursting bubbles to damage suspended cells. Scientific Reports, 2017. 7: p. 15102. 146. Huang, T.-K. and K.A. McDonald, Bioreactor engineering for recombinant protein production in plant cell suspension cultures. Biochemical Engineering Journal, 2009. 45: p. 168. 147. Ritala, A., L. Dong, N. Imseng, T. Seppänen-Laakso, N. Vasilev, S. van der Krol, H. Rischer, H. Maaheimo, A. Virkki, and J. Brändli, Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway. Journal of biotechnology, 2014. 176: p. 20. 148. Pilarek, M., P. Sobieszuk, K. Wierzchowski, and K. Dąbkowska, Impact of operating parameters on values of a volumetric mass transfer coefficient in a single-use bioreactor with wave-induced agitation. Chemical Engineering Research and Design, 2018. 149. Turner, R., A. Joseph, N. Titchener-Hooker, and J. Bender, Manufacturing of Proteins and Antibodies: Chapter Downstream Processing Technologies. 2018, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 1. 150. Seydel, P. and H. Dörnenburg, Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides. Plant Cell, Tissue and Organ Culture, 2006. 85: p. 247.

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Establishment and analysis of plant cell suspensions for cyclic peptide production

2 Establishment and analysis of plant cell suspensions for cyclic peptide production

Plant cell suspensions are useful tools for the study and production of secondary metabolites in plants. In some specialised cases they have been used for industrial pharmaceutical production. One example is the production of taliglucerase alfa, an acid β-glucosidase for the treatment of Gaucher disease [1]. Plant cell suspension-based pharmaceutical production is an emerging area of interest, and for some recombinant products offers advantages that include cost reduction and inherent protection against human pathogens over more traditional production platforms like mammalian cell cultures [2, 3]. Commonly, plant cell suspensions are initiated from calli, which consist of undifferentiated cells, and require liquid medium containing a carbon, oxygen and nitrogen source, together with salts and phytohormones. However, the exact medium composition is different for every plant species and requires optimisation. Additional factors known to influence suspension cell growth include pH, shear stress, irradiation, and temperature. In cyclotide producing plants, it is not uncommon for tens to hundreds of cyclotides to be expressed at any given time, across a multitude of plant organs [4]. Seydel & Dörnenburg were the first to test if cyclotides were also expressed in cell suspensions of a cyclotide producer, in this case Oldenlandia affinis [5, 6]. They reported yields of kalata B1 of 0.09 mg g-1 DW in an air-lift loop reactor but did not investigate long term stability and gene expression. Further studies by Slazak et al. [7] and Narayani et al. [8] demonstrated that cyclotides could also be detected in suspension cultures of Viola uliginosa and Viola odorata, where, in the case of V. ulginosa, the yield of cycloviolacin O13 reached 4 mg g-1 DW. In this species, it was demonstrated that phytohormones play a key role in the type and quantity of cyclotides produced in suspensions. To provide further insights into the biology and engineering of cyclotide production in suspension cells, the cyclotide producing plants of C. ternatea, H. enneaspermus, and O. affinis were analysed and the results are reported in this chapter. Furthermore, suspension cultures of N. benthamiana and P. hybrida were initiated to provide reference model cell lines available for transformation as presented in Chapter 5. Protocols to establish stable suspension cultures of these five species are presented here. Although callus was not considered as a production system in this work, substantial amounts of friable callus was necessary to initiate suspension cultures. Therefore, protocols for callus initiation are also presented and may prove useful for regenerating whole plants, for genetic transformation experiments and for maintenance of cell lines.

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Establishment and analysis of plant cell suspensions for cyclic peptide production

A detailed analysis of O. affinis suspension cells is reported in this chapter. Novel cyclotides were discovered, time-dependant cyclotide production in suspension was analysed, and mRNA expression linked to cyclotide production. Finally, a cyclotide class common in suspension cultures is defined and discussed.

2.1 Materials and methods

2.1.1 Plant material and in vitro culture

Seeds of Nicotiana benthamiana, Petunia hybrida, Oldenlandia affinis, and of the Clitoria ternatea composite line “Milgarra” [9] were available in house. Hybanthus enneaspermus seeds were collected as reported by Jackson et al. [10]. For seed sterilization, seeds were first rinsed to remove impurities and subsequently gently inverting for 2 minutes in 70% (v/v) ethanol. The supernatant was then decanted after centrifugation (7000 x g, 2 min), at which time the seeds were incubated in 25% (v/v) NaOCl with 0.1% (v/v) Triton-X for 5 min twice (centrifugation and decanting as before after every step). Subsequently, the seeds were rinsed three times with sterile water and dried on filter paper under aseptic conditions. In the case of H. enneaspermus, another seed sterilization method was required with seeds washed first in 5% (v/v) Triton-X detergent solution for 15 seconds, before rinsing in sterile water. Seeds were dried before a further wash in 80% (v/v) ethanol with gentle inversion for one minute. After an additional wash in sterile water, seeds were then immersed in 4 mL 20% (v/v) NaOCl with four drops Tween-20 for 5 min, 10 min, and 15 min respectively. Finally, the seeds were rinsed five times with sterile water and dried on filter paper under aseptic conditions ready for germination. For germination, sterilized seeds were placed on petri dishes containing MS medium (2.2 g L 1Murashige and Skoog medium with vitamins (M519, Phyto Technology Lab), .8 g L 1agar, 2 1 pH 6.8, ⁻no sucrose). The germination conditions were 26°C at 30 µmol m s photon density⁻ under a 16:8 light-dark cycle in an incubator (Fitotron, Simultech). The germination⁻ ⁻ efficiency in percent was calculated via the formula seeds germinated divided by total seeds times 100.

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Establishment and analysis of plant cell suspensions for cyclic peptide production

2.1.2 Callus initiation and maintenance

Callus of Nicotiana benthamiana, Petunia hybrida, Oldenlandia affinis, Hybanthus enneaspermus and Clitoria ternatea were established using explants of aseptic in vitro plants. The leaves, stems or roots were cut into small pieces (around 0.1 cm2). The aseptic explants were transferred onto callus induction medium in a Petri dish. The callus induction frequency was calculated via the formula explants developing callus tissue divided by total explant number times 100. The area of wound tissue in contact with the medium was maximized to produce as much callus per explant as possible. The incubation time was 4 weeks at 26°C with 35 µmol m 2 s 1 photon density under a 16:8 light-dark cycle in an incubator (Fitotron, Simultech). C. ternatea⁻ ⁻ and H. enneaspermus were visually screened for callus induction, growth and viability under different phytohormone concentrations. The most friable calli of all species were selected after 4 weeks, cut into small pieces and transferred onto fresh medium. The best observed callus induction medium compositions are reported in Table 3. A wide range of medium compositions was tested for C. ternatea and H. enneaspermus and is presented in Section 2.2.2. The media of the other species were adapted from the literature.

Table 3 | Callus induction media. Species NAA BAP Sucrose O. affinis 1.86 mg L-1 2.25 mg L-1 5 g L-1 P. hybrida 0.3 mg L-1 1 mg L-1 10 g L-1 H. enneaspermus 1.86 mg L-1 0.9 mg L-1 30 g L-1 N. benthamiana 2 mg L-1 0.05 mg L-1 30 g L-1 C. ternatea 1 mg L-1 1 mg L-1 20 g L-1 Note: Every medium contained full strength MS (M519, Phyto Technology Lab), 8 g L-1 Phyto agar (Duchefa Biochemie B. V.) and was adjusted to pH 5.8 using 1 M KOH.

2.1.3 Plant cell suspension initiation and maintenance

To initiate suspension cells, fresh friable calli from each tested plant species were broken up to reduce their size and transferred into 125 mL Erlenmeyer flasks with liquid growth medium. The medium contained sucrose as a carbon source and all necessary vitamins, salts, and hormones. Passaging, splitting or scale-up was performed every seven days, retaining the growth parameters. Illumination was set to 35 µmol m 2 s 1 photon density with a 16:8 light-dark cycle. The temperature was kept constant at 26°C in an incubator⁻ ⁻ (Fitotron, Simultech). The oxygen transfer rate was reliant on the rocking speed of the orbital shaker on which the culture flasks were placed and the ratio of culture volume to vessel volume. Good growth was observed for a culture volume to vessel volume

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Establishment and analysis of plant cell suspensions for cyclic peptide production ratio of 1/3 and a rocking speed of 110 rpm for 125 mL flasks, 105 rpm for 250 mL flasks, 100 rpm for 500 mL flasks and 90 rpm for 1 L flasks. Table 5 displays the medium compositions for all examined species that worked best. All cultures were supplied with 20 g L-1 sucrose as optimal concentration determined by Gamborg [11]. Passaging was performed at the onset of the stationary growth phase by discarding 4/5 of the culture volume and refilling it with fresh medium preheated to 26°C. Transfer of liquid in and out of cultures was always achieved via pouring, because insertion of pipettes or similar devices led to increased contamination rates. The growth, macroscopic and microscopic morphology of the cell lines were constantly monitored to detect contaminations, organogenesis, aggregation, wall growth and dying cultures. The dry weight of samples was determined via lyophilization, the packed cell volume was determined via centrifugation (4000 x g, 10 min, RT). The growth rate was calculated by determining the slope of the growth curve for the specified timeframe. Phytohormone concentrations in C. ternatea and H. enneaspermus suspension cultures were varied as presented in Table 4 to screen for optimised growth conditions. The best conditions found and consequently used throughout this thesis are presented in Table 5.

Table 4 | Media compositions for C. ternatea and H. enneaspermus suspension cultures. Species Condition 2,4-D NAA BAP H. enneaspermus A 1.5 mg L-1 - 1 mg L-1 G - 0.25 mg L-1 0.5 mg L-1 PC - 0.48 mg L-1 0.5 mg L-1 C. ternatea I - 1 mg L-1 1 mg L-1 J - 1 mg L-1 2 mg L-1 K - 1 mg L-1 1.5 mg L-1 Note: Every medium contained full strength MS (M519, Phyto Technology Lab), 20 g L-1 sucrose and was adjusted to pH 5.8 using 1 M KOH.

Table 5 | Optimised suspension media. Species 2,4-D NAA BAP Kinetin O. affinis 0.4 mg L-1 - - - P. hybrida 2 mg L-1 - - 0.25 mg L-1 H. enneaspermus 1.5 mg L-1 - 1 mg L-1 - - 1 mg L-1 - 0.5 mg L-1 N. benthamiana 0.2 mg L-1 - - C. ternatea - 1 mg L-1 1.5 mg L-1 - Note: Every medium contained full strength MS (M519, Phyto Technology Lab), 20 g L-1 sucrose and was adjusted to pH 5.8 using 1 M KOH. Both conditions presented for N. benthamiana worked equally well.

Growth of the C. ternatea and H. enneaspermus suspension cultures was monitored via non- invasive image analysis of settled cell volume by tilting the culture vessels by 45° and allowing the cells to settle for 1 h. Afterwards, the culture volume and the settled cell volume was determined via

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Establishment and analysis of plant cell suspensions for cyclic peptide production image analysis and comparison to a standard curve created using water in the same culture vessel as shown in Figure 4.

Figure 4 | Settled cell volume (SCV) measurement. A) Recording of a standard curve by connecting measured heights with known volumes. B) Growth measurement by determination of settled cell volume and total culture volume.

2.1.4 Extraction and purification of cyclotides from plant tissue samples

Extraction of cyclotides from biological samples was achieved via lyophilization and subsequent cell disruption. Manual grinding with mortar and pestle, automated grinding with metal balls, and sonication were tested as cell disruption methods. Manual grinding was carried out by shock freezing the samples in liquid nitrogen and subsequently grinding them with liquid nitrogen in a mortar until only a fine powder remained. This powder was milled with extraction solvent (50% acetonitrile (ACN), 1% formic acid) for 2 min. The extraction solvent also contained a known amount of spiked peptide to enable relative quantification. The concentration and monoisotopic mass of the used spiked peptides was chosen as to not coincide with peptide masses present in the sample and to produce a peak height within a 10-100% range of the strongest natural occurring cyclotide peak of the sample. The exact concentrations and sequences are reported in Section 2.1.6. Automated grinding was realized by placing 5 mg dried cells with one metal ball and 250 µL extraction solvent in a 2 mL Eppendorf tube and putting them in a cell disruptor at 1250 rpm for 1.5 min (Geno Grinder SPEX SamplePrep 2010). Cell wall rupture was achieved by sonication at 60% power output and 70% duty cycle for 5 min in 50% ACN, 1% formic acid for volumes between 5 and 50 mL. To collect larger amounts of peptides for identification, 1-5 L of plant cell suspension were cultivated in a rocking motion bioreactor. The cultivation parameters were 26°C, 16:8 light-dark cycle, 20 µmol m 2 s 1 photon density, rocking angle of 10°, 20 rpm and a gas flow rate of 0.1 L min-1 air per 1 L culture⁻ volume⁻ (supplemented with 5% (v/v) CO2). The cells were harvested after 20 days at maximum cell density in the stationary phase. Suspension cells were pelleted by centrifugation at 8,000 x g for 20 minutes and subsequently lyophilized overnight. The dry weight was recorded,

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Establishment and analysis of plant cell suspensions for cyclic peptide production before the cells were disrupted using 3 mm diameter stainless steel balls in a cell disruptor (Geno Grinder SPEX SamplePrep 2010). The cyclotides were extracted in 60 mL 50% ACN, 1% formic acid with insoluble material removed by centrifugation (5000 x g, 20 min), and filtration (0.45 µm). Peptide containing extract was then transferred into a round bottom flask for lyophilizing that resulted in 1.5 g dried crude peptide extract. After re-dissolving in 27 mL 50% ACN, 1% formic acid the sample was diluted to ~5% ACN, 1% formic acid and loaded onto a C18 solid phase extraction (SPE) column. Fractions were collected at 0, 10, 20, 30, 50, 60 and 80% ACN, 1% formic acid, and analysed for putative cyclotides using MALDI-TOF-MS. Relevant fractions were further purified using HPLC initially with a semi-preparative column (flow rate 3ml per minute; 1% ACN gradient) and later an analytical scale column (flow rate 1ml per minute; 1% ACN gradient). Fractions displaying absorption in the ultra-violet spectrum (215 nm and 280 nm) were analysed for putative cyclotides using MALDI-TOF-MS and checked for purity by LC/MS.

2.1.5 Reduction, alkylation and digestion of cyclotides

The enzymatic digestion method was adopted from literature [12]. The pure peptide samples were lyophilized and dissolved in 36 µL 100 mM NH4HCO3 in 2 mL Eppendorf tubes at pH 8. 4 µL of 100 mM dithiothreitol (DTT) was added to give a final concentration of 10 mM, then was incubated at 60°C for 30 min under a nitrogen atmosphere. After returning to RT, 4 µL 250 mM iodoacetamide was added and incubated at RT for 30 min under a nitrogen atmosphere. The volume was split into four samples and digested by trypsin, endo-GluC, chymotrypsin, and a mixture of endo-GluC plus trypsin at 37°C overnight. The reaction was quenched by adding 5 µL of 5% FA.

2.1.6 Cyclotide detection via MALDI-TOF analysis

Mass spectrometric measurements were carried out on a MALDI-TOF/TOF 4800 Analyser (AB Sciex, Canada) operated in reflector positive ion mode at a laser intensity of 3,000. The matrix consisted of 7.63 mg α-cyano-4-hydroxycinnamic acid in 1 mL of 50% ACN, 0.1% formic acid stored at RT and discarded as soon as yellow crystals started to form inside the volume. All cyclotide samples were desalted and purified via C18 Ziptips (Millipore®). Each of the following steps was executed by pipetting the column volume ten times up and down. The C18 column was activated in 100% methanol, followed by a cleaning step in 10% ACN, 1% formic acid. The extracted cyclotide samples were diluted to a final concentration of 10% ACN while maintaining 1% formic acid and

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Establishment and analysis of plant cell suspensions for cyclic peptide production loaded onto the column. A second washing step at 10% ACN, 1% formic acid was designed to get rid of impurities. The remaining cyclotides were washed of the column with 5 µL 80% ACN, 1% formic acid. The tubes containing the eluted, purified cyclotides was kept shut to prevent evaporation of ACN. 0.6 µL purified sample in 80% ACN, 1% formic acid was mixed with 0.6 µL matrix on Parafilm M. 0.6 µL of the mixture was spotted onto a MALDI plate. For relative quantification of peptide levels in O. affinis, P. hybrida, and N. benthamiana, linear kalata B1 with a Glu-Ile-Ile amino acid extension at the C-terminus was spiked into each sample according to the processed dry weight. 1 µL kalata B1-Glu-Ile-Ile at a concentration of 272 µmol L-1 was added per 1 mg dry weight of ground tissue. Because the mass of kalata B1-Glu-Ile-Ile coincides with peptides present in C. ternatea and H. enneaspermus, 1 µL kalata B1 per 1 mg dry weight of ground tissue was spiked into these samples at a concentration of 233 µmol L-1. Most O. affinis cultures expressing cycloviolacin O2 co-expressed kalata B23 at comparable intensities. When co-expressed, the monoisotopic peak of cycloviolacin O2 was easy to detect, but the monoisotopic peak of kalata B23 coincided with the fifth peak of cycloviolacin O2. The calculated isotopic pattern of cycloviolacin O2 (Figure 5) and kalata B23 (Figure 6) were used to deconvolute the combined spectra. It is customary to use the monoisotopic peak for intensity read out. This value was not directly accessible for kalata B23 whenever cycloviolacin O2 and kalata B23 were part of the same sample. Consideration of the calculated isotopic patterns revealed that the contribution of cycloviolacin O2 to the 3147 m/z+ peak is negligible. The ratio of this peak to the monoisotopic peak of kalata B23 is 0.766. Therefore, the intensity of the peak at 3147.32 m/z+ times 0.766 yields the monoisotopic peak intensity of kalata B23 and was used for relative quantification.

Figure 5 | Calculated isotopic pattern of cycloviolacin O2 with charge +1. The highest peak was set to 100 native intensity.

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Establishment and analysis of plant cell suspensions for cyclic peptide production

Figure 6 | Calculated isotopic pattern of kalata B23 with charge +1. The highest peak was set to 100 native intensity.

2.1.7 Cyclotide sequencing via LC/MS-MS analysis

Tandem mass spectrometry experiments were carried out at 1 kV laser energy without collision-induced dissociation. The sequences were elucidated by manual identification of the N-terminal b-ion and the C-terminal y-ion series and matching them to known cyclotide sequences. The LC/MS experiments included an ultra-high-pressure liquid chromatography column followed by mass spectrometry (TripleTOF 5600+, Sciex). Analyst® software (Sciex) was used for analysis and cyclotide detection. For relative quantification of peptide levels, linear kalata B1 with a Glu-Ile-Ile amino acid extension at the C-terminus was spiked into each sample according to the processed dry weight. 1 µL kalata B1-Glu-Ile-Ile at a concentration of 272 µmol L-1has been added per 1 mg dry weight.

2.1.8 In silico transcriptome analysis

The transcriptomes of O. affinis, C. ternatea and H. enneaspermus were assembled in house by Dr. Edward Gilding as described in Harris et al. (2015) [13]. Transcriptomes were searched for putative cyclotide sequences using BLAST [14]. The investigated cyclotide sequences were identified by comparison of observed peptide masses with cyclotide masses reported at cybase.org.au. The peptide masses were determined via MALDI-TOF analysis as described in Section 2.1.6.

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2.1.9 RNA extraction, purification and conversion

Two methods were used to extract RNA from plant samples. The initial extraction method was TRIzol® based. To increase RNA yield and purity, the NucleoSpin® RNA Plant kit by Macherey-Nagel was used for all samples presented in Section 2.2.5 Real-time PCR of O. affinis RNA.

2.1.9.1 TRIzol® protocol

For the TRIzol® based RNA extraction, leaf and callus tissue were ground into a fine powder under liquid nitrogen with a mortar and pestle, whereas suspension cells were centrifuged(6,000 x g, 1 min), the supernatant discarded, and the pallet ground down with a disposable polypropylene pellet pestle (DWK Life Sciences) in a 1.5 mL Eppendorf tube. 50-100 mg of the resulting tissue powder was added to a 1.5 mL Eppendorf tube, pre-cooled in liquid nitrogen. After the addition of 1 mL TRIzol® the tube was inverted vigorously, vortexed, and consequently incubated at RT for 5 minutes. The cell debris was removed by centrifuging at 17,000 x g for 5 minutes and transferring 900 µL of supernatant into a new 1.5 mL Eppendorf tube. After the addition of 200 µL chloroform, the mixture was inverted vigorously, vortexed and incubated at RT for 2 minutes. To achieve phase separation, the mixture was centrifuged at 17,000 x g for 15 minutes at 4°C. 400 µL aqueous phase were transferred into a new 1.5 mL Eppendorf tube, mixed with 400 µL isopropanol, and incubated for 10 minutes at RT. The RNA was pelleted at 17,000 x g for 10 minutes at 4°C, the liquid discarded, and the RNA pellet consequently washed by adding 1 mL chilled ethanol and incubated at RT for 1 min. Next centrifugation at 17,000 x g for 5 min pelleted the RNA again and allowed removal of the supernatant by pipetting. After air drying for 1 hour, the RNA pellet was resuspended in 50 µL DNA digestion mix (44.56 µL water, 4.46 µL DNase buffer, 0.98 µL DNase I (10% (v/v)), TURBO DNA-freeTM Kit InvitrogenTM), flicked and incubated at 37°C for 20 min to digest present DNA. To inactivate the reaction, 5 µL DNase inactivation agent were added. The mixture was centrifuged at 17,000 x g for 10 min, 40 µL supernatant transferred into a 1.5 mL Eppendorf tube and stored at - 80°C for no longer than 4 months before processing.

2.1.9.2 NucleoSpin® RNA Plant protocol

All samples were processed on a work bench and with gloves regularly cleaned with RNase away. The mortar and pestles for breaking the cells were precleaned with bleach, then RO water, then ethanol, and lastly methanol. Fresh leaves and fresh callus samples were ground directly in liquid

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Establishment and analysis of plant cell suspensions for cyclic peptide production nitrogen. Whereas suspension samples were first freed from the medium by application of vacuum to a 5 mL column including a filter inlet. The resulting dry cell pack was transferred to a mortar and ground in liquid nitrogen. For every sample, cell powder was added to a 2 mL Eppendorf tube pre- cooled in liquid nitrogen until the tube was filled to the end of the conical part (~100 mg) and put on dry ice. The following steps were taken from the manual “NucleoSpin® RNA protocols – 06/2015, Rev. 17” published by Macherey-Nagel [15]. To lyse the homogenized cells, 350 µL Buffer RA1 mixed with 3.5 µL ß-mercaptoethanol was added and vortexed vigorously. The lysate was cleared by filtration through the supplied NucleoSpin® Filter (violet ring) at 11,000 x g for 1 min. The flow through was mixed with 350 µL ethanol (70%) and filtered through a NucleoSpin® RNA Column (light blue ring) at 11,000 x g for 30 s. To prepare the column for DNA digestion, 350 µL membrane desalting buffer was applied at 11,000 x g for 1 min. 95 µL DNase reaction mixture was applied to the column and incubated at RT for 15 min to digest bound DNA. The following washing steps inactivate the rDNase, wash and dry the silica membrane. First 200 µL Buffer RAW2 was washed through the column at 11,000 x g for 30 s, second 600 µL Buffer RA3 at 11,000 x g for 30 s and last 250 µL Buffer RA3 at 11,000 x g for 2 min. The resulting pure RNA was eluted with 40 µL RNase- free water at 11,000 x g for 1 min and stored at -80°C for no longer than 4 months before processing.

2.1.9.3 RNA quality assessment

The quality of all RNA samples was checked by running agarose gels, whereas the quantity was identified using a Thermo ScientificTM NanoDropTM spectrophotometer. The gels were prepared at 1% (w/v) agarose stained with 0.001% (v/v) SYBR Safe dye (Invitrogen). 7 µL sample was mixed with 3 µL loading dye and run on the gel for 45 min at 80 V. Imaging was achieved on a Bio-Rad gel imager using the Quantity One software version 4.4.0. Every RNA sample that proofed its purity on an agarose gel was subsequently measured at the Nanodrop spectrophotometer. 1 µL sample was loaded and compared against the buffer as blank solution. The absorbance at 230, 260 and 280 nm was measured and used to determine the concentration and purity of the RNA present in the samples.

2.1.9.4 Complementary DNA synthesis

All RNA samples were converted to cDNA via reverse transcription utilizing the SuperScriptTM III Reverse Transcriptase kit by Invitrogen. First, the RNA samples stored at -80°C were thawed and diluted with RNase free water to result in 366.4 ng RNA in 11 µL. This adjustment ensures the same amount of template cDNA in all PCR wells. Second, 1 µL oligo dT and 1 µL dNTP

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Establishment and analysis of plant cell suspensions for cyclic peptide production were added and the resulting 13 µL placed in a thermocycler at 65°C for 5 min. After the addition of

4 µL 5x FS buffer (250 mM Tris-HCl (pH 8.3 at RT), 375 mM KCl, 15 mM MgCl2), 1 µL 0.1 M DTT, 1 µL RNaseOUTTM, and 1 µL SuperScriptTM III Reverse Transcriptase, the thermocycler was set to 50°C for 1 h, followed by 70°C for 15 min. To remove possible RNA impurities complementary to the produced cDNA, 1 µL of E. coli RNase H was added and kept at 37°C for 20 min.

2.1.10 Quantitative PCR analysis

The qPCR experiments were run with four biological and two technical replicates. The gene expression of plant leaf tissue, callus tissue, and four suspension time points (i.e. week 1, week 2, week 3, and week 4) was analysed. All steps except the final qPCR run were carried out on ice. The 18 µL cDNA generated via first strand synthesis was diluted by addition of 44 µL injection water to produce the necessary volume for qPCR analysis of all samples with seven primer pairs and technical replicates. Each well was filled with 4 µL diluted cDNA, 6 µL 2x SYBR® Green master mix (Applied Biosystems ®), 1 µL forward primer (10 µM), and 1 µL reverse primer (10 µM). The 96 well plate was centrifuged at 1,000 x g for 1 min before running the qPCR. The real-time PCR was run on a ViiA7TM machine (Applied Biosystems ®) and the data collected and analysed by the QuantStudioTM Real-Time PCR Software v1.3. The initial holding step started with 2 min at 50°C followed by 10 min at 95°C. The PCR stage consisted of 20 cycles of 15 sec at 95°C and 1 min of 60°C. To collect a melt curve, a final step of 15 sec at 95°C, 1 min at 60°C, and 15 sec at 95°C was added. All temperature changes were initiated at 1.6°C s-1 and were not included in the holding times. The results were analysed using the Livak method [16] with GAPDH as reference gene and the leaf tissue as calibrator sample. The relative expression levels were determined following the “Real-Time PCR Applications Guide” by Bio-Rad Laboratories, Inc. [17]. All samples were normalized against GAPDH as a reference gene,

( ) = ( , ) ( , )

𝑇𝑇 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑇𝑇 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑇𝑇 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ∆𝐶𝐶 ( ) = 𝐶𝐶 ( , ) − 𝐶𝐶 ( , )

𝑇𝑇 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 𝑡𝑡𝑡𝑡𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑇𝑇 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑇𝑇 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 𝑡𝑡𝑡𝑡𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 where is the ∆cycle𝐶𝐶 threshold, i.e.𝐶𝐶 cycle number at which− the𝐶𝐶 fluorescent signal can be detected above the𝐶𝐶𝑇𝑇 threshold. In a second step, all samples were set in relation to the calibrator sample. Here leaf tissue was chosen as calibrator sample.

= ( ) ( )

𝑇𝑇 𝑇𝑇 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑇𝑇 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 This directly translated into the∆∆ normalized𝐶𝐶 ∆𝐶𝐶 expression− ratio∆𝐶𝐶 for 100% amplification efficiency:

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Establishment and analysis of plant cell suspensions for cyclic peptide production

2 = −∆∆𝐶𝐶𝑇𝑇 This method compensates for differences𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 in sample𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 preparation𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 by𝑒𝑒 𝑟𝑟𝑟𝑟 using𝑟𝑟𝑟𝑟𝑟𝑟 a reference gene and allows for quantitative comparison with a calibrator tissue.

2.2 Results

2.2.1 Establishment of in vitro cultures for cyclotide analysis and tissue culture

Oldenlandia affinis, Petunia hybrida, Nicotiana benthamiana, and Clitoria ternatea seeds germinated after 6-8 days on solid germination medium. Shoot growth after one week is shown in Figure 7. The germination efficiency was calculated to be around 95% for O. affinis, N. benthamiana, and C. ternatea, whereas all P. hybrida seeds germinated. In contrast, germinating Hybanthus enneaspermus seeds aseptically proved difficult, as seeds swelled during the disinfection process and produced fungal contamination when placed on germination medium. Consequently, the disinfection parameters were varied to reduce fungal contamination without harming the seeds. To prevent swelling, the general contact time with the liquid compounds was shortened. The most important parameter influencing the contamination rate was the incubation time of the seeds in NaOCl. Seeds incubated for 5 minutes developed visible fungal contamination after 8 days while seeds incubated for 15 minutes failed to germinate at all. In contrast, seed that had been sterilized for 10 minutes began to germinate fungal free after 12 days, with ~50% of the seeds germinated by day 25. The slow germination rate also equated to a slow growth rate in comparison to the other cultivated plants. Of the tested plant species, P. hybrida displayed the fastest growth rate, followed by N. benthamiana. At day 25 the P. hybrida plantlets had filled the whole Petri dish, whereas O. affinis plants had reached the lid. Despite the slow germination and growth, healthy in vitro plants were established from H. enneaspermus seedlings.

