Science, Irving K. Barber Faculty of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
Nguyen, Minh Tuan Anh
Master of Science - MSc
University of British Columbia
Since the 1990s, topotecan and irinotecan, two camptothecin derivatives, have been widely used as anticancer drugs owing to their phenomenal topoisomerase-I inhibitory activity. In the pharmaceutical industry, topotecan and irinotecan are chemically synthesized from the prodrug camptothecin, a quinoline alkaloid from Camptotheca acuminata, via 10-hydroxycamptothecin (10-HCPT), using toxic reagents and harsh conditions. This project aims to develop a chemoenzymatic approach for 10-HCPT production by exploring its biosynthetic pathway in C. acuminata. Multidisciplinary techniques, including bioinformatics, molecular cloning, and organic synthesis, were implemented to discover and characterize the plant metabolic enzymes. Two cytochrome P450 monooxygenases have been discovered that regio-selectively hydroxylated camptothecin at two different positions, C-10 and C-11 on the CPT scaffold. The high regio-selectivity and conversion rate (12 mg/L – biotransformation rate) afford the production of hydroxycamptothecins (10/11-HCPT) in the baker’s yeast Saccharomyces cerevisiae. By combining cytochrome P450 enzymes and chemo-catalysts sequentially in the chemoenzymatic process, 13 camptothecin derivatives including topotecan and irinotecan have been successfully synthesized. This research is a step forward in camptothecin biochemistry and leading finally to the economical and sustainable production of more active and water-soluble camptothecin derivatives.
DISCOVERING AND HARNESSING CAMPTOTHECIN HYDROXYLASE ENZYMES IN Camptotheca acuminata FOR CHEMOENZYMATIC SYNTHESIS OF ANTICANCER CAMPTOTHECIN DERIVATIVES by Minh Tuan Anh Nguyen B.Sc., Vietnam National University Ho Chi Minh City – University of Science, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) October 2021 © Minh Tuan Anh Nguyen, 2021 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: DISCOVERING AND HARNESSING CAMPTOTHECIN HYDROXYLASE ENZYMES IN Camptotheca acuminata FOR CHEMOENZYMATIC SYNTHESIS OF ANTICANCER CAMPTOTHECIN DERIVATIVES submitted by Minh Tuan Anh Nguyen in partial fulfillment of the requirements of the degree of Master of Science. Dr. Thu-Thuy Dang, Irving K. Barber Faculty of Science, University of British Columbia Supervisor Dr. Susan Murch, Irving K. Barber Faculty of Science, University of British Columbia Supervisory Committee Member Dr. Frederic Menard, Irving K. Barber Faculty of Science, University of British Columbia Supervisory Committee Member Dr. Michael Deyholos, Irving K. Barber Faculty of Science, University of British Columbia University Examiner iii Abstract Since the 1990s, topotecan and irinotecan, two camptothecin derivatives, have been widely used as anticancer drugs owing to their phenomenal topoisomerase-I inhibitory activity. In the pharmaceutical industry, topotecan and irinotecan are chemically synthesized from the prodrug camptothecin, a quinoline alkaloid from Camptotheca acuminata, via 10-hydroxycamptothecin (10-HCPT), using toxic reagents and harsh conditions. This project aims to develop a chemoenzymatic approach for 10-HCPT production by exploring its biosynthetic pathway in C. acuminata. Multidisciplinary techniques, including bioinformatics, molecular cloning, and organic synthesis, were implemented to discover and characterize the plant metabolic enzymes. Two cytochrome P450 monooxygenases have been discovered that regio-selectively hydroxylated camptothecin at two different positions, C-10 and C-11 on the CPT scaffold. The high regio-selectivity and conversion rate (12 mg/L – biotransformation rate) afford the production of hydroxycamptothecins (10/11-HCPT) in the baker’s yeast Saccharomyces cerevisiae. By sequentially combining cytochrome P450 enzymes and chemo-catalysts in the chemoenzymatic process, 13 camptothecin derivatives including topotecan and irinotecan have been successfully synthesized. This research is a step forward in camptothecin biochemistry and leading finally to the economical and sustainable production of more active and water-soluble camptothecin derivatives. iv Lay Summary Camptothecin, a natural product isolated from the bark of Camptotheca acuminata (happy tree), has been used to produce the anticancer drugs topotecan (Hycamptin) and irinotecan (Camptosar) since the late 20th century. These therapeutic agents are mainly produced via chemical synthesis via the camptothecin derivative 10-hydroxycamptothecin. While synthetic chemistry allows the production of this compound at feasible yield, plant-derived biocatalysts (enzymes) can facilitate the sustainable production of these drugs at low cost. In this work, oxidative enzymes were discovered from the happy tree, leading to the production of two hydroxylated camptothecin products, 10-hydroxycamptothecin and 11-hydroxycamptothecin. These, in turn, serve as precursors to biochemically synthesize a suit of 13 other camptothecin-derived compounds. This proof-of-concept chemoenzymatic approach could allow the preparation of a library of camptothecin derivatives for therapeutic applications. v Preface This Master’s thesis consists of four chapters, conducted by Minh Tuan Anh Nguyen under the supervision of Dr. Thu-Thuy Dang in the Department of Chemistry, Irving K. Barber Faculty of Science, UBC Okanagan. The literature review and the experimental data were prepared by Minh Tuan Anh Nguyen with direct inputs from Dr. Thu-Thuy Dang. Chapter 2: Discovery and characterization of cytochrome P450 genes involved in camptothecin hydroxylation in Camptotheca acuminata The research idea was conceived by Dr. Thu-Thuy Dang and Dr. Trinh-Don Nguyen. The mRNA from leaves of Camptotheca acuminata was previously prepared by Dr. Thu-Thuy Dang. Minh Tuan Anh Nguyen, Yuen Yee Leung, Dr. Trinh Don Nguyen, Matthew McConnachie cloned the cytochrome P450 candidate genes. Minh Tuan Anh Nguyen conducted the in vivo and in vitro assay optimizations and collected and analyzed data from UPLC-MS/MS analysis. The purification of hydroxylated products was conducted by Minh Tuan Anh Nguyen and Matthew McConnachie. The NMR experiments were performed by Dr. Zhicheng Xia, NMR Facility Service, Department of Chemistry, Faculty of Science, UBC Vancouver. Minh Tuan Anh Nguyen analyzed and elucidated the structure of the products from the 1H- and 13CNMR and 1D-TOCSY spectra. Minh Tuan Anh Nguyen carried out the experiments in this chapter under the supervision of Drs. Dang and Nguyen. The work of this chapter is presented in this publication: Tuan-Anh Minh Nguyen, Trinh-Don Nguyen, Yuen Yee Leung, Matthew McConnachie, Oleg Sanikov, Zhicheng Xia, & Thu-Thuy T Dang, “Discovering and harnessing monooxygenase catalysts for chemoenzymatic synthesis and diversification of anticancer camptothecin analogues”, 2021. (under review). Chapter 3: Chemoenzymatic synthesis of topotecan, irinotecan, and halogenated hydroxycamptothecin analogues vi The chemical synthetic reactions were performed by Minh Tuan Anh Nguyen. The NMR experiments were conducted by Dr. Zhicheng Xia, UBC Vancouver. The NMR data were interpreted by Minh Tuan Anh Nguyen. All the work and methods in Chapter 2, Chapter 3 are presented in two publications: Tuan-Anh Minh Nguyen, Trinh-Don Nguyen, Yuen Yee Leung, Zhicheng Xia, Matthew McConnachie, & Thu-Thuy T Dang, “Discovering and harnessing monooxygenase catalysts for chemoenzymatic synthesis and diversification of anticancer camptothecin analogs”, 2021. (under review) Tuan-Anh Minh Nguyen, Matthew McConnachie, Trinh-Don Nguyen, Thu-Thuy T Dang, “Discovery and characterization of oxidative enzymes in monoterpene indole alkaloids biosynthesis”, Methods in Molecular Biology, 2021 (under review). vii Table of Contents Abstract ....................................................................................................................................................... iii Lay Summary ............................................................................................................................................. iv Preface .......................................................................................................................................................... v Table of Contents ...................................................................................................................................... vii List of Tables ............................................................................................................................................... x List of Figures ............................................................................................................................................. xi Acknowledgements .................................................................................................................................. xiv Chapter 1: Introduction ............................................................................................................................. 1 1.1 Bioactive secondary or specialized metabolites in plant ........................................................... 1 1.2 Monoterpene indole alkaloids (MIAs) ...................................................................................... 2 1.2.1 Structures, bioactivities and biosynthesis of MIAs ............................................................. 2 1.2.2 Cytochrome P450 (P450) enzyme ...................................................................................... 4 1.2. 3 Plant P450s and chemical diversity in plants ...................................................................... 7 1.2.4 Cytochrome P450 enzymes in MIA metabolism ................................................................ 8 1.3 Synthetic biology of natural products in microbes .................................................................. 13 1.4 Camptothecin and its analogues .............................................................................................. 14 1.5 Camptothecin biosynthesis ...................................................................................................... 19 1.6 Research objectives ................................................................................................................. 21 Chapter 2: Discovery and characterization of cytochrome P450 genes involved in camptothecin hydroxylation in Camptotheca acuminata ............................................................................................... 23 2.1 Summary ................................................................................................................................. 23 2.2 Introduction ............................................................................................................................. 23 2.2.1 Biosynthetic candidate genes discovery from genomic and transcriptomic datasets........ 23 2.2.2 Biochemical characterization of candidate genes in yeast ................................................ 24 2.3 Material and methods .............................................................................................................. 27 viii 2.3.1 Plant material .................................................................................................................... 27 2.3.2 Chemicals.......................................................................................................................... 28 2.3.3 Phylogenetic analysis ........................................................................................................ 28 2.3.4 Candidate identification .................................................................................................... 29 2.3.5 P450 genes cloning ........................................................................................................... 29 2.3.6 High-throughput P450 candidate genes screening assay for activities against CPT......... 32 2.3.7 Substrate specificity assay ............................................................................................... 33 2.3.8 UPLC-MS/MS analysis .................................................................................................... 33 2.3.9 In vivo assay optimizations ............................................................................................... 34 Culture media ............................................................................................................................ 34 Camptothecin and glycerol concentrations ................................................................................ 34 Different reaction time............................................................................................................... 35 In vivo assay with different yeast strains ................................................................................... 35 2.3.10 P450 microsomes preparation ...................................................................................... 35 2.3.11 In vitro microsomes assay ............................................................................................ 36 2.3.12 Western blot (SureCast HandCast system – Invitrogen) .............................................. 36 2.3.13 Structural identification of products ............................................................................. 37 2.4 Results ..................................................................................................................................... 37 2.4.1 Identification and catalytic function validation of P450 candidates ..................................... 37 2.4.2 Optimization of in vivo assay conditions .............................................................................. 43 2.5 Discussion ............................................................................................................................... 64 Chapter 3: Chemoenzymatic synthesis of high-value CPT analogues ................................................. 67 3.1 Summary ................................................................................................................................. 67 3.2 Introduction ............................................................................................................................. 67 3.3 Material and methods .............................................................................................................. 71 3.3.1 Chemicals.......................................................................................................................... 71 ix 3.3.2 Hydroxycamptothecin and 7-ethylhydroxycamptothecin preparation by using camptothecin hydroxylases ............................................................................................................ 71 3.3.3 Semi-synthesis of topotecan and its 11-hydroxy isomer: ................................................. 72 3.3.4 Semi-synthesis of irinotecan and its 11HCPT-derived isomer: ........................................ 72 3.3.5 Semi-synthesis of brominated HCPTs: ............................................................................. 72 3.4 Results ..................................................................................................................................... 73 3.5. Discussion ............................................................................................................................... 82 Chapter 4: Conclusion and future perspectives ..................................................................................... 84 4.1 Contribution of camptothecin hydroxylases in CPT biosynthesis from C. acuminata ........... 84 4.2 Chemoenzymatic process for anticancer drugs and their analogues syntheses ....................... 85 Bibliography .............................................................................................................................................. 88 Appendices ............................................................................................................................................... 101 x List of Tables Table 1.1. Metabolites from Camptotheca acuminata relating to camptothecin biosynthesis ................... 20 Table 2.1. Selected CID data of camptothecin and its analogues ............................................................... 34 Table 2.2. Nucleic acid sequence identity (%) between Ca32229 and its close homologues of from C. acuminata .................................................................................................................................................... 