Biochemical studies in the elucidation of genes involved in tropane alkaloid production in Erythroxylum coca and Erythroxylum novogranatense

by

Olga P. Estrada, B. S.

A Thesis

In

Chemical Biology

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCES

Approved

Dr. John C. D’Auria Chair of Committee

Dr. David W. Nes Co-chair of Committee

Mark Sheridan Dean of the Graduate School

May, 2017

Copyright 2017, Olga P. Estrada

Texas Tech University, Olga P. Estrada, May 2017

AKNOWLEDGMENTS I would like to thank my mentor and advisor Dr. John C. D’Auria, for providing me with the tools to become a scientist, and offering me his unconditional support. Thanks to the members of the D’Auria lab, especially Neill Kim and Benjamin Chavez for their aid during my experimental studies. And of course, thank you to my family for always giving me the strength to pursue my goals.

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TABLE OF CONTENTS

AKNOWLEDGMENTS ...... ii

ABSTRACT ...... v

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

CHAPTER I ...... 1 1.1 Alkaloids ...... 1 1.2 Ornithine-Derived Alkaloids ...... 2 1.3 Lysine-Derived Alkaloids ...... 3 1.4 Nicotinic Acid-Derived Alkaloids ...... 4 1.5 Tyrosine-Derived Alkaloids...... 4 1.6 Tryptophan-Derived Alkaloids ...... 5 1.7 Anthranilic Acid-Derived Alkaloids ...... 6 1.8 Histidine-Derived Alkaloids ...... 6 2.1 The Polymerase Chain Reaction (PCR) ...... 6 2.2 The Basic Polymerase Chain Reaction ...... 7 2.3 Specific PCR Techniques ...... 9

CHAPTER II...... 11 Abstract ...... 11 2.1 Introduction ...... 11 2.2 Similarities and Differences in Medicinal Properties ...... 13 2.3 The Scattered Distribution of Tropanes and Granatanes Amongst Angiosperms .. 15 2.4 Biosynthesis of TAs and GAs ...... 20 2.5 Tropane Alkaloid Biosynthesis ...... 20 2.6 Granatane Alkaloid Biosynthesis ...... 31 2.7 Metabolic engineering ...... 39

iii Texas Tech University, Olga P. Estrada, May 2017 2.8 Conclusions ...... 44

CHAPTER III ...... 46 3.1 Introduction ...... 46 3.2 qPCR Experimental Components ...... 48 3.3 Genes of Interest and Transcript Profiling in E. coca and E. novogranatense...... 49 3.4 Materials and Methods ...... 52 3.4.1 Plant Material ...... 52 3.4.2 RNA Extraction and cDNA Synthesis ...... 52 3.4.3 Primer Design and Optimization ...... 53 3.5 Reference Gene Selection ...... 53 3.6 Quantitative Real-Time PCR ...... 53 3.7 Data Analysis ...... 54 3.8 Results ...... 54 3.8.1 Primer Design and Optimization ...... 54 3.8.2 Melting Curve Analysis ...... 56 3.8.3 Primer Efficiencies ...... 63 3.8.4 Relative Quantification of Genes of Interests in E. coca and E. novogranatense ...... 70 3.9 Discussion ...... 72

CHAPTER IV ...... 73 4.1 Introduction ...... 73 4.2 Materials and Methods ...... 74 4.2.1 qPCR experiments ...... 74 4.2.2 Construct ...... 74 4.2.3 Enzymological experiments...... 74 4.3 Results ...... 78 4.3.1 Relative Quantification of EcSAMT Transcript Levels ...... 78 4.3.2 Expression optimization of EcSAMTpH9GW ...... 84 4.4 Discussion ...... 89

REFERENCES ...... 90

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ABSTRACT Natural products are vastly used in the modern pharmacopoeia. Among the most abundant natural products in medicine, there are tropane and granatane alkaloids. Efforts in the investigation of tropane and granatane biosynthesis are now contributing to the metabolic engineering sciences. Here, we present a portion of the most fundamental research in tropane alkaloid-producing transcriptomics with the use of qPCR techniques along with enzyme characterization experiments. In addition, a brief study on the enzyme responsible for the formation of methyl salicylate in Erythroxylum species is presented.

v Texas Tech University, Olga P. Estrada, May 2017

LIST OF TABLES 1. Summary of radiolabel feeding studies…………………………………….. 33 2. Summary of recent metabolic engineering studies using elicitors….……... 40 3. Primer Efficiency Values in E. novogranatense…….…...... 63 4. Primer Efficiency Values in E. coca.. ………………………………………63 5. Primer Efficiency Values in E. coca……………………………………….. 80 6. Primer Efficiency Values in E. novogranatense…………………………… 80

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LIST OF FIGURES 1. (a) The tropane core skeleton; (b) The bicyclic granatane core skeleton. Both structures depict their chemically accepted carbon numbering……..………12

2. Examples of granatane alkaloids and granatane alkaloid derivatives found among several plant species……………………………………………...……....17

3. The diversity and similarities of tropane and granatane producing alkaloids among angiosperms. The orders tropane alkaloids belong to are highlighted in orange. The orders granatane alkaloids belong to are highlighted in blue. Shared orders are represented by both colors. The plant families that are known to produce tropane and granatane alkaloids are branched off from the orders. The scale at the bottom represents millions of years. ……………....…....19

4. Tropane alkaloid biosynthesis up to the formation of the N-methyl-∆1-pyrrolinium cation. Arginine, ornithine and proline are interconvertable, sharing the pyrroline-5-carboxylate intermediate (highlighted in blue). Putrescine can be formed directly via decarboxylation of ornithine or indirectly through arginine. Methylation of putrescine is followed by oxidation to yield 4-methylamino butanal, which is then spontaneously cyclized to yield the N-methyl-∆1-pyrrolinium cation. Enzymes are highlight in orange and abbreviated as follows: ADC, arginine decarboxylase; AIH, agmatine imino hydrolase; NCPAH, N-carbamoylputrescine amino hydrolase; ODC, ornithine decarboxylase; PMT, putrescine N-methyl transferase; MPO, methyl putrescine oxidase...... 21

5. Tropinone formation from the N-methyl-∆1-pyrrolinium cation in which there are two possibilities for the condensation of the tropane ring. Acetyl-CoA is utilized via acetoacetate to yield hygrine-1-carboxylic acid. On the other hand, two successive decarboxylative condensations of malonyl-CoA by PKS (polyketide synthase) yields 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoyl-CoA. Enzymes are highlighted in orange and substrates are highlighted in blue………………………………….25

6. Tropinone is converted into tropine via TRI, tropinone reductase I. Tropine utilizes phenyllactoyl-CoA to yield littorine, which then uses a cytochrome p450 enzyme to for hyoscyamine. Scopolamine is epoxidized by the enzyme H6H, 6β-hydroxy hyoscyamine epoxidase, in a two-step process. Enzymes are highlighted in orange and substrates are highlighted in blue………………………..………………………………………27 vii

Texas Tech University, Olga P. Estrada, May 2017 7. Ecgonone-CoA ester undergoes a multi-step process to yield methylecgonone, which which is reduced by methylecgonne reductase, MecgoR to produce ethylecgonine. Cocaine is then formed by the acylation of methylecgonine with benzoyl-CoA. Enzymes are highlighted in orange and substrates are highlighted in blue………………………………….....29

8. Three proposed hypothetical routes to the production of granatane alkaloids. Hypothesized enzymes are presented in orange and hypothetical cofactors are presented in blue. The abbreviated enzymes and cofactor are as follows: ODC, ornithine decarboxylase; LDC, lysine decarboxylase; CMT, cadaverine N-methyl transferase; MPO, methyl putrescine oxidase; PKS, polyketide synthase; LMT, lysine N-methyl t ransferase; MLDC, N-methyllysine decarboxylase SAM, S-adenosyl-L-methionine………………………………………………………...36

9. EIC of enzyme assays performed using EcCHS with the substrates ρ-coumaroyl Co-A and malonyl Co-A. The expected product, naringenin chalcone is present with M+ 273 and compared to a known naringenin standard. EcCHS:PKS Assay was not able to make oxobutanoate…………….....50

10. Total and Extracted ion chromatograms (TIC and EIC) of enzyme assays performed using enzymes EcPKS and AbPKS and substrates malonyl-CoA and N-methylpyrrolinium cation, the expected oxobutanoate product is present (M+ 186). The no enzyme controls show no activity………………..….. 51

11. Primer optimization analysis in 4% acrylamide gels. All tested primers are labeled on top of the corresponding lane. 100 bp ladder is present at the extremities of both gels. A) Primer optimization in E. coca cDNA. B) Primer optimization in E. novogranatense cDNA. Non-template controls are denoted as NTC……………………………………………………...55

12. Melting curve analysis of reference genes in E. coca. A) Melting curve of reference gene Ec6409 in E. coca. B) Melting curve of reference gene Ec10131 in E. coca………………………………………………………….…....57

13. Melting curve analysis of EcPKS amplicons in E. coca. A) Melting curve of EcPKS in E. coca. B) Melting curve of EcPKS2 in E. coca……...... 58

14. Melting curve analysis of EcCHS amplicon in E. coca...……………...….……..59

15. Melting curve analysis of reference genes in E. novogranatense. A) Melting curve of reference gene Ec6409 in E. novogranatense. B) Melting curve of reference gene Ec10131 in E. novogranatense……………………………………………………..………60 viii

Texas Tech University, Olga P. Estrada, May 2017

16. Melting curve analysis of EcPKS amplicons in E. novogranatense. A) Melting curve of EcPKS in E. novogranatense. B) Melting curve of EcPKS2 in E. novogranatense……………………………………………….…...61

17. Melting curve analysis of EcCHS amplicon in E. novogranatense…………...…..62

18. Standard curves of reference genes in E. coca. A) Standard curve of reference gene Ec6409 in E. coca. B) Standard curve of reference gene Ec10131 in E. coca. Each point represents a dilution of template………………...64

19. Standard curves of EcPKS primers in E. coca. A) Standard curve of EcPKS primers in E. coca. B) Standard curve of EcPKS2 primers in E. coca. Each point represents a dilution of template…………………………………...…65

20. Standard curves of EcCHS primers in E. coca. Each point represents a dilution of template……………………………………………………….……...66

21. Standard curves of reference genes in E. novogranatese. A) Standard curve of reference gene Ec6409 in E. novogranatese. B) Standard curve of reference gene Ec10131 in E. novogranatense. Each point represents a dilution of template……………………………………………………….……...67

22. Standard curves of EcPKS primers in E. novogranatense. A) Standard curve of EcPKS primers in E. novogranatense. B) Standard curve of EcPKS2 primers in E. novogranatense. Each point represents a dilution of template…………………………...………………………………….68

23. Standard curves of EcCHS primers in E. novogranatense. Each point represents a dilution of template……………………………………………….....69

24. Relative quantification analysis of EcCHS, EcPKS and EcPKS2 genes. A) Relative quantification analysis in E. coca. B) Relative quantification analysis in E. novogranatense. Error bars represent standard deviation...... 71

25. Melting curve analysis of EcSAMT amplicons. A) Melting curve of EcSAMT in E. coca. B) Melting curve of EcSAMT in E. novogranatense…………………………………………………....…………..79

26. Standard curves of EcSAMT primers A) Standard curve of EcSAMT primers in E. coca. B) Standard curve of EcSAMT primers in E. novogranatense. Each point represents a dilution of template…………..……..81

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Texas Tech University, Olga P. Estrada, May 2017 27. Relative quantification analysis of EcSAMT gene. A) Relative quantification analysis in E. novogranatense. B) Relative quantification analysis in E. coca. Error bars represent standard deviation……………..……….83

28. SDS-PAGE of EcSAMTpH9 expression in E. coli BL21 (DE3) cell line………………………………………………………………..………….84

29. SDS-PAGE of EcSAMTpH9 expression in E. coli BL21 Rosetta cell line…………………………………………………………………………...85

30. SDS-PAGE of EcSAMTpH9 expression in E. coli BL21 Rosetta cell line using the OvernightExpress autoinducible expression medium……………….…86

31. SDS-PAGE of EcSAMTpH9 affinity purification of expression in E. coli BL21 ArcticExpress cell line………………………………………….….88

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CHAPTER I INTRODUCTION

1.1 Alkaloids

The secondary metabolism of living organisms produces what we know as natural products. These chemicals are used by plants mainly as defensive, reproductive, and communication means. Natural products have been extensively exploited by humans since the beginnings of civilization. A large portion of the pharmacopoeia is derived from compounds made by nature. Today, natural products are still used as a template for the development of novel pharmaceuticals in all fields of medicine. The manipulation of natural compounds by the addition or deletion of specific functional groups is performed to re-purpose or enhance their physiological activity. One of the most widely used groups of natural products in pharmaceuticals is the alkaloids. Among the alkaloids essentially used in modern medicine, there is atropine, scopolamine, cocaine, hyoscine, morphine, emetine, and ergometrine. In the nineteenth century, the isolation of alkaloids had a major impact in their use in medicine. Among the first alkaloids to be isolated and investigated were narcotine in 1803 by Derosne, opium in 1806 and morphine in 1816 by Sertürner1. The structures of the alkaloids isolated during this time were not fully understood until the next century. Modern methodology allows for the investigation of alkaloids to proceed more rapidly and precisely. The number of characterized alkaloids has been growing in direct relation to the availability of new technology. Today, there are over 27,000 different alkaloids reported in the literature, the vast majority isolated from plants 2. Although there is not a set definition for alkaloids, these compounds present key characteristics that define them. Alkaloids are nitrogen-containing compounds with low molecular weight. Although one could infer by the name that these compounds have alkaline basicity, their pH may vary greatly. The nitrogenous moiety of alkaloids is derived from several amino acids of primary metabolism. The principal amino acid

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Texas Tech University, Olga P. Estrada, May 2017 starting molecules for alkaloids are ornithine, lysine, nicotinic acid, tyrosine, tryptophan, anthranilic acid, and histidine. Their building blocks are often derived from acetate- containing biomolecules, like malonate, but can also be derived from molecules of the shikimate, or methylerythritol phosphate pathways. The following sections describe the different groups of alkaloids found in nature and examples currently used in medicine.