Figure 7 | In vitro germination after one week on agar medium. A) O. affinis. B) P. hybrida. C) H. enneaspermus.

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2.2.2 Callus formation for suspension initiation

All explants produced friable calli with clear differences in growth rate and morphology depending on species, tissue type, and phytohormone concentrations. O. affinis displayed the slowest callus growth with infrequent organogenesis symptomatic of further optimisation potential. Subculturing with selection for friable, fast growing calli improved the callus quality remarkably as shown in Figure 8.

Figure 8 | O. affinis callus. A) Three weeks after initiation from sterile explants. B) After six months of subculturing with selection for friable, fast growing calli.

The callus induction frequency of O. affinis was close to 100% for leaf, stem, and root explants. Root explants especially displayed impressive callus growth considering the small amount of available starting material and wound surface area, as the roots were very thin and short as shown in Figure 9A. The highest callus growth rate was observed for H. enneaspermus leaf explants, resulting in massive green friable calli as displayed in Figure 9C. N. benthamiana, and C. ternatea explants produced light green calli after two to four weeks as shown in Figure 9B and Figure 9D. Harvesting or subculturing was done after four weeks, when cracks started forming in the agar medium. [18]. Calli can be initiated without illumination; however, all calli generated in this work were initiated at 35 µmol m 2 s 1 photon density under a 16:8 light-dark cycle as positive effects of illumination on cyclotide production⁻ ⁻ was reported by Dörnenburg & Seydel [19].

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Figure 9 | Callus formation under 35 µmol m 2 s 1 photon density at a 16:8 light-dark cycle. This figure shows different species and timepoints to provide a general callus formation overview. The tested conditions are reported in Table 3. A) O. affinis wound tissue from different plant parts for callus initiation.⁻ B)⁻ N. benthamiana leaf callus after three weeks. C) H. enneaspermus leaf callus after eight weeks and one passage. D) C. ternatea stem callus after four weeks.

The screening conditions tested for optimised callus growth of H. enneaspermus are presented in Table 6. Callus induction from leaf explants was generally more successful compared to stem explants. However, the two best conditions induced calli from leaf and stem explants with 100% induction frequency. BA was necessary for callus induction and worked best when paired with a high concentration of either 2,4-D or NAA, with NAA producing the more friable calli.

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Table 6 | H. enneaspermus callus induction. Phytohormones Condition Leaf explants Stem explants [mg L-1] Induction Induction 2,4-D BAP NAA frequency Morphology frequency Morphology [%] [%] A - - 1.5 0 - 0 - B - - 2 0 - 0 - C - 2.5 - 0 - 0 - D 2.5 - - 0 - 0 - Bright lime Light green E 1.5 1 - 100 green hard 17 and beige hard callus callus F - 0.5 1 0 - 0 - G - 1.5 1 0 - 0 - H - 2 1 0 - 0 - Bright green I - 2 0.5 57 0 - friable callus J - 0.25 0.25 0 - 0 - Bright green K - 0.5 0.25 92 hard callus 0 - with shoots Beige and L - 2 0.25 40 green friable 0 - callus Green hard Green hard M - 0.5 0.48 100 100 nodular callus nodular callus with shoots Large green Large green N - 0.9 1.86 100 100 friable callus friable callus Note: All phytohormone concentration were tested on two separate agar plates with 15 explants each. Every medium contained full strength MS (M519, Phyto Technology Lab), 30 g L-1 sucrose, 4 g L-1 PhytagelTM (Sigma-Aldrich) and was adjusted to pH 5.8 using 1 M KOH.

C. ternatea callus induction conditions tested are presented in Table 7. Initial results produced by Dr. Georgianna Oguis [20] identified 1 mg L-1 NAA as a promising starting point. Consequently, several combinations of 1 mg L-1 NAA with either 2,4-D or BA were tried. The best calli for cell suspension induction were achieved with a combination of 1 mg L-1 NAA and 1 mg L-1 BA. As with all observed species, callus induction of leaf explants produced higher induction frequencies compared to stem explants.

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Table 7 | C. ternatea callus induction. Phytohormones Condition Leaf explants Stem explants [mg L-1] Induction Induction 2,4-D BAP NAA frequency Morphology frequency Morphology [%] [%] Beige friable Beige hard A - 1 1 100 100 callus callus Beige friable Light green B - 1.5 1 88 97 callus with hard callus shoots Light green Light green C - 2 1 100 100 and beige hard callus friable callus Light green Lime green D 1 - 1 100 and white 63 friable callus friable callus Green and Light green E 1.5 - 1 100 97 white friable hard callus callus Light green Green hard F 2 - 1 100 10 hard callus callus Note: All phytohormone concentration were tested on two separate agar plates with 15 explants each. Every medium contained full strength MS (M519, Phyto Technology Lab), 20 g L-1 sucrose, 8 g L-1 Phyto agar (Duchefa Biochemie B. V.) and was adjusted to pH 5.8 using 1 M KOH.

2.2.3 Initiation and maintenance of suspension cultures for cyclotide analysis and production

Suspension cultures of C. ternatea, H. enneaspermus, N. benthamiana, O. affinis, and P. hybrida were attained from friable calli as shown in Figure 10. The macroscopic and microscopic morphology changed drastically within the first three weeks after suspension induction. The cells started out as callus clumps and over time were sheared off into the medium to form free-floating cells and cell aggregates. After roughly eight passages in adjusted medium, as reported in Table 5, all species formed stable suspension cultures without visible changes over time. Growth was assessed visually via colour, opacity, and settling speed. Brown colour indicated dying cells, whereas high opacity and slow settling indicated healthy cultures growing in small aggregates. Growth curves were recorded via dry weight, fresh weight, optical density, packed cell volume, and settled cell volume. Non-invasive data collection was chosen to prevent contamination during the sampling process but produced large standard deviations as evident in Figure 12. Sterility was especially important for cultures that were required for multiple subsequent experiments. The caveats of the experimental design should be considered when interpreting the data. 49 | Page

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Figure 10 | Initiation of O. affinis suspension from fresh, friable green calli. The calli were picked from the agar plate, cut into small pieces and transferred into a 125 mL Erlenmeyer flask. Then the flask was filled up to 43 mL with fresh medium and placed on a shaker at 110 rpm with the lid slightly unscrewed to allow oxygen transfer.

Initial N. benthamiana cultures were treated with 1-naphthaleneacetic acid (NAA) and kinetin following reports in the literature [21, 22]. After a few trials to reduce the necessary hormones, 0.2 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) proved enough to sustain healthy suspension cultures without any obvious downside. O. affinis cultures showed high stability (i.e. no changes in morphology and cyclotide expression as assessed via microscopy and MALDI-TOF analysis) and a doubling time of 2.19 d while growing as fine cell suspension as shown in Figure 11. Aggregation into larger clumps started at a dry weight of around 7 g L 1. The maximum density of around 11 g L 1 dry weight (equivalent 1 to a packed cell volume of 60%)⁻ was reached after six days using an inoculation⁻ density of 2 g L . 1 This correlated very well with a doubling time of 2.24 d and a maximum density of around 11 g L⁻ after six days for illuminated cultures reported by Seydel et al. [23]. Remarkably, O. affinis cultures⁻ consisted of two different types of cell morphologies even after several months of subculturing. One morphology was characterized by its elongated cells that form a chain like structure, whereas the second morphology displayed round closely packed cells. Intermediate stages were also observed as displayed in Figure 11C. The 100x magnification was chosen to display many cells and show their morphology at the same time. Autofluorescence of healthy O. affinis suspension cells is shown in Figure 1B and Figure 11D. The 200x magnification was chosen to detect weak fluorescence signals and to resolve intercellular structures. The cells clearly displayed different auto fluorescence in different cell populations. Autofluorescence can potentially be used to assess the status of a culture and perhaps even be linked to growth rate or secondary metabolite production.

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Figure 11 | O. affinis cell suspension. A) Growth after initiation from illuminated leaf calli measured as dry weight. Error bars indicate standard deviation of the biological replicates, n=3. B) Growth after initiation from illuminated leaf calli measured as packed cell volume. Error bars indicate standard deviation of the biological replicates, n=3. C) Healthy culture after 8 weeks of cultivation. Brightfield, 100x magnification. D) Healthy culture after 8 weeks of cultivation. Pseudo colour multi-channel image at 200x magnification. The recorded channels were differential interference contrast (DIC), 594 nm excitation (red colour in image), 358 nm excitation (blue colour in image), and 488 nm excitation (green colour in image).

Suspension cultures of C. ternatea and H. enneaspermus were established and screened for optimised conditions, because information available in the literature was limited. Growth curves were recorded via the cell settling method. Relative growth was chosen to make cultures with different starting cell volumes more easily comparable via plotting. It was calculated by dividing the measured cell volume by the initial cell volume and multiplying the result by 100. The phytohormone compositions tested had a strong influence on growth and organogenesis shown in Figure 12. H. enneaspermus was especially prone to spontaneous organogenesis. Out of nine identical cultures treated with condition A, three formed a dense root network. Condition PC and G (reported in Table 4) induced leaf-like structures as visible in Figure 12. Selection for fast growing cultures without organogenesis in condition A and condition K resulted in cultures that were still stable after one year and were used for scale-up, elicitation and transformation as presented in Chapters 3, 4, and5.

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Figure 12 | Screening for optimised growth conditions of C. ternatea and H. enneaspermus. Growth was measured relative to the starting cell volume via cell settling and produced large variance and artifacts. The photos were taken on day 7 after passaging.

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2.2.4 Cyclotide production in plant tissues

To monitor cyclotide production in plant tissues, a reliable, efficient extraction protocol was needed. Cyclotide extraction via lyophilisation of a cell pack free from medium followed by automated grinding with steel grinding balls in a 50% ACN, 1% formic acid solution proved to be the best available method and was used for all suspension cultures. Compared to manual grinding of shock frozen cells and sonication of cells in suspension, this method was less labour intensive, allowed parallel determination of dry weights and reliably produced MALDI-TOF traces with high signal to noise ratio. All cyclotide masses reported in this chapter were obtained by reading out the one charged monoisotopic peak as observed during MALDI-TOF analysis.

2.2.4.1 Cyclotide production in Oldenlandia affinis

Cyclotide production was assessed in O. affinis leaves, stems, roots, calli and suspensions as shown in Figure 13 and Figure 14. The leaves of O. affinis plants contained many cyclotides, with kalata B2 constituting the major peak. Interestingly, the kalata B2 concentration was low in stems and not detectable in roots, calli, and suspensions. The cyclotide profile varied in individual leaves, with age, season and stress level (data not shown); however, it stayed characteristic for leaf tissue and was always easily distinguishable from stem, root, callus or suspension profiles. The highest kalata B1 concentrations were recorded in stem and callus samples. Kalata B7 was abundant in leaves and stems, but not present in roots. Cyclotide production in roots differed significantly; a peak at 3139.4 Da, later identified as cycloviolacin O2, was detected in no other plant part. A peak at 3144.4 Da, later identified as kalata B23, was also detected in the stem, but only in miniscule amounts.

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Figure 13 | Cyclotide production in O. affinis plant parts. A) MALDI-TOF traces of representative tissue samples normalized to an internal control peptide. B) Cyclotide concentrations in different tissues. The bars are normalized to 100% for the tissue that produced the highest amount of that cyclotide. Error bars indicate standard deviation of the biological replicates, n=3.

Establishment of suspension cultures started with leaf explants for callus growth to then transfer the calli into liquid medium. Each step displayed a characteristic cyclotide profile. To illustrate the tissue- and time-dependant cyclotide production of O. affinis, the five most prevalent cyclotides are presented in Figure 14 and were normalized to 100% in the most abundant sample. This depiction makes it easy to spot differences but is not quantitative between different cyclotides i. e. the same height of a kalata B1 and a kalata B15 bar does not mean that the leaf sample had the same concentration of kalata B1 and kalata B15. However, twice the height of the kalata B1 bar in the stem samples compared to the leaf samples indicates twice the concentration. The kalata B1 peak is barely visible in the leaf trace in Figure 14A due to the very strong kalata B2 peak; however, Figure 14B illustrates that there was strong kalata B1 production in leaves, by a factor of 13 higher than after one week in suspension.

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Establishment and analysis of plant cell suspensions for cyclic peptide production

Callus profiles displayed major differences compared to the leaf profiles as shown in Figure 14. Whereas the production of most cyclotides diminished in callus, kalata B1, cycloviolacin O2, and a peak at 3184.5 Da, later identified as kalata B22, were most prevalent in callus. O. affinis cell suspensions started with the exact same profile as calli. This was not surprising as small callus pieces were directly moved into liquid medium to initiate cell suspension cultures. However, as the cells adapted to the new environment, the cyclotide profile changed. After one week, the production of kalata B1, cycloviolacin O2 and kalata B22 had dropped to less than one tenth of the initial value. At the same time kalata B23 production was initiated. Over the next three weeks, kalata B1 vanished completely, while cycloviolacin O2, kalata B23, and kalata B22 production increased. This upward trend did not continue after four weeks, but rather oscillated around a low base value for at least three years (data not shown).

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Establishment and analysis of plant cell suspensions for cyclic peptide production

Figure 14 | Cyclotide concentration in different O. affinis tissues and the development over time in suspension. The leaves were harvested from in vitro plants. The callus sample was taken four weeks after callus initiation from leaves. The suspension samples were taken one, two, three, and four weeks after suspension initiation from callus. A) MALDI-TOF traces of leaf, callus, and suspension samples. The suspension samples were taken after one, two, three, and four weeks. All suspension traces (i.e. Suspension, 14 days, 21 days, and 28 days) are magnified by 100x to enable visibility of the peaks. B) Cyclotide concentrations in different tissues. The bars are normalized to display 100% for the tissue that produced the highest amount of that cyclotide. This way of presenting was chosen to plot cyclotides with very different concentrations in the same bar chart without losing visibility. Error bars indicate standard deviation of the biological replicates, n=4.

2.2.4.2 Discovery of cycloviolacin O2, kalata B22 and kalata B23 in Oldenlandia affinis

MALDI-TOF measurements revealed the presence of many different cyclotides in O. affinis plants, as reported by Jennings et al. [24]. The composition of cyclotides changed significantly in calli when compared to whole plants with the disappearance of kalata B2 as the most prevalent feature. Upon transition to suspension cultures the production of nearly all cyclotides was reduced in a culture-age dependent process, with the exception of a cyclotide detected at around 3140 Da, previously identified as kalata B18 [25]. As this peak was presumably a cyclotide with a tendency to resist downregulation in suspension cells, further investigations were warranted. On closer inspection

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Establishment and analysis of plant cell suspensions for cyclic peptide production this MS signal peak turned out to be a double peak consisting of an unknown peptide and another peptide initially recognized by mass as kalata B18 following earlier reports [25]. By fractionation and tandem MS analysis the unknown peptide was identified as cycloviolacin O2 as presented in Figure 15. Fragmentation was incomplete (e.g. b25, b26, b27 …); however, digestion by endo-GluC of bracelet cyclotides often leads to incomplete fragmentation during tandem MS [26], possibly explaining the missing b and y-ions.

Figure 15 | Tandem MS of cycloviolacin O2 enzymatically digested by endo-GluC.

Having identified the cycloviolacin O2 peptide in O. affinis extracts, the next step was the search for evidence at the transcript level. An examination of an in house generated O. affinis transcriptome revealed the sequence encoding for the 3’ part of the cycloviolacin O2 precursor and is shown in Figure 16 (transcript ID: O_affT2.1_36190_c0_g1_i1). Complementary DNA cloning for quantitative PCR experiments confirmed the full-length gene for cycloviolacin O2 in O. affinis.

Figure 16 | Transcript of cycloviolacin O2 found in O. affinis. The C-terminus displays elements typical for the Rubiaceae family and differs from the C-termini found in Violaceae transcripts.

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Establishment and analysis of plant cell suspensions for cyclic peptide production

Amplification of the genes utilizing primer pairs designed for kalata B17 and kalata B18 also revealed two new cyclotide sequences here named kalata B22 and kalata B23. Both peptides have masses identical to kalata B17 and kalata B18 respectively and can therefore not be distinguished using single mass spectrometry. The sequence of kalata B22 as found in the in-house O. affinis transcriptome is presented in Figure 17. The annotated IDs were: kalata B22 transcript ID: O_affT2.1_11355_c0_g1_i1; kalata B23 transcript ID: O_affT2.1_2212_c0_g1_i1.

Figure 17 | DNA and corresponding peptide sequence of kalata B22 as found in O. affinis.

The DNA and corresponding peptide sequence of kalata B23 is displayed in Figure 18. As the DNA sequences presented in Figure 17 and Figure 18 were the only fragments amplified when using primers designed for kalata B17 and kalata B18, it is likely that kalata B22 replaced kalata B17 and kalata B23 replaced kalata B18 in the tested cell lines.

Figure 18 | DNA and corresponding peptide sequence of kalata B23 as found in O. affinis.

2.2.4.3 Cyclotide production in Clitoria ternatea

The most promising conditions for callus and suspension growth of C. ternatea were analysed for cyclotide production and compared to leaf, branch, and root tissue. The results are presented in Figure 19. All tissue samples contained many cyclotides. The highest overall concentration was recorded in leaves, the lowest in roots. Unlike O. affinis, C. ternatea suspension cells retained a strong cyclotide production even after several weeks. Cter O, cliotide T19, Cter Q, Cter P, and Cter A were

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Establishment and analysis of plant cell suspensions for cyclic peptide production most abundant in leaves, cliotide T15 was not present in leaves and was most abundant in suspension cells. The cyclotide production in suspension cells was higher than in roots, stems or calli. The suspension samples presented here were four weeks old, the plant samples were collected from a 6 months old in vitro plant grown in solid MS medium. All MALDI traces were normalized using the monoisotopic peak of the spiked peptide at 2891.2 Da. The cyclotides were identified according to the masses reported in literature [27, 28].

Figure 19 | C. ternatea tissue dependent cyclotide concentration. The displayed suspension samples were taken four weeks after suspension initiation from callus. A) Exemplary MALDI traces of the tested tissues. The intensity is plotted relative to the intensity of an internal control peptide and therefore unitless. B) Comparison of the relative production of the six most abundant cyclotides. Error bars indicate standard deviation, n=4.

2.2.4.4 Cyclotide production in Hybanthus enneaspermus

After developing protocols to induce callus and suspension growth of H. enneaspermus, the cyclotide expression was analysed. Cyclotide production varied in H. enneaspermus leaf, branch,

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Establishment and analysis of plant cell suspensions for cyclic peptide production root, callus, and suspension samples as shown in Figure 20. The highest production of cycloviolacin O2 and cycloviolacin O13 was recorded in callus samples with suspension cells in second place. Leaves especially displayed a low cyclotide production compared to the other samples. Only roots produced Cter A and an unidentified putative cyclotide with the one-charged monoisotopic mass of 3148.4 Da in considerable quantities. Hyen E, the most prevalent cyclotide in leaves, was not present in roots, calli or suspensions. The suspension samples presented here were four weeks old, the plant samples were collected from a 6 months old in vitro plants grown in solid MS medium. All MALDI traces were normalized using the monoisotopic peak of the spiked peptide at 2891.2 Da.

Figure 20 | H. enneaspermus tissue dependent cyclotide concentration. The displayed suspension samples were taken four weeks after suspension initiation from callus. A) Exemplary MALDI traces of the tested tissues. B) Comparison of the relative production of the five most abundant cyclotides. Error bars indicate standard deviation, n=3.

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Establishment and analysis of plant cell suspensions for cyclic peptide production

One third of the suspensions cultured under condition G and PC developed white root-like structures. The MALDI analysis of the cyclotide extract of these organs compared to free-floating cells of the same suspension and in vitro roots is presented in Figure 21. The three cyclotides with the strongest MALDI signal in the organs were cycloviolacin O2, Cter A, and an unidentified putative cyclotide at m/z 3112.3 Da. Screening for stable cell lines, optimal phytohormone conditions and adjusted shear stress resulted in H. enneaspermus cell cultures that did not differentiate or change morphology over time.

Figure 21 | MALDI traces of cyclotides found in suspension cells, root-like structures formed via organogenesis in suspension, and in vitro roots of H. enneaspermus. All samples were normalized against an internal control.

The cyclotides belonging to the observed masses were identified with the help of Qingdan Du and are presented in Table 8.

Table 8 | Most abundant cyclotides detected in H. enneaspermus tissues. Cyclotide Observed monoisotopic one Sequence charged mass [Da] cyO13 3123.4 G-IPCGESCVWIPCISAAIGCSCKSKVCYRN cyO2 3139.3 G-IPCGESCVWIPCISSAIGCSCKSKVCYRN hyen E 3233.4 GV-PCGESCVYIPCFTGIINCSCRDKVCYNN Cter A 3268.5 GVIPCGESCVFIPCISTVIGCSCKNKVCYRN Conserved cysteines forming the CCK-motif are formatted bold and highlighted yellow.

The cyclotide sequences of cycloviolacin O13, cycloviolacin O2, hyen E, and Cter A were detected in the transcriptome, confirming their existence in H. enneaspermus. Cycloviolacin O2 and hyen E were additionally identified via MS/MS after digestion and NMR analysis. No known cyclotide matched the one charged mass of 3148.4 Da observed in stem and root samples.

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Like O. affinis, H. enneaspermus produced cycloviolacin O2 in callus tissue and suspension cells, whereas the production in leaf tissue was neglectable. The strong cycloviolacin O2 peak made the identification of hyen F (3144.2), hyen G (3145.2), and hyen M (3142.3) via MALDI-TOF analysis impossible, due to overlapping peaks. As in C. ternatea, the expression remained stable and at a very high level in suspension. Unlike C. ternatea, the roots produced the largest cyclotide amount of the plant tissues. Curiously, Cter A is solely produced in roots in H. enneaspermus, whereas C. ternatea displayed the largest concentration in leaves. Transcriptome search confirmed the presence in both species and revealed distinct codon usage as shown in Figure 22

Figure 22 | Alignment of Cter A sequences found in C. ternatea and H. enneaspermus.

2.2.5 Real-time PCR of O. affinis RNA

Cyclotide biosynthesis requires cyclotide and AEP gene expression. AEPs are necessary for the C-terminal processing of cyclotides as described in Section 1.2.4.1. The decline of cyclotide biosynthesis in O. affinis suspension cells could have been due to a decrease in cyclotide or AEP mRNA levels. To determine this and the great variation of cyclotide production between leaf and callus tissue, RNA was extracted from fresh plant leaves, callus tissue and suspension cells over a period of four weeks for real-time PCR analysis. Cyclotides were extracted from the same samples to compare transcript and peptide levels. Fresh plant leaves produced high RNA yields of up to 300 ng µL-1. Callus tissue on the other hand, produced very low concentrations of around 10 ng µL-1. Some RNA samples had to be concentrated via lyophilization to achieve the necessary starting mass for cDNA synthesis. Suspension samples were easy to handle and produced good RNA quantities. However, the low amount of available biomass in the first days after suspension initiation was a problem if several tests were required. All samples resulted in relatively pure RNA as depicted in Figure 23. The concentration of all RNA samples measured at the spectrophotometer were within the linear range of 10.0 ng/µL and 3700 ng/µL.

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The purified RNA was consequently translated to cDNA to be analysed in a SYBR® Green based quantitative polymerase chain reaction (PCR). Relative expression levels of OaAEP1, OaAEP2, OaAEP3, kalata B1, kalata B2, and cycloviolacin O2 mRNAs were investigated. GAPDH was selected as Figure 23 | Agarose gel of RNA extracted from O. affinis housekeeping gene and the primer pair was designed suspension cells. The leftmost lane was a 1 kB ladder. according to Gruber et al. [29]. To achieve high specificity, the 3' bases of the forward primers were anchored at the wobble base of a codon unique to the target cyclotide. The reverse primer was placed in the 3' untranslated region (UTR) of the transcript. The primer pairs were cloned, amplified and the amplification products sequenced to verify their target specificity. The final primer sequences are presented in Table 9.

Table 9 | Primer pairs used for quantitative real-time PCR of O. affinis transcripts. Targeted mRNA Direction Primer sequence Forward 5'-AATCGGACGTCTCGTTGCTA-3' GAPDH Reverse 5'-GGTAGTGCAACTGGCATTGG-3' Forward 5'-AGCCTCTTGTTGATGACTGG-3' OaAEP1 Reverse 5'-CCATCTGTTCCTCAGAGATTCC-3' Forward 5'-CAGCCTCTGGCTGATGACTGG-3' OaAEP2 Reverse 5'-CCATCTGTTCCTTCGTGATTCC-3' Forward 5'-CAACAGCATCAAACTTGTCG-3' OaAEP3 Reverse 5'-AGGACGGATTGTATTCAGAACC-3' Forward 5'-GGACTTCCAGTATGCGGTGAG-3' Oak1 Reverse 5'-TTGGTGTCGGCCTTCTCATTC-3' Forward 5'-ATGCAGCTCAAAGGTCTTCC-3' Oak4 Reverse 5'-AATTACCAGACACAAGGGCC-3' Forward 5'-GCATCAGCTCGGCGATTG-3' CycloviolacinO2 Reverse 5'-CAACCGAATCGTACTAGATACAG-3' Note: The Oak1 gene encodes kalata B1, the Oak4 gene encodes kalata B2. The GAPDH housekeeping gene was used as reference.

The results are presented in Figure 24. The data set for the cycloviolacin O2 gene is plotted on the right-hand ordinate, all other sets are plotted on the left-hand ordinate. The C-terminal cyclotide processing is aided by several enzymes in O. affinis of which OaAEP1 and OaAEP3 are the most important ones [13, 30]. OaAEP2 was also investigated to evaluate if a difference in expression of ligating to non-ligating AEP was present. The transcript levels of OaAEP1, OaAEP2, and OaAEP3 were analysed to assess the cyclization potential of the respective tissue type. As can be seen in Figure 24, all three enzymes displayed similar transcript levels. Indicating, that C-terminal processing of cyclotides was possible in all examined samples. One week after suspension initiation, OaAEP1, OaAEP2, and OaAEP3 expression experienced an upregulation by a factor of three to four compared

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Establishment and analysis of plant cell suspensions for cyclic peptide production to plant leaves. Two weeks after suspension initiation, the transcription dropped to roughly half of the plant leaf level and remained there until the end of the experiment.

6 3000 OaAEP1 OaAEP2 OaAEP3 Oak1 Oak4 CycloviolacinO2

4 2000

2 1000 Relative mRNARelative transcript expression levels [-]

0 0

Callus 14 days21 days28 days Callus 14 days21 days28 days Callus 14 days21 days28 days Callus 14 days21 days28 days Callus 14 days21 days28 days Callus 14 days21 days28 days Suspension Suspension Suspension Suspension Suspension Suspension

Figure 24 | Expression levels of mRNA transcripts necessary for cyclotide production relative to their expression level in leaf tissue and normalized to the housekeeping gene GAPDH in O. affinis. The data set for the cycloviolacin O2 gene is plotted on the right-hand ordinate, all other sets are plotted on the left-hand ordinate. The experiment included four biological and two technical replicates for each data point. Error bars indicate standard deviation.

The two cyclotide mRNAs experiencing the most obvious changes in expression between leaf, callus, and suspension were Oak1 and Oak4. Oak4 could only be detected in vanishingly small amounts in callus as well as in all suspension samples. Oak1 was still expressed in callus tissue at half the plant leaf level and subsequently ceased to be expressed in suspension. The relative expression of cycloviolacinO2 was very high in callus and suspension samples with levels of 2000 times the level detected in leaves.

2.2.6 Suspension type cyclotides

The study of cyclotide production in plant tissues revealed a striking similarity of most cyclotides present in suspensions cultures of different species investigated in this work as shown in Table 10, here named suspension type cyclotides. All suspension type cyclotides were exactly 30 amino acids long, out of which 21 were identical, four were very similar and five were different in terms of amino acid charge and size. Four not conserved amino acids were always found in position 15-18 in loop three, and the fifth not conserved amino acid was either a lysine or serine in position 23 in loop 5. Loop one and six had 100% conservation. Many of the amino acids in loop one and six contribute to the hydrophobic patch that is important for cytotoxicity [31].

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Table 10 | Cyclotide sequences alignment.

Cyclotide m/z+ Sequence Species Cter O 3084.5 GIPCGESCVFIPCITGIAGCSCKSKVCYRN Ct Cter P 3098.5 GIPCGESCVFIPCITAAIGCSCKSKVCYRN Ct cyO13 3123.4 GIPCGESCVWIPCISAAIGCSCKSKVCYRN Ct, He cyO2 3139.3 GIPCGESCVWIPCISSAIGCSCKSKVCYRN Ct, Oa, He kalata B23 3144.4 GIPCGESCVYIPCLSTVIGCSCSNKVCYRN Oa Phyb E 3152.3 GIPCGESCVWIPCISGVQGCSCSNKICYRN Ph Conservation GIPCGESCV+IPC+ GCSC +K+CYRN Note: Alignment of cyclotide sequences found in suspension cultures of C. ternatea (Ct), H. enneaspermus (He), O. affinis (Oa) and P. hybrida (Ph). Dark background indicates identical amino acids, light grey similar substitutions and white substitutions without a high degree of similarity.