41 Table 2.3. 1H-NMR data of the putative HCPT isolated from the in vivo assay of Ca32229 and CPT in comparison with previously-reported 11HCPT .......................................................................................... 47 Table 2.4. 1H-NMR data of enzymatic product in the in vivo assay of Ca32236 and CPT ........................ 50 Table 2.5. 1H-NMR data of enzymatic product in the in vivo assay of Ca32229 and 7-ECPT .................. 55 Table 2.6. 1H NMR data of enzymatically produced 10,11-dihydroxy-CPT in DMSO-d6 (298 K, 600 MHz) ........................................................................................................................................................... 61 Table 2.7. Conversion rates of in vivo feeding reactions using yeast expressing CPT hydroxylases and CPT derivatives. .......................................................................................................................................... 64 Table 3.1. 1H NMR data of the chemoenzymatic reaction product irinotecan isomer, 7-ethyl-11-[4-(1-piperidino)-1-piperidino]carbonyloxyCPT(irinotecan-11) in DMSO-d6 (298 K, 600 MHz) ..................... 78 Table 3.2. 1H NMR data of chemoenzymatic reaction product topotecan isomer 12-[(dimethylamino)methyl]-11HCPT (topotecan-11) in DMSO-d6 (298 K, 600 MHz) ................................ 80 xi List of Figures Figure 1.1. Different classes of natural products .......................................................................................... 2 Figure 1.2. MIA biosynthesis in plant........................................................................................................... 4 Figure 1.3. CPR and P450 enzymes anchoring on ER membrane ................................................................ 5 Figure 1.4. Catalytic cycle of cytochrome P450 hydroxylation reaction ..................................................... 6 Figure 1.5. Radical and concerted mechanisms of oxygen atom insertion ................................................... 7 Figure 1.6.. Vinblastine biosynthetic pathway ............................................................................................ 10 Figure 1.7. Vinorine metabolic pathway from geissochizine catalyzed by SBE. ....................................... 11 Figure 1.8. Catalytic mechanism of RsSBE with 19-E-geissochizine substrate and CrSBE with tetrahydroalstonine substrate . .................................................................................................................... 12 Figure 1.9. Vinorine reaction with vinorine hydroxylase (VH) and perakine formation mechanism from vomilenine................................................................................................................................................... 13 Figure 1.10. Camptothecin and camptothecin analogues (red arrows depict the positions of substituents increasing the bioactivity of CPT derivatives against topoisomerase-I ...................................................... 15 Figure 1.11. Proposed biosynthetic pathway of camptothecin ................................................................... 18 Figure 1.12. Enzymatic reactions of CaCYP72A565/610 and 7-deoxyloganic acid ................................. 19 Figure 1.13. Summary of research objectives ............................................................................................. 22 Figure 2.1. Brief workflow for the MIA biosynthetic candidate genes identification and characterization .................................................................................................................................................................... 25 Figure 2.2. Workflow of the biochemical characterization for cytochrome P540 candidate genes ............ 27 Figure 2.3. pESC-Leu plasmid (Agilent) .................................................................................................... 32 Figure 2.4. Identification of camptothecin hydroxylase candidates.. ......................................................... 38 Figure 2.5. In vivo assay of recombinant Ca32229 protein with camptothecin. ......................................... 40 Figure 2.6. Unrooted neighbour-joining phylogenetic tree for P450 candidates from this study and previously reported CYPs from C. acuminata and other organisms.. ........................................................ 42 xii Figure 2.7. CPT-to-putative HCPT conversion rate in in vivo assays with varied camptothecin and glycerol concentrations in YPA media. ...................................................................................................... 43 Figure 2.8. 1H-NMR spectrum of enzymatic product in the in vivo assay of Ca32229 and CPT ............... 45 Figure 2.9. 1D-TOCSY spectrum and stacked 1H-NMR spectra against 10-HCPT standard of 11-HCPT enzymatic product ....................................................................................................................................... 48 Figure 2.10. 1H-NMR spectrum of enzymatic product in the in vivo assay of Ca32236 and CPT ............. 49 Figure 2.11. 1D-TOCSY spectrum and stacked 1H-NMR spectra against 10-HCPT standard of 10-HCPT enzymatic product. ...................................................................................................................................... 51 Figure 2.12. Summary of the specific signals on 1H-NMR spectra of 10- and 11-hydroxycamptothecin.52 Figure 2.13. Substrate specificity of Ca32236 (CPT 10-hydroxylase) and Ca32229 (CPT 11-hydroxylase). ............................................................................................................................................... 54 Figure 2.14. 1D-TOCSY spectrum of enzymatic product from in the in vivo assay of Ca32229 and with 7-ECPT as the substrate. ............................................................................................................................. 56 Figure 2.15. Extracted ion chromatograms showing the in vivo activity of Ca32236 and of Ca32229 with CPT and 7-ethyl-CPT ................................................................................................................................. 57 Figure 2.16. In vivo activity of Ca32229 and Ca32236 with 9-aminoCPT ................................................. 58 Figure 2.17. In vivo activity of Ca32229 and Ca32236 with 11-HCPT ...................................................... 60 Figure 2.18. 1H NMR spectrum of products from in vivo assay of Ca32229/CPR with 10-HCPT as substrate producing 10,11-dihydroxyCPT. ................................................................................................. 61 Figure 2.19. Western blot showing the expression of Ca32229 and Ca32236 and in vitro assays using microsome extracted from the same yeasts with CPT. ............................................................................... 63 Figure 3.1. Comparison of functional group installation through traditional chemical synthesis versus C–H functionalization ...................................................................................................................................... 68 Figure 3.2. Conventional and chemoenzymatic synthesis of topotecan and its 11-isomer; irinotecan and its 11-isomer .................................................................................................................................................... 70 Figure 3.3. Chemoenzymatic reactions of 10- and 11-HCPT with NBS .................................................... 74 xiii Figure 3.4. 1D-TOCSY spectra of bromination reaction of 10-HCPT and 11-HCPT ................................ 74 Figure 3.5. Chemoenzymatic synthesis of brominated 10-HCPT and 11-HCPT ....................................... 75 Figure 3.6. Chemoenzymatic synthesis of irinotecan and irinotecan-11 .................................................... 77 Figure 3.7. The proposed mechanism of irinotecan-11 synthetic reaction ................................................. 78 Figure 3.8. 1H NMR spectrum of chemoenzymatic reaction product irinotecan-11 (7-ethyl-11-[4-(1-piperidino)-1-piperidino]carbonyloxyCPT) ................................................................................................ 79 Figure 3.9. Chemoenzymatic synthesis of topotecan and topotecan-11 ..................................................... 79 Figure 3.10. The mechanism of topotecan-11 synthetic reaction ............................................................... 81 Figure 3.11. 1H NMR spectra of chemoenzymatic reaction products topotecan-11 (12-[(dimethylamino)methyl]-11-HCPT) .......................................................................................................... 82 Figure 4.1. Biosynthetic pathway of hydroxylated camptothecin compounds. .......................................... 85 Figure 4.2. CPT analogues produced by chemoenzymatic reactions of camptothecin hydroxylases ......... 86 xiv Acknowledgements This thesis would never be successful without the kind support of many people. First and foremost, I would like to express my gratitude towards my supervisor Dr. Thu-Thuy Dang. She continuously supported me in every step of my research and encouraged me to develop my own research methodology. I am deeply grateful to all of her educational expertise, guidance on teaching me not only the fundamentals of Natural Product Biochemistry but also the crucial practical skills in the laboratory. I would like to thank Dr. Trinh-Don Nguyen for teaching me protein expression techniques and providing valuable guidance for my study, and lastly for making microsomes preparation not too tedious with all your stories and your sense of humour. My deep gratitude to Dr. Dang and Dr. Nguyen not only for their guidance in my academic research but also for their warm kindness for supporting my daily student life in Kelowna and making me feel at home with delicious Vietnamese dishes during Tết and surprising ones on long lab-working days. I want to thank my supervisory committee members, Dr. Susan Murch and Dr. Frederic Menard, for their generous support and helpful advice during my study. I would like to thank Dr. Kirsten Wolthers, Dr. Isaac Li, Dr. Wesley Zandberg, and Dr. Paul Shipley for sharing their resources. I want to thank Dr. Zhicheng Xia, UBC Vancouver, for his help on NMR experiments. And thank all my labmates Matthew, Helena, Ryland for their help and friendliness. I want to thank Dr. Nguyen Kim Phi Phung and Dr. Nguyen Tuyet Phuong for recommending and supporting my intention to pursue higher education in Canada. Thanks to my friends in Kelowna, Minh An brothers, Minh Phi for all the joy and help. To my close friends in Vietnam for their stories and friendship in all the vỉrtual gatherings. I am grateful to my family, grandparents, parents, my late stepfather and my sister for your love and encouragement. xv To my beloved family Ông nội, Bà nội, Ba Tuấn, Mẹ Lan, Ba Hưng, Chị hai and Phương, a lovely companion in my 20s for always loving me and staying by my side through ups and downs 1Chapter 1: Introduction 1.1 Bioactive secondary or specialized metabolites in plant Plant natural products, or specialized metabolites, are chemicals generated in the secondary metabolism of plants. While primary metabolism consists of the biochemical transformation of essential compounds in the living organisms such as carbohydrates, lipids, and nucleic acid, specialized metabolism (natural products metabolism) involve compounds of which functions are thought to play a role in the interactions between plants and the ecological system as defence compounds, sex pheromones, allelopathic compounds, etc. [1]. Plant natural products’ ability to interact with biological entities has been utilized for a long time, starting with traditional uses of medicinal plants, which are common across cultures worldwide for thousands of years. Higher plants have “generously” served as a primary source for most of these new chemical entities since the beginning of humanity. Owing to the leap of spectroscopic technique since the 20th century, including X-ray, nuclear magnetic resonance (NMR), mass spectrometry, etc., a vast number of specialized compounds’ structures from plants had been elucidated and tested with various biological activity assays, leading to myriad treatments for human diseases [2–4]. Hundreds of thousands of plant specialized metabolites have been characterized and can generally be categorized into three major classes, namely terpenoids, phenolic compounds, and alkaloids according to their structures and/or biosynthetic routes (Figure 1.1). Specialized metabolites are generated from the primary metabolism. Terpenoids are formed via the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathway, while phenolic compounds and alkaloids are mostly produced through the shikimate pathway [1]. Many of those are high-value chemicals, contributing more than 50% of the therapeutic compounds in the pharmaceutical industry [5]. Among these, alkaloids, the group of nitrogen-containing natural products, constitute a class of compounds with potent bioactivities, including some of the best-known drugs such as vinblastine (anticancer), quinine (antimalarial), and morphine (painkiller) [6][7]. 2 Figure 1.1. Different classes of natural products 1.2 Monoterpene indole alkaloids (MIA) 1.2.1 Structures, bioactivities, and biosynthesis of monoterpene indole alkaloids Monoterpene indole alkaloids (MIA) constitute a group of natural products commonly found in the Apocynaceae, Loganiaceae, and Rubiaceae plant families with spectacular bioactivities and a structural diversity of over 3,000 compounds identified up to date [8]. Many MIAs have been used chemotherapeutically. For instance, vinblastine and vincristine (vinca alkaloids), derived from the coupling reaction of catharanthine and vindoline from Madagascar periwinkle (Catharanthus roseus), have been approved by the US Food and Drug Administration for treating leukemia and lymphomas since 1960s [9]. Other MIAs possess distinct chemical structures and a suite of biological activities, for instance, camptothecin, a quinoline alkaloid from the bark and leaves of the Chinese happy tree (Camptotheca acuminata), has been used as an antitumour drug for its topoisomerase-I enzyme inhibition and ibogaine, an iboga-type alkaloid from Tabernanthe iboga, is an anti-opioid addiction potent drug [10][11]. Due to their high therapeutic values and unique chemistry, the biosynthesis of MIAs have been studied intensively for decades [12–17]. Preliminary studies using isotopically labelled MIA intermediates in the pathways and protein isolation from plants showed that the central precursor of MIA metabolism strictosidine is formed by the condensation of secologanin and tryptamine via strictosidine synthase (a Pictet-Spenglerase enzyme) [18][19]. Secologanin and tryptamine precursors are derived from geranyl 3pyrophosphate and tryptophan, respectively. Geranyl pyrophosphate and tryptophan are both products of the glycolysis process in the primary metabolism, yet they are produced via different pathways. Geranyl pyrophosphate is generated through the mevalonate or the methylerythritol phosphate (non-mevalonate) pathway. Meanwhile, tryptophan is produced from the shikimate pathway [1]. From strictosidine, a large number of MIA scaffolds are produced through various enzymatic reactions, including oxidation, scaffold rearrangement, hydroxylation, epoxidation, deglycosylation, and methylation (Figure 1.2) [13,14,20]. Despite numerous studies and recent advances in understanding MIAs, only a limiting number of genes accounting for the construction of MIA scaffolds have been discovered, a majority of them from Catharanthus roseus [21]. Although the functional characterization of a biosynthetic gene involves many steps such as cloning and expression in heterologous, easy-to-handle hosts (Escherichia coli bacteria, Saccharomyces cerevisiae yeast or Nicotiana benthamiana tobacco) [22], the major challenges of MIA biosynthesis elucidation are candidate genes identification and the pathway assembly. These hurdles result from the complexity of the plant genome and the fact that genes involved in the same biosynthetic pathway are not always physically clustered in the genome. Therefore, the biochemistry of MIAs in many important medicinal plants such as Camptotheca acuminata, Rauwolfia serpentina, and Gelsemium sempevirens, remains largely unelucidated [21]. 