1.2 Ornithine-Derived Alkaloids

L-ornithine is a non-protein amino acid that in the primary metabolism of plants is derived from L-glutamic acid. The polyamine alkaloids such as putrescine, spermidine and spermine are compounds derived from ornithine. These compounds play important metabolic roles in a large variety of organisms including plants, bacteria, fungi, insects, and mammals including humans. The enzyme responsible for the production of spermidine, spermidine synthase, is currently being studied as a drug target in the malaria parasite Plasmodium falciparum 3. The pyrrolidine alkaloids are those alkaloids that derive from pyrrolidine. In this manuscript, we mention in detail a pharmaceutically important subclass of the pyrrolidine alkaloids, the tropane alkaloids (for more details on tropane alkaloids please refer to Chapter II). The pyrrolidine ring system of pyrrolidine alkaloids, ∆1-pyrrolinium cation, is often derived from the four-carbon molecule putrescine. Putrescine is then N-methylated and deaminated promoting the formation of ∆1-pyrrolinium cation. The poly-carbon moieties of these alkaloids are formed from the addition of acetyl units from biological acetyl-containing molecules. Similarly to the pyrrolidine alkaloids, the core structure of pyrrolizidine alkaloids forms from putrescine which in turn derives from arginine. Via a transfer of an aminopropyl group from spermidine to putrescine, the molecule is converted into the polyamine homospermidine. Homospermidine is converted into the pyrrolizidine core structure via an oxidative deamination, an imine formation, and an intramolecular Mannich reaction. This reaction scheme is similar to the proposed mechanism for the formation of the tropane ring in tropane alkaloids. Pyrrolizidine alkaloids are found in several genera of the Boraginaceae, the Compositae, the Orchidaceae, and the

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Texas Tech University, Olga P. Estrada, May 2017 Leguminosae families 4-6. The presence of pyrrolizidine alkaloids in the pharmacopoeia is scarce because of their high hepatotoxicity due to their 1,2-unsaturation in the pyrrolizidine ring and an ester function on the side chain 7.

1.3 Lysine-Derived Alkaloids

The pathway by which L-lysine is incorporated into alkaloids is analogous to that of the previously described incorporation of L-ornithine. Lysine and ornithine only differ by one carbon atom; ornithine is capable of forming five-membered ring core structures, while lysine is capable of forming six-membered ring core structures. Retention of the nitrogen atom and carboxyl group of lysine in the core structure of the alkaloid happens as in the case of ornithine-derived alkaloids. The piperidine alkaloids are a subclass of lysine-derived alkaloids that have a core structure ring derived from a ∆1-piperidine cation. Within this group, we can find the granatane alkaloids (for more details on granatane alkaloids please refer to Chapter II). The starting molecule for the formation of the piperidine core structure is hypothesized to be cadaverine 8, and the repeating units for the formation of the poly-carbon chain in the alkaloid are believed to be derived from acetyl-containing biological molecules, such as malonyl-CoA9. The quinolizidine alkaloids, mainly found in species of the Lupinus genus, are composed of a bicyclic ring system derived from two molecules of lysine10. These alkaloids are mainly found in the family Leguminosae and are used by the plant as defense compounds 11. Cystisine, from Laburnum anagyroides, has been used for many years as an aid to stop smoking due to its nicotinic acetylcholine receptor agonist action12. Indolizidine, a nine-membered bicyclic structure, is the core of indolizidine alkaloids, which are hypothesized to be derived from L-pipecolic acid 13. These compounds are present in the Leguminosae family and in some fungi. A medically important indolizidine alkaloid is swainsonine, that has shown therapeutic activity against

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Texas Tech University, Olga P. Estrada, May 2017 the Human Immunodeficiency Virus (HIV) due to its inhibitory action against glycosidase enzymes 14.

1.4 Nicotinic Acid-Derived Alkaloids

Vitamin B3, also known as nicotinic acid and niacin, is the origin molecule for alkaloids containing a pyridine ring fused with a pyrrolidine ring or a piperidine ring core structure. These molecules are also known as pyridine alkaloids; within this sub-class, nicotine and anabasine are among the most studied compounds. Pyridine alkaloids are found in abundance in Nicotiana tabacum from the Solanaceae family. Their biosynthetic pathway has been extensively studied for several decades 15.

1.5 Tyrosine-Derived Alkaloids

For tyrosine to be utilized in the biosynthesis of phenylethylamines and simple tetrahydroisoquinoline alkaloids, it first must undergo Pyridoxal Phosphate (PLP)- dependent decarboxylation to produce tyramine, the active phenylethylamine in alkaloid biosynthesis. This group of alkaloids is produced by mammals (e.g. dopamine and adrenaline) and in several plant species of the Cactaceae (e.g. mescaline) and the Papaveraceae (e.g. reticuline) among others. Opium alkaloids such as morphine and opium are commonly synthetically derived from the natural formation of the benzyltetrahydroisoquinoline alkaloid (R)-reticuline, a process known as semi-synthetic synthesis16. Although opium alkaloids are found in nature, the quantities at which they are available are not pharmaceutically and commercially relevant. Therefore, the semi- synthetic synthesis method is preferred. Phenethylisoquinoline alkaloids are analogs to benzyltetrahydroisoquinolines in their biosynthesis, but their core structure contains one extra carbon between the tetrahydroisoquinoline and the benzyl moieties. Among the phenethylisoquinoline alkaloids, we can find compounds like colchicine, which has been tested as an anti-cancer therapy17. These compounds are mainly found in several genera of the Liliaceae family. A small class of tyrosine-derived alkaloids is the terpenoid tetrahydroisoquinoline

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Texas Tech University, Olga P. Estrada, May 2017 alkaloids. Terpenoid tetrahidroisoquinolines are found in the roots of Cepahaelis ipecacuanha from the Rubiaceae family as emetine and cephaeline. These compounds are the active ingredients of C. acuminate and ipecacuanha root preparations used by South American Indians as a remedy for amoebic dysentery 18.

1.6 Tryptophan-Derived Alkaloids

The indole moiety of tryptophan can serve as a precursor for indole alkaloid biosynthesis. One of the most ubiquitous groups of tryptophan-derived alkaloids in nature is the simple indole alkaloids. Melatonin, a simple indole alkaloid, is found in mammals as a hormone involved in the regulation of the circadian rhythm. Psilocin and psilocybin are compounds that highly resemble the molecular characteristics of melatonin. Psilocin and psilocybin are the active ingredients of hallucinogenic mushrooms. The largest group of tryptophan-derived alkaloids is the group of terpenoid indole alkaloids. The terpenoid indole alkaloids are one of the largest groups of alkaloids found in the plant kingdom. For this reason, this group of compounds has been extensively studied and many of the enzymes involved in their production have been characterized19. Reserpine, a terpenoid indole alkaloid, has been used as a mild tranquilizer 20, while vinblastine and vincristine, are present in some of the most effective anti-cancer therapies available 21. The indole characteristic of tryptophan can also serve as a precursor for the quinoline ring core of quinoline alkaloids. When the indole nucleus in terpenoid indole alkaloids has been rearranged into a quinoline system it can be incorporated into the nucleus of quinoline alkaloids. Among the quinoline alkaloids, there are important compounds used for the treatment of malaria, such as quinine 22 and anti-cancer agents such as camptothecin derivatives 23. When the indole ring is rearranged into a pyrroloindole system it can be incorporated into pyrroloindole alkaloids. The pyrroloindole alkaloid, physostigmine, has been widely used in biomedical research to study the function of acetylcholine as a neurotransmitter due to its cholinergic activity 24. Ergot alkaloids, a type of indole alkaloids, are naturally produced via a combination of fungal and plant metabolism25. Although these alkaloids have been found

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Texas Tech University, Olga P. Estrada, May 2017 in several plant genera, they seem to have been originated from fungi26. Ergot alkaloids, usually found in grain, are known for their toxic effects in humans which include nausea, vomit, vasoconstriction, vertigo, and hallucinations, among other life-threatening effects27.

1.7 Anthranilic Acid-Derived Alkaloids

Although anthranilic acid is involved as an intermediate in the biosynthesis of indole alkaloids, there is a group of alkaloids that utilizes anthranilic acid as a direct precursor. The quinazoline, the quinoline, and the acridine alkaloids have a conserved anthranilic acid moiety in their core structure. A medicinally relevant example of a quinazoline-derived alkaloid is Bromohex, a veterinary expectorant derived from the quinazoline alkaloid peganine.

1.8 Histidine-Derived Alkaloids

Histidine has a characteristic imidazole moiety. Several alkaloids that contain an imidazole moiety at their core structure are hypothesized to be derived from histidine, these alkaloids are known as imidazole alkaloids. Histidine is converted to histamine by the enzyme histidine decarboxylase in humans as a response to allergic reactions. Although experimental data is still incomplete, it has been hypothesized that a similar mechanism is present in several plants able to produce imidazole alkaloids. Pilocarpus jaborandi, known for its common name jaborandi, is responsible for the production of the imidazole alkaloid pilocarpine, a compound used in the treatment of glaucoma28.

2.1 The Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is a molecular technique that exponentially amplifies a DNA fragment to enable scientists to perform extensive research on a gene of interest. The first record of the utilization of this technique available in the literature dates back to 1985, where Randall K. Saiki and colleagues were able to “enzymatically amplify” a β-globin target sequence to aid in the diagnosis of sickle cell anemia during prenatal stages29. The second record present in the literature was a publication dated to 6

Texas Tech University, Olga P. Estrada, May 2017 1986 titled Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. In this work, K. Mullis et al, describe the novel technique in detail and highlight its remarkable benefits30. The development of the PCR granted Mullis half of the Nobel Prize in chemistry in 1993. Soon after its first appearance in the literature, the PCR has been manipulated and modified to achieve different purposes spanning all fields of life-sciences research.