It is noteworthy, that these cyclotides do not only belong to different species, but to very different plant families. C. ternatea belongs to the Fabaceae and produced Cter A, Cter O, and Cter P as primary cyclotides in suspension, of which Cter O and Cter P fit the suspension type perfectly and Cter A has minor variations and 31 instead of 30 cyclotides. H. enneaspermus belongs to the Violaceae and produces cycloviolacin O2 and cycloviolacin O13 in suspension, both of which are suspension type cyclotides. O. affinis is a member of the Rubiaceae and produces the suspension type cyclotides cycloviolacin O2 and kalata B23 in suspension. Finally, P. hybrida, part of the Solanaceae, produced only Phyb E in suspension, a cyclotide belonging to the suspension group. Modelling of the sequences in PyMOL revealed very high three-dimensional congruity and similar surface properties as shown in Figure 25. The previously solved structure of cycloviolacin O2 was used as template [32]. The charge and hydrophobicity was highlighted using the YRB scheme [33], where all carbon atoms that are not bound to nitrogen or oxygen atoms are highlighted yellow, charged oxygen atoms of glutamate and aspartate are highlighted red, charged nitrogen atoms of arginine and lysine are highlighted blue, and the rest is white. Kalata B1 is shown as example of an O. affinis cyclotide, not belonging to the suspension type.

Figure 25| Front and back surface view of the suspension type cyclotides Cter O, Cter P, cycloviolacin O2, kalata B23, and Phyb E. Additionally, kalata B1 found in O. affinis leaves, not belonging to the suspension type, is shown. Carbon atoms that are not bound to nitrogen or oxygen atoms are highlighted yellow, charged oxygen atoms of glutamate and aspartate are highlighted red, charged nitrogen atoms of arginine and lysine are highlighted blue, and the rest is white. The cyclotides were aligned by superimposing the conserved cysteine residues.

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2.3 Discussion

2.3.1 Establishment of plant cell suspensions

Plant based production systems are always subject to biological changes and can never be fully calculated and predicted. Different conditions and cell lines need to be tested and screened using parameters close to the intended application. Therefore, the first step was the initiation and optimisation of healthy and stable callus and suspension cultures of Oldenlandia affinis, Petunia hybrida, Nicotiana benthamiana, Hybanthus enneaspermus, and Clitoria ternatea. Callus growth and morphology was improved through selection and condition screening. Only limited information for H. enneaspermus suspension initiation without specific details of the medium composition and growth characteristics was available at the start of this work [34]. Parallel to the results presented here, Sathish et al. reported a H. enneaspermus cell suspension culture protocol using liquid MS medium supplemented with 2.0 mg L-1 2,4-D resulting in 0.57 g DW in 35 mL after 28 days equalling to a doubling time of 21 days [35]. The H. enneaspermus suspensions observed here grew much faster with a doubling time of 2 days. Possible influencing factors were the higher starting density used here (7 g FW callus vs. 3 g FW callus) and the different phytohormone concentrations. No published protocols for C. ternatea suspensions were available at the time of writing. One of the first detailed protocols to establish H. enneaspermus and C. ternatea cell suspensions is presented here. A cheaper and easier to prepare medium, compared to most reported media, containing 0.2 mg L-1 2,4-D as the only phytohormone without any noticeable performance drawbacks was found for N. benthamiana suspensions. Sukenik et al., for example, used 2 mg L-1 2,4-D and 0.1 mg L-1 kinetin for N. benthamiana suspensions [36]. O. affinis proved to be perfectly suited to be grown in suspension, forming fine stable suspension cultures that grew up to 11 g L 1 dry weight within six days, comparable to reports of Dörnenburg et al. [6, 23, 25]. The young cultures⁻ of C. ternatea and H. enneaspermus displayed large variations in doubling time; however, culture growth like O. affinis seemed plausible if selection for growth is carried out over several months. Cultures of all species were grown at 28°C and 35 µmol m 2 s 1 photon density under a 16:8 light-dark cycle in an incubator and passaged every seven days. ⁻ ⁻ Beyond media composition, other factors play a role in culture success and consistency. In suspension cultures, organogenesis is typically undesirable. However, organogenesis was linked to higher cyclotide production in O. affinis [6]. Increased cyclotide production was also reported for green root-like structures in Viola uliginosa [7]. Organogenesis was observed in one third of H. enneaspermus suspensions cultivated with condition G and PC. The white root-like structures did 66 | Page

Establishment and analysis of plant cell suspensions for cyclic peptide production produce a higher number and concentration of cyclotides compared to the cells floating in the same culture. Screening for adjusted conditions enabled growth of H. enneaspermus and C. ternatea in suspension without organogenesis. Contamination was a constant risk every time a suspension culture was opened for passaging or sampling. Handling without sampling is recommended, if possible. All species tested here are promising candidates for applications utilizing plant cell suspensions because of their growth, stability and handling. Growth is an important characteristic of plant cell suspensions, even though it does not always coincide with higher yields as faster growing cells often produce lower amounts of secondary metabolites. Different methods for growth quantification were tested with all species. If culture volumes allowed sampling of 2-5 mL every other day and if quantification was necessary, dry weight proved to be an easy and reliable, albeit slow, measurement to monitor growth. Packed cell volume measurements required the same sampling volumes as dry weight measurements with the advantages of fast measurements within 10 minutes and the possibility to execute further tests with the sample and the drawback of lower accuracy. Settled cell volume allowed non-invasive growth measurements without sampling but produced large variances and artefacts if large aggregates or organs were present as represented in Figure 12. It was concluded that growth measurement via settled cell volume adapted from Mustafa et al. [37] was not ideal for small volumes and non-homogeneous cultures. The larger the total cell volume and the more homogeneous the culture, the more accurate the settled cell measurement became. In conclusion, growth was measured in different ways, none of which is ideal. If no quantification of exact growth is necessary, non-invasive measures are always preferable. After maintaining cultures for several months, the familiarity with the cell line often allows rough growth estimations via visual inspection.

2.3.2 Cyclotide production in plant tissues

Cyclotides have been characterized in many plant species; however, rarely with the goal of cyclotide production in mind. Here, cyclotide production in O. affinis, C. ternatea, and H. enneaspermus tissues was investigated. O. affinis is used in African traditional medicine to induce labour, a practice that lead to the initial discovery of cyclotides [38]. This thesis on O. affinis cell suspension research builds on work of Dörnenburg et al. [23, 39-41]. C. ternatea and H. enneaspermus have economical value, possibly related to their cyclotide production. H. enneaspermus is used as aphrodisiac, treatment of urinary infections, gonorrhoea, asthma, and many more conditions in traditional Indian medicine [42]. The connection of these properties to cyclotides is currently researched in our group. Ethanol extracts of C. ternatea are marketed as a “bee- 67 | Page

Establishment and analysis of plant cell suspensions for cyclic peptide production friendly” insecticide (Sero-X®, Innovate AG). In our group, we identified the cyclotide-containing fraction as the active fraction of Sero-X®. In this work, cyclotide production analysis in wildtype C. ternatea, H. enneaspermus, and O. affinis was the first step in assessing the viability of the production of pharmaceutical and insecticidal peptides in plant tissues and yielded insights into the biosynthesis of cyclotides. Leaves, stems, roots, calli and suspensions of C. ternate, H. enneaspermus, and O. affinis were tested for their ability to produce cyclotides. Each tissue had a specific cyclotide profile, suggesting that different peptides might be best produced in different tissues and that promotor sequences might be tissue specific. Strongest cyclotide production in C. ternate was observed in leaf and suspension samples, with cliotide T15 produced exclusively in suspension. Compared to the other species, only C. ternatea produced a high number of cyclotides at high concentrations in suspension, all other species reduced the number and/or the concentration of cyclotides in suspension. Comparative promotor studies of cyclotides that are produced in suspensions and cyclotides that are not produced in suspension might help to understand cyclotide gene expression. CterO, CterP, CterA, CterQ, CliotideT15, and CliotideT19 genes in C. ternatea seem to have promotor regions that are active even in undifferentiated cells. Considering the high resistance to degradation, strong insecticidal properties, and the high concentration of cyclotides in C. ternatea suspensions, it might be possible to apply C. ternatea suspension cells directly on the field as an insecticide. This practice would circumvent the energy heavy extraction step and might add an additional fertilizing effect when the cells are broken down in the soil. Cyclotide concentrations in H. enneaspermus were highest in callus and suspension samples. However, only cycloviolacin O2 and cycloviolacin O13 were present in callus and suspension samples, whereas leaves, stems, and roots showed more than five clear cyclotide peaks. The concentration of cyclotides in leaves was suspiciously low and might have been connected to the sterile in vitro conditions. The root-like structures formed via organogenesis in suspension cultures displayed strong production of cycloviolacin O2, Cter A and several putative cyclotides similar to the strong production of cycloviolacin O2, O3, O8, O13 and Mram 8 in Viola uliginosa organogenesis reported by Slazak et al. [7]. Like in C. ternatea, a comparison of the different promotor regions and coding sequences might be informative. A comparison of Cter A sequences found in C. ternatea and H. enneaspermus revealed distinct codon usage, where polymorphisms are prevalent in the wobble position, indicating convergent evolution of Cter A. The high concentration of the hard to synthesis bracelet type cyclotide cycloviolacin O2 makes H. enneaspermus suspensions a possible system for scalable cycloviolacin O2 production. Compared to whole plants, the suspension cells of

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H. enneaspermus are easier to break open for extraction, have a higher cycloviolacin O2 concentration and produce only one other cyclotide, simplifying the purification. Cyclotide production patterns in O. affinis proved to be interesting and diverse. Every tissue had a distinct production pattern with cyclotides that were the major peak in one tissue completely missing in other tissues. In the plant, kalata B2 and kalata B15 was most prevalent in leaves, kalata B1 and kalata B7 in stems and cycloviolacin O2 and kalata B23 in roots. Initial O. affinis cell suspensions, displayed a strong kalata B1 peak and several smaller cyclotide peaks. When these cultures were scaled up in rocking motion bioreactors, the cells harvested at packed cell volume of 70% in the stationary phase contained nearly no cyclotides. Extensive testing revealed major differences in cyclotide expression and composition between different tissue types and different culture ages. O. affinis cell suspension cultures lost nearly all cyclotides over the course of three weeks. Only cycloviolacin O2 and kalata B23 were produced in low amounts after four weeks. The possible reason for this behaviour was investigated and is discussed in Section 2.3.4. Conceivable strategies to reinstate the cyclotide production are presented in Chapter 4. Another interesting behaviour was observed for kalata B2. It was the most abundant cyclotide in O. affinis leaves; however, it was not produced in roots, callus tissue and suspension cells. The cyclotide profile of roots, callus tissue and suspension cells additionally contained cycloviolacin O2. Kalata B1 was the most abundant cyclotide in calli and roots. CycloviolacinO2 mRNA expression was upregulated by a factor of two thousand in callus and suspension cells compared to leaves resulting in a stronger cycloviolacin O2 production. Expression of most other cyclotides genes was suppressed. Similar observations were made in Viola odorata, where cycloviolacin O2 was one of only two cyclotides present in roots [43] and the abundance of cycloviolacin O2 was higher in suspension cells compared to whole plants [8]. One connection of the roots and suspension cells analysed was their beige colour indicating that the photosystem was not active. Roots do not have contact with light and suspension cells are supplied with sugar as carbon source, metabolizing chemotrophically, therefore not relying on energy production of the photosystem. During the elicitation experiments presented in Chapter 4 it was shown that activation of the photosystem also brought back kalata B1 production. Light was reported before as elicitor for cyclotide production in O. affinis cell suspension [19]. In this case, light alone did not activate the photosystem in suspension, but a combination of immobilization and light did. It seems possible, that plants identify the point of attack of insects living above ground by the presence of light. This would enable some measure of site and target specificity, as insect living above ground are different from insects and nematodes living below ground that can only reach sites that light can’t reach. Cyclotides that are not coupled to the presence of light, might have high activity against pests living below ground or might have a different biological role altogether. For example,

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Establishment and analysis of plant cell suspensions for cyclic peptide production the presence of cycloviolacin O2 and kalata B23 after 55 weeks under conditions that are aseptic and ideal for growth could hint to a possibly non-defence related bioactivity. Production of native cyclotides in plant cell suspension cultures does not seem economical in wild type O. affinis cell suspensions; however, recombinant production without coeluting native cyclotides holds great promises and is tested further in Chapter 5. C. ternatea and H. enneaspermus cell suspension might be viable as insecticides or to produce cycloviolacin O2. A general trend in plant suspension cultures is reduced secondary metabolite production [44], therefore lower cyclotide concentrations were expected and some of the high concentrations observed a fortuitous find that might help understanding the regulatory pathways in undifferentiated cells and result in higher yields via elicitation or promoter region design. In summary, suspension cultures have proven to be valuable research tools for plant-based peptide production and some wild type cell lines might be practical for cyclotide production.

2.3.3 Cyclotide discovery in Oldenlandia affinis cell suspension

The analysis of cyclotide production in O. affinis suspensions revealed three unexpected cyclotide sequences that were confirmed via digestion, MS/MS, PCR, and/or transcriptome search. Cycloviolacin O2 was present in an O. affinis extract analysed by Ovesen et al. [45]. Since then, no further appearances were reported. Here, the presence of cycloviolacin O2 in O. affinis was confirmed on the peptide and transcriptome level, making O. affinis the first non-Viola species to produce cycloviolacin O2. This was an exciting find as cycloviolacin O2 has the highest cytotoxicity of all reported cyclotides [46], strong antifungal activity [47] and has so far only been reported in members of the Violaceae family (Viola odorata, Viola biflora, Viola uliginosa, Viola philippica, and Hybanthus enneaspermus). Cycloviolacin O2 is the most abundant cyclotide in V. odorata and displayed a much higher antifungal activity than the endogenous kalata B1 [47]. Like kalata B1, found in the Rubiaceae and Violaceae families, cycloviolacin O2 had an identical peptide sequence in both families, whereas the precursors were very different. This indicates convergent evolution and a strong selective pressure for this peptide sequence. Even though the CCK motif defines the scaffold of every cyclotide, there is still a high diversity in amino acid sequences, allowing many different biological functions. Considering that both species will likely face very different pests, the selective pressure for the exact same amino acid sequence might come from another function than host defence. Cycloviolacin O2 is one of the most researched cyclotides and has antifungal [47], antitumor, haemolytic [43], nematicidal activities, and moderate antibacterial properties [47]. Production of cycloviolacin O2, a member of the bracelet family, in planta is desirable as it is hard to achieve 70 | Page

Establishment and analysis of plant cell suspensions for cyclic peptide production correctly folded bracelet cyclotides via chemical synthesis [48]. Its massive upregulation in callus tissue and continued expression in cell suspension might be the first clue to biological activities that are not solely defence related. In Viola odorata, the plant cycloviolacin O2 was initially discovered in, it is one of only two cyclotides found in the roots [43], once again hinting at a special role that cycloviolacin O2 might play. Another possibility is that cycloviolacin O2 acts as a broad-spectrum antibiotic and fungicide that is active at a low concentration and is maintained for several weeks after the initial attack to prevent wound infection. This hypothesis would explain the prolonged low-level expression of cycloviolacin O2 in O. affinis after the callus was transferred into liquid medium. The overshadowing expression of many other cyclotides and cyclotide precursors might have hidden its existence in planta during mass spectrometry analysis. The specific codon usage and a C-terminus typical for Rubiaceae of CycloviolacinO2 mRNA in O. affinis suggests that future comparisons with CycloviolacinO2 mRNA from members of the Violaceae family might reveal interesting evolutionary and regulatory insights. Kalata B22 (Table 11) and kalata B23 (Table 12) have been discovered via Sanger sequencing of amplification products from primer pairs designed for kalata B17 and kalata B18. Kalata B17 and kalata B18 were chosen to investigate as they seemed to be upregulated when O. affinis suspensions were incubated with Agrobacterium tumefaciens. The detection and quantification of kalata B23 via mass spectrometry was complicated by the overlap of the isotopic pattern of cycloviolacin O2 and kalata B23. Interestingly, kalata B18 was first detected in suspension cells of O. affinis [25], under similar conditions that led to the discovery of cycloviolacin O2 in this work. The alignment of kalata B17 and kalata B22, presented in Table 11, highlights an exchange of the amino acids in position 24 and 26 as only difference.

Table 11 | BLAST alignment of the amino acid sequence of kalata B17 and kalata B22. Cyclotide Sequence Kalata B17 GIPCAESCVYIPCTITALLGCKCKDQVCYN Kalata B22 GIPCAESCVYIPCTITALLGCKCQDKVCYN Identities GIPCAESCVYIPCTITALLGCKC·D·VCYN Note: The conserved cysteines forming the CCK-motif are bold and highlighted yellow. Dark background indicates identical amino acids, light grey similar substitutions and white substitutions without a high degree of similarity.

The original report of kalata B18 [25] highlighted that isoleucine, leucine, and glutamine still had to be verified as MS cannot discriminate isobaric residues. As can be seen in Table 12, three out of the four possible amino acids with identical masses are different in kalata 23 compared to kalata B18. The isoleucine at position 11 is highly conserved in bracelet cyclotides and no switch to leucine has been observed so far. Interestingly, a switch of valine to isoleucine combined with a switch of alanine to glycine within the first five amino acids leads to ion fragments indistinguishable by mass after trypsin/endo-GluC digestion. Additional results published in Christian Gruber’s PhD

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Establishment and analysis of plant cell suspensions for cyclic peptide production thesis displayed the monoisotopic mass of the YRNGVPCAE and the CYRNGVPCAE fragment after endo-GluC digestion without showing smaller fragments [49]. However, the same masses would be observed for YRNGIPCGE and CYRNGIPCGE fragments. Transcriptome analysis is advisable to determine if kalata B18 or kalata B23 is present. This way, ambiguities inherent in mass spectrometry can be omitted. These findings highlight that even in species extensively researched for their cyclotides surprising discoveries can be made.

Table 12 | BLAST alignment of the amino acid sequence of kalata B18 and kalata B23. Cyclotide Sequence Kalata B18 GVPCAESCVYIPCISTVLGCSCSNQVCYRN Kalata B23 GIPCGESCVYIPCLSTVIGCSCSNKVCYRN Identities G+PC·ESCVYIPC·STV·GCSCSN·VCYRN Note: The conserved cysteines forming the CCK-motif are bold and highlighted yellow. Dark background indicates identical amino acids, light grey similar substitutions and white substitutions without a high degree of similarity.

2.3.4 Cyclotide and AEP mRNA translation in Oldenlandia affinis

To elucidate the cause of the cyclotide production patterns observed in O. affinis tissues, RNA extraction followed by real-time PCR was conducted for several genes related to cyclotide production. OaAEP1 and OaAEP3 were monitored because they facilitate C-terminal processing [13, 30], OaAEP2 was monitored to see if there were different patterns distinguishable between AEPs. As observed cyclotides, kalata B1, kalata B2 and cycloviolacin O2 were selected. Plant leaf tissue was chosen as calibrator sample, GAPDH as housekeeping gene and the Livak method for data analysis. The housekeeping gene GAPDH displayed high sensitivity to low quality RNA samples, resulting in qPCR runs that amplified all but the GAPDH gene. Whereas the A260/280 ratio generally was between 1.8 and 2.1, indicating no protein contamination, the A260/230 was below 1.5 for all samples that failed to amplify GAPDH. This indicates organic contamination (e.g. TRIzol, chaotropic salts, aromatic compounds…). Especially the callus extractions produced low A260/230 values. High sugar contents and in general different cytosol compositions have been reported for callus cells and might negatively influence RNA quality [50, 51]. Therefore, different extraction parameters and additional washing steps might be advantageous for RNA extraction from callus cells. The translation of AEP precursors in plant suspension cells clearly demonstrated their capability to process cyclotides. The cyclotide mRNA expression correlated nicely with the cyclotide peptide amounts detected. It was shown, that cyclotide regulation is specific for each individual cyclotide. The reason for the decline of cyclotides in suspension cells over time in O. affinis was the missing translation of cyclotide precursors, not missing processing enzymes. This indicated that the cells did not waste any energy and already blocked the first step of cyclotide expression. However, it 72 | Page

Establishment and analysis of plant cell suspensions for cyclic peptide production also indicated that the cells were still able to produce cyclic peptides if the right promoters were to be provided. Energy conservation is likely the cause for suppressing cyclotide production as the cells are floating in a nutrient rich medium without any stress factors and do not need defence molecules. Cyclotides that are still expressed in plant suspension cells might have tasks additional to plant defence and/or might be responsible for a baseline protection from pest. Recent studies have associated plant peptides with short and long-distance transmission of information about available nutrient levels [52] and growth conditions [53], a trait previously attributed to metabolites, phytohormones and microRNAs [54]. Considering these discoveries, it seems possible that some cyclotides have additional yet undiscovered roles in the inner workings of plants that might be connected to the up and down-regulation in different tissue and cell types observed in this study.

2.3.5 Suspension type cyclotides

The characterization and analysis of cyclotide production in plant, callus and suspension of C. ternatea, H. enneaspermus, O. affinis, and P. hybrida exposed a prominent similarity between the cyclotides that were present in suspension cells, regardless of species. This group, here named suspension type cyclotides, was defined by the conservation of 21 amino acids in a 30 amino acid bracelet scaffold. Two additional sites showed an amino acid substitution of valine to isoleucine or isoleucine to valine respectively in one single member of the group. This is a minor substitution with minimal impact on the properties of the peptide. The only real variation was observed in positions 15-18 in loop 3. Slazak et al. reported cycloviolacin O3 and cycloviolacin O13 in Viola uliginosa suspension [7], both suspension type cyclotides. Narayani et al. [8] reported three cyclotides, vodo I96A, vodo I97, and vodo I98 to be present only in the callus and suspension culture, not in the whole plant of Viola odorata. Vodo I98 was digested and de novo sequenced via MS/MS fragmentation. The resulting sequence was GXPCGESCVWIPCFSAAIGCSCKSKVCYRN, where X could not be distinguished between Ile and Leu [8]. Based on the results presented here, it seems likely that it is in fact Ile and vodo I98 is a suspension type cyclotide. Next to vodo I98, cycloviolacin O2 and cycloviolacin O13 were the highest accumulating cyclotides in V. odorata suspension with levels higher than in the plant [8]. In summary, the main cyclotides produced in V. uliginosa and V. odorata suspensions were suspension type cyclotides. The prediction is that cyclotides produced in suspensions of other species will also fall into this group. Recently, suspension type cyclotides were detected in Viola arcuate (viar 1, mra30, cyO2, and cyO13), Viola tonkinensis (vito 1 and cyO13), 73 | Page

Establishment and analysis of plant cell suspensions for cyclic peptide production and Viola austrosinensis (cyO2) [55]. Establishing suspension cultures of these three species would be a first step to test the prediction. A possible interpretation of the high sequence and structural similarity is an analogous evolutionary constraint imposed on this peptide group and might hint at similar biological functions. The convergent evolution theory, as presented in Section 2.3.2 for cycloviolacin O2, is therefore extended to a larger peptide group. Callus and suspension cells are less differentiated than cells in a mature plant. The continued expression might indicate strong defensive capabilities against pests critical in the early stages of development like fungi and bacteria or might indicate a different biological function altogether. This general interpretation is true for all cyclotides found in suspension, not only suspension type cyclotides. However, suspension type cyclotides seem to have a role that is useful in undifferentiated tissue and has evolved independently in multiple plant families and species. A possible function is outstanding anti-fungal efficiency as reported for cycloviolacin O2, cycloviolacin O3, cycloviolacin O13, all suspension type cyclotides, and cycloviolacin O19, not a suspension type cyclotide as defined here, but still with high similarity to cycloviolacin O13 [47]. Conduction of the same experiments with other suspension type cyclotides, could test this hypothesis. In conclusion, C. ternatea, H. enneaspermus, and O. affinis suspensions all produced cyclotides, some of which were new sequences. The recorded cyclotide peptide signals in O. affinis cells were reflected in cyclotide mRNA levels, whereas the AEP mRNA expression was not linked to cyclotide production. The analysis of the cyclotides produced in suspension cells also revealed a new group here named suspension type cyclotides. Building on the discovery that all tested species were able to produce cyclotides in suspension, the feasibility to cultivate cyclotide producing plant cell suspensions on a larger scale is examined in the following chapter.

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2.4 References

1. Grabowski, G.A., M. Golembo, and Y. Shaaltiel, Taliglucerase alfa: An enzyme replacement therapy using plant cell expression technology. Molecular Genetics and Metabolism, 2014. 112: p. 1. 2. Fischer, R. and J.F. Buyel, Molecular farming – The slope of enlightenment. Biotechnology Advances, 2020: p. 107519. 3. Schillberg, S., N. Raven, H. Spiegel, S. Rasche, and M. Buntru, Critical Analysis of the Commercial Potential of Plants for the Production of Recombinant Proteins. Front Plant Sci, 2019. 10: p. 720. 4. Narayani, M., R. Babu, A. Chadha, and S. Srivastava, Production of bioactive cyclotides: a comprehensive overview. Phytochemistry Reviews, 2020. 5. Seydel, P., Produktion von Cyclotiden in pflanzlichen Zellkulturen von Oldenlandia affinis, in Institute of Bioprocess Engineering. 2009, University of Erlangen-Nuremberg. 6. Seydel, P. and H. Dörnenburg, Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides. Plant Cell, Tissue and Organ Culture, 2006. 85: p. 247. 7. Slazak, B., E. Jacobsson, E. Kuta, and U. Goransson, Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae). Phytochemistry, 2015. 117: p. 527. 8. Narayani, M., A. Chadha, and S. Srivastava, Callus and cell suspension culture of Viola odorata as in vitro production platforms of known and novel cyclotides. Plant Cell, Tissue and Organ Culture (PCTOC), 2017. 130: p. 289. 9. Conway, M., K. McCosker, V. Osten, S. Coaker, and B. Pengelly. Butterfly pea-A legume success story in cropping lands of central Queensland. in Proceeding of the 10th Australian Agronomy Conference, Hobart. 2001. 10. Jackson, M.A., E.K. Gilding, T. Shafee, K.S. Harris, Q. Kaas, S. Poon, K. Yap, H. Jia, R. Guarino, L.Y. Chan, T. Durek, M.A. Anderson, and D.J. Craik, Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases. Nat Commun, 2018. 9: p. 2411. 11. Gamborg, O.L., Aromatic metabolism in plants. II. Enzymes of the shikimate pathway in suspension cultures of plant cells. Can J Biochem, 1966. 44: p. 791. 12. Colgrave, M.L. and D.J. Craik, Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry, 2004. 43: p. 5965. 13. Harris, K.S., T. Durek, Q. Kaas, A.G. Poth, E.K. Gilding, B.F. Conlan, I. Saska, N.L. Daly, N.L. van der Weerden, D.J. Craik, and M.A. Anderson, Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat Commun, 2015. 6: p. 10199. 14. Altschul, S.F., T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research, 1997. 25: p. 3389. 15. RNA isolation from plant. 2019, Macherey-Nagel. 16. Livak, K.J. and T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods, 2001. 25: p. 402. 17. Bio-Rad Laboratories, I., Real-Time PCR Applications Guide. 2006. 18. Ikeuchi, M., Y. Ogawa, A. Iwase, and K. Sugimoto, Plant regeneration: cellular origins and molecular mechanisms. Development, 2016. 143: p. 1442. 19. Dörnenburg, H. and P. Seydel, Effect of irradiation intensity on cell growth and kalata B1 accumulation in Oldenlandia affinis cultures. Plant Cell, Tissue and Organ Culture, 2007. 92: p. 93.