4 Figure 1.2. MIA biosynthesis in plant (TDC: tryptophan decarboxylase, STR: strictosidine synthase) 1.2.2 Cytochrome P450 enzymes Cytochrome P450 enzymes (P450s) make up one of the largest enzyme families involved in plant natural products biosynthesis [7]. P450s are well-known for their wide range of activities, such as hydroxylation, epoxidation, aromatization, and cyclization [12,14,23]. P450s require redox partners to aid in electron transfer and are categorized based on their redox partners. Class I consists of mammalian mitochondrial and bacterial P450s, which require electrons transferred by an Fe2S2-containing iron-sulfur protein. Class-II P450s are microsomal monooxygenases that gain electrons from a membrane-bound 5NADPH-cytochrome P450 oxidoreductase (CPR), the founding member of the diflavin reductase family (Figure 1.3) [24][25]. Figure 1.3. CPR and P450 enzymes anchoring on ER membrane. Adapted from [26] P450 monooxygenases break the atmospheric dioxygen molecule to introduce one oxygen atom onto the structure of the substrate and reduce the other oxygen to an H2O molecule by the oxidation of NAD(P)H. The second oxygen reduction requires two electrons from the reducing agent such as NAD(P)H (external monooxygenase) or from the substrate itself (internal monooxygenase) [27]: RH + O2 + 2e– + 2H+ → ROH + H2O P450 enzymes in eukaryotic cells such as plant P450s are membrane-anchored proteins, usually annealed on the surface of the endoplasmic reticulum (ER) via a short hydrophobic N-terminal segment. Despite the high number of P450 enzymes and low sequence identity (as low as 20%) across this superfamily, their topography and structural fold remain highly conserved. As heme-thiolate proteins, P450s’ most conserved structural features are related to their heme-binding active site. Most P450s share a mode of action of electron and proton transfer and oxygen activation (Figure 1.4) [28]. 6 Figure 1.4. Catalytic cycle of cytochrome P450 hydroxylation reaction. Adapted from [29] Two main mechanisms have been characterized depending on the nature of the substrate and on the enzyme itself (Figure 1.5). The first mechanism is the radical mechanism. The FeIV-oxo radical cation moiety abstracts a hydrogen radical from the substrate to form a carbon radical and FeIII–OH complex. Then the carbon radical combines with the hydroxyl radical from the ferric complex and produces the final alcohol product. The pentacoordinated ferric complex will coordinate with a water molecule and restarts the catalytic cycle again. This proposed mechanism goes through the radical intermediate. Although a radical intermediate is proposed in this Compound 1 7mechanism, no radical was isolated by radical trapping. Therefore, a second concerted mechanism is proposed, in which the formation of the carbon–oxygen and the cleavage of carbon–hydrogen bond occur simultaneously [30]. Figure 1.5. Radical and concerted mechanisms of oxygen atom insertion. Adapted from [30] According to the formation of compound 1 intermediate in the catalytic mechanism of P450 (Figure 1.4), beside the hydrogen abstraction from the C–H bond, P450 enzymes could also abstract hydrogen from its bond with heteroatoms, such as nitrogen, sulphur, and oxygen. The high reactivity of P450s leads to a wide range of reaction types such as hydroxylation, epoxidation, C–C bond cleavage, and sulfoxidation, resulting in a broad product spectrum [31,32]. 1.2. 3. Plant P450s and chemical diversity in plants P450s are responsible for many chemical oxidations in the living world; these proteins form the biggest superfamily of enzymes driving plant natural product diversity [33–35]. P450s are classified into families, each of which includes sequences that share at least 40% identity, and CYP71–CYP99 and CYP701–CYP999 gene families are found in plants [36]. More than hundreds of thousands of plant natural products are oxygenated compounds. Most of which were identified as the final product of the biochemical transformation of P450 enzymes [7,12,16,23,37]. Not only play a vital role in specialized metabolism, but 8P450 enzymes also manipulate the formation of primary metabolites, such as abscisic acid and gibberellins [38–40]. In recent years, explosive transcriptomic/genomic data have aided in the identification of hundreds of P450s implicated in plant specialized metabolism, demonstrating the crucial roles of these enzymes in phytochemical scaffolding and diversification [19,41–48]. To understand the chemical diversity of alkaloids, more studies on plant P450 enzymes are essential to help unveil previously unknown pathways and further generate new-to-nature derivatives with enormous therapeutic potentials. 1.2.4 Cytochrome P450 enzymes in MIA metabolism In MIA biosynthesis, cytochrome P450s play a crucial role in the scaffold formation and functional group installation [7]. Vinblastine biosynthesis is one of the most extensively studied MIA pathways. First isolated in 1957 from the leaf extract of Catharanthus roseus [49], vinblastine has played a vital role in cancer treatment, and the enzymes involved in its biosynthetic pathway have been elucidated [13,15,17,20,23,50]. Vindoline and catharanthine, monomeric precursors of vinblastine, share the same biosynthetic pathway from the central precursor strictosidine, which is bifurcated after precondylocarpine acetate [13,15,20] (Figure 1.6). Along these pathways, cytochrome P450 enzymes play critical roles in "deciding" the fate of the metabolites and pathway branching [17]. Geissochizine oxidase (GO) is an outstanding example. This P450 enzyme is responsible for converting geissochizine, via preakuammicine, to stemmadenine, the key intermediate in the biosynthesis of iboga and aspidosperma alkaloids, including catharanthine, tabersonine, and ibogaine [11,13,15,17,20]. The oxidation product of GO, preakuammicine, is also converted to akuammicine (strychnos alkaloid) via a non-enzymatic deformylation. Intriguingly, geissochizine is transformed to isositsirikine by geissochizine synthase (GS), a medium-chain alcohol dehydrogenase serving as a reductase [13]. The concerted activities of GO, GS, and other reductases highlight the crucial role of oxidative enzymes in MIA metabolic diversification [20] (Figure 1.6). 9Vindoline, a monomer constituting vinblastine, was derivatized from tabersonine. There were several oxidases involved in the biosynthesis of vindoline. The first enzyme in the array was tabersonine 16-hydroxylase, a P450. There were two different isoforms of tabersonine 16-hydroxylase, CYP71D12 and CYP71D351, that could introduce a hydroxyl group on tabersonine substrate at the C-16 position [51] [23]. Other P450s involved in this pathway include 16-methoxytabersonine 3-oxygenase enzyme that initiated the epoxidation at C2-C3 double bond [52] (Figure 1.6). 10Figure 1.6. Vinblastine biosynthetic pathway in C. roseus (SGD: β-D-glucosidase, GS: geissoschizine synthase, GO: geissoschizine oxidase, O-AT: O-acetyltransferase, PAS: precondylocarpine acetate synthase, DPAS: dihydroprecondylocarpine synthase, TS: tabersonine synthase, CS: catharanthine synthase, T16H2: tabersonine 16-hydroxylase 2, 16OMT: tabersonine 16-O-methyltransferase, 16T3O: 16-methoxytabersonine 3-oxidase, T3R: tabersonine 3- reductase, NMT: N-methyltransferase, DAT: deacetylvindoline 4-O-acetyltransferase). 11Rauwolfia serpentina (Apocynaceae), commonly called Indian snakeroot, is a traditional medicinal plant in South and Southeast Asia and has been used to reduce high blood pressure and anxiety. R. serpentina produces approximately 150 MIAs including reserpine, ajmaline, and rescinnamine. Among these, ajmaline (ajmalan alkaloid) is a powerful alkaloid classified as a class-Ia antiarrhythmic agent [12]. Ajmaline biosynthetic pathway resembles the starting compounds strictosidine and geissoschizine with catharanthine/vindoline biosynthesis [14] (Figure 1.6). A “gatekeeper” sarpagan bridge enzyme (SBE), which is a P450 enzyme, unveils the initial steps in the biosynthesis of sarpagan, ajmalan and alstophyllan alkaloids from strictosidine intermediate. Although the RsSBE’s activity was first detected in 1995[13], many years ago from the membrane-protein fraction of R. serpentina, the gene and its encoded enzyme have only been elucidated recently [14]. Figure 1.7. Vinorine metabolic pathway from geissochizine catalyzed by SBE. The SBE candidates were identified by co-expression analysis of R. serpentina transcriptomics and metabolomics data using self-organizing maps. In the heterologous protein expression in N. benthamiana and S. cerevisiae yeast cells, RsSBE and its homologue from Gelsemium sempervirens (another ajmaline-produced plant) GsSBE (61% amino acid sequence identity) could both convert geissoschizine to polyneuridine aldehyde (PNA), a sarpagan alkaloid, under the co-expression of these enzymes with strictosidine β-D-glucosidase, geissoschizine synthase, polyneuridine aldehyde esterase (PNAE), and NHNHOH OGlcHO2CNHNHOH OHMeO2CNHNHMeO2C OHNHNHMeO2C OHstrictosidine strictosidine aglycone 4,21-dehydrogeissochizine 19-E-geissochizineSGD GSNHNHCHOCOOCH3NNAcOSBENHNHCHOH PNAEVSpolyneuridine aldehyde16-epivellosiminevinorineiboga, schrychnos, aspidosperma alkaloids 12vinorine synthase (VS), and co-filtration of strictosidine substrate (Scheme 2). Intriguingly, the gene CYP71AY1 (CrSBE/AS) from C. roseus, a homologue of RsSBE and GsSBE (78% amino acid sequence identity with RsSBE) could also transform geissoschizine to polyneuridine aldehyde. However, this conversion by CrSBE is poor, and there were no sarpagan or ajmalan alkaloids detected from C. roseus. On the other hand, CrSBE enzyme was able to transform tetrahydroalstonine and ajmalicine (heteroyohimbine-type alkaloids) to alstonine and serpentine (-carboline alkaloids), respectively, via an aromatization process (Figure 1.8). The CrSBE was thus nominated as alstonine synthase (AS) [14]. There are two plausible mechanisms for polyneuridine aldehyde formation catalyzed by RsSBE and GsSBE: the hydride abstraction step at methylene group of C5, or sequential oxidation to form iminium intermediate followed by the cyclization or over-oxidation. The cyclization facilitated by the enol group on geissoschizine acts as a nucleophile reacting with the electrophile (iminium cation) to form the C5–C16 single bond. In the absence of intramolecular nucleophiles, the cyclization cannot happen. The dihydropyridinium intermediate would be spontaneously oxidized to produce the aromatic ring (-carboline) (Figure 1.8) [14]. Figure 1.8. Catalytic mechanism of (A) RsSBE with 19-E-geissochizine substrate and (B) CrSBE with tetrahydroalstonine substrate [14]. NHNHMeO2C OH19-E-geissochizine516NHNHMeO2C OH516NHNHCHOCOOCH3polyneuridine aldehyde5 16RsSBENHNHMeO2COtetrahydroalstonine516NHNHMeO2CO516“CrSBE”H H NHNMeO2COalstonine516Hspont.A B 13Another notable P450-catalyzed step in MIA biosynthetic pathway is the formation of vomilenine from vinorine, a downstream product of polyneuridine aldehyde [12]. Intriguingly, vomilenine was not the only product formed from the enzymatic assay of vinorine with vinorine hydroxylase (a cytochrome P450 enzyme). Further data analysis showed that vinorine hydroxylase not only hydroxylated vinorine to vomilenine but also catalyzed the subsequent isomerization of vomilenine to perakine. The hydroxyl group at C-21 position triggered the Michael addition via the hemiacetal ring opening. This is one of the significant pieces of evidence that depicted the crucial role of P450 enzyme in the chemical diversity in plants. Figure 1.9. (A) Vinorine reaction with vinorine hydroxylase (VH) and (B) perakine formation mechanism from vomilenine. 1.3 Synthetic biology of natural products in microbes Biosynthesis of plant natural products in microbial systems is a promising alternative method to improve supplies of highly bioactive compounds. Most bioactive natural products have complex structures with multiple stereocenters, functional groups, and side chains surrounding the core scaffold, which complicates or even precludes the total synthesis of these compounds at high titer and reproducibility. Synthesizing plant-derived potent drugs by means of microbes could generate greener and more reliable sources for natural products, as microorganisms could grow faster than plants. For instance, yeast and E. coli only take hours to grow; meanwhile, plants need months and years. Furthermore, microbes could be NNAcONNAcOvinorineOHVH VHNNAcOOvomilenine perakineA B NNAcOOHHNNHAcOOH+NNAcOHOHNNAcOOvomilenine perakineketo - enol tautomerization 14engineered to produce less complex chemical matrix than plants extracts, and could be cultivated with scalable and standardized fermentation. S. cerevisiae emerged as a remarkable platform for reconstituting the complicated biosynthetic pathway of natural products. The outstanding work of artemisinin biosynthesis by means of yeast chassis have stimulated the studies of microbial synthesis to produce plant-derived natural products [53–57]. The advances in biological synthesis of strictosidine at 0.5 mg/L [53] in S. cerevisiae yeast have highlighted a different approach to producing high-value MIA drugs. These achievements not only emphasized the strength of synthetic biology in drug synthesis, but also revealed the need for gene discovery to fully understand the biosynthesis of bioactive specialized metabolites. 1.4 Camptothecin and its analogues The MIA camptothecin (CPT; IUPAC name: (S)-4-ethyl-4-hydroxy-1H-pyrano[3’,4’:6,7] indolizino[1,2-b]quinolone-3,14-(4H,12H)-dione) was first extracted from the stems of Camptotheca acuminata Decne (Nyssaceae) in 1966 [58]. C. acuminata is widely grown and known as happy tree (“xǐ shù”/“喜树”) in many regions of China. This species is also found in India and North America. With a diploid genome of 44 chromosomes, C. acuminata is characterized as deciduous trees up to 20 m high with pale-gray and deeply fissured bark, lucid and oblong-ovate leaf blade, light green petals, and gray-brown fruits [59]. Although it is the best known, C. acuminata is not the only species that can produce CPT. By 1996, members of six plant families have been reported to be able to produce CPT, including Icacinaceae, Betulaceae, Meliaceae, Apocynaceae, Rubiaceae, Nyssaceae, and Loganiaceae [59][60]. CPT’s structure has an in-plane heteropentacyclic ring system. Rings A, B, C is a pyrrolo [3,4-beta]-quinoline moiety fused with a pyridone ring (ring D) and a six-membered α-hydroxyl lactone ring (ring E). Ring E has one asymmetric carbon at position 20 with (S) configuration. 15 Figure 1.10. Camptothecin and camptothecin analogues (red arrows depict the positions of substituents increasing the bioactivity of CPT derivatives against topoisomerase I). Camptothecin (CPT) 10-Hydroxycamptothecin (10-HCPT) Topotecan Irinotecan (S) 7 91011NNOOOHOHO11-Hydroxycamptothecin (11-HCPT) 7-Ethyl-10-hydroxycamptothecin (SN-38) 16CPT and its derivatives are potent inhibitors of DNA topoisomerase-I activity and are widely used for the treatment of lung, cervix, ovarian and colon cancers, and other diseases such as acquired immune deficiency syndrome (AIDS) and falciparum malaria. Although CPT was identified in 1966, its usage was hampered due to its low water solubility and unpredictable toxicities such as diarrhea and myelosuppression. Topoisomerase-I (topI) is an enzyme involved in DNA multiplication, cutting one DNA strand to forms a nicked DNA-topI complex required for the DNA replication process. CPT and its analogues, topotecan and irinotecan, could bind to this DNA-enzyme complex and inhibit the growth of tumours. 