2.2 The Basic Polymerase Chain Reaction

Although there is a vast array of available PCR techniques developed to meet the necessities of specific investigations, all of these techniques rely upon the basic polymerase chain reaction principles. The indispensable components of a PCR have been studied to reach optimization and detailed descriptions are available in the literature31. There are seven essential components of the basic PCR. A thermostable enzyme is needed to carry out the polymerase reaction. The most widely used enzyme in PCR is Taq polymerase, isolated from the extremophile organism Thermophilus aquaticus. Taq polymerase is the most appropriate enzyme of choice when performing traditional PCR, but other options are available for more specific methods. For example, if the fragment to be amplified will be used in cloning techniques and high fidelity is required, the enzyme of choice would be one with proofreading capabilities such as pfu polymerase from the organism Pyrococcus furiosus. If the purpose of the PCR is to synthesize first strand cDNA from RNA, the enzyme of choice would be a reverse transcriptase. The polymerization needs to be primed by a pair of designed primers corresponding to the 5’ and the 3’ ends of the fragment of interest. Primer design is one of most influential variables in PCR. Primers will determine the identity, yield, quality, and specificity of the reaction. When designing primers, several parameters should be taken into consideration. The target sequence should be analyzed and the potential primer sites should be chosen based on the following characteristics: the sites should be free of homopolymeric tracts, should not have a tendency to make secondary structures, not be self-complementary, and have no similar identity with another portion of the sequence on either strand of the target

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Texas Tech University, Olga P. Estrada, May 2017 genome. After listing the primer candidates, the selection of the most adequate primers should be based on the melting temperatures of the oligonucleotides, the G+C content should be between 40% and 60%, the length of the primer should be from 18-25 nucleotides, and if possible, the 3’ end of the primer should be G or C. To facilitate the task, several software programs are available for primer design, such as GenScript. The extender units of PCR are deoxynucleoside triphosphates (dNTPs). Usually, an equimolar mix of dNTP, dTTP, dCTP, and dGTP is used in the reaction. The presence of divalent cations in the reaction is crucial because all thermostable enzymes require them for function. The identity of the divalent cation required depends on the enzyme to be used in the reaction. All the PCR components should be suspended in a buffer solution to maintain pH. The buffer solution should be adjusted to a pH of 8.3-8.8 at room temperature, the usual selection of buffer is Tris-Cl. At 72° C the pH of this buffer will drop about one pH unit, reaching physiological pH whis is adequate for PCR. Lastly, the template DNA containing the sequence of interest should be added to the reaction. The amount of template DNA is usually about 1 ng. The process of PCR consists of three main steps, denaturation, annealing, and extension. The temperature at which double stranded DNA denaturation occurs depends on G+C content. A usual parameter used for this step is 95° C for 45 seconds, at this temperature we will be ensuring that the template gets denatured and the thermostable enzyme will not be damaged. The temperature at which the primers will anneal to the template DNA is determined by their melting temperature. The annealing temperature is usually 3° C to 5° C lower than the melting temperature of the primers. A gradient or TOUCHDOWN PCR would help in the determination of the annealing temperature for the specific pair of primers. The extension parameters are dependent on the polymerase to be used in the reaction, the optimum temperature at which the enzyme is stable and its polymerization rate. The temperature at which the thermostable enzyme Taq polymerase is able to catalyze polymerization is between 72° C and 75° C, therefore the temperature at which extension occurs is set to 72° C. To set the extension time, the rate of polymerization of the enzyme should be taken into consideration. The polymerization

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Texas Tech University, Olga P. Estrada, May 2017 rate of Taq polymerase is about 2000 nucleotides per minute at its optimal temperature, therefore 1 minute would suffice for the extension of 1000 bp. The number of cycles to be carried out to obtain a desired yield is dependent on the amount of template present in the reaction at the beginning and the primer efficiency. Usually, 30 cycles are performed before one of the reactants becomes limiting, this number of cycles is also sufficient to obtain acceptable yield of amplification by Taq polymerase.

2.3 Specific PCR Techniques

There are many PCR techniques available to achieve different purposes. To obtain cDNA amplicons from RNA samples Reverse Transcriptase-PCR (RT-PCR) is used. This method is further described in Chapter III. Rapid amplification of 5’ cDNA Ends (5’RACE) is performed when the 5’ end of the desired sequence is unknown32. The primers for this reaction are designed based on the known portion of the sequence and the addition of a homopolymeric tail to the 3’ end of the synthesized cDNA. When the sequence at the 3’ end is unknown, the technique to be used to amplify the desired fragment is Rapid Amplification of 3’ cDNA Ends (3’RACE). This technique consists of designing primers based on a poly(T) adaptor sequence attached to the 3’ end of the fragment and a partially known sequence on the 5’ end. As noted, both of these techniques require the partial knowledge of either of the termini of the fragment, and several PCR reactions need to be performed, therefore, these techniques are error prone. When the sequence of the protein of interest is known, the best method to be used in the elucidation of the corresponding DNA sequence is Mixed Oligonucleotide-Primed Amplification of cDNA (MOPAC PCR). Amplification by MOPAC PCR is achieved with degenerate primers; a mix of different possible primers designed based on the amino acid sequence of the protein of interest and the organism of origin. This method has shown to work with high efficiency33. When the fragment of interest is more than 2 kbp, Long PCR is appropriate. Conventional PCR conditions are not capable of amplifying such long fragments for several reasons (e.g. denaturation incompletion, polymerase detachment, incorporation of incorrect bases) therefore, a method able to surpass these

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Texas Tech University, Olga P. Estrada, May 2017 barriers was developed. Reaction conditions were optimized by several research groups during the early 1900’s, but the problem dealing with the incorporation of incorrect bases was solved by Wayne Barnes in 199434-35. Barnes used two different thermostable DNA polymerases, one time efficient and other with proofreading capabilities. One of the most sophisticated PCR techniques is the Quantitative PCR (qPCR) which relies on precise technical performance and several control protocols to support reliability. This method is used to precisely quantify transcript levels relatively to endogenous controls. For more on quantitative PCR please refer to Chapter III.

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CHAPTER II

TROPANE AND GRANATANE ALKALOID BIOSYNTHESIS: A SYSTEMATIC ANALYSIS

Abstract

Tropane and granatane alkaloids belong to the larger pyrroline and piperideine classes of plant alkaloids respectively. Their core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may share common ancestry in some orders while they may be independently derived in others. Tropane and granatane alkaloid diversity arises from the myriad modifications occurring to their core ring structures. Throughout much of human history, humans have cultivated tropane and granatane producing plants for their medicinal properties. This manuscript will discuss the diversity of their biological and ecological roles as well as what is known about the structural genes and enzymes responsible for their biosynthesis. In addition, modern approaches to producing some pharmaceutically important tropanes via metabolic engineering endeavors are discussed.

2.1 Introduction

Plants are sessile organisms and thus evolved natural products or “specialized metabolites” as a chemical response to both biotic and abiotic forces. Specialized metabolites are used by plants to defend themselves and communicate with other plants and organisms in their environments. Whilst the chemical diversity of plant specialized metabolites is vast with total numbers thought to exceed over 200,000 structures, common themes of structure and function have given rise to the repeated evolution of both their biosynthesis and biological roles 36. Moreover, the chemical structures and underlying biosynthetic enzymes of specialized metabolites serve as inspiration to medicinal and natural product chemists.

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Texas Tech University, Olga P. Estrada, May 2017 Many specialized metabolites are pharmacologically active and have been used by humans for therapeutic and recreational purposes since the beginning of recorded history. In particular alkaloids, pharmacologically active cyclic nitrogen containing metabolites derived from amino acids, are known for their pharmacological effects and frequently serve as the starting point for drug development 37. Some of the oldest domesticated medicinal plants have been those that produce alkaloids. For example, Erythroxylum coca, a species notable for the production of the tropane alkaloid cocaine, was used in Peruvian households at least 8,000 years ago 38. Similarly, pomegranate (Punica granatum) has a history of cultivation that goes back at least 10,000 years in Egypt and is well-known for the production of granatane alkaloids39.

Tropane (TA) and granatane (GA) alkaloids are structural homologues, sharing similar chemical compositions and core scaffolds. Despite their similarities, TAs and GAs show different distribution patterns across the plant kingdom. The N-methyl-8- azabicyclo[3.2.1]-octane core structure of TAs is found in over 200 alkaloids 37, 40-41 (Fig. 1). In contrast, N-methyl-9-azabicyclo[3.3.1]-nonane, the core scaffold of GAs, appears in considerably fewer alkaloid metabolites (Fig. 1). The bicyclic core structures of TAs and GAs differ by only one carbon atom, yet this difference alters the conformational preferences of each of the core skeletons 42. Furthermore, the presence or absence of a single carbon atom in the core rings of TAs and GAs alters their chemical and pharmacological activities.

Figure 1. (a) The tropane core skeleton; (b) The bicyclic granatane core skeleton. Both structures depict their chemically accepted carbon numbering.

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Texas Tech University, Olga P. Estrada, May 2017 2.2 Similarities and Differences in Medicinal Properties

Despite their structural similarities TAs and GAs have distinct medicinal properties. TAs have been considered panaceas throughout recorded history, especially because of their anticholinergic properties. Anticholinergics are a class of compounds used as drugs to block the action of the acetylcholine neurotransmitter to treat motion sickness and diseases such as Alzheimer’s and Parkinson’s 43. The methylated nitrogen in the core ring of cocaine (27) and other TAs serves as a structural analog of acetylcholine. TAs have been observed to attach to and inhibit muscarinic acetylcholine receptors 44. TAs found in the Solanaceae are well known for both their anticholinergic and antispasmodic properties that affect the parasympathetic nervous system 45-47. These plants have been used for pain relief, anesthesia, and as a treatment for drug addiction 45. Daturae Flos, the dried flowers of Datura metel also known as “yangjinhua” in China, has been utilized and recorded in the Chinese Pharmacopoeia as an anesthetic and was prescribed to treat cough, asthma and convulsions 48. Przewalkia tangutica is a rare medicinal solanaceous plant found in the Tibetan Plateau of China in which the roots, seeds and entire vegetative tissues are utilized 49. P. tangutica contains several biologically active TAs including anisodamine (23), scopolamine (24) and atropine (the racemic mixture of hyoscyamine) (22). The TAs present in P. tangutica are associated with many biological activities including analgesic, spasm modulation, pesticidal, and anti-inflammatory effects 49. Hyoscyamus niger, also known as henbane, has been utilized in Chinese traditional therapy as well as in Tibetan medicine 50. H. niger has been used as a sedative and sleep agent 51. Hyoscyamine (22) and scopolamine (24) are the dominant TAs of H. niger and both metabolites can cross the blood-brain barrier to effect the central nervous system 52. Scopolamine has more potent pharmaceutical activity when compared to hyoscyamine (22) and exhibits relatively fewer side effects, however the scopolamine (24) content of solanaceous plants is usually much lower than the hyoscyamine (22) content 46. Because of this, there is an ongoing effort to fully understand the biosynthesis of scopolamine (24) and other TAs within the Solanaceae (see section 4.).

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Texas Tech University, Olga P. Estrada, May 2017 The narcotic properties of cocaine (27), a TA from the non-solanaceous genus Erythroxylum, can be attributed to unique modifications of the TA core scaffold that are not present in TAs from solanaceous plants. The carboxylic acid methyl ester present at the C2 position is responsible for the binding of cocaine (27) to the dopamine transporter 53. Cocaine (27) has also been reported to block the reuptake of nor-epinephrine, serotonin (5-HT receptor) and dopamine (D-A receptor) by the binding of the aromatic ring present at the 3β position of the molecule to specific sites in these receptors, affecting the normal physiology of the central nervous system 54. This stereospecific conformation is dominant in TAs found in the Erythroxylaceae but is only a minor constituent in solanaceous plants. While no direct studies regarding the anticholinergic effects of GAs has been reported, a computational and NMR based study comparing the structures of TAs and GAs revealed that GAs adopt an N-axial form that is similar to many known TAs 55. The N-methyl group in the axial confirmation is thought to be the pharmacophore for TAs 56. Additionally, the granatane ring system provides the semisynthetic intermediate for the

potent antiemetic agents Dolasteron and Granisetron, which are serotonin 5-HT3 receptor agonists 57-58. These compounds are used as medicines to prevent acute nausea in patients undergoing chemotherapy and radiotherapy for the treatment of cancer 59.

GAs and TAs have different physiological effects beyond the central nervous system. Unlike TAs, GAs exhibit anti-proliferative effects on hepatoma cells [24]. Specifically, murine hepatoma (BNL CL.2) and human hepatoma (HepG2) cell lines were cultured with various doses of crude GA containing alkaloid fraction extracts from Sedum sarmentosum. Inhibition of excessive growth of tumor cells was observed, indicating that these compounds possess anti-cancer properties 60. Other physiological effects that distinguish GAs from TAs include the use of GAs as an anti-worm treatment. Since the isolation of the first GAs, these compounds have been claimed to possess anti-worm (anthelminthic) capabilities, which were then studied in detail by scientists in the University of Amsterdam in 1956. These studies focused on deriving which GAs possessed the highest anthelminthic activity. The anthelminthic activity of synthetic 14

Texas Tech University, Olga P. Estrada, May 2017 granatane and its derivatives were measured in liver fluke. Their results rendered the highest anthelminthic activity to the compound isopelletierine 61. These findings were later scientifically supported in 1963 with newer and better chemical methods 62. Fascioliasis, a disease caused by liver fluke Fasciola hepatica, is common in cattle. Molluscidal activity in pomegranate bark extracts was effective in killing of Lymnaea acuminata, the vector for F. hepatica 63-64. Beyond their medicinal properties, GAs also differ from TAs in that GAs have been found to be useful in the prevention of corrosion in the oil, gas and metal industries 65.