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20. Oguis, G.K., Clitoria ternatea (butterfly pea) cyclotides: Insights on functional diversity, regulation and biotechnological applications, in Institute for Molecular Bioscience. 2019, The University of Queensland. 21. Muthamilselvan, T., C.W. Lee, Y.H. Cho, F.C. Wu, C.C. Hu, Y.C. Liang, N.S. Lin, and Y.H. Hsu, A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles. Plant Biotechnol J, 2016. 14: p. 231. 22. Boivin, E.B., E. Lepage, D.P. Matton, G. De Crescenzo, and M. Jolicoeur, Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co- transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing. Biotechnol Prog, 2010. 26: p. 1534. 23. Seydel, P., C. Walter, and H. Dörnenburg, Scale-up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production. Engineering in Life Sciences, 2009. 9: p. 219. 24. Jennings, C., J. West, C. Waine, D. Craik, and M. Anderson, Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis. Proceedings of the National Academy of Sciences, 2001. 98: p. 10614. 25. Seydel, P., C.W. Gruber, D.J. Craik, and H. Dörnenburg, Formation of cyclotides and variations in cyclotide expression in Oldenlandia affinis suspension cultures. Appl Microbiol Biotechnol, 2007. 77: p. 275. 26. Hashempour, H., J. Koehbach, N.L. Daly, A. Ghassempour, and C.W. Gruber, Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry. Amino Acids, 2013. 44: p. 581. 27. Poth, A.G., M.L. Colgrave, R. Philip, B. Kerenga, N.L. Daly, M.A. Anderson, and D.J. Craik, Discovery of cyclotides in the fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins. ACS Chem Biol, 2011. 6: p. 345. 28. Nguyen, G.K., S. Zhang, N.T. Nguyen, P.Q. Nguyen, M.S. Chiu, A. Hardjojo, and J.P. Tam, Discovery and characterization of novel cyclotides originated from chimeric precursors consisting of albumin-1 chain a and cyclotide domains in the Fabaceae family. J Biol Chem, 2011. 286: p. 24275. 29. Gruber, C.W., M. Cemazar, R.J. Clark, T. Horibe, R.F. Renda, M.A. Anderson, and D.J. Craik, A novel plant protein-disulfide isomerase involved in the oxidative folding of cystine knot defense proteins. J Biol Chem, 2007. 282: p. 20435. 30. Harris, K.S., R.F. Guarino, R.S. Dissanayake, P. Quimbar, O.C. McCorkelle, S. Poon, Q. Kaas, T. Durek, E.K. Gilding, M.A. Jackson, D.J. Craik, N.L. van der Weerden, R.F. Anders, and M.A. Anderson, A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein. Sci Rep, 2019. 9: p. 10820. 31. Huang, Y.-H., M.L. Colgrave, R.J. Clark, A.C. Kotze, and D.J. Craik, Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity. Journal of Biological Chemistry, 2010. 285: p. 10797. 32. Wang, C.K., M.L. Colgrave, D.C. Ireland, Q. Kaas, and D.J. Craik, Despite a conserved cystine knot motif, different cyclotides have different membrane binding modes. Biophys J, 2009. 97: p. 1471. 33. Hagemans, D., I.A. van Belzen, T. Moran Luengo, and S.G. Rudiger, A script to highlight hydrophobicity and charge on protein surfaces. Front Mol Biosci, 2015. 2: p. 56. 34. Velayutham, P. and C. Karthi, GC-MS Profile of in vivo, in vitro and fungal elicited in vitro leaves of Hybanthus enneaspermus (L.) F. Muell. International Journal of Pharmacy and Pharmaceutical Sciences, 2015. 7: p. 260. 35. Sathish, S., V. Vasudevan, S. Karthik, G. Pavan, and M. Manickavasagam, Enhanced l-Dopa production from elicited cell suspension culture of Hybanthus enneaspermus (L.) F. Muell. Plant Biotechnology Reports, 2019. 13: p. 613. 36. Sukenik, S.C., K. Karuppanan, Q. Li, C.B. Lebrilla, S. Nandi, and K.A. McDonald, Transient Recombinant Protein Production in Glycoengineered Nicotiana benthamiana Cell Suspension Culture. Int J Mol Sci, 2018. 19. 76 | Page

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3 Mass-cultivation of plant cell suspensions for cyclic peptide production

The scale-up of benchtop experiments to industrial size production is a key engineering challenge. In the case of plant cell suspensions, it encompasses changes in thermo- and fluid- dynamics, due to the up-scaling of reactor size and type, resulting in changed diffusion properties of the supplied oxygen and carbon dioxide. Additionally, the energetic and financial implications of different production systems must be considered. Lastly, biological organisms do not grow equally on every scale, therefore a scale-up of biological production systems always includes intermediate sized experiments. The chosen reactor system has the largest impact on all these factors and must be considered carefully. Although stainless steel bioreactors are currently the industry staple for biopharmaceutical production, recent years have seen the advent of single-use bioreactors and the corresponding single- use sensors. Advantages of single-use technology include defined batch processing with minimal risk of cross contamination, pre-sterilized equipment, lower investment costs, lower set-up times, no downtime between batches, higher flexibility, and the possibility to realize novel reactor designs. As a disadvantage, the value recovery of single-use components in bioprocessing is not trivial and their end-of-life must be considered early on, as they are mostly biologically contaminated multilayered polymers that are difficult and costly to be recycled or disposed of. However, a life cycle analysis study by Whitford et al. [1] revealed lower negative impacts on climate change, human health, ecosystem quality, resource consumption, and water consumption of single-use technologies compared to traditional systems (traditional is here defined as durable technology that is reused opposed to single-use technologies that are disposed of after one use). Traditional systems require cleaning and sterilization in place resulting in high water and energy usage [2]. The transportation and end of life energy costs and environmental impacts are higher for single-use technologies, but these impacts are only a small part of the whole life cycle [1]. Single-use reactors require disposable sensors or sterilizable sensor-reactor interfaces. Although this imposes new challenges to miniaturization and production costs, it simultaneously circumvents problems of classical sensors such as their limited lifetime, resistance to steam, resistance to heat and degradation even outside of the reactor. New sensors for every batch increase the accuracy of all monitored parameters and prevent shifts that can occur over longer uptimes. This is very advantageous for reliable secondary metabolite production that often can be influenced by minimal fluctuations in pH or concentration of

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Mass-cultivation of plant cell suspensions for cyclic peptide production chemicals. Luo et al. for example, achieved a significant increase of Taxol production in Taxus chinensis suspension by controlling the dissolved oxygen level at every growth phase [3]. New advances in the field of biotechnology, including the increasing yield of secondary metabolites in optimised cell lines make smaller, cheaper and more flexible reactors available for high-value biopharmaceutical production [4]. In the light of the changing market, single-use bioreactors are becoming good options for pharmaceutical peptide production in plant cell suspensions. The engineering side benefits from highly flexible designs and operation conditions. Shear stress is especially difficult to predict and handle around the impeller tips of conventional bioreactors. For different plant suspensions a critical shear stress of 50 to 200 N m-2 has been observed [5] and bioreactors without moving parts are generally preferable if shear damage is expected [6]. However, bioreactors without moving parts provide their own challenges; for example Oldenlandia affinis cells displayed aggregation, floatation, and adherence to the vessel walls in photobioreactor screening modules (PSMs) and airlift bioreactors [7]. The cultivation of living cells requires meticulous research outside of the final production stage, especially regarding the biological properties of the cells. The small-scale growth characteristics and cyclotide profiles of C. ternatea, H. enneaspermus, and O. affinis are presented in Chapter 2. Strategies to enhance the cyclotide production are developed in Chapter 4 and Chapter 5. Three protocols to cryopreserve O. affinis suspension cells are tested in Chapter 6. Here, we utilized a wave-based agitation instead of impellers or air bubbles traveling through the culture to ensure mixing in disposable reaction vessel on a rocking motion bioreactors inheriting the advantages of single-use bioreactors and qualifying as easily scalable, low shear stress production system to assess the growth potential of plant suspension cultures to produce cyclotides.

3.1 Materials and methods

3.1.1 Suspension culture initiation and scale-up

O. affinis, H. enneaspermus and C. ternatea suspension cultures were initiated from sterile in vitro grown plant explants as detailed in Section 2.1. Briefly, freshly cut stem explants were placed on callus induction medium (see Table 3) in Petri dishes. After 30 days, the vigorously growing calli were selected and transferred onto fresh callus induction medium. After 30 more days, green friable calli were selected for suspension culture initiation and transferred into 45 mL liquid suspension

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Mass-cultivation of plant cell suspensions for cyclic peptide production medium (see Table 5) in 125 mL Erlenmeyer flasks. After 15 days, these cultures had grown to a PCV of 60% and were used to inoculate the disposable bags.

3.1.2 Reactor cultivation of suspension cultures

180 mL of 15-day-old O. affinis cultures at a density of 60% PCV at the end of the exponential growth phase were used to inoculate 800 mL fresh medium in a 2 L disposable bag to be cultivated on a rocking motion bioreactor (WAVE bioreactor system, GE Healthcare Australia). Compressed air and CO2 were supplied by gas cylinders (BOC) and were regulated by a mass flow controller unit (GE Healthcare). The air flow rate was adjusted to 0.1 vvm (volume of air per unit of medium per minute) and supplemented with 5% (v/v) CO2 for a culture volume of 1 L. The supplied gases entered and left the bag via tubing in the headspace. Agitation to induce wave formation and mass transfer was achieved by a 10° rocking angle and a rocking speed of 25 revolutions per minute (rpm) at 25°C. All other cultivations were performed on the BioStat® RM system (Sartorius). This instrument provided a high-end control unit capable of programming specific reactor parameters during cultivation (e.g. CO2 profile and rocking profile) and feedback loops for pH and dissolved oxygen. The rocking tray supports two simultaneous 2 L cultivation bags or one 20 L cultivation bag. For simultaneous cultivation, the temperature, flow rates, rocking speed and rocking angle had to be the same for both cultures. The bags and sensors fulfilled industrial grade standards for pharmaceutical production, even though no documentation was created for this research project. Cultures in 2 L bags (BioStat® RM system, Sartorius) were initiated at an inoculation density of 0.12 v/v (cell volume divided by total volume) with 1 L culture volume. Cultivations in the 10 L bags were run in a fed batch mode with an inoculation density of 0.14 v/v with 1 L culture volume. The fed batch approach was chosen to enable transfer of a limited number of cells from shaker flask cultivations directly into the reactor. After the density reached 0.7 v/v, 4 L medium was added to reach the final culture volume of 5 L. The density was measured by taking 5 mL aliquots through a screw cap sampling port that were then centrifuged (4000 x g, 10 min, RT) to determine the packed cell volume. The growth was monitored via online dissolved oxygen measurement and the rocking rate was adjusted when the oxygen concentration was dropping below 20%.

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3.1.3 Harvesting and extraction of cyclotides

For cultures up to a cell density of 0.4 v/v, sampling via the inoculation port was possible. Higher cell densities clumped the tubing and required sampling via the screwcap opening and was kept to a minimum as it introduced a high risk of contamination. Harvesting of the whole volume was also achieved via the screwcap opening. The cultures were harvested when the density reached 0.7 v/v. After harvesting, the cultures were centrifuged in 1 L vessels (4000 x g, 10 min, RT), the supernatant discarded, the cell pellet transferred into 50 mL Falcon tubes, and lyophilized at 1 mbar for 24 h. The dry cells were cracked with 25 mL 50% ACN, 1% FA and two metal bearings in each tube on the GenoGrinder® at 800 rpm for 2 min to extract the cyclotides. The resulting slur was centrifuged twice (3000 x g, 15 min, RT) and the supernatant transferred in two 1 L round bottom flasks, for subsequent layered lyophilization. The dry product was dissolved in 100 mL 50% ACN, 1% FA, split into four 50 mL Falcon tubes and centrifuged (4000 x g, 10 min, RT) to get rid of remaining macroscopic contaminants. The supernatant was consequently lyophilized and analysed for cyclotides.

3.1.4 MALDI-TOF analysis of cyclotide expression

All samples were analysed as described in Section 2.1.6.

3.1.5 LC/MS analysis of suspension extracts

The AB SCIEX Triple TOF® 5600/5600+ system was used to analyse the extracts and the consequent purification fractions for cyclotides. A small amount of the dry samples (~5 mg) was dissolved in 200 µL 50% ACN, 1% FA, centrifuged (17,700 x g, 20 min) and 20 µL transferred into a HPLC vial. The LC/MS method focused on TOF masses between 300-1800 Da with a run time of 39 min. For the first 32 min the ACN concentration was linearly increased from 1 to 40%. From minutes 32 to 36 the concentration was brought up to 80% ACN. After holding that concentration for 1.5 min it was increased to 98% within 0.5 min. That concentration was held for 1 min as final step of the method. The generated data were analysed with the Analyst® TF software.

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3.1.6 Purification of cyclotides in plant suspension extracts

As a first step to enrich cyclotides, the crude cell suspension extract was fractionated on a C18 solid-phase extraction (SPE) cartridge. The Phenomenex Strata® C18-E (55 µm, 70 Å) 50 g/150 mL Giga tube was activated with 150 mL methanol forced through the column via applied vacuum. After this initial activation step, the column was kept moist all the time. Hereinafter, the column was washed with 150 mL 80% ACN, 1% FA and equilibrated with 150 mL 1% FA. The sample was centrifuged (3900 x g, 15 min) and the supernatant filtered through 0.45 µm before loading onto the cartridge with a final concentration of 5% ACN, 1% FA. After the initial loading step, all elutes were collected and analyses for cyclotides. The next two sequential washes were implemented with 150 mL 1% FA. Fraction 1 was eluted with 150 mL 10% ACN, 1% FA, Fraction 2 with 150 mL 20% ACN, 1% FA, Fraction 3 with 150 mL 50% ACN, 1% FA, and Fraction 4 with 150 mL 80% ACN, 1% FA. The cyclotide containing Fraction 3 was further fractionated and purified during multiple HPLC runs. All fractions with strong UV absorbance were spotted 1:1 with matrix and analyses utilizing MALDI/TOF MS as described in Section 2.1.6. The cyclotides kalata B22, kalata B23, and cycloviolacin O2 were identified in O. affinis cells after cultivation on a rocking motion bioreactor. They were separated and further purified on a 1 mL analytical column. Dry weights were attempted to determine by weighing the lyophilized samples.

3.2 Results

3.2.1 Oldenlandia affinis cultivation in single-use bags – Proof of concept

As the model species for cyclotide research and the first species to be brought into suspension with the goal to produce cyclotides [8], O. affinis was chosen as a starting point to produce cyclotides in a rocking motion bioreactor. As initial proof of concept, O. affinis suspension cells were cultivated in a 2 L disposable bag on the WAVE 25 bioreactor system (Figure 26C). The culture volume was 1 L with an inoculation density of 0.9 g L 1 and a harvesting density of 50% PCV (6.3 g L 1 (DW)) after 9 days (Figure 26A). The inoculum⁻ was a 7 days old suspension culture initiated from⁻ leaf explants and was tested for cyclotide accumulation before transfer into the disposable bag (Figure 26B). Optical single-use sensors allowed the online measurement of dissolved oxygen and pH in the

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Mass-cultivation of plant cell suspensions for cyclic peptide production culture medium (Figure 26D). Rocking speed, rocking angle, and gas flow were adjusted manually to ensure sufficient oxygen supply. In this cultivation, a slow decline of the dissolved oxygen was apparent. Towards the end of the cultivation, the rocking speed, rocking angle and gas flow were increased to test their effect on the culture and to accommodate for higher cell densities.

Figure 26 | First cultivation of O. affinis in a rocking motion bioreactor. A) Growth curve of the cultivation on the Wave® reactor. The bag volume was 2 L with a culture volume of 1 L. The dry weight was determined by lyophilization. Error bars indicate standard deviation of technical replicates, n=3. B) Representative MALDI-TOF trace of the cyclotides containing fraction of the inoculum seven days after suspension initiation from callus material. C) Stages of a suspension cultivation. Sterile in vitro plant to the right, inoculum initiated from callus material in shake flask in the middle, and 2 L single-use bag on a rocking motion setup ready to be cultivated on the left. D) Online measurement of the cultivation parameters. The pH is plotted on the right-hand ordinate, all other parameters on the left-hand ordinate.

3.2.2 Optimisation and analysis of O. affinis scale-up

Having established O. affinis suspension cell cultures in 2L WAVE 25 reactor bags, the next step was to test the feasibility of using 10 L single-use bags. The reactor setup to achieve this goal was a BioStat® rocking motion bioreactor depicted in Figure 27A. The cultivation parameters are

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Mass-cultivation of plant cell suspensions for cyclic peptide production presented in Figure 27B. Initial pH measurements failed as the culture pH was out of the specified range of the sensors. After recalibration and mathematical adjustment, a pH measurement was possible but without full confidence as the sensors were not designed to measure below pH 6.

Figure 27 | O. affinis cultivation for cyclotide production on a BioStat® rocking motion bioreactor with 5 L culture volume. A) BioStat® reactor with LEDs installed to irradiate the cells at different wavelengths at up to 30 µmol m-2 s-1. B) Cultivation parameters. Dissolved oxygen was measured via an optical online sensor, the other parameters were adjusted manually.

During the planning of this scale-up, results stemming from the smaller shake flask experiments revealed a decline in expressed cyclotides over time in O. affinis suspensions as presented in Section 2.2.4. Therefore, the time from suspension cell initiation to harvesting was kept as short as possible. Additionally, suspension cells initiated from calli induced from either stem or root explants were tested. Six one week old suspension cultures initiated from stem and root explants were assessed for their cyclotide content and compared to 64 week old suspension cultures initiated from leaf explants as displayed in Table 13.

Table 13 | Comparative cyclotide expression in O. affinis suspensions. Explant Kalata B1 Kalata B7 Cycloviolacin O2 Kalata B23 Kalata B9 Kalata B22 Stem 1 2.99 0.12 0.49 0.38 0.06 0.04 Stem 2 1.54 0.08 0.35 0.45 0.04 0.04 Stem 3 18.13 0.29 4.63 3.06 0.13 0.18 Stem 4 3.55 0.17 0.88 0.86 0.05 0.06 Root 1 0.49 0.07 0.73 0.81 0.09 0.14 Root 2 1.01 0.09 0.59 0.74 0.06 0.14 Leaf 1 0.00 0.00 0.00 0.23 0.00 0.17 Leaf 2 0.00 0.00 0.00 0.25 0.00 0.19 Note: The stem and root cultures were one week old, whereas the leaf cultures were 64 weeks old. The values given are intensities of the monoisotopic peak relative to a reference peptide as observed during MALDI-TOF analysis. The peak of the reference peptide is set to 1.00. The shading compares values within a column and is blue for high cyclotide accumulation and red for low cyclotide accumulation.

The culture “Stem 3” was chosen for scale-up with the goal to extract and purify cyclotides. Figure 28 depicts the cyclotide profile of the O. affinis cells at various stages of the process. The

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Mass-cultivation of plant cell suspensions for cyclic peptide production callus trace is the “Stem 3” cell line reported in Table 13, only this time normalized for the reference peptide. Starting with 7 g (FW) callus material, 1 L of culture at 65% PCV was achieved after 15 days and transferred into the 10 L bag on the rocking motion bioreactor. Kalata B1, kalata B7, cycloviolacin O2, and kalata B23 were still present, but in much lower quantities. After 12 days, the culture reached 65% PCV and 4 L fresh medium were added to accomplish the final culture volume of 5 L. At this point, kalata B23 was the only cyclotide left clearly detectable via MALDI-TOF analysis. Six days later, 33 days after suspension initiation from callus, the 5 L O. affinis culture was ready to harvest. At this point, the cyclotide peaks were at the lower end of the MALDI-TOF detection limit, whereas the reference peptide displayed the same intensity as before.

Figure 28 | Cyclotide expression of O. affinis cells at 7g (FW) callus, 1 L suspension, 5 L suspension and harvest of 5 L suspension at a PCV of 65%. All traces were normalized using linear kalata B1-Glu-Ile-Ile as reference peptide.

Further investigation via LC/MS analysis revealed the presence of cycloviolacin O2, kalata B22, and kalata B23. Their elution times were sufficiently different to enable purification using HPLC with an ACN gradient from 1-50%. Kalata B22 was identified by its strong two charged state with the monoisotopic peak at 1592.74 Da and its slightly less strong three charged state with the monoisotopic peak at 1062.16 Da. Kalata B23 was identified by its strong two charged state with the monoisotopic peak at 1572.68 Da and its equally strong three charged state with the monoisotopic peak at 1048.81 Da. Cycloviolacin O2 was identified by its strong three charged state with the monoisotopic peak at 1047.15 Da. Its two charged state with the monoisotopic peak at 1570.23 Da was barely detectable. Under the selected conditions kalata B22 eluted at 26.79 min, kalata B23 at

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21.62 min, and cycloviolacin O2 at 20.15 min in the crude extract. These were the only cyclotides detected in the LC/MS trace via ion extraction. After three purification steps, the remaining cyclotide amounts were not enough for quantification. However, the amount of purified cycloviolacin O2 was enough to enable digestion and MS/MS sequence analysis as presented in Section 2.2.4.1. Results reported by Dörnenburg & Seydel indicated a link between chlorophyll and cyclotide expression [9]. Consequently, LEDs were installed in the lid of the bioreactor to enable irradiation of the cultures at different wavelengths at 30 µmol m-2 s-1 to support chlorophyll production. The LEDs were connected to a timer switch to set a 16:8 light-dark cycle. However, irradiated O. affinis suspension cultures grown in the single-use bags showed no signs of chlorophyll production, same as the shake flask cultures. Also, no difference in cyclotide production was observed (data not shown). Therefore, no further irradiation experiments were set up.

3.2.3 Hybanthus enneaspermus cultivation in single-use bags – Proof of concept

For comparison, the scale-up of H. enneaspermus was also tested. These cultures maintained a stable expression of cycloviolacin O2 even after cultivation in a bioreactor as shown in Panel C of Figure 29. A fed batch strategy was employed to produce 5 L culture. The inoculate of 200 mL high density H. enneaspermus culture at the end of the exponential growth phase grown in shake flasks was filled up to one litre with fresh medium in the disposable bag. After 360 h as the culture reached a density of 50% PCV four more litres of fresh medium were added to achieve the final culture volume of 5 L. The new volume required adjusted gas flow rate and rocking speed. The rocking angle was kept constant at 10° during the process. The online measurement of pH and dissolved oxygen was not reliable during the low volume phase as can be seen in Panel B) of Figure 29. Possible explanations are discussed in Section 3.3.1

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Mass-cultivation of plant cell suspensions for cyclic peptide production

Figure 29 | Fed batch cultivation of 5 L H. enneaspermus suspension in a disposable bag on a rocking motion bioreactor. A) Culture before harvesting. B) Cultivation parameters. C) MALDI trace of cyclotides in extract of harvested cells.

H. enneaspermus was displaying different cell morphologies and tissue types during the scale- up process. Out of three initial cultures grown in the single-use bags, one culture formed long white root-like structures traversing the whole culture. These structures fixed the culture in place and lead to a cultivation mode similar to a temporary immersion bioreactor, where a cycle of medium flowing through the culture and being replaced by a gas mixture is generated [10]. This morphology was also observed in several shake flask cultures and is further explored in Chapter 2.

3.2.4 Clitoria ternatea cultivation in single-use bags – Proof of concept

The first 5 L cultivation of C. ternatea that was put on the rocking motion bioreactor was lost after five days due to contamination. The backup cultures were lost at the same time; therefore, a second cultivation was not possible due to of time restraints. Up until that point, all cultures displayed strong growth and a light green colour. Despite the contamination issues, 3 non-contaminated samples were collected after one day of growth in the single-use bag. MALDI-TOF analysis revealed several expressed cyclotides even after four weeks of suspension culture as shown in Figure 30. The cyclotides identified by one-charged monoisotopic mass included Cter O (3084.5 Da), Cter P (3098.5 Da), Cter R (3227.6 Da), and Cter A (3268.6 Da). The peak at 3152.5 Da probably included 88 | Page

Mass-cultivation of plant cell suspensions for cyclic peptide production cliotide T20 (3152.5 Da) and Cter I (3155.5 Da). The peak at 3165.5 Da probably included cliotide T15 (3165.5 Da), Cter Q (3169.5 Da), and cliotide T31 (3170.5 Da). No additional LC/MS analysis was done to separate the peaks.

Figure 30 | MALDI-TOF trace of a four-week-old C. ternatea suspension culture after one day on a rocking motion bioreactor.

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3.3 Discussion

Plant cell suspensions are still a niche production system compared to mammalian or yeast systems, even though several gold rush scenarios have predicted their rise in popularity [11-13]. As a result, not many reactor systems – plant species combinations have been explored and standard protocols are often missing. Here, a non-traditional reactor system, the rocking motion bioreactor, was tested with three cyclotide producing plant species, two of which have never been cultivated on a medium scale before. Designed as proof of principle these tests did not quantify the yield and economic potential, but instead assessed the feasibility and scouted for potential roadblocks.

3.3.1 Limitations of the rocking motion bioreactor design

Sterility and homogenous sampling were the major challenges during all cultivations. Like in the shake flasks, the growing cells formed aggregates at higher densities, as is common for plant suspensions [14], and were not able to pass the small sampling openings designed for homogeneous mammalian or bacterial single cell suspension. Therefore, initial sampling was achieved by submerging 5 mL Eppendorf tubes via a screw cap port into the culture. This required the transfer of the reaction vessel into a biosafety cabinet and introduced a high risk of contamination. Three out of eight cultivations that were sampled via the screw cap port, had to be aborted due to fungal, bacterial or yeast contamination. The problem of sterile and homogeneous sampling of plant suspensions in a single-use bag was difficult to research, because most of the knowledge lies with the suppliers and there is a lack of expertise in the academic environment [15]. The suppliers were able to manufacture bags with sampling ports designed according to provided specifications, but this approach was not economical for small lot numbers. Most single-use bags, including the ones used here, were intended to host mammalian cells. Future designs might be optimised for plant-based systems and have better sampling port solutions as well as pH sensors measuring below pH 6. Large scale production lines will probably have to design their own bags specific for the cultivated species. To prevent contamination in a laboratory setup, a sampling method was devised without changing the bag design. This method ensured sampling without contact of the culture with any external material. A T-connector was introduced into the tubing of an inoculation port, with one side connected to a sterile filter and ambient pressure, the other side connected to a sampling tube. A three- clamp setup guaranteed no backflow into the reactor during the sampling process. This setup proved

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Mass-cultivation of plant cell suspensions for cyclic peptide production to be successful up to a cell density of 40% PCV. For higher cell densities, manual removal of clumps blocking the tube entry by applying pressure to the vessel wall was sometimes possible, whereas some cultures could not be sampled. This procedure did not result in homogeneous samples as the cell aggregates settled to the bottom of the culture bag and did not pass through the tubing; however, it ensured a lower contamination risk. No sampling during cultivation was the only way to prevent contamination consistently. If the necessary culture parameters are known and production can take place without sampling during the process, single-use bag bioreactors have very low contamination risks, especially because there is no risk of carry over contamination from the previous batch [6]. To discover the necessary culture parameters studies like this one will have to deal with non- homogeneous sampling prone to contamination. The online measurements of dissolved oxygen and pH enabled enough monitoring to handle the cultures without constant sampling. As a result, no growth curves were recorded for most species to minimize the risk of contamination. As an alternative to dry or wet weight growth curves, the dissolved oxygen gave a good indication of culture growth. However, the online monitoring of fed batch processes proved difficult, as the initial starting culture volume of 1 L in a 10 L bag led to unreliable readings of the online single-use sensors (Figure 29). The most likely reason, as worked out with the engineering department of the bag supplier, was the insufficient coverage of the sensor patch by the culture. The high read-out variance when rocking was probably caused by the changing fluid level above the patches situated in the middle of the bag during low culture volumes. As the sensors relied on optical measurement, the installed LEDs might have had a detrimental influence in low volume setups when the LED-light might have passed through the culture. In summary, the applied sensors need to be tested and suited for the chosen conditions. In the setup presented here it might be necessary to run the starting culture of the fed batch process at 1 L without online measurements. A dissolved oxygen percentage of 20% was chosen as minimum threshold to ensure adequate oxygen supply of the submerged cells. The online measurement allowed immediate adjustment of process parameters when the critical value was reached. The supplied gas flow was set at a constant rate according to culture volume. Consequently, in a fed patch scenario the gas flow of the 1 L growth phase was multiplied by 5 when the second 5 L growth phase was initiated. The most efficient parameter to react to dropping dissolved oxygen levels was the rocking speed. An increase of the rocking speed from 15 rpm to 17 rpm led to an increased in dissolved oxygen from 28.5% to 40% within 10 minutes in a 5 L H. enneaspermus culture (Figure 29). The rocking angle was mostly utilized to adjust the formed wave to create turbulent surface conditions to enable maximal gas exchange between liquid and gaseous phase.

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To simplify the reactor design, nullify light penetration problems at higher densities and increase the accuracy of optical sensors, plant suspension cultures can be grown chemotrophically without irradiation. One interesting optical sensors to include in disposable-bags for researched focused plant suspension cultivations is a glucose biosensors [16]. Cultivation in single-use bags in the dark including optical sensors for dissolved oxygen, pH, and glucose levels without sampling during the process seems to be a possible next step to improve plant cell suspension scale-up for cyclotide production.

3.3.2 Prospects of Oldenlandia affinis cultivation in single-use bags

O. affinis grew very well in rocking motion bioreactors and achieved a final packed cell volume of 50% after 9 days, comparable to one of the highest reported biomass productivities of plant cells in a rocking motion bioreactor: 65% PCV of BY-2 cells in a 2 L CultiBag [17]. The O. affinis cells displayed chlorophyll deficiency and changes in protein synthesis. The same trend was reported in other submerged plant cell cultures [17]. The higher shear stress conditions lead to smaller aggregates and more homogeneous cultures compared to shake flask cultures. This result was generally observed for cultures subjected to mixing conditions in the reactor (wave movement 10° rocking angle and a rocking speed of 25 revolutions per minute) and shaker flasks (orbital movement at 90 rpm). However, the wild type cultures were not able to produce enough cyclotide amount for quantification as the cyclotide production decreased over time as discussed in Section 2.2.4. Nevertheless, the 5 L cultivation of O. affinis enabled purification of enough cycloviolacin O2 to run digestion and ion fragment experiments as presented in Section 2.3.2. It was not possible to start cultures directly in the reactor bags, as that would have required impractical amounts of fresh callus tissue. Young O. affinis cells suspensions needed a starting cell density of around 0.1 g L-1 (FW). If the culture density fell below that critical value, it would die. Consequently, the amount of available callus tissue for suspension initiation limited the starting culture volume. The minimum time from 7 g (FW) callus starting material to harvest of 5 L suspension was 33 days. Unfortunately, like in the shake flask cultures, after 33 days there were nearly no cyclotides left in the O. affinis suspension cells grown on the rocking motion bioreactor regardless of the cyclotide levels in the inoculation culture. This result displayed that the modified conditions of the reactor bag compared to the shake flask did not change the cyclotide expression. However, the different rheology and shear stress matrix led to smaller aggregates, enabling higher cell densities.