3D QSAR (quantitative structure-activity relationship) study of CPT analogues revealed that more negatively-charged bulky group at positions 9, 10, and 11 would increase the inhibitory activity of CPT analogues against topI. Substituents carrying a large positively-charged group at position C-7 would enhance the inhibitory activity, but excessively increasing the large substituents at this position would reduce the activity [61]. The planar property in the structure of CPT is hypothesized as an important factor in the inhibition of this compound class against topI enzyme. The lactone ring in CPT is non-stable under basic environment and more favoured under acidic conditions. The opened-ring form of CPT cannot inhibit topI. CPT has served as a lead compound and precursor for the chemical derivatization of more clinically useful anticancer drugs, such as irinotecan (Camptosar®) and topotecan (Hycamtin®). These two derivatives have been approved by the US Food and Drug Administration and listed in the World Health Organization’s Model List of Essential Medicine [62]. Topotecan (9-[(dimethylamino)methyl]-10-hydroxycamptothecin) and irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) are currently approved in many countries for the treatment of metastatic ovarian cancer, cervical, uterine, and brain cancers among others [63]. Sales of irinotecan and topotecan have totalled over fifteen million US dollars as of 2018 [64], and the pharmaceutical industry has been relying on camptothecin and 10-hydroxycamptothecin (10-HCPT), natural products from C. acuminata, for the semi-synthesis of these high-value compounds. While CPT is relatively accessible, C. acuminata produces 10-HCPT at a very low abundance (<0.00005 % dry weight) [63] in bark and seeds [65]. Moreover, the CPT derivatives such as 1711-hydroxycamptothecin (11-HCPT), which exhibits a much greater therapeutic index than CPT, occur in even lower quantities [66], limiting its clinical use. Isolation of CPT from plants, followed by chemical derivatization in harsh conditions, remains the major source for the semi-synthesis of irinotecan, topotecan, and other chemotherapeutic agents [67–71]. Demands of using CPT increase every year worldwide. Despite the efforts for CPT total synthesis studies from commercially available chemicals, plants remain the main supply of CPT for the pharmaceutical industry. The price of 1 ton of crude extract of CPT has risen up to 3 billion US dollars per year, which not only escalates the cost of CPT-derived antitumour drugs production but also destroys a large number of natural resources [72]. Conventional production strategies present challenges associated with purity and scale-up of the compounds, contributing to high costs and inaccessibility. For instance, topotecan semi-synthesis from CPT involves partial hydrogenation and oxidation with the catalysts Pd and Pb(OAc)4 to yield 10-HCPT [73], followed by condensation with formaldehyde and dimethylamine [74–77]. Although this method can deliver topotecan at economically feasible yields, it involves labour-intensive processes and the use of toxic Pb(OAc)4 and H2O2 on industrial scales [73]. 18 Figure 1.11. Proposed biosynthetic pathway of camptothecin (numbers indicate the plausible order of enzymatic reactions in the biosynthetic pathway of camptothecin). OHOHOHHOHOHN HNHOHO2CO-GlcOOHHHOHOHHO2CHOHOGlcHO2CHOHOGlcHO2CHOOHO2CHOGlcHO7-DLHG8OIO7-DLGTgeraniol10-hydroxygeraniol9,10-dioxogeranialiridodialstrictosidinic acidsecologanic acid1234568G8HN HtryptamineISN HNOONON HOOstrictosamide3-(S)-pumilosideHHHHOGlcOGlc791011121314strictosamide epoxidestrictosamide diolstrictosamide ketolactamoxidaseaddition of wateroxidasereductasedehydrationdehydrationoxidasecyclase/Pictet-Spenglerase+HN HNOOHHOGlcON HNOOHHOGlcHOOHN HNOOHOGlcOOHNONOHHNONO3-(S)-deoxypumilosideOGlcOGlcNONOOHNONOOcamptothecin1516allylic isomerizationglucosidaseoxidoreductase hydroxylase/oxidase/P450NONOOOH18NONOOGlcreduction19N HNH2NH2COOHTDCNONOOGlcoxidase1720loganic acid7-deoxyloganetic acid7-deoxyloganic acid 19 The poorly understood chemical steps observed in this system make it exceptionally challenging to identify the biosynthetic enzymes involved. Therefore, the elucidation of any single step in the biosynthesis of quinoline alkaloids in C. acuminata will be critical towards understanding and engineering of this biosynthetic pathway. It will also have significant implications in finding a more economical way of producing these anticancer drugs. 1.5 Camptothecin biosynthesis The biosynthetic pathway of CPT in C. acuminata is proposed in Figure 1.11 based on metabolomics and isotopic labelling studies [78]. CPT biosynthesis starts from the seco-iridoid pathway, resembling the upper stream of vinblastine biosynthesis. Nevertheless, based on the chemical composition studies of C. acuminata [60,78,79], instead of strictosidine, another derivative, strictosidinic acid, is proposed to be the actual biosynthetic precursor of CPT. Strictosidinic acid is the condensed product of tryptamine and secologanic acid via a Pictet-Spengler-type reaction, while strictosidine is generated from tryptamine and secologanin (Figure 1.11). Interestingly, two C. acuminata P450s, CaCYP72A565 and CaCYP72A610, catalyze the intriguing dual reactions of hydroxylating 7-deoxyloganetic acid to loganic acid, the cleaving C7 –C8 bond to yield secologanic acid, which entered the downstream pathway leading to CPT [80] (Figure 1.12). These steps in the biosynthetic pathway of CPT in C. acuminata are similar to those in the seco-iridoid pathway of vinblastine in C. roseus, in which secologanin, a methylated form of loganic acid, is produced by the C7–C8 bond breakage of loganin and catalyzed by secologanin synthase (SLS) enzyme, a P450. Up to date, no genes involved in the downstream pathway of CPT biosynthesis have been elucidated. The related compounds involved in camptothecin biosynthesis were determined via the deuterium labelling experiments on tryptamine (Table 1.1) [78]. Figure 1.12. Enzymatic reactions of CaCYP72A565/610 and 7-deoxyloganic acid 20Early studies on the accumulation of CPT and its derivatives showed that while CPT is accumulated in most tissues, its oxidized and methylated derivatives (HCPT and 10-methoxycamptothecin) collectively account for up to 50% of the total MIAs in young bark, and up to 30% in mature leaves [65]. Although CPT accumulation is seen mostly in young leaves and decreases in mature tissues, HCPT does not accumulate according to plant height or age, but it is always highest in the bark of any given section of the plants [81]. Table 1.1. Metabolites from C. acuminata relating to CPT biosynthesis No. Name Formula Molecular weight (g/mol) 1 Loganic acid C16H24O10 376 2 Tryptamine C10H12N2 160 3 Secologanic acid C16H22O10 374 4 Strictosidinic acid C26H32N2O9 516 5 Strictosamide C26H30N2O8 498 6 Strictosamide epoxide C26H30N2O9 514 7 Strictosamide diol C26H32N2O10 532 8 Strictosamide ketolactam C26H30N2O10 530 9 Pumiloside C26H28N2O9 512 10 Deoxypumiloside C26H28N2O8 496 11 Camptothecin C20H16N2O4 348 12 10- or 11-Hydroxycamptothecin C20H16N2O5 364 13 9- or 10-Methoxycamptothecin C21H18N2O5 378 211.6 Research objectives The primary objective of this research was to discover enzymes from C. acuminata involved in the hydroxylations of CPT and to utilize these hydroxylases as biocatalysts in chemo-enzymatic reactions to synthesize anticancer drugs topotecan, irinotecan, and SN-38, and other CPT analogues (Figure 1.13). Specific objectives are: 1. To identify P450 candidate genes involved in CPT hydroxylation reactions; 2. To biochemically characterize the putative camptothecin hydroxylase enzymes; 3. To optimize the conditions in yeast whole-cell in vivo assay for hydroxycamptothecin bioproduction and elucidate the structure of enzymatic assays’ product by NMR analysis; and 4. To combine biocatalysts (hydroxylases) with chemo-catalysts to synthesize a suite of anticancer compounds and other potential drugs having the CPT scaffold. Experimental data for objectives 1, 2, and 3 are described in Chapter 2, and data for objective 4 are described in Chapter 3. At the beginning of this project, no biosynthetic genes involved in the later stage of CPT biosynthesis from C. acuminata had been reported. 22 Figure 1.13. Summary of research objectives. Created with BioRender.com. 23Chapter 2: Discovery and characterization of cytochrome P450 genes involved in camptothecin hydroxylation in Camptotheca acuminata 2.1 Summary P450 candidate genes identified from the genome and transcriptome of C. acuminata were cloned into the pESC-Leu2d expression vector, harbouring cytochrome P450 reductase (CPR). The plasmids were in turn transformed into YPL 150 C:PEP4KO S. cerevisiae yeast strain to characterize their catalytic activities with an array of alkaloid substrates. A high-throughput bioactivity screening assay was developed for simultaneous identification of the P450s’ activity. This study resulted in the discovery of two new P450s involved in the regio-specific hydroxylations of camptothecin, camptothecin 10-hydroxylase (CPT10H) – Ca32236 and camptothecin 11-hydroxylase (CPT11H) – Ca32229. These enzymes revealed selective catalytic function toward compounds bearing camptothecin scaffolds. High quantities of hydroxycamptothecin products were produced via optimized in vivo procedure, which could be further used to synthesize potent high-value derivatives. 2.2 Introduction 2.2.1 Biosynthetic candidate genes discovery from genomic and transcriptomic datasets Plant genomes and transcriptomes are highly complex, containing tens of thousands of protein-encoding sequences and thousands of genes expressed at any moment. A transcriptomic study of C. roseus, for instance, revealed roughly 33,000 genes [82]. A manageable set of gene candidates is thus necessary for the effective elucidation of each biosynthetic step or pathway. The transcriptomes/genomes of many MIA-producing plants have been sequenced, including C. roseus, R. serpentina, C. acuminata, Nothaphodytes nimmoniana, Ophiorrhiza pumila, and Tabernanthe iboga [62,82–86]. These data have become the backbone of bioinformatic analysis for candidate gene identification. The candidate genes discovery involves a co-analysis of genomic/transcriptomic expression and metabolomic production/accumulation patterns. Among the co-expression analysis methods, self-organizing map (SOM) emerged as a form of unsupervised machine learning that groups data points 24together based on how similar their expression patterns are. The greatest advantage is that multiple different nodes, depicting similar expression patterns of candidate genes, can be selected from the same map. Successful examples include the identification of a sarpagan bridge enzyme and vinorine hydroxylase from R. serpentina, and an alkaloid transporter in C. roseus [12,14,87]. Homology search is employed when the same or similar compound is produced in multiple different species of plants. A homology-based screening can be performed using a simple BLAST search of the known gene against the genome or transcriptome of the plant of interest to identify candidate genes. Examples for successful application of this method in MIA biosynthesis include the identification of ibogamine 10-hydroxylase from the Tabernanthe iboga, which showed homology to tabersonine 16-hydroxylase from C. Roseus [16]. 2.2.2 Biochemical characterization of candidate genes in yeast 25Figure 2.1. Brief workflow for the MIA biosynthetic candidate genes identification and characterization (Nguyen et al., 2021). Created with BioRender.com. 26Identification of new candidate genes could be achieved using one of those methods mentioned above. From a list of candidates, gene(s) of interest could be biochemically characterized in vitro or in vivo using different heterologous hosts (including but not limited to bacteria, yeast, and tobacco) depending on the type of enzymes, substrate availability, and other factors (Figure 2.1). For cytochrome P450 enzymes, eukaryotic hosts such as S. cerevisiae yeast and N. benthamiana tobacco are usually employed as they display less codon bias towards plant genes compared to prokaryotic hosts, as well as provide subcellular supports for membrane-bound enzymes such as cytochrome P450-based oxidases. Yeast is often a preferred first choice for its well-understood genetics and physiology as well as relatively simple growth conditions [22]. Enzymes functionally expressed in such hosts could be used to reconstitute biosynthetic pathways or combined with chemical synthesis in a chemoenzymatic pipeline. This study focuses on the discovery and characterization of recombinant plant cytochrome P450 enzymes in yeast. The general workflow for biochemical characterization of cytochrome P450 genes starts from the acquisition of a candidate genes list. Then, molecular cloning techniques are applied to express the recombinant protein in a heterologous microbe, such as yeast. The recombinant protein is then used in enzymatic in vivo and in vitro assays for activity validation against specialized metabolites. The enzymatic reaction progress is analyzed by analytical methods, such as LC-MS. The stereochemistry of the final enzymatic products could be further elucidated by the nuclear magnetic resonance (NMR) techniques (Figure 2.2). 27 Figure 2.2. Workflow of the biochemical characterization for cytochrome P540 candidate genes. Created with BioRender.com. In this part of the research, multidisciplinary approaches that includes the use of self-organizing map for candidate identification, high throughput in vivo yeast assays for enzyme activity discovery and analytical chemistry were used to identify and characterize new monooxygenases involved in the metabolism of CPT in C. acuminata. 2.3 Material and methods 2.3.1 Plant material Camptotheca acuminata cuttings were obtained from Mr. Howard Higson at Quarryhill Botanical Garden (CA, USA) and Mr. Sean C. Lahmeyer at the Huntington Library, Art Collections, and Botanical Gardens (CA, USA). The cuttings were frozen upon receipt for RNA extraction and purification. 282.3.2 Chemicals Camptothecin was purchased from Sigma-Aldrich and Alfa Aesar. The authentic standard of 10-hydroxycamptothecin was purchased from Sigma-Aldrich and MCE Inc. 11-Hydroxycamptothecin was purified from the in vivo yeast whole-cell assay of Ca32229 gene with camptothecin substrate. 7-Ethylcamptothecin, 7-ethyl-10-hydroxycamptothecin, NADPH were purchased from Sigma-Aldrich. Secologanin, tetrahydroalstonine, yohimbine, ajmalicine, corynanthine, strictosidine, and strictosamide were purchased from Northernchem Inc. Yeast media–Drop-out mix synthetic minus leucine (Bioshop). Agarose, tryptone, peptone, yeast extract, were purchased from Fisherbrand 2.3.3 Phylogenetic analysis Unrooted neighbor-joining phylogenetic tree for P450 candidates from this study and other reported P450s from other organisms were conducted using the Geneious Tree Builder program in the Geneious software package (Biomatters). Abbreviations and accession numbers: C. acuminata camptothecin 10 hydroxylase, CaCPT10H, MN631049; C. acuminata camptothecin 11 hydroxylase, CaCPT11H Arabidopsis thaliana CYP81D1, AtCTP81D1, NP_568533.2; A. thaliana CYP81F1, AtCTP81F1, O65790.2; A. thaliana CYP81H1, AtCTP81H1, NC_003075.7; A. thaliana CYP81K1, AtCTP81K1, NC_003076.8; C. roseus CYP71AY1, KF309243.1; C. roseus tabersonine 16-hydroxylase, CrCYP71D12, FJ647194.1; C. roseus geraniol 10-hydroxylase, CrG10H, Q8VWZ7.1; C. roseus CYP72A224 7-deoxyloganic acid 7-hydroxylase, Cr7DLH, AGX93062.1; C. roseus CrCYP71BT1, AHK60840.1; C. roseus secologanin synthase, CrSLS, Q05047; C. roseus tabersonine 19-hydroxylase CrCYP71BJ1 (T19H), ADZ48681; C. roseus CrCYP71D1V1, JN613015.1; C. roseus tabersonine 16-hydroxylase, ACM92061; C. roseus tabersonine 6,7-epoxidase, CrCYP71D521, AVH80640; Camellia sinensis cytochrome P450 81D11, CsCYP81D11, XP_028101205.1; Echinochloa phyllopogon cytochrome P450 CYP81A12.R, EpCYP81A12, BAO73908.1; Hypericum calycinum, HcCYP81AA1, ANC33509.1; Rauwolfia serpentine sarpagan bridge enzyme, RsSBE, P0DO13.1; Sesamum alatum cytochrome P450 CYP81Q3, SaCYP81Q3; BAE48236.1; Papaver somniferum cytochrome P450 CYP82X1, PsCYP82X1; AFB74614.1; P. somniferum cytochrome P450 CYP82Y1, PsCYP82Y1, AFB74617.1; P. somniferum 29cytochrome P450 CYP82X2, PsCYP82X2, AFB74617.1; Sesamum indicum cytochrome P450 81E8-like, SiCYP81E8, NP_001306620.1; Salvia miltiorrhiza cytochrome P450 CYP82V2, SmCYP82V2, KP337709.1L; Sesamum radiatum cytochrome P450 CYP81Q2, SrCYP81Q2, AB194715.1; Theobroma cacao cytochrome P450 71D9, TcCYP71D9, XM_018120397.1; Tabernanthe iboga ibogamine 10-hydroxylase (I10H), TiCYP76 (I10H), MH454074.1. 2.3.4 Candidate identification Publicly available transcriptomic and metabolomic data of seven different organs of C. acuminata (http://medicinalplantgenomics.msu.edu/contacts.shtml) were analyzed for contigs with FPKM (fragments per kilobase of exon per million fragments mapped) expression values higher than zero (FPKM expression values of zero for more than half of the treatments or with zero expression variance throughout the samples were eliminated). Self-organizing maps were used and visualized in R (RStudio 1.4.1717, RStudio, Inc) with the Kohonen package as reported before [12]. The map was created to give about 50 contigs per node. P450 candidates that are in the same nodes or neighbouring nodes that have similar expression patterns with previously reported genes were selected for cloning and testing for activity. More than 10 P450 candidates belonging to different P450 families, including CYP71, CYP72, CYP76, CYP81 and CYP82, were identified. [62]. 