2.3 The Scattered Distribution of Tropanes and Granatanes Amongst Angiosperms

Tropane alkaloids are commonly found in the genus Erythroxylum of the Erythroxylaceae family. The Erythroxylum genus includes at least 230 species that are distributed throughout the tropical regions of the Americas 38, 66-67. Erythroxylum coca was one of the first domesticated plant species that provided nutritional, medicinal, and digestive properties to ancient civilizations by chewing the leaves of the plant 68. Most of the cultivated coca used for cocaine (27) production comes from this species 69. Albert Neimann first isolated cocaine (27) as a pure substance in 1860 70 and its use exploded in popularity following an endorsement by Sigmund Freud 71. The leaves of Erythroxylum novogranatense were also chewed by the elite class for their high content of methyl salicylate, which imparts a minty taste 72-73. This species is known as “Colombian coca” and is found to be cultivated in the mountains of present day Colombia. “Trujillo coca” (E. novogranatense var. truxillense) is a cultivar that is grown in dry and arid regions. This species is also rich in methyl salicylate and contains other flavoring qualities that are still used in the production of Coca Cola, however today the extracts are decocainized 69. Atropine, scopolamine (24), and hyoscyamine (22) are a few well-known TAs from the Solanaceae family. As discussed above, these compounds are commonly found in species such as Atropa belladonna, H. niger, and many members of the genus Datura. Scopolamine (24) was first isolated in 1888 from Scopolia japonica 74. In medieval

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Texas Tech University, Olga P. Estrada, May 2017 Europe, extracts from A. belladonna, were used as poisons, hallucinogens and aphrodisiacs 52. Five to ten berries of A. belladonna could kill a person. The toxicity of the extracts has also been used on arrows to poison victims 75. When extracts of A. belladonna are applied to the eyes, dilation of the pupils occurs 76. For this reason, women used A. belladonna as a cosmetic drug during the Renaissance. Women of the 15th century who were devoted to witchcraft also exploited the psychoactive effects of A. belladonna 51. Mucous membranes, such as those found in the walls of the oral cavity and the vulva, are readily susceptible to drug absorption. It is believed that the application of alkaloid-containing salves to the skin or vulva was achieved by the use of brooms. It gave users the feeling of being able to fly, feeding the folkloric associations of witches with brooms 52. Atropine was first isolated in 1833 from A. belladonna 77-78. The correct structure of atropine was obtained by Willstätter in 1889 after much deliberation and structural studies 52. Leaves of D. metel from solanaceous plants were used as herbal cigarettes in the 19th and 20th centuries to treat patients with asthma or other respiratory conditions 52. GAs include pelletierine (1), isopelletierine, pseudopelletierine (3), and N- methylpelletierine (2) and their derivatives anabasine (4) and anaferine (5) (Fig. 2). GAs are predominantly found in P. granatum although they have been characterized in other species such as S. sarmentosum, and Withania somnifera 79-82. The pomegranate tree is native to Iran, Afghanistan, Baluchistan, and Himalayas in Northern India. Pomegranate can also be found in the Mediterranean and Caucasus regions due to its ancient cultivation. Today, pomegranate is cultivated all over India, Southeast Asia, Malaysia, the East Indies, tropical Africa, and the United States 83. In 1879, French chemists Charles Tanret and Pierre J. Pelletier isolated a basic substance from the root bark of the pomegranate tree and characterized the salt 80. As of this writing, no studies regarding characterization of any structural genes or the enzymes responsible for the production of GAs has been reported. Interestingly, the isolation of pseudopelletierine (3) has been reported in the species Erythroxylum lucidum 84 suggesting that GAs and TAs may use similar biosynthetic machinery.

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Texas Tech University, Olga P. Estrada, May 2017 Scientific reports on the occurrence and distribution of TAs from angiosperm families other than the Solanaceae and Erythoxylaceae are few. When viewed against a phylogeny of angiosperms, the results reveal a scattered and non-contiguous distribution (Fig. 3). For example, TAs have been reported in members of the family Proteaceae. Compounds found in the family Proteaceae include the pyranotropanes, strobamine and bellendine as well as the compounds ferruginine and ferugine 85. In addition, a few members within the families Brassicaceae and Convolvulaceae have been found to produce calystegines, which are heavily hydroxylated forms of TAs 86. The main TA producing species are scattered among four orders, which include the Solanales, Malpighiales, Proteales and Brassicales. In the case of GAs, P. granatum from the family Lythraceae is the main producing species. However, the appearance of GAs has also been reported in members of the Solanaceae, Crassulaceae, and Erythroxylaceae families. These families belong to the orders Myrtales, Solanales, Malpighiales, and Saxifragales. This distribution pattern raises the important question about the biosynthetic origin of the respective GA and TA pathway. Namely, have these biosynthetic pathways arisen from a common ancestor or have they arisen independently in several cases? Recent evidence in TA biosynthesis suggests that certain biosynthetic steps have multiple origins 40. By extension, this could also mean that GA biosynthesis has arisen independently more than once.

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Figure 2. Examples of granatane alkaloids and granatane alkaloid derivatives found among several plant species.

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Figure 3. The diversity and similarities of tropane and granatane producing alkaloids among angiosperms. The orders tropane alkaloids belong to are highlighted in orange. The orders granatane alkaloids belong to are highlighted in blue. Shared orders are represented by both colors. The plant families that are known to produce tropane and granatane alkaloids are branched off from the orders. The scale at the bottom represents millions of years. Modifications of the phylogenetic tree is republished with permission from the Botanical Society of America, from Angiosperm diversification though time, Susana Magallon and Amanda Castillo, 96, 1, 2009.

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Texas Tech University, Olga P. Estrada, May 2017 2.4 Biosynthesis of TAs and GAs

Diversity among tropane and granatane alkaloids can also be seen at the biochemical level. Most data available regarding enzymes involved in TA biosynthesis comes from species within the Solanaceae family. Despite recent advancements, some critical steps in TA biosynthesis remain ambiguous. Additionally, recent advances in high throughput sequencing technologies, plant genomics, and biochemical methods have aided the progress of elucidating TA biosynthesis in other non-model species, specifically within the Erythroxylaceae. Lastly, even though the biosynthesis of GAs is predicted to be similar to TAs, plausibly a whole new set of enzymes were recruited for the biosynthesis of GAs. This would mean that untapped gene and enzyme diversity is present in the GA and TA biosynthetic pathways. It is therefore important to elucidate the biosynthesis of these alkaloids to help further understand specific parts of their mechanisms and their metabolic processes.

2.5 Tropane Alkaloid Biosynthesis

Overall, the process of producing either TAs or GAs in plants begins with the diversion of specific amino acids from primary metabolism into the formation of an initial nitrogen containing ringed compound. This heterocycle will in most cases proceed on to form a bicyclic structure in which the core skeletons are created (Fig. 1). Further modifications will add diverse functional groups to the core structure yielding the final end product. Ornithine (7) and arginine (12) are the amino acids predicted to be the starting substrates of TA biosynthesis (Fig. 4) 87. Feeding studies using 14C-proline into the roots of A. belladonna also suggest that proline (6) may be a starting amino acid for incorporation into the tropane moiety. Other studies using D. metel and D. stramonium have also shown the incorporation of proline (6) into TA compounds, such as scopolamine (24) and tropine (20) 88. Biosynthetically each of these three amino acids can generate a pyrroline-5-carboxylate intermediate, which can be interconvertible

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Texas Tech University, Olga P. Estrada, May 2017 between these amino acids 89. Consequently, feeding studies of labeled amino acids are difficult to interpret without further enzymological data.

Figure 4. Tropane alkaloid biosynthesis up to the formation of the N-methyl-1- pyrrolinium cation. Arginine, ornithine and proline are interconvertable, sharing the pyrroline-5-carboxylate intermediate (highlighted in blue). Putrescine can be formed directly via decarboxylation of ornithine or indirectly through arginine. Methylation of putrescine is followed by oxidation to yield 4-methylamino butanal, which is then spontaneously cyclized to yield the N-methyl-∆1-pyrrolinium cation. Enzymes are highlight in orange and abbreviated as follows: ADC, arginine decarboxylase; AIH, agmatine imino hydrolase; NCPAH, N-carbamoylputrescine amino hydrolase; ODC, ornithine decarboxylase; PMT, putrescine N-methyl transferase; MPO, methyl putrescine oxidase.

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Texas Tech University, Olga P. Estrada, May 2017 A nonsymmetrical intermediate has been proposed for the production of the pyrrolidine ring, if the amino acid ornithine (7) is first methylated at the γ-N position. A proposed alternative route includes the decarboxylation of ornithine (7) to form the polyamine putrescine (8) as the first step 90-91. Feeding studies of radioactively labeled ornithine-2-14C have shown that several Datura species incorporate a nonsymmetrical intermediate 90. However, a symmetrical intermediate has been reported for Nicotiana, Erythroxylum, and Hyoscyamus species 92. Symmetrical incorporation showed activity at positions C1 and C5 of the tropane ring and has been reported for Nicotiana, E. coca, and Hyoscyamus albus 90-91. A one-step enzymatic approach is possible to reach a symmetrical intermediate by converting ornithine (7) into putrescine (8) catalyzed by ornithine decarboxylase (ODC) 93. Another route to putrescine (8) can be taken by starting with the amino acid arginine (12). The decarboxylation of arginine (12) into agmatine (13) is catalyzed by arginine decarboxylase (ADC). Agmatine (13) can then be converted into N-carbamoyl putrescine (14) via agmatine imino hydrolase (AIH). The enzyme N-carbamoyl putrescine amido hydrolase (NCPAH) then yields putrescine (8). Both the ADC and ODC directed pathways have different and diverse outcomes in regards to primary and secondary metabolism. Putrescine (8) that was produced by ODC is important for the supply of polyamines for primary metabolic processes such as cellular differentiation, development and division 94. On the other hand, putrescine (8) that was produced by ADC in the Solanaceae is thought to be required for environmental stress related responses. The formation of N-methylputrescine (9) catalyzed by putrescine N- methyltransferase (PMT) is considered the first rate-limiting step in the TA biosynthetic pathway 95. PMT is a S-adenosylmethionine (SAM) dependent methyltransferase that attaches a methyl group to the nitrogen atom that ultimately appears in the tropane skeleton. The first PMT sequence to be isolated from plants came from tobacco (Nicotiana tabacum) 96. Since then, many other PMT related sequences have been isolated and characterized from other pyrrolidine alkaloid producing plant species. PMT belongs to a large family of enzymes that are involved in polyamine production. These

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Texas Tech University, Olga P. Estrada, May 2017 primary metabolites play an important role in stress physiology, senescence, and morphogenesis 97. Several lines of evidence suggest that PMT function has evolved from an ancestral spermidine or spermine synthases 98. This would suggest that an enzyme from primary metabolism was recruited for the production of secondary metabolites. Localization of the PMT protein is root specific for both nicotine and tropane producing solanaceous plants 99-101. While N-methylputrescine (9) is the predominant intermediate for solanaceous TAs, at least one alternative has been suggested. [6-14C]-1,5,10- triazadecane fed to Nicotiana glutinosa plants revealed that N-methylspermidine can also be incorporated into the pyrrolidine ring of nicotine 102. The next step in TA biosynthesis is the formation of 4-methylamino-butanal (10) from N-methylputrescine (9) catalyzed by methylputrescine oxidase (MPO). This enzyme coexists with a class of copper-dependent diamine oxidases that uses copper as a cofactor to oxidize a conserved tyrosine residue into a topaquinone, essential for enzyme catalysis 103. MPO was first characterized from the species N. tabacum 104-105. Although an MPO like gene has yet to be discovered in E. coca, the corresponding activity from intact plants fed 4-monodeuterated N-methylputrescine shows the same enantioselectivity as solanaceous plants that produce either nicotine or TAs 106. Using 13C fractionation techniques, researchers have found that both nicotine and hyoscyamine (22) share the same biosynthetic pathway, at least up to the N-methyl-∆1-pyrrolinium ion (11) 107. Subsequently, a hypothesis has been proposed that a metabolic channel prevails in which a multi-enzyme complex is active. The product of MPO, 4-methylamino-butanal (10), spontaneously cyclizes to form the N-methyl-∆1-pyrrolinium cation (11) 90. Feeding studies using ornithine-2-14C detected labeled 4-methylamino-butanal in D. stramonium plants 90. The N-methyl-∆1-pyrrolinium cation (11) serves as the first ring in the bicyclic tropane skeleton. While the biochemical role of PMTs and MPOs are generally accepted steps leading to the first ring closure of tropane intermediates in the Solanaceae, the enzymatic basis of the second ring closure is controversial. For many years, the compound hygrine (16), (R)- 1-(1-methylpyrrolidin-2-yl)-propan-2-one was thought to be a direct intermediate based

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Texas Tech University, Olga P. Estrada, May 2017 on feeding studies, however more recent studies have demonstrated that previous results are likely experimental artifacts 108-110. In solanaceous plants the best incorporation into 13 the second ring was achieved from racemic ethyl [2,3- C2]-4-(N-methyl-2-pyrrolidinyl)- 3-oxobutanoate 108, 111, a polyketide based molecule (Fig 5). Further evidence for the 13 involvement of a polyketide was demonstrated by the feeding of methyl (RS)-[1,2- C2,1- 14C]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate to the leaves of E. coca 112. This strongly implicates the involvement of acetate-derived metabolites (e.g. polyketides) during the formation of the second ring in TAs. There are limited types of enzymes capable of catalyzing this type of condensation reaction. In the case of benzylisoquinoline alkaloid biosynthesis a Pictet–Spenglerase enzyme was shown to condense dopamine with 3,4-dihydroxyphenylacetaldehyde 113. However, based on more recent isotope and radiolabeled feeding studies it is hypothesized that a type III polyketide synthase (PKS) reaction is involved in condensing acetate units onto the N-methyl-∆1-pyrrolinium cation (11) leading to the second ring closure of TA biosynthesis 108, 111, 114. Type III PKSs are a family of enzymes known to catalyze the iterative decarboxylative condensation of malonyl-CoA onto CoA-tethered substrates. Type III PKSs are promiscuous enzymes that have a broad tolerance for diverse substrates and are able to catalyze multiple reactions 115-116. Type III PKSs that use cyclic nitrogen-containing substrates have been previously characterized for their roles in alkaloid production 117-119. However, unlike these previous studies the predicted substrate in TA metabolism, N-methyl-∆1- pyrrolinium cation (11) is charged and lacks a CoA thioester.