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A strong influence of the inoculation culture on cyclotide production and cell growth was observed. Cultures from the same explant tissue type varied by a factor of up to 4 and cultures from different explant tissue types varied by a factor of up to 18 in cyclotide production levels. A strong influence of the inoculation culture on cell growth was reported before, however the influence on cyclotide production was not tested [18]. The data gathered here show that cyclotide production in young O. affinis suspension cultures varies greatly; however, after a few weeks all suspension cultures displayed very similar, low cyclotide production. Even though the cyclotide production level varied in young O. affinis cultures, the produced cyclotides were always the same with kalata B1, cycloviolacin O2 and kalata B23 as the three most prominent. The same relative cyclotide profile was observed in 9 days old O. affinis cultures cultivated in an irradiated 25 L bioreactor [19]. It was not beneficial to screen for high producing young shake flask-grown suspension cultures to inoculate the single-use bags, as the cyclotide production diminished for all cultures before a volume of 5 L could be harvested. After an initial drop in culture pH from 6.0 to 5.3, there was a slight increase to a harvesting pH of 6.1. The culture settled around pH 6 with no changes during the last 100 hours of cultivation. Similar pH autoregulation was reported by Arya et al. for Dioscorea deltoidea suspensions [20]. These values must be considered carefully, as the single use sensors were not designed to work at pH lower than 6.0. The pH measurements below 6.0 presented here were mathematically interpolated after discussions with the supplier and should be within a 10% error margin. The morphology and viability of the cells was not impacted by the changes in pH observed during the cultivation. Future experiments could research a possible link of culture pH to growth and secondary metabolite production. The dissolved oxygen fell during the exponential growth phase from the initial saturation to under 10% in a 5 L culture on the BioStat® RMB (Figure 27). Suggesting that a higher rocking angle and/or speed will be necessary at the end of a cultivation, when higher cell densities are reached. The oxygen mass transfer (kLa) is mainly determined by the surface aeration mechanism at rocking speeds up to 20 rpm, whereas breaking wave mechanisms are predominant at 40 rpm [21]. This might enable even higher cell densities if the cells can withstand the resulting shear stress. Seydel et al. reported a doubling time of 1.12 days for O. affinis suspension cells in a 25 L air-lift loop photobioreactor with an accumulation of 0.09 mg g-1 DW of kalata B1 at a photon density of 17 µmol m-2 s-1 [18]. Here the same medium composition and irradiation was tested in a 5 L rocking motion bioreactor. The observed doubling time was 1.51 days, but only minute kalata B1 production was detected. The same stifled kalata B1 production was observed in cultures grown in shake flasks at 100 rpm, 26°C, and a 16-h photoperiod at 35 µmol m-2 s-1 for the same time as the reactor cultures. Seydel et al. reported a kalata B1 production of up to 0.37 mg g-1 in cultures grown 93 | Page

Mass-cultivation of plant cell suspensions for cyclic peptide production in shake flasks at 100 rpm, 24°C, and a 16-h photoperiod at 35 µmol m-2 s-1 after 7 days; however, after 9 days the kalata B1 concentration decreased to 0.07 mg g-1 [18]. Although the cultures were different, as evident in the chlorophyll production of the cells of the previous study, there could have been a time dependent downregulation of cyclotide production present in both cultures. The air-lift loop photobioreactor used by Seydel et al. experienced strong cell attachment and wall-growth in the head space resulting in reduced controllability and productivity [18]. The reactor tested here did not have any of these problems and performed well at all cell densities. The only observed wall-growth was in the corners of the single-use bags and did not have any perceived influence on the energy input into the culture or the oxygen transfer rate. The low observed cyclotide concentrations indicate that wild type O. affinis suspensions are not suitable to produce cyclotides under the tested conditions. However, their growth characteristics and low amounts of required phytohormones make them well suited for biomass generation. Lower doubling times and higher cell densities than observed in the well-studied model species N. benthamiana were achieved in shake flasks (data not shown). The same cultures grew equally well at the 5 L scale in single-use bags. Cyclotide production in O. affinis suspensions might be possible if transformation or reactivation of the cyclotide expression is achieved. In this thesis, elicitation strategies are presented in Chapter 4, whilst transient transformation experiments are presented in Chapter 5.

3.3.3 Prospects of Hybanthus enneaspermus cultivation in single-use bags

H. enneaspermus retained a light green colour and a strong cycloviolacin O2 production during cultivation in a rocking motion bioreactor with a 16 h photoperiod at 17 µmol m-2 s-1 supplemented with 1.5 mg L-1 2,4-D and 1.0 mg L-1 6-benzylaminopurine (BA). H. enneaspermus cell suspensions used for L-Dopa production presented in literature did not display green colour when cultivated in the dark and supplemented with 2.0 mg L-1 2,4-D [22]. The growth characteristics, as assessed here via online dissolved oxygen measurement, seemed akin to O. affinis although slower. And, like in O. affinis suspensions, the pH stabilized around 6. H. enneaspermus displayed spontaneous organogenesis and formed white root-like structures in one out of three single-use bag cultivations. This was especially impressive considering that these were free-floating cells affected by constant shear stress. Similar structures, albeit green, were reported in V. uliginosa calli in the presence of the auxins 2,4-D and 1-naphthaleneacetic acid (NAA) and suspensions in the presence of the cytokinin thidiazuron (TDZ) [23]. Here, the auxin NAA and the cytokinin 6-benzylaminopurine (BA) was used for callus induction, whereas the auxin 2,4-D and

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Mass-cultivation of plant cell suspensions for cyclic peptide production the cytokinin BA was added to the liquid suspension medium of H. enneaspermus. It was not possible to determine the root cause of the one to three ratio observed in single-use bags and shake flasks. The cyclotide production of the root-like structures differed significantly from the free-floating cells, with generally higher cyclotide levels of a large cyclotide number. The recent discovery and sequencing of H. enneaspermus cyclotides [24] allowed the identification of cycloviolacin O2 and Cter A as major components. In this case the immobilization and differentiation seemed to be the influencing factors for cyclotide production, not chlorophyll content. As cycloviolacin O2 is a bracelet type cyclotide and therefore hard to chemically synthesize, while at the same time highly studied in the last few years due to its outstanding biological activities [24-27], production of cycloviolacin O2 in H. enneaspermus suspension in single-use bags could be an option.

3.3.4 Prospects of Clitoria ternatea cultivation in single-use bags

C. ternatea is especially relevant for cyclotide research as it is the first plant industrially harvested for its cyclotide content. Innovate AG is marketing C. ternatea extract as “bee-friendly insecticide” with cyclotides as probable active compound [28]. The extract is collected from fresh whole plant biomass. Cultivation of C. ternatea in suspension might help to identify the active compound(s) and enable targeted production of active components. Even if this production does not prove to be viable, C. ternatea is a very interesting species to study cyclotide expression in different tissue types and cell differentiation states, as it contains a plethora of different cyclotides with unique gene setups [29, 30] and is widely used in commercial food and cosmetic products [31-36]. The C. ternatea suspensions grown in a single-use bag on a rocking motion bioreactor displayed light green colour, medium sized aggregates and a healthy growth. Quantification was not achieved as all cultures were lost to contamination and setting up new cultures was not possible in the given time as it would have required 3 months to reach the reactor stage starting from fresh seeds. The contamination had an incubation time of seven days, before any visible signs appeared. This led to the contamination of the backup line that was sub-cultured four days after the main cultures. However, this study supplies all the necessary tools to establish stable C. ternatea suspension cultures up to a 5 L scale, enabling future studies to tie on. This experience signifies the importance of cell banking and redundant culture handling when dealing with plant suspensions and highlights the challenges of plant cell suspension sampling in single-use bags. No cryopreservation protocol was available for C. ternatea at the point of writing; however, suspension culture protocols like the ones presented here are vital for effective development of preservation protocols in the future.

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Mass-cultivation of plant cell suspensions for cyclic peptide production

One interesting application of C. ternatea hairy root cultures, organ cultures caused by Agrobacterium rhizogenes infection, is the production the anti-cancer compound taraxerol [37]. Hairy root cultures normally produce higher concentrations of secondary metabolites, have higher long- term genetic and biosynthetic stability than suspension cultures [10], and could therefore also be interesting for cyclotide production. Like the H. enneaspermus root-like cultures formed via organogenesis, C. ternatea roots cultures and possibly even transgenic hair roots, could be cultivated in the single-use bag system presented here. In summary, all three species showed a lot of promise for cultivation in a rocking motion bioreactor, however yield enhancement was required for efficient cyclotide production in O. affinis suspensions. Therefore, the next chapter explores elicitation to boost cyclotide production in O. affinis suspensions.

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3.4 References

1. Whitford, W.G., M.A. Petrich, and W.P. Flanagan, Environmental Impacts of Single-Use Systems, in Single‐Use Technology in Biopharmaceutical Manufacture, D. Eibl and R. Eibl, Editors. 2019, John Wiley & Sons, Inc. p. 169. 2. Rawlings, B. and H. Pora, Environmental impact of single-use and reusable bioprocess systems. BioProcess Int, 2009. 7: p. 18. 3. Luo, J., F. Yu, L. Liu, C. Wu, and X. Mei, Effect of dissolved oxygen on the suspension culture of Taxus chinensis. Sheng wu gong cheng xue bao= Chinese journal of biotechnology, 2001. 17: p. 215. 4. Georgiev, M.I., R. Eibl, and J.-J. Zhong, Hosting the plant cells in vitro: recent trends in bioreactors. Applied microbiology and biotechnology, 2013. 97: p. 3787. 5. Kieran, P.M., P.F. Macloughlin, and D.M. Malone, Plant cell suspension cultures: some engineering considerations. Journal of Biotechnology, 1997. 59: p. 39. 6. Srikantan, C. and S. Srivastava, Bioreactor Design and Analysis for Large-Scale Plant Cell and Hairy Root Cultivation, in Hairy Roots, V. Srivastava and S. Mehrotra, Editors. 2018, Springer, Singapore. p. 147. 7. Seydel, P., Produktion von Cyclotiden in pflanzlichen Zellkulturen von Oldenlandia affinis, in Institute of Bioprocess Engineering. 2009, University of Erlangen-Nuremberg. 8. Seydel, P. and H. Dörnenburg, Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides. Plant Cell, Tissue and Organ Culture, 2006. 85: p. 247. 9. Dörnenburg, H. and P. Seydel, Effect of irradiation intensity on cell growth and kalata B1 accumulation in Oldenlandia affinis cultures. Plant Cell, Tissue and Organ Culture, 2007. 92: p. 93. 10. Huang, T.K. and K.A. McDonald, Bioreactor systems for in vitro production of foreign proteins using plant cell cultures. Biotechnol Adv, 2012. 30: p. 398. 11. Santos, R.B., R. Abranches, R. Fischer, M. Sack, and T. Holland, Putting the Spotlight Back on Plant Suspension Cultures. Front Plant Sci, 2016. 7: p. 297. 12. Fischer, R. and J.F. Buyel, Molecular farming – The slope of enlightenment. Biotechnology Advances, 2020: p. 107519. 13. Komarova, T.V., S. Baschieri, M. Donini, C. Marusic, E. Benvenuto, and Y.L. Dorokhov, Transient expression systems for plant-derived biopharmaceuticals. Expert Review of Vaccines, 2010. 9: p. 859. 14. Su, R., M. Sujarani, P. Shalini, and N. Prabhu, A Review on Bioreactor Technology Assisted Plant Suspension Culture. Asian Journal of Biotechnology and Bioresource Technology, 2019. 5: p. 1. 15. Lopes, A.G., Single-use in the biopharmaceutical industry: A review of current technology impact, challenges and limitations. Food and Bioproducts Processing, 2015. 93: p. 98. 16. Tric, M., M. Lederle, L. Neuner, I. Dolgowjasow, P. Wiedemann, S. Wolfl, and T. Werner, Optical biosensor optimized for continuous in-line glucose monitoring in animal cell culture. Anal Bioanal Chem, 2017. 409: p. 5711. 17. Eibl, R., S. Werner, and D. Eibl, Disposable bioreactors for plant liquid cultures at Litre‐ scale. Engineering in Life Sciences, 2009. 9: p. 156. 18. Seydel, P., C. Walter, and H. Dörnenburg, Scale-up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production. Engineering in Life Sciences, 2009. 9: p. 219. 19. Seydel, P., C.W. Gruber, D.J. Craik, and H. Dörnenburg, Formation of cyclotides and variations in cyclotide expression in Oldenlandia affinis suspension cultures. Appl Microbiol Biotechnol, 2007. 77: p. 275.

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20. Butenko, R.G., A.K. Lipsky, N.D. Chernyak, and H.C. Arya, Changes in culture medium pH by cell suspension cultures of Dioscorea deltoidea. Plant Science Letters, 1984. 35: p. 207. 21. Bai, Y., M. Moo-Young, and W.A. Anderson, A mechanistic model for gas-liquid mass transfer prediction in a rocking disposable bioreactor. Biotechnol Bioeng, 2019. 116: p. 1986. 22. Sathish, S., V. Vasudevan, S. Karthik, G. Pavan, and M. Manickavasagam, Enhanced l-Dopa production from elicited cell suspension culture of Hybanthus enneaspermus (L.) F. Muell. Plant Biotechnology Reports, 2019. 13: p. 613. 23. Slazak, B., E. Jacobsson, E. Kuta, and U. Goransson, Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae). Phytochemistry, 2015. 117: p. 527. 24. Du, Q., L.Y. Chan, E.K. Gilding, S.T. Henriques, N.D. Condon, A.S. Ravipati, Q. Kaas, Y.H. Huang, and D.J. Craik, Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus. J Biol Chem, 2020. 25. Parsley, N.C., C.L. Kirkpatrick, C.M. Crittenden, J.G. Rad, D.W. Hoskin, J.S. Brodbelt, and L.M. Hicks, PepSAVI-MS reveals anticancer and antifungal cycloviolacins in Viola odorata. Phytochemistry, 2018. 152: p. 61. 26. Slazak, B., M. Kapusta, A.A. Stromstedt, A. Slomka, M. Krychowiak, M. Shariatgorji, P.E. Andren, J. Bohdanowicz, E. Kuta, and U. Goransson, How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System. Front Plant Sci, 2018. 9: p. 1296. 27. Slazak, B., M. Kapusta, S. Malik, J. Bohdanowicz, E. Kuta, P. Malec, and U. Göransson, Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense. Planta, 2016. 244: p. 1029. 28. On the evaluation of the new active clitoria ternatea in the product Sero-X Insecticide. 2016, Australian Pesticides and Veterinary Medicines Authority, ISSN: 1443-1335 (electronic). 29. Oguis, G.K., E.K. Gilding, Y.-H. Huang, A.G. Poth, M.A. Jackson, and D.J. Craik, Insecticidal diversity of butterfly pea (Clitoria ternatea) accessions. Industrial Crops and Products, 2020. 147: p. 112214. 30. Oguis, G.K., E.K. Gilding, M.A. Jackson, and D.J. Craik, Butterfly Pea (Clitoria ternatea), a Cyclotide-Bearing Plant With Applications in Agriculture and Medicine. Front Plant Sci, 2019. 10: p. 645. 31. Chusak, C., C.J. Henry, P. Chantarasinlapin, V. Techasukthavorn, and S. Adisakwattana, Influence of Clitoria ternatea flower extract on the in vitro enzymatic digestibility of starch and its application in bread. Foods, 2018. 7: p. 102. 32. Chusak, C., T. Thilavech, C.J. Henry, and S. Adisakwattana, Acute effect of Clitoria ternatea flower beverage on glycemic response and antioxidant capacity in healthy subjects: a randomized crossover trial. BMC complementary and alternative medicine, 2018. 18: p. 1. 33. Pasukamonset, P., O. Kwon, and S. Adisakwattana, Alginate-based encapsulation of polyphenols from Clitoria ternatea petal flower extract enhances stability and biological activity under simulated gastrointestinal conditions. Food Hydrocolloids, 2016. 61: p. 772. 34. Pasukamonset, P., O. Kwon, and S. Adisakwattana, Oxidative stability of cooked pork patties incorporated with Clitoria ternatea extract (blue pea flower petal) during refrigerated storage. Journal of Food Processing and Preservation, 2017. 41: p. e12751. 35. Pasukamonset, P., T. Pumalee, N. Sanguansuk, C. Chumyen, P. Wongvasu, S. Adisakwattana, and S. Ngamukote, Physicochemical, antioxidant and sensory characteristics of sponge cakes fortified with Clitoria ternatea extract. Journal of Food Science and Technology, 2018. 55: p. 2881. 36. Adisakwattana, S., P. Pasukamonset, and C. Chusak, Clitoria ternatea beverages and antioxidant usage, in Pathology, V.R. Preedy, Editor. 2020, Academic Press. p. 189. 37. Swain, S.S., K.K. Rout, and P.K. Chand, Production of triterpenoid anti-cancer compound taraxerol in Agrobacterium-transformed root cultures of butterfly pea (Clitoria ternatea L.). Appl Biochem Biotechnol, 2012. 168: p. 487. 98 | Page

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Cyclotide elicitation in suspension cultures 4 Cyclotide elicitation in suspension cultures

In a biotechnological framework, elicitation describes the induction, ideally at high levels, of a target metabolite in a biological system induced by an external signal (i.e. elicitor). Considering a cell in a suspension culture, chemicals, peptides, proteins, shear stress, osmotic pressure, pH, illumination, and many more are possible elicitors that may induce a cell to make a product [1]. The gathered data can also further the understanding of the influence of stress factors on biochemical pathways [1]. Elicitors often prompt a complex response in the target system [2]. Yu et al. (2019) demonstrated, that the treatment of Salvia miltiorrhiza suspension cells with sodium chloride significantly reduced the production of the primary metabolites fructose and glucose, whereas 10 secondary metabolites were upregulated [3]. De-Xian Kok et al. reported that rice calli can be switched from high growth rates to enhanced production of secondary metabolites by altering the elicitor (Pluronic F-68) concentration in the medium [4]. These examples demonstrate that beneficial elicitation of secondary metabolites in suspension cells is feasible, even though it can induce an energetic burden for the host that can result in slower growth or reduced cell viability. Therefore, a promising strategy for elicited biological production consists of two stages; a first stage optimised for growth and a second stage optimised for production. In this work, methyl jasmonate, salicylic acid and sodium chloride were chosen as candidates for eliciting cyclotide production. These elicitors have been previously linked to plant defence [5-7] or stress response [8, 9]. This is relevant, as cyclotides are believed to be an important part of the innate plant immune system [10]. Three cyclotide producing species were chosen for cyclotide elicitation in suspension. No previous elicitation attempts have been reported for Clitoria ternatea and Hybanthus enneaspermus suspension cultures, whereas Oldenlandia affinis suspensions have been shown to be influenced by irradiation at different intensities [11]. Additionally, cyclotide production in O. affinis suspension cells and organized tissue was enhanced by chitosan [12]. The fast-growing suspension cultures of the three species used in this work were monitored for up to seven days after elicitation. Suspension cultures enable a large set of identical samples experiencing identical conditions and elicitor concentrations. The experiments presented here were designed to identify conditions that elicit cyclotide production and might enable a two-stage process for plant suspension based cyclotide production.

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Cyclotide elicitation in suspension cultures 4.1 Materials and methods

Maintenance, elicitation and sampling of suspension cells was done under sterile conditions in a biosafety cabinet. All treatments were done under identical elicitor concentrations in triplicates for each species. Ethanol, a chemical damaging plant cells, was needed to dissolve methyl jasmonate and salicylic acid. 2.5 µL ethanol per 1 mL of culture volume was chosen as final ethanol concentration to not harm the cultures.

4.1.1 Oldenlandia affinis

The following subsections describe the preparation of Oldenlandia affinis suspensions for treatment with selected elicitors. Additionally, the sampling and analysis methods are presented.

4.1.1.1 Suspension culture

A two-year-old Oldenlandia affinis suspension culture (80 mL in 250 mL Erlenmeyer flask) was monitored for homogeneity and stability via microscopy and MALDI-TOF analysis for two weeks. Subsequently, the suspension culture was split into four 250 mL Erlenmeyer flasks and fresh growth medium was added to reach 80 mL in each flask. The growth medium composition is presented in Table 14 for quick reference. After one week, this procedure was repeated, resulting in 12, 80 mL cultures. After another week, these cultures were split to achieve 24 cultures. These cultures were subcultured after one week by replacing 75% of the culture with fresh medium. After another week, 75% of the culture was again replaced with fresh medium. Then the first samples of day 0 without elicitation were taken.

Table 14 | Optimised O. affinis suspension medium. Species 2,4-D pH Sucrose Salts & Vitamins O. affinis 0.4 mg L-1 5.8 20 g L-1 Murashige & Skoog [13] Note: The pH was adjusted to 5.8 using 1 M KOH.

4.1.1.2 Elicitation

One hour after the replacement of 75% of the culture with fresh medium the elicitors were added.

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Cyclotide elicitation in suspension cultures

4.1.1.2.1 Control

The control solution was prepared by mixing 2 mL sterile water with 2 mL ethanol and filtering through a 0.45 µm sterile filter. 400 µL of the control solution was added to three O. affinis cultures.

4.1.1.2.2 Methyl jasmonate

0.5 µL, 8.3 µL and 16.6 µL of 95% methyl jasmonate (Sigma Aldrich) were mixed with 4 mL 50% (v/v) ethanol each and filtered through a 0.45 µm sterile filter. 400 µL of each mixture was added to three O. affinis cultures each to achieve a final concentration of 3 µM, 50 µM, and 100 µM methyl jasmonate respectively.

4.1.1.2.3 Salicylic acid

100 µL water were added to 1.9 mL ethanol. 33.3 mg salicylic acid (Sigma Aldrich) was added and mixed until dissolved completely. Then 2 mL water was added, and the mixture filtered through a 0.45 µm sterile filter. 400 µL of the mixture were added to three O. affinis cultures each to achieve a final concentration of 300 µM

4.1.1.2.4 Sodium chloride

0.142 g, 2.367 g, and 3.156 g sodium chloride were dissolved in 10 mL water each and consequently filtered through a 0.45 µm sterile filter. 1 mL of the first solution was added to three O. affinis cultures each to achieve a final concentration of 3 mM. 1 mL of the second solution was added to three O. affinis cultures each to achieve a final concentration of 50 mM. 3 mL of the third solution was added to three O. affinis cultures each to achieve a final concentration of 200 mM.

4.1.1.2.5 Immobilization

Oldenlandia affinis suspension cells were immobilized within growth at the vessel walls. After the central cells turned green, these were tested for cyclotides. Additionally, suspension cells were plated in small aggregates on solid callus induction medium. Table 15 lists the callus induction medium for quick reference. These cells were grown on solid agar medium for 63 days.

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Table 15 | O. affinis callus induction medium. Species NAA BAP Sucrose O. affinis 1.86 mg L-1 2.25 mg L-1 5 g L-1 Note: The basis was ultrapure water. Additional ingredients were full strength MS (M519, Phyto Technology Lab) and 8 g L-1 Phyto agar (Duchefa Biochemie B. V.). The pH was adjusted to 5.7 using 1 M KOH.

4.1.1.2.6 Starvation

O. affinis cell suspensions were starved by reducing or leaving sucrose and/or phytohormones out of the supplied medium.

4.1.1.2.7 Illumination

O. affinis cell suspensions were cultivated in the dark and with a 16h photoperiod under 35 µmol m 2 s 1 photon density using RGB LEDs with a wavelength mix optimised for photosystem

I and photosystem⁻ ⁻ II. The relative cyclotide concentration compared via MALDI-TOF MS measurements including an internal control.

4.1.1.3 Sampling

Samples of all cultures were taken on day 0 (before elicitation, after subculturing), day 1, day 4, and day 7. To achieve a homogeneous and representative sample set, samples of each culture were taken by transferring 1.5 mL culture into a RNase free 2 mL Eppendorf tube.

4.1.1.4 Microscopy

All samples were imaged at 50x and 200x magnification at a stereo light microscope using bright field, differential interference contrast and fluorescence at 353 nm, 493 nm, 593 nm, and 633 nm excitation wavelength. The samples were prepared fresh and wet, without fixation on glass slides with a plastic cover slip.

4.1.1.5 MALDI-TOF analysis

All samples were analysed as described in Section 2.1.6.

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Cyclotide elicitation in suspension cultures 4.1.2 Clitoria ternatea

The following subchapters describe the preparation of Clitoria ternatea suspensions for treatment with selected elicitors. Additionally, the sampling and analysis methods are presented.

4.1.2.1 Suspension culture

Clitoria ternatea callus cultures were induced from sterile leaf explants as described in Section 2.1.2 and grown for four weeks. Cell suspension cultures were initiated from the callus cultures as described in Section 2.1.3 with a small variation in phytohormone concentration; here, after short screening of the conditions presented in Table 16 for convenience, 2 mg L-1 BA and 1 mg L-1 NAA were used, as this produced the best growth for this cell line.

Table 16 | C. ternatea suspension initiation conditions. Condition NAA [mg L-1] BAP [mg L-1] Explant tissue I 1 1 Leaf J 1 2 Stem K 1 1.5 Stem

The cells were grown for three weeks before elicitation with weekly replacement of 75% of the culture with fresh medium. All cultures were kept at a volume of 40 mL in 125 mL Erlenmeyer flasks.

4.1.2.2 Elicitation

Two days after the replacement of 75% of the culture with fresh medium the elicitors were added. The culture volume was determined by weight and used to calculate the elicitor volumes to be added.

4.1.2.2.1 Control

The three negative control cultures were treated with ethanol to achieve a final concentration of 0.1 % (v/v) ethanol. The cultures treated with methyl jasmonate had the same final ethanol concentration.

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4.1.2.2.2 Methyl jasmonate

95% methyl jasmonate (Sigma Aldrich) was mixed with 50% (v/v) ethanol and filtered through a 0.45 µm sterile filter. Three C. ternatea cultures were adjusted to 50 µM, and three cultures were adjusted to100 µM methyl jasmonate.

4.1.2.3 Sampling

Samples of all cultures were taken on day 0 (after subculturing). Additional samples were taken 6 hours, 1 day, 3 days and 5 days after elicitation on day 2. To achieve a homogeneous and representative sample set, samples of each culture were taken by transferring 1.5 mL culture into a RNase free 2 mL Eppendorf tube.

4.1.2.4 MALDI-TOF analysis

The same protocol as described in Section 2.1.6 was used except for spiking in cyclic kalata B1 instead of linear kalata B1-Glu-Ile-Ile to not overlap with native cyclotides present in C. ternatea.

4.1.3 Hybanthus enneaspermus

The following subchapters describe the preparation of Hybanthus enneaspermus suspensions for treatment with selected elicitors. Additionally, the sampling and analysis methods are presented.

4.1.3.1 Suspension culture

Hybanthus enneaspermus callus cultures were induced from sterile leaf explants as described in Section 2.1.2 and grown for four weeks. Cell suspension cultures were initiated from the callus cultures as described in Section 2.1.3, except a variation in phytohormone concentration. 1 mg L-1 BA and 1.5 mg L-1 2,4-D were used, as this produced the best growth for this cell line. The cells were grown for three weeks before elicitation with weekly replacement of 75% of the culture with fresh medium. All cultures were kept at a volume of 40 mL in 125 mL Erlenmeyer flasks.

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4.1.3.2 Elicitation

Two days after the replacement of 75% of the culture with fresh medium the elicitors were added. The culture volume was determined by weight and used to calculate the elicitor volumes to be added.

4.1.3.2.1 Control

4.1.3.2.2 Methyl jasmonate

95% methyl jasmonate (Sigma Aldrich) was mixed with 50% (v/v) ethanol and filtered through a 0.45 µm sterile filter. Three H. enneaspermus cultures were adjusted to 50 µM, and three cultures were adjusted to100 µM methyl jasmonate.

4.1.3.3 Sampling

Samples of all cultures were taken on day 0 (after subculturing). Additional samples were taken 6 hours, 1 day, 2 days and 3 days after elicitation on day 2. To achieve a homogeneous and representative sample set, samples of each culture were taken by transferring 1.5 mL culture into a RNase free 2 mL Eppendorf tube.

4.1.3.4 MALDI-TOF analysis

The same protocol as described in Section 2.1.6 was used except for spiking in cyclic kalata B1 instead of linear kalata B1-Glu-Ile-Ile to not overlap with native cyclotides present in H. enneaspermus.