2.3.5 P450 genes cloning The cDNA templates were synthesized from the reverse-transcription of the mRNA extracted from young leaves of C. acuminata by using LunaScript RT SuperMix (New England Biolab). Full-length open-reading frame regions of Ca23831, Ca23838, Ca000754, Ca12175, CaCYP12176, Ca23839, Ca17137, Ca6009, Ca6007, Ca6006, Ca32229, Ca19403, Ca30731, Ca24044, Ca12282, Ca27984, Ca23088, Ca4097, Ca4921, Ca32236, Ca32709, Ca17987, Ca12663, Ca15564, Ca15529, Ca32245 candidate genes were amplified from the cDNA templates with the forward primer GGGCGGCCGCACTAGAACAAAATG-5’-gene-sequence and the reverse primer ATCCATCGATACTAG-reverse-complement-3’-gene-sequence without-stop-codon using the following reaction mixture: 30Reagents Volume (μL) Nuclease–free water 8 GC enhancer 10 Forward primer 2.5 Reverse primer 2.5 cDNA 2 2X Platinum™ SuperFi™ II PCR Master Mix (ThermoFisher Scientific) 25 The polymerase chain reaction (PCR) was performed in a MiniAmp Plus thermal cycler (ThermoFisher Scientific) using the following thermal cycling program: Cycle step Temperature (oC) Time Cycles Initial denaturation 98 30 seconds 1 Denaturation Annealing Extension 98 60 72 10 seconds 10 seconds 30 seconds per 1 kb 35 Final extension 72 10 5 minutes Hold 1 – The amplicons were purified using gel extraction Nucleospin kit (Macherey-Nagel). The high-copy expression plasmid pESC-Leu2d harbouring cytochrome P450 reductase (CPR) was used for the initial high-throughput bioactivity of P450s screening assay[88]. The pESC-Leu2d::CPR plasmid was digested by two restriction enzymes SpeI-HF and NotI-HF (New England Biolab) for 2 hours prior to inserting the P450 genes into the multiple cloning site 1 of the plasmid under the GAL10 promoter (Figure 2.3). The P450 genes were inserted into pESC-Leu2d::CPR plasmid using 5X In-Fusion cloning system (Takara Bio USA Inc.). 31The resulted plasmids were then transformed into Stellar E. coli competent cells (Takara Bio USA Inc.) and grown on the Luria-Bertani (LB) with carbenicillin agar plate at 37 oC overnight for plasmids’ copies amplification. The positive colonies were identified by colony PCR with 2X Frogga Taq polymerase (FroggaBio) and GAL10-forward (GGTGGTAATGCCATGTAATATG) and -reverse (GGCAAGGTAGACAAGCCGACAAC) primers. The positive clones were inoculated in 5 mL LB with carbenicillin medium at 37 oC overnight for plasmid extraction using plasmid mini-prep kit (Bioneer, South Korea). The cloned sequences were confirmed by Sanger sequencing (EurofinGenomics). A plasmid quantity of 0.1–0.5 g was transformed into the YPL 150 C:PEP4KO (PEP4KO) yeast strain for protein expression. The yeast cells were dispersed in the 1.5-mL centrifuge tubes with 100 L frozen competent cell (FCC) solution (5 % v/v glycerol, 10 % v/v DMSO) for one transformation and stored at –80 oC in advance [89]. The yeast cells were aggregated by centrifugation to remove the FCC solution and dispersed in the master mix solution consisting of 120 L 50 % polyethylene glycol (PEG), 18 L 1.0 M lithium acetate (LiOAc), 25 L boiled carrier DNA prior to adding the plasmid. The yeast cells were spread onto SC-Leu with 2 % glucose agar plate and incubated at 30 oC for 4–5 days. The colonies carrying plasmids were identified by performing colony PCR with 2X Frogga Taq polymerase (FroggaBio) and GAL10 forward and reverse primers. 32 Figure 2.3. pESC-Leu plasmid (Agilent) Five genes that share very high sequence identity, namely Ca32229, Ca32234, Ca32235, Ca32236, Ca32245 were synthesized by TwistBioscience. These sequences were then amplified by PCR using 2X Platinum™ SuperFi™ II PCR Master Mix (Invitrogen), and the pESC forward (ACCCTCACTAAAGGGCGGCC) and pESC reverse (CTTGTAATCCATCGATACTAG) primers to clone into the SpeI and NotI restriction sites of multi-cloning site 1 in pESC-Leu2d::CPR plasmid (Figure 2.3). The resulted constructs were transformed into pESC-Leu2d::CPR plasmid and PEP4KO yeast strain for recombinant protein expression. 2.3.6 High-throughput P450 candidate genes screening assay for activities against CPT Yeast cells containing pESC-Leu2d::CPR_CYPs plasmid were inoculated in a 96-well plate with 100 L SC-Leu and 2 % glucose medium per well at 30 oC, 750 rpm for 18–20 hours. For protein expression induction, 10 L of the inoculation was transferred to 90 L SC-Leu with 0.2 % glucose, 1.8 % galactose medium. Simultaneously, 1 L of 1 mM alkaloid substrate was fed into each culture well and incubated at 30 oC, 750 rpm. After 48 hours, the assay was stopped by adding 100 L of methanol and transferred into 331.5-mL microcentrifuge tubes and centrifuged at 16,000 g for 30 minutes. The supernatant was filtered through the 0.2 m syringe filter and analyzed by UPLC-MS/MS. 2.3.7 Substrate specificity assay Twenty-two monoterpene indole alkaloids, including those from C. acuminata such as strictosamide, CPT, 10-HCPT, etc., and other compounds with distinct ring configurations (bicyclic to pentacyclic) were screened with various P450 candidates using the high-throughput assay as described in the previous section 2.3.6. 2.3.8 UPLC-MS/MS analysis The yeast assay extracts were analyzed by a Waters ACQUITY UPLC I-Class (SM-FTN) ultra-performance liquid chromatography (UPLC) system coupled with a Xevo TQ-S Cronos Triple Quadrupole Mass Spectrometer. Chromatography was performed on an XBridge BEH XP (10 x 2.1 mm, 1.7 m) column at a flow rate of 0.6 mL.min-1. The column was pre-equilibrated in 90 % solvent A (water + 0.1% formic acid), and 10 % solvent B (acetonitrile + 0.01% formic acid). The following elution conditions were used: 0–8 min, 10–50% B; 8.0–8.5 min, 50–100% B; 8.5–9.5 min, 100% B; and 9.5–11 min, 100–10% B to re-equilibrate the column. The 10 M authentic standards were used to compare the mass to charge (m/z) ratios and retention times with the substrates, products. The commercially available authentic standards camptothecin and 10-hydroxycamptothecin were analyzed in the multiple reaction monitoring (MRM) experiments to selectively identify the product via the daughter ions in the enzymatic assay matrix. The adducts [M+H]+ with m/z 365.2 and 349.2 were used in the selected ion recording (SIR) profile. The collision-induced dissociation (CID) experiments of 100 M 10-HCPT and CPT were conducted, and the fragmented moieties’ mass values were filled into the MRM profile to selectively detect the compounds (Table 2.1). The conversion of the hydroxylation reaction was calculated based on the peak area of both substrate and product by using TargetLynx with corresponding compounds’ retention time. 34Table 2.1. Selected CID data of camptothecin and its analogues Compound name Parent (m/z) Daughter (m/z) Dwell(s) Cone (V) Collision (V) Ionization mode Camptothecin 349.1596 305.1500 0.001 44 22 ESI(+) 10-Hydroxycamptothecin 365.1596 321.1500 0.001 36 22 ESI(+) 7-Ethylcamptothecin 377.1128 195.0661 0.025 92 50 ESI(+) 7-Ethyl-10-hydroxycamptothecin 393.1128 212.0293 0.025 86 32 ESI(+) 2.3.9 In vivo assay optimizations Culture media As Ca32229 was discovered in the early of this study, all the optimizations of in vivo assay condition were conducted on the pESC-Leu2d::CPR_Ca32229 transformed yeast against CPT. The PEP4KO yeast cells containing pESC-Leu::CPR/Ca32229 plasmids were inoculated in 1 mL SC-Leu + 2% glucose medium at 30 oC, 200 rpm for 18–20 hours. The yeast culture was subsequently induced for protein expression by diluting 100 L of the inoculation into 900 L of one of the following three media: SC-Leu + 0.2 % glucose + 1.8 % galactose medium; YPA (supplemented with adenine sulfate 0.04 mg/mL) + 2 % galactose; and YPA (adenine sulfate 0.04 mg/mL) + 2 % galactose + 10 % glycerol at 30 oC, 200 rpm. The three cultures were fed with 50 L of 1 mM camptothecin and incubated at 30 oC, 200 rpm for 48 hours. Finally, the supernatants were collected and analyzed by UPLC-MS/MS to generate the conversion rate. Camptothecin and glycerol concentrations To optimize the assay conditions, five concentrations of glycerol (0, 5, 10, 15, 20 %) and seven concentrations of the substrate CPT (1, 5, 10, 20, 50, 75, and 100 μM) were tested for a total of 35 combinations. 35Reaction time The pESC-Leu2d::CPR_Ca32229 transformed PEP4KO yeast cells were incubated with 50 µM CPT substrate in the 1-mL scale assay. The YPA with 10% glycerol media was used to obtain high conversion rate. The reaction time was altered from 1 day to 4 days. After each day, the reaction supernatants from the cultures were collected by centrifugation, filtered by 0.2 m filter, and extracted by methanol for UPLC-MS/MS analysis. In vivo assay with different yeast strains The plasmid pESC-Leu::CPR/Ca32229 was transformed into three yeast cell lines beside PEP4KO strain. The first one was Δerg6 Δtop1 double mutant strain SMY75-1.4A [90]. The remaining yeast cell lines were YPH499 and wild-type JHY6-1C strains. The frozen yeast cells transformations were performed as demonstrated in section 2.2.3. The in vivo assays of pESC-Leu::CPR/Ca32229 in 4 distinct yeast strains with camptothecin as substrate were conducted to evaluate the performance of 4 strains of the same gene on camptothecin hydroxylation. The positive yeast colony was picked from the SC-Leu + 2 % glucose agar plate and inoculated in 1 mL SC-Leu + 2 % glucose medium at 30 oC, 200 rpm for 18–20 hours. The yeast culture was subsequently induced for protein expression by diluting 100 L of the culture into 900 L YPA (supplemented with adenine sulfate 0.04 mg/mL) with 2 % galactose and 10 % glycerol media. CPT substrate was added into the culture to a final concentration of 50 µM and incubate the culture at 30 oC, 200 rpm for 48 hours. The experiments were performed in the sterile test tubes. After 48 hours, the reaction mixture supernatants from the cultures were collected by centrifugation, filtered by 0.2 m filter, and extracted by methanol for UPLC-MS/MS analysis. 2.3.10 P450 microsomes preparation The yeast cells harbouring expression construct were inoculated in 5 mL sterile SC-Leu + 2 % glucose medium at 30 oC for 18–20 hours, before transferred to 250 mL with the same medium and incubated at 30 oC for another 18–20 hours. Yeast cells were harvested by centrifugation at 4,000 g for 5 minutes, resuspended in 250 mL YPA medium supplemented with 2% galactose medium and grown at 30 36oC for 24 hours. Microsome was prepared according to previously described protocol [12]. Specifically, yeast cells were collected by centrifugation at 6,000 g for 15 minutes at 4oC, and washed by 15 mL TE (50 mM Tris, 1 mM EDTA, pH 7.4) supplemented with 0.1 M KCl. The washed yeast cells were resuspended in 15 mL ice-cold TE buffer supplemented with 0.6 M sorbitol, and aliquoted into the 3-mL NALGENE tubes with pre-chilled 0.5-mm glass beads. The tubes were subjected to a VWR bead mill homogenizer at level 3 for 45 seconds to disrupt the yeast cell wall. The lysate was collected and centrifuged at 10,000 g for 15 minutes at 4 oC. The supernatant was further centrifuged at 40,000 g for 1 hour, and insoluble pellet containing microsomal fraction was collected. The pellet was resuspended in 750 L ice-cold TE buffer containing 20% glycerol. 2.3.11 In vitro microsomes assay For a 100 L in vitro assay, 5–10 mg microsomes was used in 100 mM HEPES pH 7.5. NADPH cofactor was added to final concentration 250 M. One L of 1 mM camptothecin substrate was added into the mixture. The reaction mixture was incubated at 37 oC. After 30 minutes, the reaction was stopped by adding methanol, and analyzed with UPLC-MS/MS. 2.3.12 Western blot (SureCast HandCast system – Invitrogen) Five mg of the microsomal protein was mixed in the 2X loading buffer (Tris-glycine) and 5% -mercaptoethanol to a final volume of 60 L. The mixture was boiled at 100 oC for 5 minutes, and loaded into an 10 % SDS-PAGE gel. The gel was run at 150 V for 75 minutes. The gel was then washed with water and soaked in transfer buffer (25 mM Tris, 92 mM glycine, 20 % methanol) for 10 seconds. The transfer step with a PVDF membrane was proceeded in the 4 oC fridge at 25 V for 1 hour 40 minutes. After that, the membrane was gently removed and washed with cold 1X TSB-T (25 mM Tris-HCl, 150 mM NaCl, 0.05 % Tween-20, pH 7.5) 3 times. After the wash, the membrane was blocked in 10 mL TSB-T complemented with 5 % skim milk (m/w) for 1–2 hours at room temperature. Then, the membrane was washed by TSB-T buffer 3 times and immersed in 10 mL 3 % skim milk in 1X TSB-T buffer. Two to 5 L of -FLAG M2 (1 mg/mL) antibodies (MilliporeSigma) were added into the mixture, and the 37membrane was incubated at 4 oC overnight. The membrane was then washed with 2X TSB-T 3 times, 10 minutes per wash. Finally, 750 L peroxide solution and 750 L enhancer solution (SuperSignal™ West Atto Ultimate Sensitivity Substrate) were pipetted onto the surface of the membrane. The membrane was soaked in the mixture for 5 minutes. The protein bands were visualized by Bio-Rad ChemiDocTM Touch Imaging System. 2.3.13 Structural identification of products To achieve sufficient yields of enzymatic products for structural elucidation with NMR analysis, I scaled up transgenic yeast cultures to 65 mL and fed them with 50 µM substrates. The six replicates were grown under the same conditions for yeast culture as described earlier. After 48 hours, fed cultures were centrifuged and subjected to liquid-liquid extraction with ethyl acetate or chloroform. The organic phase was concentrated using a rotary evaporator and separated using a Varian semi-preparative HPLC system with a Kinetex® 5 μm EVO C18 column (100 Å, 100 x 250 mm) at a flow rate of 1.5 mL.min-1. The column was equilibrated in 90 % solvent A (water, 0.1% formic acid) and 10% solvent B (acetonitrile, 0.1% formic acid). The following elution conditions were used: 0–5 min, 10–20 % B; 5–25 min, 20–70% B; 25–27 min, 70–90% B; 27–30 min, 90 % B; 30–31 min, 90–10 % B; and 31–34 min, 10% B to re-equilibrate the column. Approximately 1.0 mg of each product was independently dissolved in 600 μL DMSO-d6 and subjected to 1H NMR and 1D-TOCSY NMR analyses on a Bruker Avance 600 NMR spectrometer. 2.4 Results 2.4.1 Identification and characterization of P450 candidates Using the available C. acuminata transcriptome and genome data [62,82], gene expression patterns were analyzed using the self-organizing map. 26 candidates that displayed similar expression and accumulation patterns with 10-hydroxycamptothecin out of 300 annotated P450s (Figure 2.4) were identified. Co-expression patterns of selected P450 candidates from Figure 2.4B illustrated the distinction of the distribution of the candidates in different organs. Due to the high expression of 10-HCPT in the bark 38(Figure 2.4A), the candidate genes were assessed based on the high distribution of the P450 candidates in the bark organ. Figure 2.4. Identification of camptothecin hydroxylase candidates. (A) Relative abundance of 10-HCPT in different C. acuminata organs (n = 3). (B) Relative abundance of selected P450 candidates in the corresponding organs. Data adapted from http://medicinalplantgenomics.msu.edu/contacts.shtml To test for enzymatic activities, I cloned the P450 coding regions of the following candidates, Ca23831, Ca23838, Ca000754, Ca12175, CaCYP12176, Ca23839, Ca17137, Ca6009, Ca6007, Ca6006, Ca32229, Ca19403, Ca30731, Ca24044, Ca12282, Ca27984, Ca23088, Ca4097, Ca4921, Ca32236, Ca32709, Ca17987, Ca12663, Ca15564, Ca15529, Ca32245 into the galactose-inducible dual expression vector pESC-Leu2d with a redox partner cytochrome P450 reductase (CPR). Initially, the protease-deficient A B 39YPL 150 C:PEP4KO yeast strain was chosen to be the heterologous host to validate the function of the cloned candidate genes [91]. High-throughput assays showed that among the candidates tested, only the yeast culture containing the Ca32229 construct could convert camptothecin substrate (m/z 349.2) to a new product (m/z 365.2). This showed the same mass to charge (m/z) ratio 365.2 and was observed to be eluted at the same retention time with that of the 10-hydroxycamptothecin authentic standard. The conversion was calculated at 9% (Figure 2.5). Several alkaloids involved in the proposed biosynthetic pathway of camptothecin, such as strictosidine, and strictosamide were also tested with the P450 candidates, but there was no hydroxylated product detected. 