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- CoA. Enzymes are - oxobutanoyl 3 - - CoA is utilized via acetoacetate to yield to yield acetoacetate via utilized is CoA -

pyrrolinium cation in which there are are pyrrolinium there two in which cation - 1 pyrrolidinyl) ∆ - 2 - - methyl - methyl - N (1 - id. On the other hand, two successive decarboxylative condensations of malonyl of decarboxylative condensations two successive hand, other id. the On Tropinone Tropinone formation the from

carboxylic ac 1 - - Figure 5. Figure tropane Acetyl ring. the of possibilities condensation the for hygrine PKS 4 (polyketide CoA by synthase) yields inand orange highlighted are highlighted in substrates blue.

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Texas Tech University, Olga P. Estrada, May 2017 Spontaneous decarboxylation at the C2 position following ring closure can be prevented by the formation of the methyl ester. This would explain the presence of the carbomethoxy group found in cocaine (27) as well as other TA producing plants found in the Erythroxylaceae to protect the carboxylic acid found in the ecgonone-CoA ester (19). The resulting compound, after the second ring closure, contains a keto function at the C3 position 40. This position must undergo a reduction in order for ester formation to occur. For the members of the Solanaceae, tropinone reductase (TR) enzymes catalyze the reduction of keto groups in the tropane ring. These enzymes are part of the short chain dehydrogenase/reductase (SDR) family that catalyzes NAD(P)(H)-dependent monomeric oxidoreductase reactions 120. Its activity controls the metabolic flux towards TA biosynthesis downstream 121. In solanaceous plants, two types of tropinone reductases exist, tropinone reductase I (TR I) and tropinone reductase II (TR II). They share a common tertiary “Rossman” fold structure, a conserved motif that consists of two pairs of α-helices and six parallel β-sheets, a catalytic active site with the motif YxxxK, and a dinucleotide cofactor-binding motif 122. These enzymes share more than 50% of amino acid sequence similarity and are also assumed to have evolved from a common ancestor 121. As little as five amino acid differences are needed to change the stereospecificity of the product 123. Although there is much similarity between these two enzymes, they can lead to two very different products at the end of its biosynthesis. TR I only converts the 3-keto function to a product that has a 3α-configuration (Fig. 6). This produces tropine (20), or 3α-tropanol, which is a precursor to a wide range of esterified TAs, such as the important solanaceous compound atropine 40. On the contrary, TR II produces an alcohol solely with a 3β-configuration called pseudotropine (3β-tropanol). This is then converted to different nonesterified TAs called calystegines. A gene duplication event is attributed to these two different TRs in the Solanaceae family 124.

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Figure 6. Tropinone is converted into tropine via TRI, tropinone reductase I. Tropine utilizes phenyllactoyl-CoA to yield littorine, which then uses a cytochrome p450 enzyme to for hyoscyamine. Scopolamine is epoxidized by the enzyme H6H, 6β- hydroxy hyoscyamine epoxidase, in a two-step process. Enzymes are highlighted in orange and substrates are highlighted in blue.

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Texas Tech University, Olga P. Estrada, May 2017 However in E. coca, a very different type of reductase enzyme was found for the reduction of the 3-keto function of methylecgonone (25) (Fig. 7). This is the first evidence of a polyphyletic origin for TA biosynthesis in plants. Methylecgonone reductase (MecgoR) was purified from crude protein extracts from coca leaves using classical biochemical techniques 40. This enzyme was found to be very different than the TR enzymes that catalyzed the reduction reaction of the 3-keto function in solanaceous plants. MecgoR shares an overall identity of less than 10% at the amino acid level when compared to any TRs. MecgoR belongs to an aldo-keto reductase (AKR) superfamily of enzymes. AKRs that have been characterized so far share a common α/β-barrel motif, using either NADH or NADPH as a cofactor 125. The MecgoR protein is localized in the palisade parenchyma of young developing leaves. This is in contrast to the localization of roots in TRs of solanaceous plants. This enzyme is also similar to other enzymes of alkaloid metabolism such as codeinone reductase and an enzyme of flavonoid biosynthesis, chalcone reductase. The stereospecific enzyme MecgoR catalyzes the conversion of methylecgonone (25) to the 3β-hydroxy-containing compound methylecgonine (26). MecgoR can also use tropinone (17) as a substrate but this only produces pseudotropine, which is consistent in E. coca with the presence of only 3β- hydroxy esters. Common TAs are esterified with aromatic or aliphatic acids, with the stereochemistry of the hydroxyl group dependent on the reductase used.

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Figure 7. Ecgonone-CoA ester undergoes a multi-step process to yield methylecgonone, which which is reduced by methylecgonne reductase, MecgoR to produce ethylecgonine. Cocaine is then formed by the acylation of methylecgonine with benzoyl-CoA. Enzymes are highlighted in orange and substrates are highlighted in blue.

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Texas Tech University, Olga P. Estrada, May 2017 The benzoic ester of methylecgonine (26) is cocaine (27). Methylecgonine (26) is a molecule that has little physiological activity until it is converted into cocaine (27) 126. There has been a prediction that an acyltransferase in E. coca utilizes benzoyl-CoA as the activated acid. This prediction was based on feeding studies using trans-[3-13C,14C]- cinnamic acid and the N-acetylcysteamine thioester of [3-13C,14C]-trans-cinnamic acid 127. Methylecgonine (26) undergoes esterification with a benzoyl moiety that was predicted to utilize benzoyl-CoA as the activated acyl donor 128. It was not determined if it arises from benzoyl-CoA or benzaldehyde, but the moiety was found to be derived from cinnamic acid 127-128. Acylation reactions of secondary metabolites in plants are catalyzed by several acyltransferase families, however only the BAHD acyltransferase is known to utilize the activated acyl-CoA thioesters 129. TAs are modified through the esterification of the hydroxyl function at the C3 position in the tropane ring. It has been established that in E. coca, the cocaine synthase reaction uses benzoyl-CoA and methylecgonine (26) as substrates. With the CoA-dependent nature of this enzyme along with the reported properties for the tigloyl-CoA:pseudotropine acyltransferase from D. stramonium, it was hypothesized that cocaine synthase is a member of the BAHD acyltransferase superfamily 130. This superfamily of enzymes is well known to participate in secondary metabolite modification of esters and amides 129. Cocaine synthase was found to be capable of producing both cocaine (27) via activated benzoyl-CoA thioester and cinnamoylcocaine via activated cinnamoyl-CoA thioesters 131. It has been determined that the acylation of the 3β-hydroxyl function of methylecgonine (26), catalyzed by cocaine synthase, forms cocaine (28) and other TAs in E. coca. The accumulation and biosynthesis of TAs in E. coca occur within the same tissue. Cocaine synthase is found localized in the parenchyma and spongy mesophyll of the leaves. These tissues are both responsible for the biosynthesis and storage of TAs in this species. 4-coumaroylquinate has been reported to assist in the storage of cocaine (28) and cinnamoylcocaine in E. coca 132. This is in contrast to TAs produced in the Solanaceae family where the core biosynthetic pathway is in the roots, while the metabolites accumulate in the aboveground portions of the plants 101.

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Texas Tech University, Olga P. Estrada, May 2017 The rearrangement of the hydroxyl group of the phenyllactic acid moiety of littorine (21) in TA side chain biosynthesis has drawn interest. The phenomenon occurs in both atropine and scopolamine (24) biosynthesis. A branched-chain residue, tropic acid, is formed from the linear chain phenyllactic acid. To better understand this process, feeding studies of radiolabeled compounds have tried to elucidate the mechanism of this reaction 133-137. Feeding studies and quantum chemistry calculations by Sandala et al. (2008) have led to the hypothesis that a cytochrome p450 coupled with an alcohol dehydrogenase is involved in the conversions of the littorine (21) precursor into hyoscyamine (22) 138. Li et al. (2006) were able to suppress cytochrome p450 CYP80F1 expression by using virus- induced gene silencing techniques that caused reduced levels of hyoscyamine (22) and promoted the accumulation of littorine (21) 139. Enzyme assays with synthetic deuteron and arylfluoro analogues of littorine (21) have successfully examined the role of a p450 140. Hyoscyamine (22) is converted into the epoxide scopolamine (24) via hyoscyamine 6β-hydroxylase (H6H). This enzyme was shown to be a 2-oxoglutarate-dependent dioxygenase from purified H. niger 141-142. Hydroxylation at the C6 position of hyoscyamine (22) followed by the epoxidation of anisodamine (23) (6β-hydroxy hyoscyamus) (23) is catalyzed by H6H. The localization of this enzyme was also determined to be exclusively in the pericycle of roots 143. New studies in the networking analysis of metabolites involved with TAs in Solanaceae are more complex and finely controlled than expected 144.

2.6 Granatane Alkaloid Biosynthesis

In this review, our definition of GAs includes piperideine derived compounds that incorporate a pelletierine (1) or N-methylpelletierine (2) core structure. There has been some confusion among scientists regarding the naming and configuration of pelletierine (1). Older reports sometimes use the name isopelletierine incorrectly to refer to the compound [R-1-(2-piperidyl) propan-2-one], which in reality corresponds to pelletierine (1). The name isopelletierine is the optically inactive racemate of pelletierine (1) 145. The

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Texas Tech University, Olga P. Estrada, May 2017 correct chemical name for the compound N-methylpelletierine (2) is 1-[(2R)-1-methyl- 2piperdinyl]-2-propanone. Pseudopelletierine (3), also referred to as granatanone, contains a bicyclic core and can be referred to as [9-methyl-9-azabicyclo [3,3,1] nonan-3- one]. Additionally, the presence of anabasine (4), in Nicotiana species would by extension mean that select members of the Solanaceae can be classified as granatane producing members. Furthermore, the solanaceous species W. somifera produces anabasine (4), which could also be classified as a GA. In addition, several Sedum species (family Crassulaceae) produce the compounds pelletierine (1), N-methylpelletierine (2), and pseudopelletierine (3). All of the species discussed above are members of the order Solanales and are more closely related to one another than they are to other granatane producing angiosperms (Fig. 3). This may suggest that, at least within this order of angiosperms, the biosynthetic pathways leading to either tropanes or granatanes are commonly derived. The last time members of the Solanales shared a common ancestor with granatane producing lines in the Myrtales is approximately 120 million years ago. This is similar to the distance between the tropane producing Erythroxylaceae and Solanaceae. The only research studies performed with the aim to demonstrate the biochemical precursors of GAs have been performed by feeding plants radiolabeled precursors. Table 1 provides a comprehensive summary of feeding studies performed in granatane- producing species as of this review. The labeled products were then subjected to analysis via chemical breakdown and modification. While these studies were informative, they also can be misleading when attempting to interpret them through the viewpoint of biochemical enzymes and mechanisms. The general consensus is that lysine (28) is the starting substrate for the entry into granatane biosynthesis. In several cases, different forms of labeled lysine were found incorporated into the core granatane structure 8-9, 146- 149.

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Texas Tech University, Olga P. Estrada, May 2017 Table 1. Summary of radiolabel feeding studies Species Studied Radiolabeled Where label was found Reference Compound

Punica granatum 1-14C-acetate N-methyl isopelletierine 148 14 Punica granatum 2- C-lysine N-methyl isopelletierine 148 Withania 2 -14C-lysine anaferine 148

somnifera

14 147 Punica granatum [1- C] acetate Isopelletierine, N- methylisopelletierine,

and pseudopelletierine Withania [1-14C] acetate anaferine 147

somnifera Punica granatum DL-[2-14C] lysine N-methylisopelletierine 147 asymmetrically

14 147 Withania DL-[2- C] lysine Anaferine asymmetrically somnifera

Punica granatum [N-methyl -14 C, 814 C] pseudopelletierine 147

methylisopelletierine Withania [8-14C] anaferine 147

somnif era isopelletierine Punica granatum [1,515C] cadaverine N-methylisopelletierine 150

And [3H] cadaverine and pseudopelletierine

nonrandomly

Punica granatum [14C] methionine N-methylisopelletierine 150 and pseudopelletierine Sedum Sodium [1,2,3,4 - N- methylpelletierine 9

13 sarmentosum C 4] acetate and 13 sodium [1,2- C2]

acetate

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Texas Tech University, Olga P. Estrada, May 2017 Table 1. Continued Species Studied Radiolabeled Where label was found Reference Compound

Sedum acre and DL-[6-14C] lysine, Only L enantiomer of 146 8 Sedum L-[4,5- H2] lysine, lysine was incorporated sarmentosum and D- [6-14C] lysine into N -methylpelletierine.