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Cyclotide elicitation in suspension cultures 4.2 Results

4.2.1 Elicitation of cyclotide production in Oldenlandia affinis suspensions

4.2.1.1 Methyl jasmonate, sodium chloride and salicylic acid

O. affinis suspension cultures were treated with methyl jasmonate, sodium chloride, and salicylic acid. The cultures were elicited one hour after subculturing. Samples were taken after one, four, and seven days as biological triplicates. Water and ethanol were added to the negative control cultures to achieve the same total volume and ethanol concentration in all cultures. This treatment ensured identical conditions between all cultures with the elicitor as the only difference. Figure 31A shows the growth behaviour of untreated O. affinis cultures during the elicitation period. The peptide production presented in Figure 31B, C, and D was normalized against the production in the respective culture before elicitation. All cultures displayed a strong drop in cyclotide production after passaging. Among the seven tested conditions, only 3 mM sodium chloride (NaCl) elicited a cyclotide production significantly higher than the control cultures and the cultures before treatment as shown in Figure 31. Higher concentrations of sodium chloride did not lead to a higher cyclotide expression. At 200 mM sodium chloride the cells died within four days. Up until their death however, they showed the same trend of cyclotide expression as the 50 mM treatment (data not shown here). 3 µM, 50 µM, or 200 µM methyl jasmonate did not lead to a significant deviation from the trend observed in the control cultures. Treatment with 300 µM salicylic acid did not result in meaningfully different cyclotide production compared to the negative control (data not shown here).

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Figure 31 | Cyclotide elicitation in O. affinis suspension cells. Water and ethanol were added to the negative control cultures to achieve the same total volume and ethanol concentration in all cultures. Error bars indicate standard deviation, biological replicates n=3. A) O. affinis growth curve. The first sample was taken before replacing 75% of culture medium with fresh medium for subculturing. The second sample was taken after the replacement. B) Normalized peptide production of cycloviolacin O2. C) Normalized peptide production of kalata B22. D) Normalized peptide production of kalata B23.

Two different cell morphologies were distinguished in O. affinis culture before elicitation as can be seen in Figure 32H. The elongated bar-like cells formed chains where the border cells divided, elongating the chain, whereas the ellipsoid cells formed round 3-dimensional aggregates. In both cell populations, the nucleus and some thread like structures starting from the nucleus could be distinguished. Most cells were healthy with intact cell wall, although some malformed cells and cell debris were present. As depicted in Figure 32A noteworthy change in morphology was observed one day after elicitation with 3 mM sodium chloride. Most cells showed dark spots close to and inside the nucleus. Many cells started to coil up and formed a spiral like structure. By day four, most cells had aggregated into large super-structures with only a few single-free cells around them. Most cells showed dark spots within the cytoplasm and the nucleus as can be seen in Figure 32B. The salt treatment clearly induced a stress response in the culture. After seven days, many cells seemed to revert to their original appearance. However, there were still cells with strong dark spots present. Nearly all cells had adopted the elongated morphology, with a few exceptions that seemed stuck in between the two morphologies (Figure 32C). 108 | Page

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A higher sodium chloride concentration had immediate influence on the cell morphology as shown in Figure 32D taken one day after treatment with 50 mM. The cells lost volume and were thinner. Additionally, many more dark spots were observed. The same morphology change and large amounts of extracellular particles were clearly apparent in samples treated with 200 mM sodium chloride. Many cells were already dead, as evident by their ruptured cell wall and missing cell organelles, at day one and their debris was visible in the culture. Cells that still possessed an intact cell wall and nucleus displayed many, strong dark spots as evident in Figure 32E. Overall, the sample contained way fewer living cells and cell aggregates. Interestingly, cells treated with 100 µM methyl jasmonate displayed similar morphology to the 200 mM sodium chloride treatment, without dying subsequently (Figure 32F). The treatment with 300 µM salicylic acid resulted in a reduced number of ellipsoid cells as seen in Figure 32G, however the present elongated cells were not distinguishable different from untreated cells.

Figure 32 | Microscopy images of selected O. affinis suspension cultures during elicitation experiments. A) One day after treatment with 3 mM sodium chloride. B) Four days after treatment with 3 mM sodium chloride. C) Seven days after treatment with 3 mM sodium chloride. D) One day after treatment with 50 mM sodium chloride. E) One days after treatment with 200 mM sodium chloride. F) One days after treatment with 100 µM methyl jasmonate. G) One days after treatment with 300 µM salicylic acid. H) | Untreated O. affinis suspension culture before elicitation.

The peptide levels of cycloviolacin O2, kalata B22, and kalata B23 after NaCl elicitation were significantly higher than in leaf tissue or non-treated suspension cells as shown in Figure 33. This figure highlights the potential of cyclotide production in O. affinis suspension cells compared the whole plant cultivation. Kalata B1 production could not be reinstated. Callus tissue produced the largest amount of cycloviolacin O2 and kalata B1.

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4 Kalata B1 Kalata B22 Kalata B23 Cycloviolacin O2

2 Relative peptide Relative concentration [-] 1

0 Leaf Callus Suspension Suspension + 3 mM NaCl

Figure 33 | Relative cyclotide expression in O. affinis. 'Leaf': leaf samples grown in the wild. 'Callus': callus tissue eight weeks after initiation. 'Suspension': four weeks old suspension cultures. 'Suspension + 3 mM NaCl': four weeks old suspension cultures treated with 3 mM sodium chloride. Error bars indicate standard deviation, n=4.

4.2.1.2 Illumination

Illumination did not result in observable changes in the cyclotide profile of O. affinis suspension cultures (data not visualized here). Illuminated and non-illuminated cultures equally reduced their cyclotide production as presented in Section 2.2.4.1.

4.2.1.3 Immobilization

Following incidental observations of culture growth, the cells adhering to the flask wall were also analysed for cyclotide expression. This wall growth was observed for all species if grown in the same vessel for more than two weeks. Some of these immobilized cells turned green in O. affinis cultures, apparently reactivating photosynthesis to overcome diffusion based nutrient starvation. Immobilized cells subjected to diffusion based nutrient starvation that did not turn green, turned brown and slowly died. Cells in suspension that were subjected to nutrient starvation did not turn green and did not show increased cyclotide levels as presented in Section 4.2.1.4. High cyclotide levels were detected in cells isolated from these green wall growth patches, as shown in Figure 34. In contrast, Figure 35 highlights that the floating cells of the same culture displayed nearly no cyclotides with only cycloviolacin O2 and kalata B23 detected at the lower limits of detection.

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Figure 34 | Cyclotide profile of green O. affinis wall growth of 77 days old suspension cells.

Figure 35 | Cyclotide profile of floating O. affinis suspension cells of the same culture as the wall growth in Figure 34.

To test if cyclotide production could artificially and reproducibly be re-instated, suspension cell aliquots were placed back onto solid agar medium optimised for callus growth. These cells developed a strong callus like cyclotide profile after two weeks with kalata B1 as the major cyclotide peak. The cyclotide production was stable for at least 63 days (see Figure 36).

Figure 36 | Cyclotide profile of O. affinis callus initiated from suspension cells. 111 | Page

Cyclotide elicitation in suspension cultures

4.2.1.4 Starvation

Starvation was a possible explanation of the green colour observed in immobilized cells indicating a switch from heterotroph to phototroph. To investigate, suspension cultures were stressed under various conditions including sucrose depletion, vitamin depletion, different levels of growth hormones, and different levels of shear stress including no agitation. None of these conditions resulted in an upregulation of cyclotide production. However, some cultures exhibited increased cycloviolacin O2 and kalata B23 production after two weeks without hormones combined with one week without agitation as displayed in Figure 37. Under these conditions, the cells were settling to the bottom of the flask and their visual health was declining, as assessed by their brown colour and cells with broken cell walls visible under the microscope (data not shown).

Figure 37 | Cyclotide profile of O. affinis suspension after two weeks without phytohormones and one week without agitation.

4.2.2 Elicitation of cyclotide production in Clitoria ternatea suspensions

As a pre-requisite for testing elicitation of C. ternatea suspension cultures, optimal explant starting material and growth media for cyclotide production were identified using 60 explants for each of 3 growth conditions. The growth conditions are listed in Table 17 for quick reference.

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Table 17 | C. ternatea culture conditions. Condition NAA BAP 2,4-D I 1 mg L-1 1 mg L-1 - J 1 mg L-1 2 mg L-1 - K 1 mg L-1 1.5 mg L-1 - Note: Phytohormone concentrations tested for their influence on cyclotide production in C. ternatea suspension cultures.

The different phytohormone concentrations were initially screened to optimise growth as no data for C. ternatea suspension cells was available in literature. The hormone treatments influenced cyclotide production in C. ternatea callus and suspension culture, whereas the tissue type of the explant had little influence as presented in Figure 38. Condition J resulted in the highest cyclotide production in callus and suspension. Consequently, this condition was used for the methyl jasmonate elicitation experiment.

Figure 38 | Influence of hormone treatment and explant tissue type on cyclotide expression in C. ternatea callus and suspension cells. A) Condition I initiated from leaf explants. B) Condition J initiated from stem explants. C) Condition K initiated from stem explants. The phytohormone concentrations of the different conditions are listed in Table 16. Error bars indicate standard deviation of the biological replicates, n=3.

C. ternatea suspension cultures were treated with 50 µM and 100 µM methyl jasmonate two days after subculturing. Samples were taken six hours, one day, three days, and five days after elicitation. The response in cyclotide production was monitored via MALDI-TOF analysis and utilised an internal peptide control enabling relative quantification. The cyclotide production was normalized against the initial level in the untreated cells to highlight the trend and enable comparability. The most prevalent cyclotides found in the tested C. ternatea suspensions were cliotide T1, Cter A, and Cter P. No statistically significant difference in production was observed for 50 and 100 µM methyl jasmonate compared to the negative control as shown in Figure 39.

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Figure 39 | C. ternatea suspension elicitation with 50 and 100 µM methyl jasmonate. A) Normalized cliotide T1 production. B) Normalized Cter A production. C) Normalized Cter P production. Error bars indicate standard deviation of the biological replicates, n=3.

4.2.3 Elicitation of cyclotide production in Hybanthus enneaspermus suspensions

Three phytohormone treatments, of H. enneaspermus callus and suspension culture were tested using 60 explants each. The treatments are presented in Table 18 for quick reference.

Table 18 | H. enneaspermus culture conditions. Condition 2,4-D NAA BA A 1.5 mg L-1 - 1 mg L-1 G - 0.25 mg L-1 0.5 mg L-1 PC - 0.48 mg L-1 0.5 mg L-1 Note: Phytohormone concentrations tested for their influence on cyclotide production in H. enneaspermus suspension cultures.

No significant influence on cyclotide production was observed as presented in Figure 40. Condition A resulted in stable growth without organogenesis and was subsequently used for the methyl jasmonate elicitation experiment. Leaves did produce many cyclotides, but not cycloviolacin O13 and Cter A. Cycloviolacin O2 was present in minute amounts in leaves. The callus and young suspension cultures contained cycloviolacin O2 and cycloviolacin O13 as most prevalent cyclotides. The relative cyclotide levels were higher in callus and suspension cells compared to leaves (data not shown).

Figure 40 | Influence of hormone treatment and explant tissue type on cyclotide expression in H. enneaspermus callus and suspension cells. A) Condition A initiated from leaf explants. B) Condition G initiated from leaf explants. C) Condition PC initiated from leaf explants. The phytohormone concentrations of the different conditions are listed in Table 16. Error bars indicate standard deviation of three biological replicates.

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Analogous to the C. ternatea elicitation experiments, H. enneaspermus suspension cells were treated with 50 µM and 100 µM methyl jasmonate two days after subculturing. Samples were taken six hours, one day, two days, and three days after elicitation. The response in cyclotide expression was assessed via relative MALDI-TOF analysis. The cyclotide production was normalized against the initial level in the untreated cells to highlight the trend and enable comparability. Only cycloviolacin O2 was detected in measurable amounts in the tested H. enneaspermus suspensions. As shown in Figure 41A, no significant change in cycloviolacin O2 expression was recorded after methyl jasmonate elicitation. The cultures remained healthy and displayed a slight green colour as can be seen in Figure 41B.

Figure 41 | H. enneaspermus suspension elicitation with 50 and 100 µM methyl jasmonate. A) Normalized cycloviolacin O2 production. B) Healthy H. enneaspermus suspension cultures used for elicitation. Error bars indicate standard deviation of three biological replicates.

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Cyclotide elicitation in suspension cultures 4.3 Discussion

Elicitation of plant cell suspensions was assessed as a possible strategy to enhance cyclotide production. The two main elicitors tested were methyl jasmonate and sodium chloride. Plant cell suspensions allow for the handling of large sample numbers, with identical cell densities and homogeneous distribution of the tested substance throughout the culture, a clear advantage compared to plants. However, the standard deviation between biological replicates was still very high and difficult to explain. This could have negative effects on consistent high yields in a production process. Nevertheless, C. ternatea, H. enneaspermus, and O. affinis suspensions have proven to be valuable tools for screening experiments, either to enhance secondary metabolite production or to elucidate biosynthetic pathways. Their homogeneous growth in suspension might enable automatized screening assays in 24 or 96 well plates, a large advantage considering the vast amount of possible elicitors and need to test combinatorial effects [1].

4.3.1 Enhancing cyclotide production in Oldenlandia affinis

Only sodium chloride at a concentration of 3 mM led to a statistically significant upregulation of cycloviolacin O2, kalata B22 and kalata B23 production. None of the tested elicitor concentrations activated the production of a cyclotide not present before the treatment. The observed changes in all three were very similar, possibly indicating a common regulatory pathway. The simplified and normalized influence of the tested elicitors on cycloviolacin O2 expression is depicted in Figure 42.

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Figure 42 | Summary of cycloviolacin O2 production in O. affinis suspension after elicitation. Shown here are the arithmetic means for cycloviolacin O2 production of all tested treatments normalized against the cycloviolacin O2 production before treatment. Error bars are not shown to highlight the trend. The full data sets are presented in Section 4.2.

The general cyclotide production trend was reminiscent of the observed cell growth in suspension as presented in Figure 31A. That neither the cell density nor the cyclotide levels reached the initial value was most likely a sampling artefact, because taking samples out of a relatively small culture volume changes the culture conditions. Considering the strong cyclotide production in callus tissue, cell-cell interactions could play an important role. At the end of the growth cycle in suspension, the cell density in the medium is much higher than in the beginning resulting in stronger and more frequent cell-cell interactions and consequently in higher cyclotide production. The textbook definition of cell growth in suspension divides it into lag-, log-, and stationary- phase [14]. The lag-phase is characterized by an initial very slow growth. During this time, the cells adjust to the changed environment. In the log-phase (i.e. logarithmic), this change is successfully completed, and the cells grow exponentially. Finally, in the stationary phase, the cell growth becomes limited by the availability of nutrients, oxygen, and/or space until the maximal cell number is achieved. If nothing is changed a final death-phase follows in which the cells start dying due to nutrient depletion. These growth phases were not very pronounced in O. affinis suspensions. Especially the lag-phase was missing as reported before for O. affinis cultures with high inoculation density [15]. This indicates that O. affinis was able to adapt quickly to changes in cell density, sucrose availability, and pH. Future experiments could try to link cyclotide production to growth phase or cell

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Cyclotide elicitation in suspension cultures density. However, cyclotide levels were so low compared to plant or callus tissue that economical cyclotide production in wild type O. affinis cells without elicitation seemed unlikely. Even though treatment with 3 mM sodium chloride had a strong positive influence on cyclotide levels, treatment with 50 mM sodium chloride did not result in any upregulation of cyclotide production, and 200 mM sodium chloride killed the cells within four days. The dark spots, that were recorded inside the cells after sodium chloride and methyl jasmonate treatment, were hypothesized to be protein storage vacuoles as reported before [16]. The measured cyclotide production did not correlate with visual stress indicators, demonstrating that cyclotides are probably not part of a general stress response. Generally, suspension cells seem to be more susceptible to salt stress than whole plants and the inhibitory concentrations observed here correlate well with the concentrations observed previously in potato cultivars [17]. Sodium chloride induces osmotic pressure, similar to drought stress in plants, and has been reported to change cell physiology and enhance secondary metabolism in plant cells [18]. Orthosiphon stamineus cell suspensions, for example, have been treated with sodium chloride concentrations of up to 85.6 mM before and increased production of phenolic content was observed as result of increased osmotic pressure [19]. As another example, sodium chloride enhanced the production of steviol glycosides in Stevia rebaudiana suspension cells by 92% with minor impact on growth and morphology [8]. However, glycosides can actively counteract the osmotic pressure whereas cyclotides are not known to be compatible solutes. Next steps would include mRNA extraction to assess cyclotide and AEP gene expression during sodium chloride treatments to elucidate the regulatory pathways. Methyl jasmonate was demonstrated to elicit the production of paclitaxel in Taxus cell suspension culture 5.1-fold [20]. The same study confirmed that 2.5 µL of ethanol per 1 mL of culture volume was tolerated by suspension cells. Additionally, there was a lag of 4 days before the onset of paclitaxel accumulation. The overall culture time of the Taxus cells was 14 days within which a 2.5-fold growth was observed. Here, methyl jasmonate had a clear influence on cell morphology, but not on cyclotide production. 2.5 µL mL-1 ethanol was used for all experiments to be comparable and to not damage the suspension cells. Also, all cultures were monitored for at least eight days to catch a delayed response like the one observed in the Taxus cell suspensions. Future studies could examine the gene response to methyl jasmonate in O. affinis suspension and see if other proteins are affected by the treatment. The strong change in cell morphology indicates that there should be significant changes in the secondary metabolite profile, but cyclotides have not been one of them. Joseph et al. observed that undifferentiated suspension cells of Hypericum perforatum were highly amenable to elicitation with salicylic acid. They achieved doubling of the production of hypericin and pseudohypericin without visibly affecting the growth and development [21]. In this work, treatment with 300 µM salicylic acid did not affect cell morphology, only the ratio of ellipsoid 118 | Page

Cyclotide elicitation in suspension cultures to elongated cells, as shown in Figure 32G and had no influence on recorded cyclotide levels. This result, in combination with low response in cyclotide expression C. ternatea leaves reported before [22], indicates that cyclotide production is at least partially independent of the salicylic acid systemic disease resistance pathways [7]. Following the microscopic observations, it was concluded that the morphology of O. affinis suspension cells is a good indicator of their stress level; however, neither their cyclotide production nor their ability to survive can be deduced by it. Other studies have linked the plant cell wall to pathogen response [23], possibly explaining the morphology changes observed here. Some of these stress-induced responses are also causing alterations in the cell differentiation status [24]. As shown in this work, cell differentiation has a large influence on cyclotide production. However, it was also shown that most stresses do not directly cause enhanced cyclotide production. Even though there was a large deviation in cyclotide production under seemingly identical conditions, field grown plants display even larger deviations depending on season, soil, available sunlight and many more influencing factors [25]. Significant increase in cyclotide production in O. affinis has been reported by utilizing low light conditions as elicitation during the callus and suspension stage, resulting in 0.49 mg kalata B1 per gram dry weight [11]. In this study, these results could not be reproduced, as illuminated and not illuminated cultures lost their cyclotide profile over time in the same way. The two most produced cyclotides in plant leaves; kalata B1 and kalata B2 were never observed in a suspension culture older than four weeks. However, elicitation of suspension cells brought the production of three cyclotides that were barely synthesised in plant leaves nearly to the level of kalata B1 in leaves. Cyclotide production seemed to be tissue specific in all tested species and elicitation could only boost cyclotides already present within this tissue. This indicates that elicitors are epistatic to patterns of permissive cyclotide expression in each tissue/cell type. Immobilization of suspension cells on solid agar medium resulted green tissue growth returning kalata B1 production. A close relation between visually assessed chlorophyll production and kalata B1 synthesis was observed confirming the results obtained by Dörnenburg & Seydel [11]. These results suggest that cyclotide production is coupled to a combination of differentiation, the available chemical signals and the perceived physical surrounding. This observation might also explain the inability to elicit kalata B1 production via irradiation in this study, as all O. affinis suspension cultures remained white and did not produce chlorophyll, even under irradiation. Although these results are biologically intriguing, the scale-up of callus production on solid medium is unlikely to represent an economical approach, unless the target product is of very high value. A more scalable way is immobilization of suspension cells within alginate beads. Alginate immobilization was reported to enhance production and promote secretion of secondary metabolites 119 | Page

Cyclotide elicitation in suspension cultures in plant cell suspensions [26, 27] and is a promising next step to enhance cyclotide production in O. affinis suspensions.

4.3.2 Enhancing cyclotide production in Clitoria ternatea

C. ternatea suspensions displayed the strongest cyclotide production of all tested species. Here, it was shown that variations in the phytohormones NAA and BA changed the cyclotide production by a factor of two. Phytohormones of the auxin and cytokinin subgroups, to which NAA and BA belong respectively, are necessary for fine suspension growth without organogenesis and can therefore only be changed within certain limits. Auxins and cytokinins are also known to influence secondary metabolite production [28], adding to the complexity of cyclotide elicitation and production. The immense potential of suspension cultures to screen many parameters can help to find optimised conditions in C. ternatea. After screening, a two-stage process can be envisioned with initial biomass production, followed by cyclotide production. This would include a low stress environment with a medium composition optimised for biomass generation in the first step and introduction of stresses designed to elicit cyclotide production in a second step that will not support fast growth. Treatment with 50 and 100 µM methyl jasmonate did not increase cyclotide production. The protocol presented here should enable testing of additional elicitors that consequently might enhance the cyclotide yield also in whole plants, which could be beneficial for insecticide production [29]. No other studies have characterized C. ternatea suspensions so far, but the strong cyclotide production observed even without elicitation makes them prime targets for cyclotide production in plant cell suspensions.

4.3.3 Enhancing cyclotide production in Hybanthus enneaspermus

Different phytohormone concentrations had a minor effect on cyclotide synthesis in fine H. enneaspermus suspensions and often lead to undesired organogenesis. A closer look at the conditions inducing organogenesis and a discussion of reports of organogenesis in Violaceae suspensions is given in Section 3.3.3. Condition A allowed cultivation of stable suspension cells, but further optimisation potential of the phytohormone concentrations to achieve better growth or higher cyclotide yields is likely.

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Treatment of H. enneaspermus with 50 and 100 µM methyl jasmonate did not yield significant changes in cyclotide expression. Additionally, H. enneaspermus and C. ternatea did not display the same strong pronounced correlation between cell density and cyclotide expression as O. affinis did (data not shown). This behaviour might be useful for cyclotide production in suspension cells in the future. Parallel to the results presented here, Sathish et al. reported the influence of salicylic acid, yeast extract, and methyl jasmonate on L-Dopa production, a drug for the treatment of Parkinson’s disease, in H. enneaspermus cell suspensions cultured in MS medium containing 2.0 mg L-1 2,4 -D [30]. They achieved a 9.25-fold increase in L-Dopa production after treatment with 150µM salicylic acid, verifying that elicited secondary metabolite production in H. enneaspermus suspensions is feasible. This is the first work to characterize cyclotide production in H. enneaspermus cell suspensions, therefore there is much to be discovered and optimised. Next steps could include further medium optimisation, transcriptome analysis to understand the regulatory pathways of cyclotide production in H. enneaspermus, and testing of different extraction methods like sonication, crossflow filtration followed by chemical digestion, or the possibility of cyclotide secretion. At present, production of bracelet cyclotides is difficult via the solid-phase peptide synthesis route [31]. In contrast, the production of cycloviolacin O2, a bracelet cyclotide with outstanding antifungal and cytotoxic and moderate antibacterial properties [32, 33], in H. enneaspermus suspension is promising. To broaden the range of possible products, transformation of plant cell suspensions is investigated in the following chapter.

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Cyclotide elicitation in suspension cultures 4.4 References

1. Narayani, M. and S. Srivastava, Elicitation: a stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production. Fundamentals and Perspectives of Natural Products Research, 2017. 16: p. 1227. 2. Long, T.A., Many needles in a haystack: cell-type specific abiotic stress responses. Current Opinion in Plant Biology, 2011. 14: p. 325. 3. Yu, Y., T. Wang, Y. Wu, Y. Zhou, Y. Jiang, and L. Zhang, Effect of elicitors on the metabolites in the suspension cell culture of Salvia miltiorrhiza Bunge. Physiology and molecular biology of plants, 2019. 25: p. 229. 4. Kok, A.D.-X., W.M.A.N. Wan Abdullah, N.-P. Tan, J. Ong-Abdullah, R. Sekeli, C.-Y. Wee, and K.-S. Lai, Growth promoting effects of Pluronic F-68 on callus proliferation of recalcitrant rice cultivar. 3 Biotech, 2020. 10: p. 116. 5. Farmer, E.E. and C.A. Ryan, Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Proceedings of the National Academy of Sciences, 1990. 87: p. 7713. 6. Klessig, D.F., J. Durner, R. Noad, D.A. Navarre, D. Wendehenne, D. Kumar, J.M. Zhou, J. Shah, S. Zhang, P. Kachroo, Y. Trifa, D. Pontier, E. Lam, and H. Silva, Nitric oxide and salicylic acid signaling in plant defense. Proceedings of the National Academy of Sciences, 2000. 97: p. 8849. 7. Durner, J., J. Shah, and D.F. Klessig, Salicylic acid and disease resistance in plants. Trends in Plant Science, 1997. 2: p. 266. 8. Gupta, P., S. Sharma, and S. Saxena, Effect of Salts (NaCl and Na2CO3) on Callus and Suspension Culture of Stevia rebaudiana for Steviol glycoside Production. Applied Biochemistry and Biotechnology, 2014. 172: p. 2894. 9. Mendoza, D., O. Cuaspud, J.P. Arias, O. Ruiz, and M. Arias, Effect of salicylic acid and methyl jasmonate in the production of phenolic compounds in plant cell suspension cultures of Thevetia peruviana. Biotechnol Rep (Amst), 2018. 19: p. e00273. 10. Huang, Y.H., Q. Du, and D.J. Craik, Cyclotides: Disulfide-rich peptide toxins in plants. Toxicon, 2019. 172: p. 33. 11. Dörnenburg, H. and P. Seydel, Effect of irradiation intensity on cell growth and kalata B1 accumulation in Oldenlandia affinis cultures. Plant Cell, Tissue and Organ Culture, 2007. 92: p. 93. 12. Dörnenburg, H., Cyclotide synthesis and supply: from plant to bioprocess. Biopolymers, 2010. 94: p. 602. 13. Murashige, T. and F. Skoog, A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 1962. 15: p. 473. 14. Mohamad Puad, N.I. and T.A. Abdullah, Monitoring the Growth of Plant Cells in Suspension Culture, in Multifaceted Protocol in Biotechnology, A. Amid, et al., Editors. 2018, Springer Singapore. p. 203. 15. Seydel, P., C. Walter, and H. Dörnenburg, Scale-up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production. Engineering in Life Sciences, 2009. 9: p. 219. 16. Hoh, B., G. Hinz, B.K. Jeong, and D.G. Robinson, Protein storage vacuoles form de novo during pea cotyledon development. Journal of Cell Science, 1995. 108: p. 299. 17. Naik, P.S. and J.M. Widholm, Comparison of tissue culture and whole plant responses to salinity in potato. Plant Cell, Tissue and Organ Culture, 1993. 33: p. 273. 18. Zhong, J.-J., Plant cell culture for production of paclitaxel and other taxanes. Journal of Bioscience and Bioengineering, 2002. 94: p. 591.

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19. Lim, F.L., M.F. Yam, M.Z. Asmawi, and L.-K. Chan, Elicitation of Orthosiphon stamineus cell suspension culture for enhancement of phenolic compounds biosynthesis and antioxidant activity. Industrial Crops and Products, 2013. 50: p. 436. 20. Yukimune, Y., H. Tabata, Y. Higashi, and Y. Hara, Methyl jasmonate-induced overproduction of paclitaxel and baccatin III in Taxus cell suspension cultures. Nature Biotechnology, 1996. 14: p. 1129. 21. Gadzovska, S., S. Maury, A. Delaunay, M. Spasenoski, D. Hagège, D. Courtois, and C. Joseph, The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L. Plant Cell, Tissue and Organ Culture (PCTOC), 2013. 113: p. 25. 22. Oguis, G.K., Clitoria ternatea (butterfly pea) cyclotides: Insights on functional diversity, regulation and biotechnological applications, in Institute for Molecular Bioscience. 2019, The University of Queensland. 23. Hamann, T., Plant cell wall integrity maintenance as an essential component of biotic stress response mechanisms. Front Plant Sci, 2012. 3: p. 77. 24. Patakas, A., Abiotic Stress-Induced Morphological and Anatomical Changes in Plants, in Abiotic Stress Responses in Plants, P. Ahmad and M.N.V. Prasad, Editors. 2012, Springer New York. p. 21. 25. Ovesen, R.G., U. Goransson, S.H. Hansen, J. Nielsen, and H.C. Hansen, A liquid chromatography-electrospray ionization-mass spectrometry method for quantification of cyclotides in plants avoiding sorption during sample preparation. J Chromatogr A, 2011. 1218: p. 7964. 26. Gilleta, F., C. Roisin, M.A. Fliniaux, A. Jacquin–Dubreuil, J.N. Barbotin, and J.E. Nava– Saucedo, Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin. Enzyme and Microbial Technology, 2000. 26: p. 229. 27. Inyai, C., P. Boonsnongcheep, J. Komaikul, B. Sritularak, H. Tanaka, and W. Putalun, Alginate immobilization of Morus alba L. cell suspension cultures improved the accumulation and secretion of stilbenoids. Bioprocess Biosyst Eng, 2019. 42: p. 131. 28. Pawar, K.D. and S.R. Thengane, Influence of hormones and medium components on expression of dipyranocoumarins in cell suspension cultures of Calophyllum inophyllum L. Process Biochemistry, 2009. 44: p. 916. 29. On the evaluation of the new active clitoria ternatea in the product Sero-X Insecticide. 2016, Australian Pesticides and Veterinary Medicines Authority, ISSN: 1443-1335 (electronic). 30. Sathish, S., V. Vasudevan, S. Karthik, G. Pavan, and M. Manickavasagam, Enhanced l-Dopa production from elicited cell suspension culture of Hybanthus enneaspermus (L.) F. Muell. Plant Biotechnology Reports, 2019. 13: p. 613. 31. de Veer, S.J., M.W. Kan, and D.J. Craik, Cyclotides: From Structure to Function. Chem Rev, 2019. 119: p. 12375. 32. Niyomploy, P., L.Y. Chan, P.J. Harvey, A.G. Poth, M.L. Colgrave, and D.J. Craik, Discovery and Characterization of Cyclotides from Rinorea Species. J Nat Prod, 2018. 81: p. 2512. 33. Slazak, B., M. Kapusta, A.A. Stromstedt, A. Slomka, M. Krychowiak, M. Shariatgorji, P.E. Andren, J. Bohdanowicz, E. Kuta, and U. Goransson, How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System. Front Plant Sci, 2018. 9: p. 1296.