40 Figure 2.5. In vivo assay of recombinant Ca32229 protein with camptothecin. After the discovery of Ca32229 as a CPT hydroxylase, this sequence was used as the query to perform the BLAST search against whole C. acuminata genome sequences to identify other camptothecin hydroxylase homologues that may catalyze the formation of different HCPTs, including those that are 41hydroxylated at C-9, 10, or 11 on ring A of CPT. The analysis revealed four P450 homologues with > 90% gene sequence identity with Ca32229 (Table 2.2). Table 2.2. Nucleic acid sequence identity (%) between Ca32229 and its close homologues from C. acuminata Of these four additional candidates, only Ca32236 was able to hydroxylate CPT. A peak with m/z 365.2 [M+H]+ with respect to the adduct of 10-HCPT was observed and eluted at a similar retention time with 10-HCPT standard. While the conversion of synthetic Ca32229 with CPT substrate was 9%, the conversion of CPT with synthetic Ca32236 was at 5%. The other three candidates, Ca32234, Ca32235, and Ca32245, showed no activity towards CPT. The phylogenetic analysis of the two putative CPT hydroxylases Ca32229 and Ca32236 together with 36 P450 sequences from monoterpene indole alkaloid-producing plants including such as C. roseus and R. serpentina revealed that these two new P450s form a completely separate clade that is distinct from other P450s (Figure 2.6A). Ca32229 Ca32234 Ca32235 Ca32236 Ca32245 Ca32229 - 94.3 93.2 93.9 92.9 Ca32234 94.3 - 92.8 92.0 95.8 Ca32235 93.2 92.8 - 96.0 92.2 Ca32236 93.9 92.0 96.0 - 91.5 Ca32245 92.9 95.8 92.2 91.5 - 42 Figure 2.6. (A) Unrooted neighbour-joining phylogenetic tree for P450 candidates from this study (red and blue) and previously reported P450s from C. acuminata and other organisms. Active CPTHs are shown in blue. Bootstrap frequencies for each clade were based on 1000 iterations. (B) Protein alignment of Ca32229, Ca32245, Ca32234, Ca32235, and Ca32236. A B 432.4.2 Optimization of in vivo production of hydroxylated product As Ca32229 was discovered in the early stage of this study, all the optimizations of in vivo assay condition were initially conducted on the PEP4KO yeast harbouring pESC-Leu2d::CPR_Ca32229. The selective dropout media SC-Leu allowed sufficient quantity of product (~1 µM) for LC-MS screening. However, the hydroxylation yield in the selective media was only 4–9 %, when the in vivo assay was upscaled to 1 mL in test tubes. When rich YPA medium was used, the conversion rate increased to ~10 %. Intriguingly, when glycerol was added into the YPA media, there was a big leap of the conversion from 10% to 25% (LC-MS conversion). Preliminary results of the experiments with various glycerol and camptothecin concentrations were assessed (Figure 2.7). The results demonstrated that higher substrate concentration might not necessarily afford better transformation, and the optimal conversion rate (~25%) was achieved with 15% glycerol and 10 μM CPT. Nevertheless, to obtain a higher product quantity for further purification and derivatization with fewer replicates, the CPT concentration was used at 50 μM in the YPA medium containing 10% glycerol to yield ~20% hydroxycamptothecin conversion. Figure 2.7. CPT-to-putative HCPT conversion rate in in vivo assays with varied camptothecin and glycerol concentrations (bars) in YPA media. 0510152025301 5 10 20 50 75 100Conversion (%)Camptothecin concentration (μM)0 5 10 15 20 44 Furthermore, the reaction time was surveyed by testing Ca32229/PEP4KO yeast strain reactivity in different intervals from 1 day to 4 days. The highest conversion rate was observed from day 2 to day 3 at 22–25%, and the conversion rate significantly dropped to 12% on day 4. No byproducts were detected after 4 days of reaction by UPLC-MS/MS. All the conversion rate data were acquired by using TargetLynx. In addition, different yeast strains were tested. CPT’s poor solubility in water and several common organic solvents, such as acetone, and chloroform hinders its permeability into microbial cells such as yeast in the assay. Furthermore, as CPT is a topoisomerase-I (topI) inhibitor, it would interfere the growth of the cells in the media, resulting in a low expression level and/or catalytic activity of the intracellular Ca32229 enzyme. To improve the efficiency of in vivo hydroxylation of CPT in the yeast cell, I employed the Δerg6 Δtop1 yeast strain, a double mutant yeast with a more permeable cell wall and knocked-out topoisomerase-I[90]. It was expected that CPT could go through the cell wall more easily and have less effect on cell growth. The results show that Δerg6 Δtop1 yeast strain expressing Ca32229 and CPR incubated with CPT in YPA with 10% glycerol media allowed markedly higher conversion, up to 62%. Transgenic Δerg6 Δtop1 yeast in YPA with 10% glycerol was thus replicated in 1-mL scale and large-scale assays to generate ~ 60% conversion rate toward putative hydroxycamptothecin product. The pESC-Leu2d::CPR_Ca32236 transformed in Δerg6 Δtop1 yeast was implemented with the same procedure had the highest conversion rate at 67%. In summary, feeding 50 μM CPT substrate into transgenic yeast culture in YPA with 10% glycerol media at 30 oC for 48 hours yielded the optimal results. Under these conditions, yeast expressing Ca32229 and Ca32236 yielded 62% and 67% putative HCPT products, respectively. 2.4.3 Large scale production of enzymatic products Using the optimized conditions above, six large-scale yeast cultures were grown in 65 mL medium shaken in a 2 L baffled Erlenmeyer flask to collect sufficient amount for NMR analysis to elucidate the structure of products from the enzymatic assays of Ca32229 and Ca32236. After purification using semi-preparative HPLC, approximately 1–3 mg putative hydroxycamptothecin products could be obtained from 6.8 mg camptothecin substrate in six incubating cultures. 452.4.4 Structural elucidation of enzymatic hydroxylated products Approximately 1 mg purified putative hydroxylated products of Ca32229 and Ca32236 were used for structural elucidation by 1H-NMR and 1D-TOCSY NMR. For the product of Ca32229, the camptothecin scaffold of the product was confirmed by comparing its 1H-NMR spectrum to that of previous report of the camptothecin structure [92]. Figure 2. 8. 1H-NMR spectrum of enzymatic product in the in vivo assay of Ca32229 and CPT To verify the hydroxylation position, I analyzed the authentic standard 10-hydroxycamptothecin in the same condition as the reference data and focused on protons of the aromatic proton region (6.5–8.0 ppm) as any hydroxyl groups installed on the ring A of camptothecin would generate a specific spin system of aromatic protons. When Ca32229 was incubated with CPT substrate for longer than 48 hours, the 46dihydroxycamptothecin compound has not been detected by LC-MS. Based on these observations, the Ca32229 was hypothesized to be a monohydroxylase enzyme. Due to the impurity signals in the region of 7.00–7.50 ppm that overlapped the signals of the aromatic protons (Figure 2.8), 1D-TOCSY analysis was conducted by irradiating at 8.02 ppm to resolve these proton signals. 1D-TOCSY spectrum of Ca32229’s product displayed one doublet of doublets (dd) signal at 7.28 ppm (J = 8.7 Hz, 2.4 Hz; ortho and meta coupling), one doublet (d) signal at 7.38 ppm (J = 3.0 Hz; meta coupling), and one doublet (d) signal at 7.97 ppm (J = 7.8 Hz; meta coupling). These data indicated that the hydroxyl group was at two possible positions, C10 (10-HCPT) or C11 (11-HCPT). A comparison of the expanded 1H-NMR spectra from 7.00–8.15 ppm of the authentic standard 10-hydroxycamptothecin with the 1D-TOCSY spectrum of Ca32229 (Figure 2.9) showed that the distinct chemical shifts of the dd signal at 7.28 ppm and the doublet signal at 7.38 ppm of the Ca32229’s product were different from those of the 10-HCPT (7.42 ppm and 7.28 ppm, respectively), indicating that Ca32229’s product is not 10-HCPT. Further comparison with published structural data showed that Ca32229’s product was 11-hydroxycamptothecin (Table 2.3) [66]. 47Table 2.3. 1H-NMR data of the putative HCPT isolated from the in vivo assay of Ca32229 and CPT in comparison with previously-reported 11-HCPT H Ca32229’s product 11-HCPT [66] 5 5.22 (s) 5.20 (s) 7 8.54 (s) 8.82 (s) 9 7.97 (d, 7.8) 7.95 (d, 9.0) 10 11 (-OH) 7.28 (dd, 8.7, 2.4) 10.45 (s) 7.26 (dd, 9.0, 2.5) 10.43 (s) 12 7.38 (d, 3.0) 7.36 (d, 2.5) 14 7.30 (s) 7.28 (s) 17 5.42 (s) 5.41 (s) 18 0.88 (t, 7.2) 0.88 (t) 19 1.87 (m) 1.85 (m) 20 6.52 (s) 6.51 (s) 48 Figure 2. 9. (A) 1D-TOCSY spectrum and (B) stacked 1H-NMR spectra against 10-HCPT standard of 11-HCPT enzymatic product. A B 49Similar 1H-NMR and 1D-TOCSY analyses were performed for the enzymatic product of Ca32236. Specifically, a camptothecin scaffold was confirmed in 1H-NMR spectrum of Ca32236’s product. The 1D-TOCSY spectrum of Ca32236’s product showed one doublet signal at 7.29 ppm (J = 2.4 Hz; meta coupling), one dd signal at 7.44 ppm (J = 9.0 Hz, 2.4 Hz; ortho and meta coupling), and one d signal at 8.03 ppm (J = 9.0 Hz; ortho coupling). Comparing these data with the authentic standard and literature reference of 10-HCPT [93] confirmed that Ca32236’s product was 10-HCPT (Figure 2.11; Table 2.4). Ca32236 and Ca32229 were thus named CPT 10-hydroxylase (CPT10H) and CPT 11-hydroxylase (CPT11H), respectively. Figure 2.10. 1H-NMR spectrum of enzymatic product in the in vivo assay of Ca32236 and CPT 50Table 2.4. 1H-NMR data of enzymatic product in the in vivo assay of Ca32236 and CPT H Ca32236 Commercial 10-HCPT 10-HCPT [94] 5 5.24 (s) 5.23 (s) 5.23 (s) 7 8.46 (s) 8.45 (s) 8.45 (s) 9 10(-OH) 7.29 (d, 2.4) 10.33 (s) 7.28 (d, 3.0) 10.37 (s) 7.28 (d, 2.6) 10.37 (s) 11 7.44 (dd, 9.0, 2.4) 7.42 (dd, 9.0, 3.0) 7.43 (dd, 9.1, 2.6) 12 8.03 (d, 9.0) 8.02 (d, 9.0) 8.03 (d, 9.1) 14 7.26 (s) 7.26 (s) 7.27 (s) 17 5.41 (s) 5.41 (s) 5.41 (s) 18 0.88 (t, 7.2) 0.88 (t, 7.2) 0.88 (t, 7.2) 19 1.87 (m) 1.86 (m) 1.81 – 1.90 (m) 20 6.49 (s) 6.51 (s) 6.52 (s) 51 Figure 2.11. (A) 1D-TOCSY spectrum and (B) stacked 1H-NMR spectra against 10-HCPT standard of 10-HCPT enzymatic product. B A 52 Figure 2.12. Summary of the specific signals on 1H-NMR spectra of 10- and 11-hydroxycamptothecin. 532.4.4 Substrate specificity To evaluate the substrate specificity of CPT10H and CPT11H, their activities against MIAs with various scaffolds including yohimbine, tetrahydroalstonine, reserpine, serpentine, corynanthine, ajmalicine, strictosidine, and strictosamide were tested using the in vivo yeast feeding assays (Figure 2.15). LC-MS and NMR analyses showed that the enzymes solely converted CPT-scaffold alkaloids, namely CPT, 7-ethylcamptothecin, 10-HCPT and 9-aminocamptothecin to the respective hydroxylated products (Figures 2.13–2.17). The new P450s’ selectivity appeared to be restricted to the flat and hydrophobic structures of CPT and its analogues. CPT10H could convert 7-ethylcamptothecin substrate to the 7-ethyl-10-HCPT product, or SN-38, a commonly used anticancer agent (Figure 2.15). Another product, 7-ethyl-11-hydroxycamptothein, resulted from the activity of CPT11H against 7-ethylCPT, is a new-to-nature compound (Figures 2.14, 2.15; Table 2.5). This newly-established activity of CPT11H could pave the way for new studies on anticancer activity of this compound, considering its similarity in structure with 7-ethyl-10-HCPT. 54 Figure 2.13. Substrate specificity of CPTHs, Ca32236 (CPT 10-hydroxylase) and Ca32229 (CPT 11-hydroxylase). Substrates from different subgroups of monoterpenoid indole alkaloids (MIA) include simple secoiridoid (secologanin), central precursors of MIA biosynthetic pathway (strictosidine, 55strictosamide), heteroyohimbanes (ajmalicine, tetrahydroalstonine), yohimbane (yohimbine), ajmalan (ajmaline), β-carboline (harmalol, harmaline), and CPT and CPT analogues (10-HCPT, 11-HCPT, 7-ethylCPT, 9-aminoCPT, 9-nitroCPT). Only CPT, 7-ethylCPT, 10-HCPT, and 9-aminoCPT were accepted as substrates with different conversion rates. Numbers in brackets are conversion rates (blue for 32236 and pink for 32229), and [-] for non-detected rates. Table 2. 5. 1H-NMR data of enzymatic product in the in vivo assay of Ca32229 and 7-ECPT H This study 7-Ethyl-10-hydroxycamptothecin (SN-38) 5 5.27 (s) 5.28 (s) 9 8.14 (d, 9.0) 7.40 (d, 2.4) 10 11 (-OH) 7.29 (dd, 9.0, 2.4) 10.39 (s) 10.31 (s) (-OH) 7.42 (dd, 8.4, 2.4) 12 7.38 (d, 2.4) 8.03 (d, 9.0) 14 7.29 (s) 7.24 (s) 17 5.43 (s) 5.42 (s) 18 0.88 (t, 7.2) 0.87 (t, 7.2) 19 1.86 (m) 1.86 (m) 20 6.52 (s) 6.52 (s) -CH2- of ethyl chain 3.24 (m) 3.08 (q, 7.8) -CH3 of ethyl chain 1.30 (t, 7.8) 1.30 (t, 7.8) 56 Figure 2.14. 1D-TOCSY spectrum of enzymatic product from in the in vivo assay of Ca32229 and with 7-ECPT as substrate. 57 Figure 2.15. Extracted ion chromatograms showing the in vivo activity of Ca32236 with CPT (A) and 7-ECPT (B), and of Ca32229 with CPT (C) and 7-ethyl-CPT (D). CPT: camptothecin; HCPT: hydroxy-CPT; ECPT: ethyl-CPT; EV: empty vector (negative control). A B C D 58Several CPT analogues were included in the alkaloids tested with CPT10H and CPT11H (Figure 2.13) with an aim to produce new-to-nature compounds having CPT scaffold for topI inhibitory activity screening. These analogues, such as 9-aminoCPT and 9-nitroCPT, have been previously reported to have antitumor activity and tested in clinal trials. UPLC-MS/MS analysis showed that Ca32229 and Ca32236 could convert 9-aminocamptothecin to products with SIR detection for adduct [M+H]+ m/z 380, corresponding to hydroxylated 9-aminoCPT, most likely 9-amino-11-hydroxyCPT, and 9-amino-10-hydroxyCPT, respectively (Figure 2.16). The poor conversion rate (9–10%) hindered their structural identification by NMR. Figure 2.16. In vivo activity of Ca32229 and Ca32236 with 9-aminoCPT 59A yeast in vivo assay of CPT11H with 10-HCPT showed that this compound could hydroxylate 10-HCPT (Figures 2.17, 2.18; Table 2.6). The 1H-NMR spectrum of the new product showed signals for the two labile protons of the hydroxyl groups at low field region (from 10–11 ppm), and two singlet signals of H-9 and H-12 on ring A at 7.26 and 7.37 ppm, respectively, which indicate that the structure was 10,11-dihydroxycamptothecin. Nevertheless, Ca32236 (CPT10H) could not transform 11-HCPT to dihydroxycamptothecin yeast feeding assay. Another HCPT isomer, 9-HCPT, was assayed with both Ca32229 and Ca32236, but no dihydroxycamptothecin signal was detected from UPLC-MS/MS analysis. These data showed that although camptothecin hydroxylases are selective against flat pentacyclic structure of the CPT scaffold, the polarity of the substrate was an important factor. Ca32229 displayed high conversion rates with the nonpolar substrates CPT and 7-ECPT substrates and lower activities against compounds having higher polarity such as 9-aminocamptothecin and 10-HCPT. Ca32236 was even more selective with nonpolar substrates since it could accept CPT and -7ECPT but became inert with more polar CPT analogues, including 11-HCPT and 9-HCPT. 60 Figure 2.17. In vivo activity of Ca32229 and Ca32236 with 11-HCPT A B 61Table 2.6. 1H NMR data of enzymatically produced 10,11-dihydroxy-CPT in DMSO-d6 (298 K, 600 MHz) Figure 2.18. 1H NMR spectrum of products from in vivo assay of Ca32229/CPR with 10-HCPT as substrate producing 10,11-dihydroxyCPT. H This study 5 5.18 (s) 7 8.34 (s) 9 7.26 (s) 10 (-OH) 11 (-OH) 10.15 (s) 10.35 (s) 12 7.37 (s) 14 7.28 (s) 17 5.40 (s) 18 0.88 (m) 19 1.88 (m) 20 (-OH) 6.46 (s) 62The in vitro enzyme assays were conducted by using microsomal fractions of yeast transformed with pESC-Leu2d::CPR/Ca32229 or Ca32236 in the presence of NADPH cofactor. While the yeast feeding in vivo reactions could generate abundant hydroxylated CPT products, the in vitro assays only produced trace quantity (< 1% conversion). As immune-blotting with α-FLAG antibodies confirmed that CPT10H and CPT11H were well expressed in the microsome (Figure 2.19A), the low in vitro activities could be due to the instability of the P450s when the proteins were removed out of the yeast cells. 63 Figure 2.19. (A) Western blot showing the expression of Ca32229 and Ca32236 in Saccharomyces cerevisiae harbouring pESC-Leu2d::CPR (EV: empty vector control), pESC-Leu2d::CPR_Ca32236 and pESC-Leu2d::CPR_Ca32229. (B) In vitro assays using microsome extracted from the same yeasts with CPT. A B 64In conclusion, this research has unambiguously identified two P450s in C. acuminata that catalyze the regio-specific oxidations of CPT. Ca32236 converted CPT to 10-HCPT while Ca32229 catalyzed C-11 hydroxylation to yield 11-HCPT. These CPTHs selectively catalyzed CPT-scaffold compounds in the substrate specificity experiments (Table 2.7). Table 2.7. Conversion rates of in vivo feeding reactions using yeast expressing CPT hydroxylases and CPT derivatives. aconversion rate calculated based on LC-MS analysis byield of biotransformation from the yeast in vivo assay was obtained from 1 L yeast culture incubated with 17 mg CPT starting material. c due to the low yield and low recovery rate of our semipreparative system, these products could not be recovered for further structural elucidation. 