Nicotiana glauca DL-[6-14C] lysine, Only L enantiomer of 146 8 L-[4,5 - H2] lysine, lysine was incorporated and D-[6-14 C] lysine into anabasine.

Sedum [6-14C] lysine N-methylisopelletierine 149 sarmentosum

Sedum 6-14C -DL-lysine N-methylpelletierine 8 sarmentosum asymmetrically

Over the course of studies regarding granatane and piperideine alkaloid biosynthesis, there has been some controversy over the origin of the intermediates in the biosynthetic pathway. One of the main questions that has repeatedly been tested is

whether or not cadaverine ( ) is present in the pathway to form pelletierine ( ) and other 29 1 granatane derivatives. Several contradictory observations have been made depending on the type of label of the compound fed to plants as well as which species was used. The majority of the debate regarding the beginning intermediates arises when older radiolabel feeding studies in the Sedum species are used as a reference. These studies report the asymmetrical incorporation of the starting precursors into their respective alkaloids. However, other studies performed on pomegranate report that a symmetrical intermediate

(such as cadaverine (29)) must exist 151. Possible explanations for this problem could be that the members of the family Crassulaceae (e.g. Sedum species) do not utilize the same

enzymatic steps as other granatane producing members such as those found in the family

Lythraceae.

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Texas Tech University, Olga P. Estrada, May 2017 The three hypothesized biosynthetic pathways for the production of GAs will be referred to as Hypothesis I, Hypothesis II, and Hypothesis III, their schematic representation is shown in Figure 8. Hypothesis I is based on the observation that P. 14 14 3 granatum plants fed [1.5- C] and [1.5- C H2] cadaverine yields incorporation of the labels into N-methylpelletierine (2) and pseudopelletierine (3) 150. In this pathway, lysine (28) is first decarboxylated to form cadaverine (29) which would subsequently be methylated to form N-methylcadaverine (30) 148. N-methylcadaverine (30) would then be oxidized to form 5-methylaminopentanal (31) promoting a spontaneous cyclization to form the N-methyl-∆1-piperidinium cation (32). This product would then ultimately form N-methylpelletierine (2) or be converted to the corresponding oxobutanoate by the decarboxylative condensation of two malonyl-CoA which would then cyclize to form pseudopelletierine (3). Feeding studies performed in Sedum species report that cadaverine (29) is not present as an intermediate, giving rise to Hypothesis II. By feeding [1-14C] cadaverine, Leistner et al. (1990) have predicted that there should be a lysine decarboxylase enzyme that also contains oxidase activity such that the only cadaverine (29) intermediate that exists is always enzyme bound 151. Therefore, according to this hypothesis, lysine (28) is catalyzed in one enzyme or enzyme complex to produce 5- aminopentanal (34). The 5-aminopentanal (34) intermediate would cyclize to form a ∆1- piperidinium cation (35), which can then be methylated at the nitrogen atom to form the N-methyl-∆1 -piperidinium cation (32) or remain unmethylated and ultimately go on to form pelletierine (1).

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Texas Tech University, Olga P. Estrada, May 2017 methyl methyl - verine N methyl transferase; -

methionine. - L - adenosyl - to the production of granatane alkaloids. Hypothesized enzymes granatane Hypothesized of alkaloids. enzymes to production the methyllysine decarboxylase SAM, S - Three proposed hypothetical routes routes proposedThree hypothetical transferase; MPO, methyllysine putrescine oxidase; synthase;transferase; PKS,N polyketide LMT, Figure 8. Figure are presented in orange and hypothetical cofactors are presented in blue. The abbreviated enzymes and cofactor are as follows: ODC, ornithine decarboxylase; lysineLDC, decarboxylase; CMT, cada MLDC, N

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Texas Tech University, Olga P. Estrada, May 2017

In all three hypotheses the origin of the N-methyl group is via labeled methionine 150. The biochemical methyl donor for this incorporation would be S-adenosylmethionine (SAM). Hypothesis III also involves an early asymmetrical intermediate. In this hypothesis, the first biosynthetic step is the methylation of lysine (28) which occurs at the ε-N position producing ε -N-methyl-lysine (37). This compound would then be decarboxylated to form N-methylcadaverine (30) which would undergo oxidation to form 5-methylaminopentanal (31), promoting a spontaneous cyclization to from the N-methyl- ∆1-piperidinium cation (32). As described in Hypothesis I, the N-methyl-∆1-piperidinium cation (32) would ultimately form N-methylpelletierine (2) and pseudopelletierine (3). Radioactive acetate is incorporated into GAs regardless of which species is fed 8-9, 147-148. As is the case with tropane alkaloid biosynthesis, the enzymes involved in the extension and cyclization reactions of granatanes includes a putative polyketide synthase. Plants readily convert acetate into malonyl-CoA via the enzyme acetyl-CoA carboxylase 152. A type III polyketide synthase utilizing 2 units of malonyl-CoA and the N-methyl-∆1- piperidinium cation (32) would produce 4-(1-methyl-2-piperinidyl)-3-oxobutanoyl-CoA (33). The prediction of the presence of a type III polyketide synthase in GA biosynthesis 13 14 is supported by radiolabeling studies which fed ethyl (R,S)-[2,3- C2,3- C-]-4-(l-methyl- 2-pyrrolidinyl)-3-oxobutanoate to D. stramonium plants 108, 111, 114. The oxobutanoyl compound may then have several fates; its conversion to pseudopelletierine (3) by intermolecular interaction of the positively charged nitrogen and the carboxyl CoA, or its conversion to N-methylpelletierine (2) or pelletierine (1) by losing the carboxyl as CO2. Bicyclic GA producing species are similar to solanaceous plants producing tropanes, namely the loss of the carboxyl group due to a lack of methyl ester protection. As mentioned earlier in this review, the protection of the carboxyl group of oxobutanoate by methylation gives rise to the moiety responsible for the narcotic effects of cocaine (27), which would be an explanation why GAs do not exhibit narcotic effects 53. The enzymes responsible for carrying out the biochemical reactions described above are based on the reactions described and an extension of similar reactions carried out in 37

Texas Tech University, Olga P. Estrada, May 2017 tropane producing species. The decarboxylation of lysine (28) described in Hypothesis I would be performed by a P. granatum lysine decarboxylase (LDC). The presence of a lysine decarboxylase in the synthesis of anabasine (4) in Nicotiana species has been studied by the incorporation of [15N]-lysine into anabasine 153. The methylation of cadaverine (29) would be achieved with the help of a P. granatum cadaverine N- methyltransferase (CMT). CMTs have not been isolated and characterized in other species, but this enzyme is predicted to be related to putrescine N-methyltransferases (PMT) and spermidine synthases (SPDS). PMT cDNA sequences have been found in Nicotiana species by the sequencing of large genomic libraries 154-155. The enzymes SPDS and PMT have been found to share substrate specificity, but PMT is dependent of the decarboxylated S-adenosylmethionine (dcSAM) as a co-substrate. The oxidation of N-methylcadaverine (30) in P. granatum and Sedum species could be performed with the aid of an enzyme similar to the methylputrescine oxygenase (MPO) present in the biosynthesis of nicotine in N. tabacum. While a copper dependent oxidase is used for tropane alkaloid biosynthesis, it is not possible to rule out alternative enzymes such as polyamine oxidases that require FAD as a cofactor 156. The methylation of lysine (28) described in Hypothesis III would be performed by a lysine methyltransferase (LMT). The responsible enzyme may be related to the lysine methyltransferases ubiquitously present in eukaryotic primary metabolism for gene access regulation to chromatin 157. In both granatane and tropane alkaloid producing species, dimerized versions of intermediates within their respective pathways have been found. For example, cuscohygrine is the dimerized form of hygrine (16) 158. Originally it was believed that hygrine (16) was a true intermediate of the biosynthesis of TAs. However, it is most likely that cuscohygrine is a dimerized product of hygrine (16) which in turn is a breakdown product of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoyl-CoA (18). If this compound is present as a free acid under physiological conditions a β-ketoester is formed, β-ketoesters very often spontaneously decarboxylate 159. In the case of GA biosynthesis, anaferine (5) is the dimerization product of pelletierine (1). This also supports the presence of an oxobutanoate intermediate.

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Texas Tech University, Olga P. Estrada, May 2017 2.7 Metabolic engineering

There has been an increasing interest in the biosynthesis of tropane alkaloids (TAs), especially to up-regulate the production of valued compounds, such as atropine and scopolamine (24). The World Health Organization (WHO) includes these important pharmaceutical compounds on their list of essential drugs 160. In normal plant biosynthesis, the yields of the final compounds are in low quantities. Synthesizing TAs chemically in the lab has also been difficult and costly because of their stereochemical nature. Nocquet et al. (2016) attempted a total synthesis approach to produce the compound scopolamine (24), however their low yield of 16% does not make this method economically feasible 37. A major problem for the commercial production of scopolamine (24) in hairy root cultures is achieving industrial level yields 161. Researchers are now focused on metabolic engineering plants that produce these important compounds to increase final yields, or engineering microorganisms that will be able to produce the compounds from simple sugars or common precursors. A comprehensive table of the most recent metabolic engineering studies targeting specific genes can be seen on Table 2.

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Texas Tech University, Olga P. Estrada, May 2017 Table 2. Summary of recent metabolic engineering studies using elicitors.

Species Target Elicitor Effect Reference

compound

Anisodus Scopolamine Acetylsalicylic acid Increased scopolamine 162

luridus (ASA) content

Anisodus Scopolamine Ultraviolet ray-B Increased scopolamine 162

luridus (UV-B) content

45 Datura metel Atropine Staphylococcus aureus Decreased atropine content, increased root

biomass

Datura metel Atropine Bacillus cereus Decreased atropine 45

content, increased root

biomass

Datura metel Atropine Silver nitrate Decreased atropine 45

content, increased root biomass Datura metel Atropine Nanosilver Increased atropine 45 content, increased root

biomass

Datura Hyoscyamine Agrobacterium Increased 144

innoxia rhizogenes hyoscyamine content, increased root

biomass

163 Erythroxylum Cocaine Anderson Increased cocaine coca rhododendron content in calli

medium (ARM)

Erythroxylum Chlorogenic Salicylic acid Decreased CGA 163 coca acid (CGA) content

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Texas Tech University, Olga P. Estrada, May 2017

Due to its high demand in medicine, scopolamine (24) is the most popular choice for increasing yields via metabolic engineering. Past methods such as genetic breeding, polyploid breeding and radiation breeding have failed to yield a higher content of scopolamine (24) in A. belladonna 164. Researchers are now focused on the genes encoding rate-limiting enzymes in TA biosynthesis that can be genetically modified in planta. A common focal point centers on what is considered the first and last rate-limiting enzymes in the TA pathway, putrescine N-methyltransferase (PMT) and hyoscyamine 6- hydroxylase (H6H). The overexpression of only one PMT gene in transgenic hairy root cultures of A. belladonna did not change the total TA content 165. If the same PMT gene in D. metel was overexpressed, the TA content was significantly increased by almost four times that of the control 166. H6H has a high catalytic efficiency for converting hyoscyamine (22) to scopolamine (24) 167. The overexpression of the H6H gene resulted in an increase in the biosynthesis of scopolamine (24) in the transformed TA producing plant, A. belladonna. Another successful use of H6H was in transgenic Hyoscyamus muticus hairy root cultures, where scopolamine (24) levels increased to over 100 times that of the controls 168. Furthermore, overexpression of H6H in transgenic A. belladonna plants resulted in the leaf and stem alkaloid contents to be exclusively scopolamine (24) 167. Metabolic engineering endeavors are becoming more complex and are moving away from only modifying one gene at a time. When both PMT and H6H were overexpressed simultaneously in transgenic H. niger root cultures, scopolamine (24) biosynthesis increased and yielded a scopolamine (24) content of over nine times more than the wild type 169. In an important experiment testing whether metabolic engineering can occur between genes isolated from different species, overexpression of NtPMT and HnH6H in A. belladonna significantly increased scopolamine (24) content of secondary roots when compared to wild-type plants 46. More studies are needed to understand flux through the tropane biosynthetic pathway in order to increase the overproduction of alkaloids. It is clear that the network of contributing metabolites and pathways are complex and need to be understood on a broader level 170. 41