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5 Transformation of plant suspension cells using cell pack technology

The employment of a natural cyclotide producer that is equipped with ligase-type AEPs could prove advantageous for producing high quantities of recombinant cyclic peptides in plants. In this chapter the ability to transform O. affinis, a plant with two genetically and functionally characterized ligase-type AEPs [1] is tested, and the possible transformation and subsequent production of cyclotides in N. benthamiana, H. enneaspermus, and C. ternatea investigated. Transformation of plants can be defined as the transient or permanent expression of foreign genes in plant cells. One of the most widely used techniques is Agrobacterium-mediated transformation, which utilizes the natural ability of Agrobacterium tumefaciens to insert transfer DNA (T-DNA) into plant cells [2]. In nature, A. tumefaciens infection of plants causes tumours via the integration of bacterial tumorigenesis genes into the host plant’s genome. These genes induce the observed pathology by upregulating cytokinin and auxin synthesis. In turn, these hyper-proliferating cells produce opines or nopalines, which are used by A. tumefaciens as an energy source [2]. In the laboratory, A. tumefaciens can be disarmed by removing genes important for tumorigenesis and opine production that are normally present on the tumour inducing plasmid (Ti plasmid). Importantly, this step does not influence the ability of A. tumefaciens to transfer DNA. To enable recombinant protein expression in planta, genes of interest can be added to the disarmed Ti plasmid. Modern transformation systems do not use the modified Agrobacterium Ti plasmid, but smaller binary vector systems that can be replicated in A. tumefaciens and Escherichia coli. This enables manipulation and multiplication of the vector in E. coli with consequent purification and introduction into A. tumefaciens. In addition to the genes of interest, at least two selectable markers must be present on the binary vector, one to enable selection of transformed bacteria and another for selection in plant cells. The latter requirement is only necessarily when stably transformed cells are sought. Transient transformation describes recombinant protein expression without integration of the foreign genes into the host genome, but still employing the ability for Agrobacterium to introduce T- DNAs into host plant cells. It produces higher yields and requires lower production times compared to stable transformation [3]. The higher yields are partly contributed to the fact that the number of T- DNA molecules transferred is much higher than the number that ends up integrated into the genome. The short time frame coupled with high transient transformation efficiency enables flexible production systems as well as possibilities to address basic research questions, such as determining gene function, protein-protein interactions and regulatory mechanisms [4, 5]. Additionally, the risk

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Transformation of plant suspension cells using cell pack technology of unintentional release into the open environment is minimal as the genetic changes are not inherited. In summary, transient transformation is the ideal tool to investigate cyclotide expression in plants and plant cell suspensions. Plant cell suspensions come with several advantages for transient transformation, including but not limited to, inherent containment and sterility, control over all process parameters and accessibility of every cell for infiltration. The accessibility is necessary because Agrobacterium needs direct contact with the target cell to transfer its T-DNA [2]. However, the conditions in liquid suspensions are not ideal, presumably because of the natural optimisation of the transfer mechanism for plant tissue. To circumvent this problem and to enable scalability, Rademacher et al. developed a plant cell pack (PCP) protocol that resulted in a transfection efficiency of over 50%, whereas cells in suspension had less than 5% [6]. PCPs are porous, three-dimensional cell packages devoid of liquid medium. The major advantage of PCPs is the ability to screen many parameters under constant biological conditions because every plant species might require different incubation times, bacterial concentrations, promotor systems, vector designs, genes, gene combinations humidity, temperature, and irradiation. Here, PCPs were deployed to screen for parameters required to transform O. affinis that could enable recombinant production of engineered cyclotides. Additionally, first PCP transformations of H. enneaspermus and C. ternatea were attempted and PCPs were compared to leaf-based agroinfiltration for production of cyclotides in N. benthamiana.

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5.1 Materials and methods

5.1.1 Agrobacterium transformation

Two disarmed Agrobacterium tumefaciens strains, LBA4404 and EHA105 were tested for their ability to transform plant cells. Initial proof of concept experiments used the pProGG construct as shown in Figure 43. The vector was developed from pGreenII in-house by Dr. Edward Gilding. To create a functional promoter-reporter fusion vector, the constitutive CaMV 35S promoter was cloned into pProGG using Gateway® technology to yield pProGG-35S. This vector expression system allowed detection of positively transformed cells via β-glucuronidase (GUS) activity staining or visualisation of green fluorescent protein (GFP) fluorescence. Further experiments were conducted using the pEAQ-DEST1 expression vector system [7] with constructs designed for expression of native Oak1 (encoding kB1), an engineered Oak6 gene (where the three kB2 domains were replaced with [T20K]kB1), butelase 1 (AEP found in C. ternatea), HeAEP3 (AEP found in H. enneaspermus), and OaAEP3 (AEP found in O. affinis). Finally, OaAEP1b (AEP found in O. affinis) within the pBin19 vector was kindly provided by Dr Simon Poon [8]. All plasmids were purified from E. coli using the PureLink® Quick Plasmid Miniprep kit and sent to the AGRF (Australian Genome Research Facility, Brisbane) for sequencing. A. tumefaciens strains were then transformed by electroporation with cell lines containing the target vector stored at -80°C in 20% (v/v) glycerol.

Figure 43 | pProGG vector map.

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5.1.2 Agrobacterium-mediated infiltration of suspension cells in plant cell packs

The cell pack method used for infiltration was adapted from Rademacher et al. [6]. The A. tumefaciens cultures were washed, resuspended in Murashige and Skoog medium with vitamins (M519, Phyto Technology Lab) and incubated with 200 µM acetosyringone for 1 h. Suspension cultures of O. affinis, H. enneaspermus, C. ternatea, and N. benthamiana were harvested during the exponential growth phase at around 40% PCV. Cell packs of around 1 mL were formed by adding 2.5 mL of culture to a 5 mL disposable filter column and removing the liquid via applying a vacuum.

The PCPs were incubated with 1 mL A. tumefaciens adjusted to an OD600 (optical density at 600 nm) of 0.5 unless indicated otherwise. After 30 min incubation at RT, the liquid was removed by applying vacuum and the PCPs were sealed with a small water reservoir supplied underneath the cell pack to prevent the cells from drying out and maintain high humidity in the cell pack. During the transient expression phase the PCPs were incubated for 10 days under constant high humidity at 26°C with 30 µmol m-2 s-1 photon density. The humidity was assessed indirectly by visual screening for cell browning. Samples were taken every third day from day 3 to 10 to monitor the transformation rate.

5.1.2.1 Transient cyclotide expression in Nicotiana benthamiana PCPs and leaves

The different vector-gene combinations used for PCP transformation are listed in Table 19. The domains encoding kB1 and kB1[T20K] were codon optimised and named “opt kB1” and “opt T20K” respectively. The leaves were infiltrated with combination 3 and 4. Each PCP combination was done in 3 replicates; leaf infiltration was done in 5 different leaves of different plants as replicates.

The Agrobacterium OD600 was adjusted to 0.2 prior to 3 h incubation with 200 µM acetosyringone and 45 min incubation of the PCP.

Table 19 | Vector-gene combinations used for N. benthamiana transformation. Enzyme Cyclotide Combination Agrobacterium Vector Gene Vector Gene 1 EHA105 pBin19 OaAEP1b pEAQ Oak1[opt kB1] 2 EHA105 pBin19 OaAEP1b pEAQ Oak6[T20K] 3 LBA4404 pEAQ Butelase-1 pEAQ Oak6[opt kB1] 4 LBA4404 pEAQ Butelase-1 pEAQ Oak6[opt T20K]

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5.1.3 GUS staining of transformed plant cell packs

To identify positively transformed cells, GUS staining was performed as per Glazebrook and Weigel [9]. In brief, the staining buffer was freshly prepared for each experiment. 1.7 mL DI water was mixed with 200 µL 0.5 M sodium phosphate buffer (pH 7.2), 40 µL 10% (v/v) Triton X-100, and 40 µL of a solution containing 100 mM potassium ferrocyanide and 100 mM potassium ferricyanide. In the next step, 20 µL100 mM X-Gluc in DMF was added. A representative cross section of the PCP after transformation was transferred into a 2 mL Eppendorf tube or a well on a 96 well plate and incubated with GUS staining solution for 24 h in the dark at 37°C. Transformation rates were determined by visual assessment of GUS stained PCP sections. Blue staining of more than 5% but less than 30% of visible cells was interpreted as positive transformation with low transformation rate. Blue staining of more than 60% of visible cells was interpreted as positive transformation with high transformation rate.

5.1.4 GFP detection in transformed plant cells

Cells at different growth and transformation stages were suspended in growth medium and imaged under a stereo wide-field microscope for a visual assessment of health and GFP expression. Plant cells in suspension tend to aggregate and block pipette tips. To achieve preparation of a representative sample, 42 µL were taken up by a 200 µL pipette tip that was cut at a 30° angle 1/3 away from the tip. The volume was placed on a glass slide and covered by a plastic slip. This slip was used to apply gentle pressure to flatten the plant cell aggregates and result in a monolayer of cells. If necessary, the slip was sealed and fixed with nail polish.

5.1.5 MADLI-TOF analysis

MADLI-TOF analysis was done as described in Section 2.1.6.

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5.2 Results

5.2.1 Transient transformation of Oldenlandia affinis cell packs

5.2.1.1 First positive transformation of O. affinis

Two Agrobacterium tumefaciens strains (LBA4404 and EHA105) were tested for their ability to transfer DNA into O. affinis cell packs. Of the two, EHA105 is reported to have enhanced virulence, which is sometimes an advantage, thus this strain was used alongside LBA4404 [10, 11]. Both strains were armed with the pProGG-35S vector (Figure 43) to enable detection of transient reporter protein production via GFP or GUS assays. It is noteworthy that GUS and GFP signals used to detected positive transformation would not be seen if the transgene was transcribed in the non- target host cell due to the presence of an intron at the start of the GUS-Plus moiety. O. affinis cell suspensions were prepared for transformation by removal of liquid by vacuum and arrangement into plant cell packs (PCPs) [6]. PCP volumes of 2 mL enabled high throughput screening and parallel processing of up to 21 samples. O. affinis formed homogeneous, porous, white PCPs as can be seen in Figure 44A. For an initial assessment, O. affinis cell cultures were infiltrated with EHA105 or LBA4404 cells containing pProGG-35S and sampled after 4, 7, and 10 days. In the case of LBA4404, no GUS or GFP reporter activity could be detected; however, for the more virulent EHA105 some GUS and GFP reporter activity was observed in cells assayed at day 7 as shown in Figure 44B. Between cultures and biological replicates, this homogeneous transient expression throughout the PCP was not reproducible. In many cases only small patches of cells or layers within the PCP were positive for GUS reporter activity. Furthermore, monitoring transformation via GFP observation was sometimes ambiguous as the cells displayed a weak autofluorescence at 488 nm visible under the microscope and the GFP intensity was not very strong as seen in Figure 44C. In contrast, the positive control N. benthamiana PCPs consistently produced GUS reporter activity for both Agrobacterium strains on all sampling days (Figure 44B). N. benthamiana was chosen as positive control following the initial reports by Rademacher et al. [6].

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Figure 44 | O. affinis PCP transformation. A) Plant cell packs of N. benthamiana (left row) and O. affinis (other rows) after seven days of incubation with Agrobacterium tumefaciens. No dried-out cells and only minimal suffocated (dark) cells at the bottom were visible. B) GUS staining of cell packs infiltrated with EHA105 after seven days. O. affinis cell pack number 2 displayed a high cell transformation efficiency, whereas number 1 displayed only a single transformed spot. N. benthamiana cell packs were used as positive control and displayed high transformation efficiency. The cells became dislodged during ethanol treatment and started floating but showed otherwise no different behaviour than O. affinis cells. The O. affinis negative control PCPs displayed no transformation. C) Merger of bright field and fluorescence (488 nm) microscopy of O. affinis cells expressing GFP at 200x.

5.2.1.2 Screening of parameters influencing PCP transformation efficiency

As initial experiments to transform O. affinis cell packs were not reproducible, experiments were set up to screen for parameters influencing the transformation efficiency. Utilizing a setup as shown in Figure 45A, various PCP heights, EHA105 densities, humidity and expression times were tested. The aim was to reproducibly achieve the same transformation rate as culture number 2 in Figure 44B. Screening parameters and the range of parameters within which positive transformation was observed are presented in Table 20.

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Table 20 | Screened conditions for EHA105 transformation of O. affinis. Parameter Screening range Positive transformation

OD600(EHA105) 0.1-2.0 0.1-1.0 Expression time 3-10 days 5-9 days PCP height 1-50 mm 10-40 mm Humidity Drowned-dry Constant high No direct relation between screening parameter and transformation rate was observed. However, a range within which positive transformation was possible was identified. Here, positive transformation was assumed if more than 5% of visible cells were blue after GUS staining. The optical density of EHA105 was measured in the cultivation medium directly before incubation at 600 nm. The humidity was assessed visually. The expression time describes the time from incubation to harvest. All experiments were carried out in biological triplicates.

Despite testing multiple parameters, the transformation rate, as evaluated by visible examination of GUS stained tissues did not reach the level observed previously as shown in Figure 44B. Furthermore, expression levels as determined by GUS staining intensity were highly variable. For example, O. affinis PCP vials #1, #2 and #3 (Figure 45C) only differed in inoculum density

(OD600 0.5, 0.25 and 0.1 respectively) but vial #2 failed to express reporter protein and appeared to dry out. Furthermore, a clear spatial influence on transformation efficiency was observed for O. affinis PCPs. The uppermost layer displayed very high transformation rates, whereas the lowest layer displayed no transformation. The body of the PCP in-between these two layers was characterized by rare point like colonies of transformed cells as shown in Figure 45B. When the height of the PCP was reduced to create a single layer, no transformation was observed, and the PCPs appeared to dry out, as shown in vials 4, 5, and 6 in Figure 45C. One observed norm was that cell packs displaying browning of cells or dried out cells, signs of low PCP quality, did not transform. The PCP quality was difficult to quantify as it was not possible to determine the fluid level and the packing density without disrupting the PCP. Therefore, differences in PCP quality could only be observed after several days of incubation. An important factor influencing the PCP quality was the humidity during the expression period following the incubation. Exact measurement of the humidity inside the PCPs was not possible, but low humidity resulted in cell death by drying out the cell pack, while high humidity and retained liquid inside the PCP suffocated the cells. In saying this, other efficiency factors likely exist, as even visually healthy and identical PCPs produced different transformation rates. Another example is shown in Figure 45D where even N. benthamiana, that normally transformed reliably, displayed high variation in transformation under identical conditions. Well 1 (top left) was the negative control, the next six wells in the top row were incubated with LBA4404 under the same conditions, and top right and the bottom left well were incubated with EHA105. As an additional influencing factor, different concentrations of A. tumefaciens strain EHA105 in the incubation medium were tested. OD600 variation from 0.1 to 1.0 did not have an immediate effect on the observed GUS staining. An OD600 higher than 1.0 was detrimental for cell viability.

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Harvesting after 3-10 days indicated that the transformation rate increased until day 5-7, to then level out. If the cells started to dry out, the transformation rate dropped significantly.

Figure 45 | Plant cell pack transformation parameter screening. A) Setup to screen 21 PCPs per batch. B) Observed spatial transformation efficiency. The liquid visible above the PCP is 80% ethanol used for preservation. C) Influence of O. affinis PCP height and EHA105 incubation density on transformation efficiency. Vial 1: Height = 4 cm, OD600 = 0.5. Vial 2: Height = 4 cm, OD600 = 0.25. Vial 3: Height = 4 cm, OD600 = 0.1. Vial 4: Height = 0.25 cm, OD600 = 0.5. Vial 6: Height = 0.25 cm, OD600 = 0.25. Vial 6: Height = 0.25 cm, OD600 = 0.1. D) GUS staining of N. benthamiana PCP sections in a 96-well plate.

5.2.1.3 Cyclotide elicitation during Agrobacterium-mediated transformation in O. affinis

MALDI-TOF analysis of O. affinis cells extracts during Agrobacterium-mediated transformation in PCPs revealed a strong increase in concentration over time of three cyclotides as shown in Figure 46. This effect was not related to the presence of the Agrobacterium or the pProGG- 35S vector as the negative control displayed the same trend (data not shown).

Figure 46 | Cyclotide elicitation in O. affinis PCPs during Agrobacterium-mediated transformation. A) Cyclotide production in O. affinis PCPs over time. All samples were normalized against a known amount of reference peptide (kalata B1-Glu-Ile-Ile). Error bars indicate standard deviation of biological replicates, n=3. B) MALDI trace of O. affinis PCP after 9 days.

5.2.2 Scale-up of plant cell pack transformation

To achieve a higher total number of transformed cells, PCPs were prepared in larger filter setups than the 2 mL screening columns. The setup is shown in Figure 47. These PCPs were difficult

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Transformation of plant suspension cells using cell pack technology to handle because the large surface area led to a small pressure difference resulting in up to two hours needed to remove the liquid. After the liquid was removed, the large ratio of surface area to total volume increased the risk of drying out. To prevent drying out, evaporation was minimized via sealing of the incubation chamber with a plastic lid. Figure 47C shows that high transformation rates in O. affinis were possible under these conditions. However, out of six identical samples, only one showed high transient expression with over 60% transformed cells, whereas five showed very low transformation rates with less than 5% transformed cells. Condition screening as described in Section 5.2.1.2 did not reliably improve the transformation consistency in the larger PCPs.

Figure 47 | Scale-up of O. affinis transformation. A) Side view of the PCP during liquid removal step. B) Top view of the PCP after liquid removal. C) Part of the O. affinis PCP after five days of incubation with EHA105 and 24 hours GUS staining. Dish diameter is 4 cm.

5.2.3 Transient expression in Hybanthus enneaspermus and Clitoria ternatea PCPs

H. enneaspermus and C. ternatea PCPs were incubated with EHA105 containing the pProGG-35S vector at 0.5 OD600 to assess if the conditions that enabled O. affinis transformation could also transform H. enneaspermus and C. ternatea. All 18 PCPs tested negative for GFP fluorescence and GUS staining. The same Agrobacterium conditions were also applied to attempt transformation of H. enneaspermus and C. ternatea, calli and internodes respectively, on solid medium. No explants with clear GFP signals or positive GUS staining were observed.

5.2.4 Transient cyclotide expression in Nicotiana benthamiana PCPs

N. benthamiana PCPs displayed consistently high transformation rates with EHA105 and LBA4404 using the pProGG-35S vector. Building on these results and to quantitatively compare PCP 134 | Page

Transformation of plant suspension cells using cell pack technology transformation with agroinfiltration in leaves, both systems were set up to transiently express Oak1 and Oak1[T20K] alongside AEP encoding genes required for post-translational cyclisation. The used Agrobacterium strain was LBA4404 with cyclotide precursor-encoding genes Oak1, Oak1[T20K] and the butelase-1 gene, encoding the peptide ligase AEP butelase-1, inserted in the pEAQ vector [7]. Butelase-1 was chosen as the AEP with the ability to cyclise the [T20K]kB1 precursor. As shown in Figure 48A, the PCPs failed to produce any recombinant peptide, whereas the leaves produced predominantly cyclic kB1 and [T20K]kB1 peptide. The percentage of correctly processed circular peptide was 98.2% for kalata B1 and 98.3% for T20K. Leaves infiltrated with infiltration medium not containing Agrobacterium were used as negative control.

Figure 48 | Transient co-expression of butelase-1 with Oak1 or Oak1[T20K] in N. benthamiana analysed via MALDI-TOF and normalized per dry weight using a reference peptide. A) Comparison of expression between leaves and PCPs. B) MALDI trace of leaf extract highlighting the strong cyclic peptide signal and the ratio of cyclic to linear peptide.

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5.3 Discussion

5.3.1 Agrobacterium-based transformation of plant cell packs

This work established a proof of concept for Agrobacterium-based O. affinis PCP transformation, but there are further steps that need to be optimised to use this tool for research or production. It was not possible to obtain predictable and reproducible results from the chosen experimental parameters. Nevertheless, vital knowledge was gained on important influencing factors of PCP transformation. The packing density and humidity during the expression phase were identified as crucial parameters for positive transformation, but even seemingly ideal PCPs could not always be transformed. This was irrespective of location in the rack of packs or handling time and other procedural effects. It was noted that especially if leftover liquid was present then it resulted in cell suffocation within one day, similar to the results reported by Rademacher et al. [6]. Additionally, a strong spatial influence was observed as the surface layer was highly transformable, whereas the bottom layer was never transformed in O. affinis PCPs. A possible explanation is the availability of oxygen and/or Agrobacterium. Both diffuse through the PCP from top to bottom and are necessary for transient expression. N. benthamiana did not display the same characteristics, probably because the N. benthamiana suspension cultures formed larger aggregates and therefore less densely packed PCPs compared to O. affinis. Very thin O. affinis PCPs, to enable easy diffusion, did not work as they dried out too quickly. Other strategies that might be applied in future studies to achieve lower packing densities include introduction of scaffolds or dissolvable fillers and different cell pack geometries. Other studies casted Nicotiana tabacum Bright Yellow 2 PCPs into many different formats without diffusion limitations [6, 12]. Perhaps another explanation is that tobacco species naturally form PCPs well suited for Agrobacterium-mediated transformation or are more susceptible to transformation outright. Furthering that point, there are reports of genotype effects in transformation efficiency [13] and some plants, like sorghum, have been notoriously difficult to transform [14]. The fact, that some O. affinis cultures displayed very high transformation efficiencies indicates that O. affinis transformation is not hindered by genotype effects regarding PCP transformation. Perhaps it might even be possible to mix N. benthamiana cells and O. affinis cells to create PCPs with better packing properties and consequently higher transformation efficiencies.

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5.3.2 Cyclotide elicitation in O. affinis PCPs

O. affinis PCPs with or without Agrobacterium treatment displayed the strong elicitation of cycloviolacin O2, kalata B22, and kalata B23 within the first 9 days. Building on the knowledge gathered in Chapter 4 and ruling out cell-bacteria interaction as source, cell immobilization resulting in cell-cell contact and communication was identified as the most likely cause. Interestingly, the cells did not turn green and kalata B1 was not expressed as was the case in O. affinis suspension cells immobilized on callus induction medium. The elicitation observed in immobilized PCPs was more alike to the elicitation in suspension cells treated with 3 mM sodium chloride. Future experiments might find the commonality of sodium chloride and immobilization and elucidate the biological function of the suspension type cyclotides cycloviolacin O2, kalata B22, and kalata B23. The results presented here underscore the strong influence of immobilization on cyclotide production in O. affinis.

5.3.3 Plant cell packs for research and production of cyclic peptides

If the reproducibility problem is solved, one possible application of PCPs could be in the screening and transient production of designer cyclic peptides in O. affinis. The time to harvest is within weeks for cell suspensions, whereas it takes months or years for transformed plants, making PCPs advantageous for screening or production on a laboratory scale. As the example of viscumin production in Nicotiana tabacum PCPs shows, the production of pharmaceutical proteins in plants can lead to cost savings of more than 80% and 3-fold higher bioactivity compared to production in E. coli [12]. To achieve these cost savings, Gengenbach et al. used PCPs as a screening platform and leaf-based agroinfiltration for scale-up [12]. A similar approach might be feasible for O. affinis. As described in Chapter 2, O. affinis suspension cultures ceased cyclotide production after only a few weeks of culture. This was shown to be due to a sharp decrease in the expression level of the endogenous cyclotides. In contrast, transcripts remained steady for the known cyclotide processing enzymes OaAEP1and OaAEP3. Consequently, O. affinis suspension cells do not need co- expression of ligase-type AEPs to process designer cyclotides [15]. This might increase the cyclotide yield compared to species not normally expressing cyclotides because no second transformation is needed, and the cellular environment is suited for cyclotide processing. These findings fueled the idea to introduce cyclotide genes with a different promoter, one which will not be shut down in suspension, into an environment that is natively able to fold and ligate cyclotides correctly (i.e. the O. affinis cell).

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This would result in expression of only the target cyclic peptide in a species that normally expresses over 20 cyclotides, simplifying the purification. The scale-up of transformation in PCPs proved difficult due to the handling of the cells and cell packs. A large percentage of transformed cells was achieved in a setup using 50 mL of an O. affinis culture at 40% PCV to form the PCP; however, many transformations using an identical setup failed. Based on the experience gathered here in handling PCPs on a milli-liter scale, increasing this to a liter scale will be very challenging. Rademacher et al. reported PCPs of up to 100 g cells in a column [6] and Gengenbach et al. reported PCPs from 300 µL culture in 96-well plates [12] for recombinant protein production. Considering these reports and the results collected here, PCPs might be suitable for cyclotide production on a small to medium scale, but on larger scales leaf-based agroinfiltration is preferable. Previous transient expression attempts in O. affinis utilizing vacuum agroinfiltration of young shoots were unsuccessful in preliminary tests (unpublished work by Dr. Edward Gilding). A possible hinderance for transformation of O. affinis plants using Agrobacterium is the presence of cyclotides that have been shown to have antibacterial properties [16]. Cells in suspension are undifferentiated and do not have the full complement of peptides, especially some cyclotides which are implicated in defense, possibly resulting in a higher transformation efficiency. Therefore, conditions that worked in PCPs are not guaranteed to work in planta, but they are a promising advance and starting point. In summary, results achieved in transforming O. affinis PCPs could enable the first transformation of O. affinis cells regenerated into whole plants. At this point, cyclotide production in N. benthamiana PCPs seems unfavourable as no product could be detected and transformation was not as reliable as leaf infiltration. This result was unexpected, and experiments will be necessary to elucidate the cause. The only obvious difference between leaf cells and suspension cells was the differentiation state, indicating that there are further unidentified requirements for cyclotide production that are met in leaves but not in suspension cells. Comparative transcriptome analysis could help identify these requirements. Unless significant advances are made, the classical leaf-based agroinfiltration is superior in yield and scalability for peptide production. Transient expression in leaves can easily by scaled-up by increasing the number of plants with recent facilities reaching up to 3500 kg of plant biomass per week [17]. One hurdle is the fact that post-translational processing of cyclotide precursors requires specific enzymes, not all of which are present in N. benthamiana. However, to produce correctly processed kalata B1 in

N. benthamiana leaves the co-expression of OaAEP1b and Oak1 was enough [18, 19]. Even though not yet economically competitive, N. benthamiana suspensions and PCPs are valuable screening tools for transformation parameters and might become economical production systems in the future [20].

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Transformation of H. enneaspermus PCPs was not successful. However, the sample number was low and PCP transformation of O. affinis showed that this method might have large variations in species not transformed in PCPs previously. Reports of Agrobacterium-mediated transformation in leaves indicate that PCP transformation of H. enneaspermus should be possible and further parameter screening is necessary. Sivanandhan et al., for example, reported a transformation efficiency of 28% with LBA4404 at 0.5 OD600 [21]. Suspension cells of C. ternatea have never been researched before and here the ambitious first Agrobacterium tumefaciens-mediated PCP transformation was tested. Unfortunately, the attempt failed. Previously, it was shown that Agrobacterium rhizogenes was able to induce hairy roots from C. ternatea internodes and Agrobacterium tumefaciens was able to transform leaf explants [22, 23]. Therefore, like in the case of H. enneaspermus, PCP transformation of C. ternatea might be possible after additional parameter screening. In conclusion, transformation of O. affinis and N. benthamiana PCPs was successful, paving the way for recombinant cyclotide production in PCPs. The possibility to transform O. affinis also enables the labelling of cyclotide precursors to elucidate their processing within the cell, the knock down of enzymes and cyclotides to monitor the consequences and the recombinant expression of engineered cyclic peptides are scientific questions we are now equipped to address. H. enneaspermus and C. ternatea were tested to evaluate if the transformation parameters observed for O. affinis PCPs were transferable and generalizable between species. The gathered results indicate that each species might need specific parameters; however, more testing is necessary to validate this hypothesis. In the following chapter cryopreservation of O. affinis cells is investigated to preserve valuable cell suspension cultures.