2.5 Discussion Omics-based strategies emerged as a potent method for elucidating plant natural products biosynthetic pathways. Recent advances in identifying missing biosynthetic enzymes have highlighted the power of combining transcriptomics and metabolites profiling data [11,12,78,95]. In this study, candidate genes involved in the metabolism of camptothecin were identified by using publicly available Gene Substrate Hydroxylated product Conversion rate (%)a Yield of biotransformation (mg)b Isolated products after semipreparative HPLC (mg) Ca32236 CPT10H Camptothecin (CPT) 10-HydroxyCPT 67 12.0 8.4 7-EthylCPT 7-Ethyl-10-hydroxyCPT 8 1.5 0.6 9-AminoCPT 9-Amino-10-hydroxyCPT 9 1.7 n/a c Ca32229 CPT11H Camptothecin (CPT) 11-HydroxyCPT 62 11.0 8.1 7-EthylCPT 7-Ethyl-11-hydroxyCPT 32 6.1 3.5 9-AminoCPT 9-amino-10-hydroxyCPT 9 1.7 n/a c 10-HydroxyCPT 10,11-DihydroxyCPT 11 2.0 0.6 65transcriptomics and metabolomics data of C. acuminata. The two genes CPT10H and CPT11H discovered from this approach emphasized the tight correlation and high accuracy of the co-expression analysis applied in plant biosynthetic genes discovery. To the best of our knowledge, these P450s are the first genes reported in the downstream metabolism of CPT in C. acuminata. These genes could serve as the baits for unravelling other CPT metabolic genes. Notably, these enzymes are the first P450s found to hydroxylate the quinoline ring of monoterpene indole alkaloids. Therefore, they could be used to facilitate the discovery of other quinoline-hydroxylating enzymes using homology search as seen in studies on homologous oxidases in MIA biosynthesis in C. roseus and T. iboga [16]. Hence, the discovery of these camptothecin hydroxylases greatly contributed to the library of plant P450 enzymes that target this unique quinoline scaffold. As CPT10H and CPT11H exhibited selective hydroxylating function with camptothecin scaffolds, further structural protein studies would be necessary to elucidate the structural elements underlying this regioselectivity. Additional mutagenesis studies on these enzymes could expand the substrate spectra and potentially produce more new-to-nature compounds for the quest of high bioactive compounds for pharmaceutical industry. The success of the bioproduction of HCPT compounds from yeast platform has overcome the hurdles of cascade redox reactions via the chemical synthesis of these compounds with high titers (5–8 mg purified compound/L). The engineered high-permeable yeast strain was the crucial factor leading to the high titer production of hydroxycamptothecin products. The PEP4KO yeast strain is a suitable microbial host to overexpress cytochrome P450 enzymes, yet the specific topI inhibitory activity affecting cell growth and low water-solubility of CPT substrate hampered the catalytic activity of CPT hydroxylases expressed in this strain. The reported double-mutant erg6 top1 yeast strain exhibited high tolerance against CPT, due to the topoisomerase-I gene knockout and the more permeable cell wall resulting in effective penetration of CPT to the active site of endoplasmic reticulum-anchoring CPTHs [90]. Culture conditions also play a critical role in the yeast feeding reaction as YPA medium supplemented with 10% glycerol improved the CPT-to-HCPT conversion rates nearly 60-fold to 12 mg/L (biotransformation conversion) compared to standard culture using SC medium without glycerol. The NADPH balance in the yeast culture 66was reported as a factor improving the natural products biosynthesis, and the addition of 10% glycerol might have aided the NADPH reproduction as glycerol is required for the activity of the NADP-dependent glycerol dehydrogenase Gcy1p in generating the reduced product NADPH. Apart from the NADPH regeneration function, glycerol has been reported to stabilize the recombinant protein and the cellular membrane, and keep the correct protein folding [54]. This work showed that 10% glycerol was sufficient to improve the conversion rate of the yeast biotransformation of CPT. These results also highlighted the feasibility of using baker’s yeast fermentation for supplying 10-HCPT, a crucial compound for anticancer drugs production. Most importantly, the conversion rates are equivalent to those of chemical synthesis conversion (approximately 60%) [96], and afforded abundant putative HCPT structural elucidation and subsequent chemoenzymatic processes. Further yeast engineering efforts with stable integration of the CPTHs into the yeast genome via CRISPR/Cas9 techniques could further improve the yields [97]. This finding, therefore, allows for a more sustainable and economical approach to synthesize 10-hydroxycamptothecin and 11-HCPT by using plant cytochrome P450 biocatalysts. The high regio-selectivity and conversion rate (12 mg/L – biotransformation rate) afford the production of hydroxycamptothecins and their derivatives by chemical transformation at selective positions. The biosynthesis carried out at mild conditions (physiological pH, aqueous medium, 30 oC) surpassed the use of toxic chemo-catalysts, organic solvents, and harsh reaction conditions. 67Chapter 3: Chemoenzymatic synthesis of high-value CPT analogues 3.1 Summary Using CPTHs as the biocatalysts, this study produced water-soluble topI inhibitors such as topotecan, irinotecan, and their structural isomers via chemoenzymatic synthesis. While total asymmetric syntheses of topotecan and irinotecan have been reported, these drugs can now be preferentially prepared by short chemoenzymatic steps starting from natural camptothecin using the approach reported herein. In these reactions, the enzymes responsible for the oxidation of camptothecin were used to accomplish selective and specific chemical transformations in mild conditions of pH and temperature (pH 7, 30 oC), which are difficult to achieve by conventional chemical synthesis routes. From the enzymatic products reported in Chapter 2, including 10-HCPT, SN-38, and 11-HCPT under neutral aqueous conditions, a suite of CPT analogues was generated, including the clinically approved anticancer drugs topotecan and irinotecan and their new-to-chemist isomers [98]. This discovery will stimulate significant progress in the production of camptothecin derivatives leading finally to the development of more sustainable ways of producing active and water-soluble camptothecin-based pharmaceuticals. 3.2 Introduction C–H bonds are the most abundant and inert functional groups in natural products. Many efforts have been made to functionalize C–H bonds but there are several drawbacks that chemical synthesis could encounter, including laborious procedures and chemo- and regio-selectivity control. The C–H bond functionalization using enzymes as catalysts emerged as a promising alternative method to synthesize high-value natural products. The application of enzymes in synthesis can be utilized in either the early-stage starting material synthesis or the late-stage functionalization [99]. While natural products are the most valuable source for the discovery of therapeutic agents, the derivatization of natural products by biochemical means to generate molecules for biological screenings has only been developed recently. Cytochrome P450 (P450s) are ideal enzyme candidates for biocatalytic synthesis of pharmaceuticals as they are involved in the scaffolding and decoration of many biosynthetic pathways in plants [7]. The most 68common reaction conducted by P450s is the activation of the unreactive C–H bond via hydroxylation, which is of particular interest in the pharmaceutical industry as it is essential for the biotransformation of compounds, especially those that lack functional groups useful for conjugations. The hydroxyl group serves as the intermediate for further derivatizations or as a directing group for other substituents on the aromatic substitution reactions. Late-stage functionalizations to increase the activity, selectivity, and solubility of a drug candidate, via regiospecific hydroxylation, for example, remain a challenge but important step in organic chemistry. For instance, the derivatization of an outstanding antimalarial drug artemisinin into the hydroxylated artemisinin by the mutated cytochrome P450BM3 avoids the drug resistance of the nonderivatized form artemisinin. Mutated P450BM3 could selectively functionalize Csp3–H bonds by introducing the –OH hydroxyl group onto the ground scaffold of artemisinin at different positions C-7 and C-6. The hydroxyl groups were then replaced by the fluorine substituents yielding fluorinated artemisinin, which exhibited better in vivo bioactivity by extending the half-life of these potential drugs in vivo [100]. Functional group installation could be conducted by a plethora of organic reactions. However, chemical reactions tend to work less selectively on molecules with complex structures and multi-reactive centers, which leads to the need for extra protection or derivatization steps. Meanwhile, enzymes could selectively perform chemical transformation at specific sites, thus simplifying the whole synthetic route by decreasing the synthetic steps and minimizing byproducts (Figure 3.1). Figure 3.1. Comparison of functional group installation through traditional chemical synthesis versus C–H functionalization. Adapted from Fessner, 2019 [101]. 69Moreover, enzymes also play a crucial role in the scaffold formation steps. For example, (-)-englerin A, a terpenoid isolated from Phyllanthus engleri, is a renal cancer cells growth inhibitor that has been successfully synthesized by integrating biocatalysts and chemical synthesis. The microbial enzymes expressed step by step in the E.coli and yeast platforms were able to synthesize guaia-6,10(14)-diene from acetyl-CoA via the engineered mevalonate pathway at high titer 0.8 g/L. Guaia-6,10(14)-diene intermediate then entered the chemistry phase and was converted to (-)-englerin A [102]. In the production of camptothecin-related drugs, conventional production of topotecan and irinotecan relies on the chemical synthesis of 10-HCPT and 7-ethyl-10-HCPT from camptothecin and 7-ethylcamptothecin. Although these methods could deliver topotecan and irinotecan at economically feasible yield, the processes involve several laborious steps. For instance, the hydroxylation of camptothecin requires two cascade redox reactions with the heterogeneous catalyst Pd for hydrogenation, which is difficult to manipulate. Our discovery of camptothecin hydroxylases CPT10H and CPT11H not only contributes to the production of the 10-hydroxycamptothecin derivatives at mild condition with high conversion as compared to chemical synthesis but also broaden the scope of chemo-enzymatic research field using bio- and chemo-catalysts to synthesize high-value compounds with less cost and environmentally friendly. The optimal conditions for the majority of enzymes are aqueous medium and physiological pH, which are simple to manipulate and control. Using Ca32236 enzyme to hydroxylate CPT and 7-ECPT, 10-HCPT and SN-38 could be produced in yeast at high titer, which could then be used as the precursors for the one-step synthetic reactions afterwards (Figure 3.2). 70 Figure 3.2. Conventional and chemoenzymatic synthesis of (A) topotecan and its 11-isomer (10-hydroxycamptothecin R1 = -OH, R2 = -H; 11-hydroxycamptothecin R1 = -H, R2 = -OH; topotecan R3 = -OH, R4 = -H; topotecan-11 isomer R3 = -H, R4 = -OH) and (B) irinotecan and its 11-isomer (7-ethyl-10-hydroxycamptothecin R1 = -OH, R2 = -H; 7-ethyl-11-hydroxycamptothecin R1 = -H, R2 = -OH; irinotecan R3 = , R4 = -H; irinotecan-11 isomer R3 = -H, R4 = ) A B 71An important approach in producing bioactive chemicals is using halogenated analogues as they can have not only great effects on the bioactivity of the compounds but also provide chemical handles that can be used for further functionalization. As no natural halogenated MIAs have been reported to the best of our knowledge, I envisage that the introduction of halogens to the MIA scaffolds will make them suitable substrates for post-biosynthetic chemoselective derivatization via cross-coupling reactions. The halogenated hydroxycamptothecin analogues could serve as promising compounds improving their pharmacokinetics and potency as medical agents [103]. 3.3 Material and methods 3.3.1 Chemicals N-bromosuccinimide, N,N-dimethylmethyleneiminium chloride, pyridine, and triethylamine were purchased from Sigma-Aldrich. 4-Piperidinopiperidine-1-carbonyl chloride was purchased from Toronto Research Chemicals. DMSO was purchased from Fisherbrand. 10-HCPT, 11-HCPT, SN-38, and 7-ethyl-11-HCPT were prepared from the in vivo assays with CPT11H and CPT10H. 3.3.2 Hydroxycamptothecin and 7-ethylhydroxycamptothecin preparation by using camptothecin hydroxylases To generate sufficient amounts of HCPT and 7EHCPT for the synthesis of topotecan and irinotecan and other compounds, I scaled up enzymatic reactions. The transgenic erg6top1 yeast strain was inoculated in 2 mL of SC-Leu medium containing 2% (w/v) glucose overnight at 30 oC and 275 rpm. The culture was subsequently diluted 10-fold to an OD of 0.05 in SC-Leu supplemented with 2% (w/v) glucose and cultured for 16 hours. Yeast was then harvested and sub-cultured for 48 hours in YPA medium containing 2% (w/v) galactose, and 10% glycerol to induce the production of recombinant P450s. CPT or 7ECPT was fed into the culture to reach the final concentration of 50 μM as soon as the yeast was switched from SC-Leu to YPA medium. After 48-hour inoculation, the supernatant was collected by centrifugation at 4000 rpm, for 5 minutes. HCPT and 7EHCPT were extracted out of reaction matrix by liquid-liquid 72extraction with ethyl acetate and chloroform, respectively. The solvent was removed by using a rotatory evaporator to obtain crude HCPT and 7EHCPT extracts as substrates for further chemical synthesis. 3.3.3 Semi-synthesis of topotecan and its 11-hydroxy isomer: Fifteen mg of solid N,N-dimethylmethyleneiminium chloride (0.16 mmol), 6 mg of enzymatically-produced HCPT (0.016 mmol) dissolved in 1 mL isopropanol:chloroform (1:1), and 2 μL triethylamine were added into a 4-mL reaction flask. The mixture was magnetically stirred at room temperature for 24 hours, followed by acidification to pH 3–4 with 1 N HCl [74]. The reaction mixture was analyzed by LC-MS/MS. To isolate putative topotecan for structural identity confirmation, the reaction mixture was dried in vacuo, dissolved in methanol, and subjected to semi-preparative HPLC to isolate putative topotecan. The isolated product was dried, resuspended in DMSO-d6, and subjected to 1H NMR analysis on Bruker Avance 600 NMR spectrometer. 3.3.4 Semi-synthesis of irinotecan and its 11HCPT-derived isomer: Six mg of solid 4-piperidinopiperidine-1-carbonyl chloride (0.026 mmol) was added into a 4 mL reaction flask. One mg of 7-ethyl-HCPT (0.0026 mmol) substrates from the enzymatic reaction was dissolved by 200 μL pyridine and transferred into the reaction flask. The reaction mixture was magnetically stirred at room temperature for 2 hours, and analyzed by LC-MS/MS to detect the irinotecan product. To obtain products for NMR analysis, pyridine was removed by using rotatory evaporator for 2 hours. The dried crude mixture was dissolved by 300 μL water, and extracted with 1.5 mL dichloromethane to obtain the irinotecan product. Dichloromethane layer was dried in vacuo to obtain 1.5 mg dried product. The dried product was dissolved in DMSO-d6 and subjected to 1H NMR analysis on a Bruker Avance 600 NMR spectrometer to elucidate the structure of the final product. 3.3.5 Semi-synthesis of brominated HCPTs: Fifteen mg of solid N-bromosuccinimide (NBS) (0.084 mmol), and 3 mg of dried enzymatically-produced HCPT (0.0082 mmol) dissolved in 200 μL DMSO (pre-cooled at 4 oC) were added to a 4-mL reaction flask on ice. The mixture was magnetically stirred at room temperature in the dark for 2 hr. The reaction progress was monitored by LC-MS/MS to detect the brominated HCPT product. The reaction 73mixture was then transferred into 5 mL cold water, and adjusted to pH 3–4 with 1 N HCl [104]. Water and organic solvent were removed by GeneVac evaporator at lower than 40 oC. The dried reaction mixture was then dissolved in methanol and the brominated products was purified by semi-preparative HPLC. The dried product was dissolved in DMSO-d6 and subjected to 1H NMR and 1D-TOCSY (50 ms spin-lock time, irradiation frequency set at 8.02 ppm) analyses on a Bruker Avance 600 NMR spectrometer to determine the position of the bromine substituent position. 