Texas Tech University, Olga P. Estrada, May 2017 D. metel produces important medicinal tropanes and is used by researchers because of its tractable hairy root culture system. Agrobacterium rhizogenes can transform plant roots to hairy roots by utilizing the Ri T-DNA plasmid it carries. Hairy roots that have been induced by A. rhizogenes have high growth rates, are genetically stable, and produce copious amounts of lateral roots 171. Increased TA biosynthesis correlated with an increase in root biomass. Biotic elicitors, such as yeast extract, bacteria, fungi and viruses, as well as abiotic elicitors, such as metal ions or inorganic components, are used and studied to increase the productivity of hairy roots. These elicitors can trigger different defense responses and phytoalexins in plants as well as improve the release of metabolites into the medium 45. Shakeran et al. (2015) focused on using Staphylococcus aureus and Bacillus cereus as biotic elicitors and silver nitrate and nanosilver as abiotic elicitors on the hairy root cultures of D. metel to see their effects on biomass and atropine production. When live bacteria are present in transformed root cultures, there is a considerable influence on secondary metabolite accumulation 172. Contrary to this, atropine content in the hairy roots of D. metel infected by B. cereus and S. aureus was reduced more than half when compared to the control. The authors hypothesize that this may be a cause of atropine secretion into the culture medium that was then converted into scopolamine (24) 45. However, scopolamine (24) was not analyzed in the spent media. The living bacteria can cause various influences on roots, effecting enzymes in the TA pathway to produce alkaloids in D. metel roots 172. Although atropine accumulation decreased with these biotic elicitors, the biomass of the roots slightly increased approximately 15% when compared to the control 45. Other attempts to engineer higher TA contents in plants include those that use abiotic elicitors. A summary of recent metabolic engineering studies using elicitors can be seen on Table 3. Silver nitrate can increase anisodamine (23) content in Anisodus acutangulus hairy root cultures 173. In addition, calcium and nitrate can increase hyoscyamine (22) content in D. stramonium hairy root cultures 174. Silver nitrate can inhibit the activation of ethylene and in doing so, promotes polyamine synthesis 175-176. As a consequence, the overall effect of silver nitrate treatment can be seen in the increase of root

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Texas Tech University, Olga P. Estrada, May 2017 biomass 175-177. Furthermore, silver nitrate has been demonstrated to elicit the production of phytoalexins which in turn increases TA levels in the root 178. Shakeran et al. (2015) used silver nitrate as an abiotic elicitor in D. metel and observed an increase in the transformed hairy root biomass of approximately 16%. However, only half of the expected atropine accumulation was observed in these treatments. Although secretion of atropine was not measured in this study, subsequent reports corroborated this hypothesis by finding a three-fold increase in alkaloids in the spent culture media following silver nitrate treatment 179. Still, further attempts at increasing alkaloid levels include the treatment using nanosilver particles. These differ from silver nitrate in their physicochemical properties 170, 180. Nanosilver particles adhere strongly to plant tissues and cause an increase in the activation of enzymes involved in secondary metabolite production. This treatment was used successfully in Artemisia annua and D. metel hairy roots increasing tropanes by at least 2.4 fold 45. Initial polyamine substrates such as putrescine (8) must be present in abundance during TA production for a high yield of the final alkaloid. Currently there are only a few studies attempting to engineer the TA pathway in microorganisms. Qian et al. (2009) engineered a strain of Escherichia coli capable of efficiently producing putrescine (8) 181. However, it was first necessary to reduce the flux of polyamine precursors through a competing pathway. Metabolic pathways for putrescine (8) degradation, uptake and utilization were also deleted. Stress to cells by the overproduction of putrescine (8) was handled by the deletion of RpoS, a stress responsive RNA polymerase sigma factor. To increase the conversion of ornithine (7) to putrescine (8), overexpression of ornithine biosynthetic enzymes and ornithine decarboxylase (ODC) was also necessary. The final metabolically engineered E. coli strain produced 1.68 g/L of putrescine (8) and high cell density cultures (HCDCs) produced 24.2 g/L of putrescine (8). This would be the first step for engineering alkaloid biosynthesis that relies on putrescine (8) in microorganisms. If metabolic engineers in the future wish to engineer these pathways in other organisms, such as bacteria or yeast, it will be necessary to up-regulate the beginning precursor pathways such that pools of these primary metabolites do not get depleted. Depletion of these

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Texas Tech University, Olga P. Estrada, May 2017 essential metabolites would result in the death of the organism during the biosynthesis of these compounds. Using plant in vitro cell culture or tissue culture is an important tool when studying the regulation and biosynthesis of secondary metabolites. Large quantities of plant material can be produced under controlled and sterile conditions. In tissue cultures of solanaceous plant species, using elicitors mimicking stress hormones can increase important secondary metabolite production 182. Recently, cell cultures of E. coca in the Erythroxylaceae were used to study TA biosynthesis 163. Various culture media were tested on their ability to support callus formation as well as cocaine (27) production. The jasmonic acid-isoleucine (JA-Ile) analogue coronalon and salicylic acid (SA) were also used as elicitors to observed their effects on calli metabolism. All three culture media growing calli accumulated cocaine (27). The medium used to grow calli also significantly affected natural product metabolism. The only treatments that yielded higher amounts of cocaine (27) were dependent upon culture media, not upon elicitor treatment. For example, Anderson rhododendron medium (ARM) produced cocaine (27) an order of a magnitude greater than both Gamborg B5 (GB5) and modified Murashige-Tucker medium (MMT), but lower levels of hydroxycinnamate-quinate esters such as chlorogenic acid (CGA) were detected. Interestingly the elicitors coronalon and salicylic acid did not yield any increase in TA production suggesting that TAs, at least in E. coca, may not be regulated by common plant defense hormones.

2.8 Conclusions

Recent advances in genomics, transcriptomic and metabolomic technologies are poised to illuminate the biosynthetic foundations of TAs and GAs. Future research on the biosynthesis of TAs and GAs will affect multiple fields of research. First, enzymes involved in TA and GA biosynthesis will expand our fundamental knowledge of chemistry and enzymology. Second, elucidation of the genes and enzymes underlying TA and GA biosynthesis will expand the molecular tools available to synthetic biologists. With these additional tools, scientists can look to not only produce TAs and GAs in

44

Texas Tech University, Olga P. Estrada, May 2017 heterologous hosts but also to engineer novel molecules 183. Lastly, future discoveries of the function and structure of genes and enzymes in TA and GA biosynthesis will strengthen our understanding of the evolution of plant metabolism. Plant metabolism, especially specialized metabolites such as TAs and GAs, evolved in response to dynamic environments. Detailed knowledge of the catalytic properties of TA and GA biosynthetic enzymes as well as their biophysical properties are critical to elucidating extant, ancestral, vestigial and promiscuous biochemical activities and chemical diversity of these metabolic pathways. This fundamental knowledge will be useful in predicting and engineering plants to withstand ongoing changes in the environment.

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Texas Tech University, Olga P. Estrada, May 2017

CHAPTER III TRANSCRIPT PROFILE OF GENES INVOLVED IN TROPANE ALKALOID BIOSYNTHESIS IN ERYTHROXYLUM COCA AND ERYTHROXYLUM NOVOGRANATENSE

3.1 Introduction

The real-time quantitative Polymerase Chain Reaction (qPCR) is a method designed to study gene expression in any given tissue. Although this technique is a powerful tool in molecular research, its reliability lies upon technical skills, preliminary control studies, the selection of the appropriate reference genes, and a robust statistical analysis. This technique is based on the principle that PCR is an exponential process. Therefore, the analysis of qPCR data should take into consideration all the factors that may affect the exponential characteristic of the process. Real-time qPCR differs from traditional qPCR, in that the former is a more sensitive and time-efficient technique. With traditional qPCR we are able to measure final products, while with real-time PCR techniques we are able to measure reaction products right after each cycle. The high sensitivity of real-time qPCR has numerous advantages, but if the appropriate controls are not implemented, the presentation of results may be erroneous. The components of each reaction are essentially the same as in traditional PCR with the addition of molecules able to report the presence of PCR products. The cDNA template is obtained from reverse transcription of total RNA extraction from the tissue of interest. cDNA first strand synthesis is performed by Reverse Transcriptase PCR (RT-PCR), where the activity of a reverse transcriptase enzyme enables the synthesis of cDNA from the extracted RNA. Several methods for the quantification of PCR products in real time have been developed. One of the most popular methods for PCR product quantification is the use of fluorescently labeled oligonucleotide probes. In this case, the fluorescence is generated only by the incorporation of the labeled oligonucleotide by Taq polymerase, preventing unwanted

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Texas Tech University, Olga P. Estrada, May 2017 signal emitted by nonspecific products. TaqMan probe is used as a method for quantification of PCR products in real time, it depends on a specific oligonucleotide that is able to anneal to an internal sequence of the gene of interest, this probe is different from the primers in that Taq polymerase in not able to extend this sequence. The TaqMan probe consists of the mentioned oligonucleotide sequence, a reporter molecule attached to the 5’ end of the probe, and a quencher molecule at the 3’ end of the probe. When the probe is present intact in the mixture the quencher comes in proximity to the reporter molecule, preventing fluorescence emission by florescence resonance energy transfer (FRET). As the probe gets incorporated into the amplicon, the exonuclease activity of Taq polymerase cleaves off the reporter and the quencher molecules. This way the reporter molecule is physically separated from the probe, allowing for emission of fluorescence. Therefore, in TaqMan qPCR fluorescence emission is proportional to amplicon quantity. The method for quantification of PCR products in real time that was selected for the experiments carried out throughout this project is PowerUp™ SYBR® green master mix. This master mix consists of company proprietary optimized buffers, a heat-optimized Uracyl N-Glycosilase (UNG) to prevent carry-over DNA contamination, and SYBR® green DNA biding dye. SYBR green is a DNA biding dye that specifically binds to the minor grove of double stranded DNA, therefore it is capable of fluorescently report the presence of PCR products. This dye is also capable of reporting the presence of unspecific hybridization products to a minor extent, this is the reason why the compendium of preliminary studies for real time qPCR includes Melting Curve data (further discussed in this chapter). As mentioned above, qPCR is a sensitive technique that requires optimal sample preparation and handling along with precise technical performance. The report of qPCR experiment details and analysis in the literature has been subject to standardization through guidelines to ensure reliability and repeatability184. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines address nomenclature, sample preparation, quantification of nucleic acids, qPCR assay requirements, and data analysis requirements, among other critical points that ensure the

47

Texas Tech University, Olga P. Estrada, May 2017 proper report of qPCR experiments in the literature. The MIQE guidelines also provide a detailed checklist for authors preparing qPCR study manuscripts for publication.

3.2 qPCR Experimental Components

Each step in the development of real time quantitative PCR should be carefully designed and performed to meet standards and guidelines to ensure reliability. The most critical step in the development of qPCR experiments is primer design and optimization, as this will determine the specificity of amplicons. Primers are designed to be highly specific to the gene of interest. In order to ensure primer optimization, amplification of the gene of interest is performed by a legacy PCR using cDNA derived from RNA isolated from the tissue of interest as a template and analyzed by agarose-gel electrophoresis. In this analysis, one aims to visualize the gel band size corresponding to the gene of interest and the absence of any other band, which would indicate poor primer specificity and/or primer dimer formation. After the data generated shows primer optimization, experiments to demonstrate primer efficiency are designed. An additional indicator for primer specificity is melting curve data, which is analyzed after every qPCR run. A single melting point present in the analysis is an indicator of primer specificity. Primer efficiency is calculated from a standard curve obtained from the real- time amplification of a series of dilutions of the template. The efficiencies are derived by plotting the cycle threshold (Ct) of the reaction against the log of the relative concentration of template. The cycle threshold for these experiments is set at the linear dynamic range of amplification, above background noise levels and below plateau phase. The slope of the standard curve is used to calculate efficiency as E=10-1/slope 185. Usually, 5 to 7 points corresponding to dilutions of the template are analyzed and plotted to calculate efficiency. For every additional point added to the plot, uncertainty is reduced by 25%. Each reaction is performed in replicate to obtain statistically relevant data and accounts for variation. When calculating relative expression, is important to normalize measurements for the gene of interest with its particular primer efficiency for every experiment to minimize uncertainty. The quantity of template to be used in qPCR

48

Texas Tech University, Olga P. Estrada, May 2017 reactions is calculated based on the primer efficiency data. Generally, a quantity at which amplification occurs at around 20 to 30 cycles is chosen. Each qPCR reaction contains cDNA template, the specific pair of primers, and a master mix made up of Taq polymerase, divalent cations, buffer, and a fluorescent DNA binding agent, such as SYBR green. Each analysis should be performed in three technical replicates and three biological replicates, which yields a total of nine reactions per gene to be analyzed. In addition to this, a non-template control (NTC) and an RNA only control (ROC) should be included for each tissue to be analyzed. Each run should include the analysis of two or more reference genes. Reference genes should be stably expressed throughout all tissues to be tested at all times and their relative abundances should be close to the abundance of the genes of interest. The stability of candidate genes is addressed experimentally for the determination of appropriate reference genes. Analysis of real-time quantitative PCR is then performed by ∆∆Ct relative quantification, a mathematical model devised to obtain fold quantities of expression for the genes of interest relative to reference gene transcript abundance186.