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5.4 References

1. Harris, K.S., R.F. Guarino, R.S. Dissanayake, P. Quimbar, O.C. McCorkelle, S. Poon, Q. Kaas, T. Durek, E.K. Gilding, M.A. Jackson, D.J. Craik, N.L. van der Weerden, R.F. Anders, and M.A. Anderson, A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein. Sci Rep, 2019. 9: p. 10820. 2. Guo, M., J. Ye, D. Gao, N. Xu, and J. Yang, Agrobacterium-mediated horizontal gene transfer: Mechanism, biotechnological application, potential risk and forestalling strategy. Biotechnol Adv, 2019. 37: p. 259. 3. Komarova, T.V., S. Baschieri, M. Donini, C. Marusic, E. Benvenuto, and Y.L. Dorokhov, Transient expression systems for plant-derived biopharmaceuticals. Expert Review of Vaccines, 2010. 9: p. 859. 4. Lin, Y., F. Meng, C. Fang, B. Zhu, and J. Jiang, Rapid validation of transcriptional enhancers using agrobacterium-mediated transient assay. Plant Methods, 2019. 15: p. 21. 5. Guidarelli, M. and E. Baraldi, Transient transformation meets gene function discovery: the strawberry fruit case. Front Plant Sci, 2015. 6: p. 444. 6. Rademacher, T., M. Sack, D. Blessing, R. Fischer, T. Holland, and J. Buyel, Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering. Plant Biotechnol J, 2019. 17: p. 1560. 7. Sainsbury, F., E.C. Thuenemann, and G.P. Lomonossoff, pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J, 2009. 7: p. 682. 8. Poon, S., K.S. Harris, M.A. Jackson, O.C. McCorkelle, E.K. Gilding, T. Durek, N.L. van der Weerden, D.J. Craik, and M.A. Anderson, Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. J Exp Bot, 2018. 69: p. 633. 9. Weigel, D. and J. Glazebrook, How to study gene expression. Arabidopsis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2002: p. 170. 10. Hood, E., S. Gelvin, L. Melchers, and A. Hoekema, New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Research, 1993. 2: p. 208. 11. Lazo, G.R., P.A. Stein, and R.A. Ludwig, A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium, 1991: p. 963. 12. Gengenbach, B.B., L.L. Keil, P. Opdensteinen, C.R. Müschen, G. Melmer, H. Lentzen, J. Bührmann, and J.F. Buyel, Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin. Biotechnology and Bioengineering, 2019. 116: p. 2236. 13. Cheng, M., B.A. Lowe, T.M. Spencer, X. Ye, and C.L. Armstrong, Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cellular & Developmental Biology - Plant, 2004. 40: p. 31. 14. Che, P., A. Anand, E. Wu, J.D. Sander, M.K. Simon, W. Zhu, A.L. Sigmund, G. Zastrow- Hayes, M. Miller, D. Liu, S.J. Lawit, Z.Y. Zhao, M.C. Albertsen, and T.J. Jones, Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application. Plant Biotechnol J, 2018. 16: p. 1388. 15. Jackson, M.A., E.K. Gilding, T. Shafee, K.S. Harris, Q. Kaas, S. Poon, K. Yap, H. Jia, R. Guarino, L.Y. Chan, T. Durek, M.A. Anderson, and D.J. Craik, Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases. Nat Commun, 2018. 9: p. 2411.

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16. Craik, D.J., M. Čemažar, C.K. Wang, and N.L. Daly, The cyclotide family of circular miniproteins: nature's combinatorial peptide template. Peptide Science: Original Research on Biomolecules, 2006. 84: p. 250. 17. Holtz, B.R., B.R. Berquist, L.D. Bennett, V.J. Kommineni, R.K. Munigunti, E.L. White, D.C. Wilkerson, K.Y. Wong, L.H. Ly, and S. Marcel, Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals. Plant Biotechnol J, 2015. 13: p. 1180. 18. Rehm, F.B.H., M.A. Jackson, E. De Geyter, K. Yap, E.K. Gilding, T. Durek, and D.J. Craik, Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization. Proc Natl Acad Sci U S A, 2019. 116: p. 7831. 19. Harris, K.S., S. Poon, P. Quimbar, and M.A. Anderson, In Vitro and In Planta Cyclization of Target Peptides Using an Asparaginyl Endopeptidase from Oldenlandia affinis, in Enzyme- Mediated Ligation Methods, T. Nuijens and M. Schmidt, Editors. 2019, Springer New York. p. 211. 20. Sukenik, S.C., K. Karuppanan, Q. Li, C.B. Lebrilla, S. Nandi, and K.A. McDonald, Transient Recombinant Protein Production in Glycoengineered Nicotiana benthamiana Cell Suspension Culture. Int J Mol Sci, 2018. 19. 21. Sivanandhan, G., C. Arunachalam, V. Vasudevan, G. Kapildev, A.A. Sulaiman, N. Selvaraj, A. Ganapathi, and Y.P. Lim, Factors affecting Agrobacterium-mediated transformation in Hybanthus enneaspermus (L.) F. Muell. Plant Biotechnology Reports, 2016. 10: p. 49. 22. Swain, S.S., L. Sahu, A. Pal, D.P. Barik, C. Pradhan, and P.K. Chand, Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium x plant factors influencing transformation. World J Microbiol Biotechnol, 2012. 28: p. 729. 23. Buddharak, P. and R. Chundet, Isolation and characterization of flavonoid 3′ Hydroxylase (F3′H) gene and genetic transformation in butterfly pea (Clitoria ternatea Linn.) via Agrobacterium tumefaciens. Acta Horticulturae, 2009. 836: p. 247.

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6 Cryopreservation of Oldenlandia affinis cell suspensions

Cell suspension lines are products of exacting and careful culture. As time in culture increases so does the risk of contamination, genetic changes and morphological shifts [1], thus developing strategies for their preservation are essential. Cryopreservation is the storage of biological material at ultra-low temperatures and for cell lines is the only option for long-term storage. Thankfully, plant cells possess the ability to be frozen, stored indefinitely and thawed whilst maintaining their viability. However, the necessary conditions vary greatly between species and tissue types [2]. General methods for cryopreservation of plant cell suspensions include two-step freezing, vitrification and encapsulation [3]. As a rule of thumb, intracellular ice formation has to be avoided and small, dense cytoplasmic cells are more likely to survive freezing [2]. Cryopreservation protocols are available for several plant species including Arabidopsis thaliana, Daucus carota, Nicotiana tabacum, Oryza sativa, and Petunia hybrida [3, 4]. Many less researched species, like Oldenlandia affinis, do not have cryopreservation protocols available. Finding species specific protocols is not trivial, as it is hard to predict if a species is cryopreservable and the required conditions are often difficult to find. For example, in his Diplomarbeit (German equivalent of a master’s thesis) Christian Gassen aimed to establish a cryopreservation protocol for Vitis vinifera but was unable to produce any growth after freezing [5]. It is unclear how many protocols for plant cryopreservation have failed over the years, as negative results are usually not published, but the number is probably large. In the work described in this chapter, the ability to preserve O. affinis utilizing SGD-based two-step freezing and vitrification as classical approaches were tested. SGD is a general term for a cryoprotectant containing sucrose, glycerol and dimethyl sulfoxide to hinder intracellular ice formation via increasing the solute concentration inside the cell [2, 3]. The chosen cryoprotectants must penetrate the cell membrane and must be non-toxic to the cell. Vitrification describes the process of transitioning from a liquid to a glass like state without formation of crystals. In plant suspensions, this can be achieved by administering high concentrations of cryoprotectants for dehydration followed by rapid freezing in liquid nitrogen to vitrify the medium and cells [3]. Additionally to the two aforementioned approaches, an innovative methanol-based two-step freezing method is tested here. Methanol-based cryopreservation, a well-known technique to cryoprotect algae [6], fish [7] and mammalian cells [8], has not been reported for cryopreservation of plant cell suspensions before.

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6.1 Materials and methods

Three different methods, namely two-step freezing using methanol (protocol by Dr. Ian Ross), vitrification [2], and two-step freezing using a SGD solution [3] were adapted from literature and evaluated for cell viability and morphology. All tests were carried out in biological triplicates.

6.1.1 Cryopreservation

6.1.1.1 Two-step freezing using methanol as cryoprotectant

This protocol was adapted from discussions with Dr. Ian Ross from the University of Queensland’s Centre for Solar Biotechnology. All steps were performed under sterile conditions. 1 mL O. affinis growth medium (full strength MS (M519, Phyto Technology Lab), 20 g L-1 sucrose, pH 5.8, 0.4 mg L-1 2,4-D) was supplemented with 10% (v/v) methanol. 500 µL of this mixture was added to a cryopreservation vial and mixed with 500 µL O. affinis cell suspension in the exponential growth phase, three days after subculturing, by gently inverting multiple times. The vial was then placed in a Mr. FrostyTM Freezing Container and put into a stable -80°C environment for 6 h. Consequently, the vial was transferred into liquid nitrogen and stored for at least 24 h before thawing.

6.1.1.2 Two-step freezing using an SGD cryoprotectant solution

The second two-step freezing experiment used a solution of 2 M sucrose, 1 M glycerol, 1 M dimethyl sulfoxide, and 1% (w/v) L-proline as cryoprotectant and followed the protocol reported by Mustafa et al. (2011) [3].

6.1.1.3 Vitrification

The vitrification experiments were carried out following the protocol published by Grout (2007) with no variation to the protocol [2].

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6.1.2 Regrowth of frozen cells

Regrowth experiments were conducted according to Ogawa et al. with minor modifications [4]. After thawing, the cells were dried on filter paper, transferred onto agar with growth medium and incubated for one day in the dark. Subsequently, the cells were transferred into 1 mL liquid growth medium and monitored for regrowth over 20 days in the dark. It was not possible to determine the inoculation density. The O. affinis growth medium used for regrowth contained full strength MS (M519, Phyto Technology Lab), 20 g L-1 sucrose, pH 5.8, and 0.4 mg L-1 2,4-D.

6.1.3 Cell viability assays

All samples were checked via microscopy after cryopreservation treatment to assess cell viability and condition. Random samples were tested using a 3-[4,5-dimethylthiazol-2yl]-2,5- diphenyl tetrazolium bromide (MTT)-based cell viability assay adopted from Castro-Concha et al. [9]. Cells frozen directly in culture media, i.e. without cryoprotective measures applied and pure medium without cells, were used as negative control, fresh cells were used as positive control. Cell growth in the dark, to avoid damaging the cells by irradiation, was visually monitored for 20 days after thawing. Visual growth determination for 10-14 days after thawing is standard procedure for plant cell lines [3].

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6.2 Results and discussion

Two step freezing protocols using methanol and SGD cryoprotectants and a vitrification protocol using several cryoprotectants were tested for cryopreservation of O. affinis suspension cells. No regrowth of O. affinis suspension cells after freezing was achieved for any of the tested protocols. Thawed cells displayed similar cell morphologies after SGD two-step freezing and vitrification, as shown in Figure 49 and were both unable to grow on agar or in liquid medium.

Figure 49 | Microscopy images of O. affinis cryopreservation experiments.

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Cryopreservation of Oldenlandia affinis cell suspensions

The microscopy images revealed an intact cell wall of the O. affinis suspension cells after thawing for both two-step freezing and vitrification protocols; however, the inner cell compartments were condensed into small spheres. Cells frozen and thawed without cryoprotectant also displayed an intact cell wall, but the cell compartments were condensed into random shapes suggesting profound disruption to intracellular structures. Similar random shapes were reported after intracellular ice formation in Hypericum perforatum cell suspensions [10]. The autofluorescence and cell viability assay showed no viable cells after any of the tested protocols. Consequently, not the regrowth parameters, but the absence of living cells was the likely reason for the lack of observable regrowth. The different shaped cell compartments confirmed an influence of the tested cryoprotectants on intracellular ice formation and indicate that different cryoprotectant concentrations and compositions might lead to more promising results. The MTT assay of cells treated according to the SGD two-step freezing protocol showed some enzyme activity, the measure of cell viability in this test, in the protected samples as shown in Figure 50. Nevertheless, these cells were not able to grow in suspension after freezing and thawing.

0.10

0.08

0.06

0.04

Enzyme activity [-] activity Enzyme 0.02

0.00

Positive controlProtected cells UnprotectedNegative cells control

Figure 50 | MTT assay of O. affinis cells after cryopreservation. Positive control was fresh non-frozen cells, protected cells were treated according to the SGD two-step freezing protocol, unprotected cells were frozen in medium without cryoprotectant, and the negative control was medium without cells. Error bars indicate standard deviation of the three biological replicates.

Methanol was reported to induce light and temperature sensitivity [6] and fragile cells are often sensitive to light; therefore, all regrowth experiments took place in the dark. Initial attempts to establish O. affinis cell suspensions from callus showed that no growth was observed if the initial cell density was below a certain threshold. The regrowth of O. affinis after cryopreservation might have been affected by this phenomenon as most of the visible cells were dead and the apparent cell density was much higher than the actual viable cell density. Possible solutions for this problem include a

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Cryopreservation of Oldenlandia affinis cell suspensions higher percentage of surviving cells due to optimised freezing conditions, a cell sorting step before regrowth, plating on solid medium before transfer into liquid medium, and initial regrowth in very small volumes with subsequent scale-up steps. Unlike bacteria, all plant species need a certain density of living cells for initiation of suspension growth [11]. To optimise the freezing conditions, sampling after every stage of the cryopreservation process to assess the influences on the cells and adjust accordingly could be an option. The microscopy images presented for stress responses (Section 4.2.1) and cryopreservation in O. affinis can be used as comparison. The fact that other plant species grow after thawing without additional steps, suggests that regrowth might also be possible for O. affinis if the right cryopreservation conditions are met. Cryopreservation of plant suspensions has been used for many years and several aspects influencing success have been identified [2]. Factors of major importance are the chosen cryoprotectant, the incubation time, the recovery conditions, and of course the biological species. Additional influencing factors include pre-treatment, cooling rate, and subzero holding times [2]. Obviously, all factors can be varied resulting in a multidimensional parameter space to be tested. The workload required to screen for viable conditions is only justified if a high value target needs to be preserved. The cells used in this work did not produce high value products, and consequently, no subsequent screening after the three initial experiments was initiated. However, should heterologous expression of high-value products be successful in O. affinis, revisiting cryopreservation would be desirable. The ability to store cell lines is an important consideration for biotechnological production systems. Since no cryopreservation protocol for O. affinis is available at present, it might be preferable to produce cyclotide pharmaceuticals in other species. P. hybrida has the necessary enzymes for cyclotide production and can be cryopreserved [3], and BY2 cells can easily be transformed and cryopreserved [3] to name two examples. Both P. hybrida and BY2 cells are promising starting points to produce grafted cyclotides. P. hybrida suspension cultures tested in this work also displayed strong similarities with O. affinis in terms of downregulation of cyclotide production over time, indicating that production of recombinant cyclotides without coelution of natural cyclotides might be possible. In summary, cryopreservation of O. affinis cell suspensions was not yet successful, but further advances are possible by designing experiments to vary cryoprotectant compositions and concentrations. If O. affinis remains recalcitrant to cryopreservation, then other systems that already possess cryopreservation protocols should be chosen as cyclotide production platforms.

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6.3 References

1. Bui, T.V., I.L. Ross, G. Jakob, and B. Hankamer, Impact of procedural steps and cryopreservation agents in the cryopreservation of chlorophyte microalgae. PLoS One, 2013. 8: p. e78668. 2. Grout, B.W.W., Cryopreservation of Plant Cell Suspensions, in Cryopreservation and Freeze-Drying Protocols, J.G. Day and G.N. Stacey, Editors. 2007, Humana Press: Totowa, NJ. p. 153. 3. Mustafa, N.R., W. de Winter, F. van Iren, and R. Verpoorte, Initiation, growth and cryopreservation of plant cell suspension cultures. Nat. Protocols, 2011. 6: p. 715. 4. Ogawa, Y., N. Sakurai, A. Oikawa, K. Kai, Y. Morishita, K. Mori, K. Moriya, F. Fujii, K. Aoki, H. Suzuki, D. Ohta, K. Saito, and D. Shibata, High-throughput cryopreservation of plant cell cultures for functional genomics. Plant Cell Physiol, 2012. 53: p. 943. 5. Gassen, C., Untersuchungen zur Gefrierkonservierung von Vitis vinifera- Pflanzenzellkulturen, in Institute of Bioprocess Engineering. 2009, University Erlangen- Nuremberg. 6. Crutchfield, A., K. Diller, and J. Brand, Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta). European Journal of Phycology, 1999. 34: p. 43. 7. Harvey, B., R.N. Kelley, and M. Ashwood-Smith, Cryopreservation of zebra fish spermatozoa using methanol. Canadian Journal of Zoology, 1982. 60: p. 1867. 8. Rall, W.F., M. Czlonkowska, S.C. Barton, and C. Polge, Cryoprotection of Day-4 mouse embryos by methanol. Reproduction, 1984. 70: p. 293. 9. Castro-Concha, L.A., R.M. Escobedo, and M.L. de Miranda-Ham, Measurement of cell viability in in vitro cultures, in Plant Cell Culture Protocols. 2006, Springer. p. 71. 10. Mišianiková, A., D. Zubrická, L. Petijová, K. Bruňáková, and E. Čellárová, Effect of Cryoprotectant Solution and of Cooling Rate on Crystallization Temperature in Cryopreserved Hypericum Perforatum Cell Suspension Cultures. Cryoletters, 2016. 37: p. 173. 11. Murthy, H.N., E.-J. Lee, and K.-Y. Paek, Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell, Tissue and Organ Culture, 2014. 118: p. 1.

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Conclusions

7 Conclusions

This thesis aimed to take plant pharmaceutical peptides from research to production. As with most scientific endeavours, the search for answers led to more questions and interesting results. This chapter summarizes the discoveries made, puts them in context and glimpses into the future.

7.1 Disappearing cyclotides

Initial attempts to produce cyclotides in O. affinis cell suspensions revealed a decline of cyclotide concentration over time. Reduced secondary metabolite concentration is often cited as a problem in plant cell suspensions and the molecular mechanisms are poorly understood [1, 2]. Cyclotides, like many secondary metabolites, are produced to protect plants against herbivores, pathogens, and pests [3, 4]. Plant suspension cells are dedifferentiated and therefore do not experience stress factors related to plant pests [5], possibly explaining the reduced cyclotide production. Quantitative PCR experiments showed that the necessary enzymes for posttranslational processing of cyclotides were still present in O. affinis suspension cells, but many of the cyclotide genes were no longer expressed. Consequently, cyclotide production in O. affinis suspension might be possible, if gene expression is restored. Cell cultures of P. hybrida also had reduced cyclotide production, suggesting that this phenomenon is not unique to O. affinis. H. enneaspermus, and C. ternatea suspensions displayed strong cyclotide production without time dependent decline. All suspension cultures produced a small number of cyclotides with very high sequence similarity. This observation was surprising, considering that all species stemmed from different plant families belonging to different biological orders. The widespread occurrence and high sequence similarity indicate strong evolutionary constraints on these peptides to increase plant fitness. Possible explanations include a biological function not related to plant defence or a baseline defence against pests and pathogens normally endangering young, undifferentiated tissue. Further experiments are needed to probe these hypotheses and generate insights that can verify the suspension type as a new universal subgroup of cyclotides.

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Conclusions

7.2 Elicitation to bring back cyclotides

Elicitation, a common tool to increase secondary metabolite yields in plant cell cultures [6-8], was chosen as the first experiment to boost the cyclotide yield. Dörnenburg et al. reported significantly increased cyclotide production in O. affinis tissue and suspension cultures by elicitation via irradiation, agitation, or chitosan treatment [9, 10]. In the current study, starving, immobilization, methyl jasmonate, and sodium chloride were tested to increase cyclotide production in O. affinis suspension cultures. 3 mM sodium chloride enhanced the production of cycloviolacin O2, kalata B22, and kalata B23 by 800%, 200% and 500% respectively seven days after treatment. Cell immobilization on the vessel wall or on solid medium resulted in chlorophyll production and a cyclotide profile very similar to callus with a strong kalata B1 peak present in the MALDI trace of the cyclotide extract. A common strategy in molecular farming is to uncouple growth from production to improve yields [8]. The insights gathered in this work enable growth of O. affinis in suspension under optimised conditions with subsequent immobilization or elicitation to produce cyclotides in a two- step approach. Another promising method to be tested is the immobilization of suspension cells in alginate beads to enhance cyclotide production and possibly secret them into the medium circumventing the energy expensive extraction steps [11].

7.3 Transgenic plant cell cultures to produce cyclotides

The second experiment set up to boost cyclotide yield and enable production of engineered cyclotides in plant cell suspension was recombinant production. A. tumefaciens was added to plant cell packs, resulting in the first reported transformation of O. affinis. This technique will be of great benefit for O. affinis molecular biology studies and might allow for flexible on demand production of engineered cyclotides if the limitations in reproducibility reported in this study can be solved. One strategy to tackle reproducibility problems is to employ automation assisted mass testing, for example using a programmable robotic workflow. Possible follow-up experiments include: (1) testing the influence of a newly discovered cystatin dual-inhibitor of papain and AEP cysteine proteases [12] on the regulation of cyclotide production and (2) testing the heterologous expression of cyclotide precursor genes and/or grafted variants to assess if O. affinis suspension cells are still capable of peptide cyclisation. In general, commercial success of transgenic plant cell suspensions is still 152 | Page

Conclusions hindered by the limited understanding of most secondary metabolite pathways in plants [7]. To overcome this hurdle, the knowledge of cyclotide pathways was advanced in this work and might be advanced further by follow-up experiments enabled by O. affinis transformation, perhaps cumulating in commercial applications.

7.4 Cryopreservation as a roadblock

The industrial application of stable transgenic cell lines requires long-term storage to ensure continued operation in case of contamination, genetic mutations or epigenetic changes. For plant cell cultures long-term storage is only possible via a process called cryopreservation [13]. In this work three protocols were tested to assess O. affinis cryopreservation. No cell survival was observed, but valuable insights for future experiments were gathered. Transient transformation of wild type cell lines and regeneration of stable transgenic plants from callus were explored as production strategies that do not require cryopreservation.

7.5 From farm to pharm

This study aimed to realize cyclic peptide production in plant cell suspensions. High biomass accumulation of wildtype O. affinis was achieved on a 5 L scale, using GMP guidelines. Similar systems with reactor volumes of 100-2000 L are already deployed for other plant species [14], indicating good scale-up potential. O. affinis elicitation and transformation presented here might enable highly flexible systems with decoupled growth and production. One possible strategy is O. affinis suspension inoculation in several single-use bags, followed by transient transformation before the stationary phase, and harvesting 7 days later. The multiple bag setup allows pseudo- continuous production and the switch of target product without any down time but is limited by the volume that can be transformed transiently. Another option is the establishment of stable transformed cell lines to either be grown in reactors or to regrow transgenic plants. C. ternatea and H. enneaspermus suspension cultures were successfully initiated and the toolset developed for O. affinis can be easily transferred to these new species. C. ternatea is of industrial importance as it is the basis for a new, bee-friendly insecticide Sero-X launched in Australia

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Conclusions in 2017 and currently undergoing regulatory approval elsewhere in the world. A better molecular understanding of C. ternatea could lead to higher production efficiency, discovery of the active compound(s) and possibly development of new production methods. H. enneaspermus is a well- known Indian medicinal herb with many possible pharmaceutical applications [15]. H. enneaspermus cell suspensions have been shown to produce L-3,4-dihyroxyphenylalanine, an amino acid used for the treatment of Parkinson’s disease, at 8.88 mg g-1 DW when elicited with 150 µM salicylic acid [16]. These reports combined with the results presented here mark H. enneaspermus as a good candidate for cyclic peptide production in suspension. The discovery and optimisation of recombinant protein production in plants led to some very optimistic predictions of “a world in which any protein, either naturally occurring or designed by man, could be produced safely, inexpensively and in almost unlimited quantities using only simple nutrients, water and sunlight” [17]. Seventeen years later, the hope is still alive, but the optimism is tempered with a greater understanding of the challenges involved. The techniques developed have brought great advances in laboratory research and agriculture, however the unlimited supply of cheap pharmaceuticals has never eventuated. Nevertheless, there are a few success stories like the transient ZMappTM production in tobacco leaves to battle the Ebola crisis or the stable expression of taliglucerase alfa in carrot and tobacco cell culture [18, 19]. The potential is still there; however, more research is required to bring about the predicted advances. The experiments and results presented in this thesis contribute to this endeavour and take us closer to moving from the farm to pharm.

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Conclusions

7.6 References

1. Hellwig, S., J. Drossard, R.M. Twyman, and R. Fischer, Plant cell cultures for the production of recombinant proteins. Nat Biotech, 2004. 22: p. 1415. 2. Xu, J., X. Ge, and M.C. Dolan, Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures. Biotechnol Adv, 2011. 29: p. 278. 3. Bennett, R.N. and R.M. Wallsgrove, Secondary metabolites in plant defence mechanisms. New Phytologist, 1994. 127: p. 617. 4. Slazak, B., M. Kapusta, A.A. Stromstedt, A. Slomka, M. Krychowiak, M. Shariatgorji, P.E. Andren, J. Bohdanowicz, E. Kuta, and U. Goransson, How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System. Front Plant Sci, 2018. 9: p. 1296. 5. Tanurdzic, M., M.W. Vaughn, H. Jiang, T.J. Lee, R.K. Slotkin, B. Sosinski, W.F. Thompson, R.W. Doerge, and R.A. Martienssen, Epigenomic consequences of immortalized plant cell suspension culture. PLoS Biol, 2008. 6: p. 2880. 6. Ramirez-Estrada, K., H. Vidal-Limon, D. Hidalgo, E. Moyano, M. Golenioswki, R. Cusidó, and J. Palazon, Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules, 2016. 21: p. 182. 7. Nadeem, H. and F. Ahmad, Prospects for the Use of Plant Cell Culture as Alternatives to Produce Secondary Metabolites, in Natural Bio-active Compounds, M.S. Akhtar and M.K. Swamy, Editors. 2019, Springer Singapore. p. 153. 8. Murthy, H.N., E.-J. Lee, and K.-Y. Paek, Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell, Tissue and Organ Culture, 2014. 118: p. 1. 9. Dörnenburg, H. and P. Seydel, Effect of irradiation intensity on cell growth and kalata B1 accumulation in Oldenlandia affinis cultures. Plant Cell, Tissue and Organ Culture, 2007. 92: p. 93. 10. Dörnenburg, H., Progress in kalata peptide production via plant cell bioprocessing. Biotechnol J, 2009. 4: p. 632. 11. Inyai, C., P. Boonsnongcheep, J. Komaikul, B. Sritularak, H. Tanaka, and W. Putalun, Alginate immobilization of Morus alba L. cell suspension cultures improved the accumulation and secretion of stilbenoids. Bioprocess Biosyst Eng, 2019. 42: p. 131. 12. Vorster, B.J., C.A. Cullis, and K.J. Kunert, Plant Vacuolar Processing Enzymes. Front Plant Sci, 2019. 10: p. 479. 13. Schumacher, H.M., M. Westphal, and E. Heine-Dobbernack, Cryopreservation of Plant Cell Lines, in Cryopreservation and Freeze-Drying Protocols, W.F. Wolkers and H. Oldenhof, Editors. 2015, Springer New York. p. 423. 14. Lopes, A.G., Single-use in the biopharmaceutical industry: A review of current technology impact, challenges and limitations. Food and Bioproducts Processing, 2015. 93: p. 98. 15. Patel, D.K., R. Kumar, K. Sairam, and S. Hemalatha, Hybanthus enneaspermus (L.) F. Muell: a concise report on its phytopharmacological aspects. Chin J Nat Med, 2013. 11: p. 199. 16. Sathish, S., V. Vasudevan, S. Karthik, G. Pavan, and M. Manickavasagam, Enhanced l-Dopa production from elicited cell suspension culture of Hybanthus enneaspermus (L.) F. Muell. Plant Biotechnology Reports, 2019. 13: p. 613. 17. Ma, J.K.C., P.M.W. Drake, and P. Christou, The production of recombinant pharmaceutical proteins in plants. Nature Reviews Genetics, 2003. 4: p. 794. 18. Qiu, X., G. Wong, J. Audet, A. Bello, L. Fernando, J.B. Alimonti, H. Fausther-Bovendo, H. Wei, J. Aviles, E. Hiatt, A. Johnson, J. Morton, K. Swope, O. Bohorov, N. Bohorova, C. Goodman, D. Kim, M.H. Pauly, J. Velasco, J. Pettitt, G.G. Olinger, K. Whaley, B. Xu, J.E.

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Conclusions

Strong, L. Zeitlin, and G.P. Kobinger, Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature, 2014. 514: p. 47. 19. Grabowski, G.A., M. Golembo, and Y. Shaaltiel, Taliglucerase alfa: An enzyme replacement therapy using plant cell expression technology. Molecular Genetics and Metabolism, 2014. 112: p. 1.

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