3.4 Results 3.4.1 Production of 10-HCPT derivatives Treatment of enzymatically produced 10-HCPT with an appropriate iminium reagent, N,N-dimethylmethyleneiminium chloride, yielded 9-[(dialkylamino)methyl]-10-HCPT, commonly known as topotecan [74]. When the enzymatic product 11-HCPT was allowed to react with the same iminium reagent, I obtained a total conversion to the new product 12-[(dialkylamino)methyl]-11-HCPT. Likewise, using the enzymatic products 7-ethyl-10-HCPT and 7-ethyl-11-HCPT with [1,4′]bipiperidinyl-1′-carbonyl chloride in pyridine, I achieved conversion to the clinically important drug irinotecan and its 11-HCPT-derived isomer. Furthermore, using a halogenated reagent such as N-bromosuccinimide (NBS) on 10-HCPT and 11-HCPT derived from in vivo biosynthesis afforded approximately 1 mg of 9-bromo-10-HCPT and a monobromo-11-HCPT, which can be further derivatized (Figure 3.3). The products were confirmed as mono-brominated derivatives by means of UPLC-MS/MS (Figure 3.5). 1D-TOCSY NMR of the reaction mixtures of HCPTs with NBS with irradiation at 8 ppm to identify the position of the bromine substituent. showed two doublet signals in each spectrum (J = 9.0 Hz), indicating that there were two protons at ortho position to each other on ring A (Figure 3.4). These data demonstrated that the bromination reactions of 10-HCPT and 11-HCPT yielded 9-bromo-10-HCPT, and 12-bromo-11-HCPT, respectively. 74 Figure 3.3. Chemoenzymatic reactions of 10- and 11-HCPT with NBS B Figure 3.4. 1D-TOCSY spectra of bromination products of (A) 10-HCPT and (B) 11-HCPT A 75NONOOcamptothecinNNOOOHO10-hydroxycamptothecinHONNOOOHO9-bromo-10-hydroxycamptothecinHOCPT10H1 2NBS, DMSOBrHO NONOOcamptothecinNNOOOHO11-hydroxycamptothecinNNOOOHO12-bromo-11-hydroxycamptothecinCPT11H1 2NBS, DMSOHO HOHOBr Figure 3.5. Chemoenzymatic synthesis of brominated 10-HCPT (A) and 11-HCPT (B) B A 76Enzymatically-produced HCPTs were allowed to react with N,N-dimethylmethyleneiminium chloride and 4-piperidinopiperidine-1-carbonyl chloride to yield topotecan, irinotecan, and their 11HCPT-derived isomers (Figures 3.6 to 3.9). All new products were confirmed by LC-MS analysis, and topotecan and irinotecan products were verified by comparison with authentic standards. All the chemoenzymatic transformations were completed at room temperature, and no substrates or decomposition products were detected at the end. The structures of the novel 7-ethyl-11HCPT-derived isomer of irinotecan, 7-ethyl-11-[4-(1-piperidino)-1-piperidino]carbonyloxyCPT (irinotecan-11), and 11HCPT’s derivative 12-[(dimethylamino)methyl]-11HCPT (topotecan-11) were confirmed by 1H-NMR analysis (Figures 3.8, 3.11; Tables 3.1, 3.2). In the irinotecan-11 synthesis, approximately 1.5 mg of the pure compound was obtained. The purity of the compound after liquid-liquid extraction was assessed by thin-layer chromatography compared to the authentic irinotecan standard. The 1H NMR data (Table 3.1; Figure 3.8) demonstrated the substitution of the hydroxyl group at C-11 position of ring A. 77 Figure 3.6. Chemoenzymatic synthesis of irinotecan (left) and irinotecan-11 (right). The retention times and m/z values of synthetic products were compared to irinotecan authentic standard. Irinotecan Irinotecan-11 78Table 3.1. 1H NMR data of the chemoenzymatic reaction product irinotecan isomer, 7-ethyl-11-[4-(1-piperidino)-1-piperidino]carbonyloxyCPT (irinotecan-11) in DMSO-d6 (298 K, 600 MHz) Figure 3. 7. The proposed mechanism of irinotecan-11 (7-ethyl-11-[4-(1-piperidino)-1-piperidino]carbonyloxyCPT) synthetic reaction H This study 5 5.34 (s) 9 8.31 (d, 9.6) 10 11 7.56 (dd, 9.0, 2.4) 1.23, 2.02, 2.38, 3.17, 3.24, 3.51, 3.57, 4.08 12 7.88 (d, 2.4) 14 7.32 (s) 17 5.44 (s) 18 0.88 (t, 7.2) 19 1.86 (m) 20 (-OH) 6.52 (s) -CH2- of ethyl chain 3.24 (m) -CH3 of ethyl chain 1.32 (t, 7.8) 79In the topotecan-11 synthesis, approximately 3 mg of pure compound was obtained. The hydroxyl group on ring A directed the substituent group to its ortho position. The substituent group –CH2N(CH3)2 position on ring A of topotecan-11 product was identified by the ortho coupling signals of H-9 and H-10 (J = 9.0 Hz) on the 1H NMR spectrum (Table 3.2; Figure 3.11) Figure 3. 8. 1H NMR spectrum of chemoenzymatic reaction product irinotecan-11 (7-ethyl-11-[4-(1-piperidino)-1-piperidino]carbonyloxyCPT) 80 Figure 3. 9. Chemoenzymatic synthesis of topotecan (left) and topotecan-11 (right). The retention times and m/z values of synthetic products were compared to topotecan authentic standard. Topotecan Topotecan-11 81Table 3. 2. 1H NMR data of chemoenzymatic reaction product topotecan isomer 12-[(dimethylamino)methyl]-11HCPT (topotecan-11) in DMSO-d6 (298 K, 600 MHz) Figure 3. 10. The proposed mechanism of topotecan-11 (12-[(dimethylamino)methyl]-11HCPT) synthetic reaction H This study 5 5.27 7 8.65 (s) 9 8.12 (d, 9.0) 10 11 (-OH) 7.64 (d, 9.0) - 12 (-CH2N(CH3)2) 4.59, 2.85 14 7.48 17 5.44 18 0.89 (t, 7.2) 19 1.89 (m) 20 (-OH) - Cl- 82 Figure 3. 11. 1H NMR spectra of chemoenzymatic reaction products topotecan-11 (12-[(dimethylamino)methyl]-11-HCPT) 3.5. Discussion The hydroxylated products obtained from the in vivo whole-cell enzymatic reactions were used in the chemical reactions to generate high-value derivatives. With this method, the enzymes would selectively introduce the crucial hydroxyl group onto the CPT scaffold. The attached hydroxyl group served as the intermediate for further functionalization or the directing group to attach more substituents onto the ring A of CPT scaffold. The low reactive Csp2–H bonds of camptothecin impeded the chemical substitutions on the aromatic rings of quinoline skeleton. Furthermore, the substitutions are non-specific and hard to control because of the deficiency of the directing functional group, such as -OH hydroxyl group. In addition, 83substitutions on ring A of the CPT scaffold have been shown to display powerful bioactivities [61]. Hence, the hydroxylations at C-10 and C-11 of the camptothecin scaffold are critical features in designing active camptothecin derivatives. The late-stage chemical decorations of hydroxycamptothecins could be carried out on the hydroxyl group or on ring A via the electrophilic substitution under the direction of the hydroxyl group. Although C–H bonds functionalization through C–O bond formation could be achieved by using palladium-catalyzed oxidation [105], the synthetic routes required complex manipulations. “Natural chemists” such as plants could overcome these hurdles by providing elegant enzymatic tools that help to selectively hydroxylate specific substrates. The CPTHs facilitated the accessibility of enzymatic products 10-hydroxycamptothecin and 7-ethyl-10-HCPT, crucial precursors to synthesize prevailing anti-cancer drugs topotecan and irinotecan (Figure 3.6, 3.9). To the best of our knowledge, the biosynthesis of 11-HCPT in this research is the first study on the production of this compound in both chemical synthesis and synthetic biology fields. The production of 5–8 mg/L allowed us to further utilize to synthesize C-11-derivatized products. As 11-HCPT was reported to exhibit greater bioactivity than CPT [66], the chemo-enzymatic products from this compound could potentially serve as potent chemotherapeutic agents. 84Chapter 4: Conclusion and future perspectives Although CPT and its derivatives are important anticancer agents, CPT biosynthesis remains largely elusive. Furthermore, the production of clinically important CPT derivatives such as topotecan and irinotecan depends on chemical synthesis from CPT extracted from plants. The main goal of this research is to discover the genes encoding for enzymes from C. acuminata that catalyze the hydroxylation of CPT. Two regio-specific CPT hydroxylases were discovered, which allowed for the establishment of a chemoenzymatic pipeline to produce a suite of 13 CPT derivatives, including the high-value anticancer drugs topotecan and irinotecan as well as their new-to-nature analogues. 4.1 Camptothecin hydroxylases in camptothecin biosynthesis from C. acuminata While the transcriptomic, genomic and metabolomic data of C. acuminata have been available for the past 10 years, limited work has been reported on the biosynthetic pathway of camptothecin downstream of strictosidinic acid [82]. To the best of our knowledge, this study is the first to report the enzymes in camptothecin metabolism, thereby paving the way for more discovery of the enzymes involved in this pathway. Specifically, these CPTHs could be used as queries to facilitate the discovery of other candidate genes upstream of the pathway. The discovery of CPTH allows future development of alternative biocatalysts-based methods to produce HCPTs instead of chemical synthesis. The enzymatic approach developed here facilitates the HCPT production by expressing CPTH gene in yeast and feeding the transgenic yeast with CPT at 30 oC and in aqueous medium. This research is also the first study on the synthesis of 11-HCPT via either chemical or biological synthesis opening possibilities of more sustainable supplies of this highly therapeutic compound. The discovery of the two regio-specific CPTHs paved the way into the largely elusive CPT biosynthesis in C. acuminata. The CPTHs’ narrow substrate spectrum suggests their dedicated metabolic roles and was used to develop a proof-of-concept chemoenzymatic pipeline leading to potent anti-tumour CPT derivatives. 85 Figure 4.1. Biosynthetic pathway of hydroxylated camptothecin compounds. 4.2 Chemoenzymatic procedures for anticancer drugs and their analogues syntheses Despite its relative accessibility, CPT is not an ideal anticancer drug owing to its low solubility, side effects, and drug resistance [63]. Chemical substitutions on the CPT scaffold are required to improve its potency [103]. Hydroxylation at C-10 and C-11 is a critical feature in designing active CPT derivatives [66], and commercial synthesis of the anticancer drugs topotecan and irinotecan utilize 10-HCPT [74,77]. Moreover, CPT analogues harbouring the 11-hydroxyl group exhibit a greater therapeutic index with much less toxicity than CPT [106], and 11HCPT derivatives such as 7-ethyl-11HCPT overcome interpatient variability and drug resistance compared to irinotecan [107]. Unfortunately, access to HCPTs is limited because of their low abundance in planta (<0.00001–0.002% dry weight) [81] [66]. The first synthesis of 10-HCPT from CPT was pioneered in 1991 [73], and followed by many attempts to improve its efficiency [108] [96]. The work presented here thus provides an excellent alternative to chemical synthesis of 10-HCPT, and a new possibility for 11HCPT production. The high regio-selectivity and high conversion rate 86of these enzymes afford specific HCPT on demand, facilitating further chemical decorations at desired positions. Overall, by implementing chemoenzymatic process, 13 compounds have been successfully synthesized (Figure 4.2). Among these, three compounds irinotecan, topotecan, and SN-38 have been clinically used to treat cancer. The derivatives synthesized from the 11-hydroxycamptothecin are new-to-nature compounds. The rising demand for topotecan and irinotecan provides a demand for short, chemoenzymatic steps starting from CPT using CPT10H and CPT11H at mild conditions (pH 7, 30 oC). In addition, these enzymes can accept substituted CPT derivatives. Halogenated organic compounds are extremely rare in nature, yet they constitute up to 15% of the pharmaceutical products on the market [103], and the halo-HCPTs produced in this work could potentially serve as starting handles for regio-selective and regio-divergent arylation via cross-coupling [109] to further diversify the products with new bioactivity potentials. Figure 4. 2. CPT analogues produced by chemoenzymatic reactions of camptothecin hydroxylases 4.3 Future directions C. acuminata and other species produce three natural HCPTs, namely 9-, 10- and 11-HCPT [60]. The next step will focus on identifying the remaining enzyme that regio-specifically hydroxylates CPT at C-9 to make 9-HCPT. The discovery of CPT 9-hydroxylase will be essential to fully understand the 87structure-function relationships of all CPT hydroxylases and allow further protein and metabolic engineering approaches to facilitate the C–H bond functionalization of resembling CPT scaffold compounds. Hydroxyl group could serve as either a directing group on aromatic ring substitution or an intermediate functional group for derivatization (for the synthesis of brominated hydroxycamptothecins, topotecan, irinotecan, and their analogues). In addition, the hydroxyl group could increase the molecular polarity in pharmacological studies. Furthermore, the improvement of the bioproduction of hydroxycamptothecins in yeast fermentation, such as integrating the CPTH gene into the yeast genome by CRISPR/Cas9 technique, could push the flux to further synthesize camptothecin derivatives. The catalytic activity of the microsomal protein fractions from yeast of these genes currently exhibited low activity towards camptothecin. Another expressing host, such as tobacco, could be a promising heterologous host to express CPTH proteins with high stability and bioactivity retention. 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A B 103Appendix 3. 1H NMR data of chemoenzymatic reaction product 9-bromo-10-HCPT in DMSO-d6 (298 K, 600 MHz) H This study 5 5.30 (s) 7 8.74 (s) 9 - 10 (-OH) 11 11.18 (s) 7.63 (d, 9.0) 12 8.08 (d, 9.0) 14 7.29 (s) 17 5.42 (s) 18 0.88 (m) 19 1.86 (m) 20 (-OH) - 104Appendix 4. 1H NMR data of chemoenzymatic reaction product 12-bromo-11HCPT in DMSO-d6 (298 K, 600 MHz) H This study 5 5.27 (s) 7 8.62 (s) 9 8.00 (d, 9.0) 10 11 (-OH) 7.46 (d, 9.0) 11.05 (s) 12 - 14 7.36 (s) 17 5.44 (s) 18 0.89 (m) 19 1.87 (m) 20 (-OH) 4.73 (s) 105Appendix 5. 1H NMR data of CPT substrate in DMSO-d6 (298 K, 600 MHz) H This study Xu et al. 2015 [94] 5 5.29 (s) 5.31 (s) 7 8.70 (s) 8.71 (s) 9 10 8.13 (d, 8.4) 7.72 8.15 (d, 7.6) 7.73 (t, 8.0) 11 7.87 7.88 (t, 8.4) 12 8.18 (d, 8.4) 8.19 (d, 8.8) 14 7.35 (s) 7.36 (s) 17 5.43 (s) 5.44 (s) 18 0.88 (t, 7.2) 0.89 (t, 7.2) 19 1.88 (m) 1.87 (q, 7.6) 20 (-OH) 6.55 (s) 6.56 (s) 106Appendix 6. 1H NMR spectrum of camptothecin 107Appendix 7. Primers used for the construction of expression vectors Primers used to assemble P450 candidates in pESC-leu2d expression vector Vector name Forward primer (5’-3’) Reverse primer (5’-3’) pESC-leu2d-12175 CACTAAAGGGCGGCCAACAAAATGGAGTGGACATTGAGCACAC ATCCATCGATACTAGTGCAAACACTCCTCGTCT pESC-leu2d-17987 CACTAAAGGGCGGCCAACAAAATGGATTTCTTTAGCTTTTTGTTG ATCCATCGATACTAGAACAAGGGTTGGAACAGCACG pESC-leu2d-32709 CACTAAAGGGCGGCCAACAAAATGGAGTGGACATTGAGCACA ATCCATCGATACTAGTGCAAACACTCGTCGTCTTTTG pESC-leu2d-32236 CACTAAAGGGCGGCCAACAAAATGGAGAACTTGTACTACTGCCT ATCCATCGATACTAGACGGAAACAAGTGCCTTCA pESC-leu2d-32245 CACTAAAGGGCGGCCAACAAAATGGAGAAGTTGTACTACTGC ATCCATCGATACTAGAATAAAGGAAGTGTCTTCAAGCTGG pESC-leu2d-15664 CACTAAAGGGCGGCCAACAAAATGACTTGGTTTCTCTTTCTTCTT GC ATCCATCGATACTAGGAGTTTGTGTAAAATCAAGTGAGCAC pESC-leu2d-32229 CACTAAAGGGCGGCCAACAAAATGGAGAACTTGTACTACTGCCT ATCCATCGATACTAGACTGAAACAAGTGTCTTCAAGCTG 108Appendix 7. Primers used for the construction of expression vectors pESC-leu2d-15529 CACTAAAGGGCGGCCAACAAAATGGCTCCCCTTCTACTGCT ATCCATCGATACTAGTTCAACGATTGAAGGAACAT pESC-leu2d-12663 CACTAAAGGGCGGCCAACAAAATGGAGATGGAAATAATGAACTCA ATCCATCGATACTAGGAGTTTGTGTAAAATCAAGTGAGCAC pESC-leu2d-23831 CACTAAAGGGCGGCCAACAAAATGGTCATTACTGGCGAAG ATCCATCGATACTAGACTGAAACAAGTGTCTTCAAGCTG pESC-leu2d-23838 CACTAAAGGGCGGCCAACAAAATGGACACACTGTATACAT ATCCATCGATACTAGAATTTGAGACAGAAGGTTC pESC-leu2d-33614 CACTAAAGGGCGGCCAACAAAATGTTGGATCTCAACACAGC ATCCATCGATACTAGACCTCCATCAATGACCCCAACCCT pESC-leu2d-2739 CACTAAAGGGCGGCCAACAAAATGGAATCTACTTGGGCGTTC ATCCATCGATACTAGGAAAGAAACTTTTTTCATGACTT pESC-leu2d-6009 CACTAAAGGGCGGCCAACAAAATGGAGAACATATACTACTACCTTGCT ATCCATCGATACTAGATCAGAACAATGGCTTTCAAGTTGG 109Appendix 7. Primers used for the construction of expression vectors pESC-leu2d-23839 CACTAAAGGGCGGCCAACAAAATGGACACATTAATATGCCTGTACT ATCCATCGATACTAGAAGTTGAGAAAGAATGTTCACCA pESC-leu2d-4097 CACTAAAGGGCGGCCAACAAAATGTACATCTTTGTTATCCTCTCTTTGGT ATCCATCGATACTAGCTGAAGAAGATGTTTGATCATGGCA pESC-leu2d-4921 CACTAAAGGGCGGCCAACAAAATGGAGCTTCAATATCCTTCCATCTCT ATCCATCGATACTAGTTTCCCATCTTTGTAACAGCAGC
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Dang, Thu Thuy
Discovering and harnessing camptothecin hydroxylase enzymes in Camptotheca acuminata for chemoenzymatic synthesis of anticancer camptothecin derivatives
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