3.3 Genes of Interest and Transcript Profiling in E. coca and E. novogranatense

Biochemical studies for the elucidation of tropane alkaloid biosynthesis in the Erythroxylaceae family have been performed for more than a decade. In these studies, researchers have been able to isolate and characterize most of the enzymes involved in the pathway. Today, efforts are focused on the characterization of the secondary ring closure of tropane alkaloids in members of the Erythroxylaceae family. It has been hypothesized that a type III polyketide synthase (PKS) is responsible for this biochemical reaction. Two putative PKS were first identified from an E. coca young leaf cDNA library. These sequences were cloned into a pEP-Strep vector and expressed for further enzyme assays. Experiments have identified one of these enzymes as an authentic chalcone synthase (CHS) that catalyzes the production of naringenin chalcone using malonyl-CoA and 4-coumaroyl-CoA as substrates, refer to figure 9. The other putative

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Texas Tech University, Olga P. Estrada, May 2017 enzyme has shown PKS activity with N-methylpyrrolinium and malonyl-CoA as substrates and showed no activity with 4-coumaroyl-CoA or acetoacetyl-CoA, refer to figure 10. Other attempts to find a novel PKS in E. coca have been performed, an in- house E. coca young leaf 454 library was searched and a third putative PKS was found. E. coca PKS2 also has shown PKS activity with N-methylpyrrolinium and malonyl-CoA as substrates. The genes corresponding to these enzymes have been named as follows; EcCHS for E. coca chalcone synthase, EcPKS for the putative PKS in E. coca first identified, and EcPKS2 for the secondly identified putative E. coca PKS.

9.0e7

1.0e7 1.1e6

1.0e5 5.5e5

1.0e5

8.4e7

5.0e6

M+ 84da M+ 273da

Figure 9. EIC of enzyme assays performed using EcCHS with the substrates ρ- coumaroyl Co-A and malonyl Co-A. The expected product, naringenin chalcone is present with M+ 273 and compared to a known naringenin standard. EcCHS:PKS Assay was not able to make oxobutanoate. 50

Texas Tech University, Olga P. Estrada, May 2017

M+ 84da M+ 186da

Figure 10. Total and Extracted ion chromatograms (TIC and EIC) of enzyme assays performed using enzymes EcPKS and AbPKS and substrates malonyl-CoA and N- methylpyrrolinium cation, the expected oxobutanoate product is present (M+ 186). The no enzyme controls show no activity.

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Identified genes involved in tropane alkaloid production and storage in Erythroxylum species have shown to be highly transcribed in the young leaf stage compared to mature leaves and other plant organs132, 187. Based on these findings, we expect to find EcCHS, EcPKS and EcPKS2 transcripts in abundance in the young leaf stages. We have conducted real-time qPCR experiments in three leaf stages to study transcript abundance of tropane alkaloid production genes in E. coca and E. novogranatense. Two reference genes were used for normalization, Ec6409 and Ec10131, experimentally determined as appropriate reference genes for qPCR analysis of leaf tissue from E. coca by Docimo et al188.

3.4 Materials and Methods

3.4.1 Plant Material Erythroxylum coca and Erythroxylum novogranatense were grown in an Environmental Growth Chamber at 22° C under a photoperiod of 12 hr light/12 hr dark with relative humidity of 65% constant. Plant tissue used for RNA extraction was obtained from 4-year-old E. coca and E. novogranatense plants. Leaves in three developmental stages were obtained; young expanding leaves in a rolled state (L1); young expanded (unrolled) leaves (L2); and fully mature leaves (L3).

3.4.2 RNA Extraction and cDNA Synthesis Total RNA was extracted from 100 mg of fresh plant tissue using a Quiagen plant RNA extraction kit (Qiagen, Germany). Genomic DNA was degraded by in-column treatment with RNAse-free DNAse I (Qiagen, Germany). RNA quality and quantity was addressed by spectrophotometric readings using a micro-volume spectrophotometer DeNovix DS-11 (DeNovix, Wilmington, USA). Using 2μg of total RNA, cDNA was

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Texas Tech University, Olga P. Estrada, May 2017 synthesized using a Super Script II First Strand Kit (Invitrogen, Germany) according to manufacturer instructions. The product was diluted 1:2 (vol:vol) and stored at -20° C.

3.4.3 Primer Design and Optimization Primers were designed for each gene of interest and synthesized and purified by Integrated DNA Technologies (IDT, USA). Legacy PCR was performed for each primer set to identify single amplicon products. Each reaction was composed of 2 μl Taq polymerase, 10 μl 5X GoTaq buffer (Promega, USA), 1 μl 10mM dNTP mix, 1 μl primers and 1 μl cDNA brought to a total of 50 μl with sterile double distilled water. PCR parameters were as follows; 94° C for 45 seconds, 30 cycles of denaturation at 94° C for 45 seconds, annealing at 60° C for 30 seconds, extension at 72° C for 30 seconds, and an additional extension at 72° C for 7 minutes. Amplification products were analyzed by ethidium bromide supplemented 4% agarose gel electrophoresis. Images of gels were taken using a white/dual UV Transilluminator photoimager (VWR, US).

3.5 Reference Gene Selection

Reference genes, Ec6409 and Ec10131, were selected based on experimental evidence from Docimo et al.188

3.6 Quantitative Real-Time PCR

qPCR experiments were performed on a QuantStudio3 instrument (Thermo Fisher Scientific, USA). Reaction components were as follows; 5 μl PowerUp SYBR green Master Mix (Thermo Fisher Scientific, USA), 0.5 μl primers and 2 μl cDNA in a final volume of 10 μl. All samples were run in three biological replicates and three technical replicates. The thermocycling conditions were as follows; denaturation at 95° C for 10 minutes; 40 cycles of denaturation at 95° C for 15 seconds and annealing/extension at 60° C for 1 minute. Melting curve analyses were performed at the end of each run to rule out the existence of primer dimers or non-specific amplicons. Non-template controls and RNA only controls were included for each primer set to ensure the absence of

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Texas Tech University, Olga P. Estrada, May 2017 contamination. Primer efficiencies were determined by a standard curve based on seven different two-fold dilutions of cDNA cloned amplicon for each primer set.

3.7 Data Analysis

qPCR raw data was exported to Microsoft Excel using the qBase v1.3.5 macro189. Analysis of primer efficiencies was performed by linear regression in the qBase software. All relative quantities we calculated in the qBase software accounting for each primer efficiency and normalizing to reference genes Ec10131 and Ec6409.

3.8 Results

3.8.1 Primer Design and Optimization Two putative sequences from an E. coca young leaf cDNA library190 were identified and denoted as EcPKS and EcCHS based on tested activity. A third sequence was identified from the young E. coca leaf 454 library and denoted as EcPKS2 based on activity. qPCR-grade primers were designed for EcCHS, EcPKS, and EcPKS2 sequences, see Tables 3 and 4. Single bands corresponding to the size of the studied amplicons were present in a 4% agarose gel electrophoresis indicating the presence of specific PCR products, see figure 11. A) for experiments in E. coca and figure 11. B) for experiments in E. novogranatense.

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A)

B)

Figure 11. Primer optimization analysis in 4% acrylamide gels. All tested primers are labeled on top of the corresponding lane. 100 bp ladder is present at the extremities of both gels. A) Primer optimization in E. coca cDNA. B) Primer optimization in E. novogranatense cDNA. Non-template controls are denoted as NTC.

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Texas Tech University, Olga P. Estrada, May 2017 3.8.2 Melting Curve Analysis Melting curve analyses showed the presence of specific amplicons for reference genes and all genes of interest in both organisms, E. coca and E. novogranatense. In figures 12 to 14, we are able to observe the presence of a single peak, indicative of a single amplification product.

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A)

B)

Figure 12. Melting curve analysis of reference genes in E. coca. A) Melting curve of reference gene Ec6409 in E. coca. B) Melting curve of reference gene Ec10131 in E. coca.

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A)

B)

Figure 13. Melting curve analysis of EcPKS amplicons in E. coca. A) Melting curve of EcPKS in E. coca. B) Melting curve of EcPKS2 in E. coca.

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Figure 14. Melting curve analysis of EcCHS amplicon in E. coca.

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Texas Tech University, Olga P. Estrada, May 2017 A)

B)

Figure 15. Melting curve analysis of reference genes in E. novogranatense. A) Melting curve of reference gene Ec6409 in E. novogranatense. B) Melting curve of reference gene Ec10131 in E. novogranatense.

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Texas Tech University, Olga P. Estrada, May 2017 A)

B)

Figure 16. Melting curve analysis of EcPKS amplicons in E. novogranatense. A) Melting curve of EcPKS in E. novogranatense. B) Melting curve of EcPKS2 in E. novogranatense. 61

Texas Tech University, Olga P. Estrada, May 2017

Figure 17. Melting curve analysis of EcCHS amplicon in E. novogranatense.

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Texas Tech University, Olga P. Estrada, May 2017 3.8.3 Primer Efficiencies Each primer set specific to each gene of interest was tested for amplification efficiency in both, E. coca and E. novogranatense. Standard curves were created for reference genes and genes of interest in both organisms of interest, see figures 18 to 23. Primer efficiencies were calculated from standard curve linear regression plot by the qBase analysis program, all efficiencies were close to 2 (Tables 3 and 4) and used for normalization in relative quantification analysis. The coefficients of correlation (R2) values were also calculated by the qBase software and listed in Tables 3 and 4. These values are indicative of linearity within the range of tested parameters, necessary for relative quantification of the genes of interest.

Table 3. Primer Efficiency Values in E. coca

Table 4. Primer Efficiency Values in E. novogranate nse

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A)

B)

Figure 18. Standard curves of reference genes in E. coca. A) Standard curve of reference gene Ec6409 in E. coca. B) Standard curve of reference gene Ec10131 in E. coca. Each point represents a dilution of template. 64

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A)

B)

Figure 19. Standard curves of EcPKS primers in E. coca. A) Standard curve of EcPKS primers in E. coca. B) Standard curve of EcPKS2 primers in E. coca. Each point represents a dilution of template.65

Texas Tech University, Olga P. Estrada, May 2017

Figure 20. Standard curves of EcCHS primers in E. coca. Each point represents a dilution of template.

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Texas Tech University, Olga P. Estrada, May 2017

A)

B)

Figure 21. Standard curves of reference genes in E. novogranatese. A) Standard curve of reference gene Ec6409 in E. novogranatese. B) Standard curve of reference gene Ec10131 in E. novogranatense. Each point represents a dilution of template. 67

Texas Tech University, Olga P. Estrada, May 2017

A)

B)

Figure 22. Standard curves of EcPKS primers in E. novogranatense. A) Standard curve of EcPKS primers in E. novogranatense. B) Standard curve of EcPKS2 primers in E. novogranatense. Each point represents a dilution of template. 68

Texas Tech University, Olga P. Estrada, May 2017

Figure 23. Standard curves of EcCHS primers in E. novogranatense. Each point represents a dilution of template.

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Texas Tech University, Olga P. Estrada, May 2017 3.8.4 Relative Quantification of Genes of Interests in E. coca and E. novogranatense To determine significance among Ct values obtained from qPCR analysis, t-tests were performed for all comparisons made. P values greater than .05 were determined to have no significance. Significant differences were determined for E. coca EcPKS in all three tissues tested. In E. coca experiments for the determination of relative gene quantities, presented in figure 24 A), we see a relative expression level of about 90 times greater for EcPKS in L1 tissue relative to L2 and L3 tissue. We were also able to determine a significant difference of EcPKS2 in E. coca expression levels between L1 and L2 tissues and between L1 and L3 tissues, but no significant difference in EcPKS2 expression levels between L2 and L3 tissues. It was found that EcPKS2 relative expression levels were about 5 times higher in L1 than in L2 and L3. For the EcCHS gene in E. coca, significant differences between L1 and L3 tissues and between L2 and L3 tissues were determined, although the fold relative expression difference between L1 and L3 and between L2 and L3 is less than 1. For relative expression experiments performed in E. novogranatense, presented in figure 24 B), we were able to determine a significant difference in relative expression of EcPKS in all three tissues tested. The relative expression of EcPKS in L1 was determined to be about 9 times greater than in L3 tissue, and about 3 times greater than in L2 tissue. No significant differences between tested tissues were determined in the relative expression of EcPKS2 in E. novograntense. For the EcCHS gene in E. novogranatense, significant differences were determined between tissues L1 and L3 and between tissues L2 and L3. The relative expression of EcCHS in E. novogranatense was determined to be about 4 times higher in L1 tissue than in L3 tissue and about 5 times higher in L2 tissue than in L3 tissue.

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Texas Tech University, Olga P. Estrada, May 2017 A)

B)

Figure 24. Relative quantification analysis of EcCHS, EcPKS and EcPKS2 genes. A) Relative quantification analysis in E. coca. B) Relative quantification analysis in E. novogranatense. Error bars represent standard deviation. 71

Texas Tech University, Olga P. Estrada, May 2017 3.9 Discussion

With the use of modern qPCR techniques, proper controls, and reference genes, we are able to report statistically robust data about the relative expression levels of genes involved in tropane alkaloid production. Although subtle differences were found for EcCHS and EcPKS among the three tested tissues in E. coca and E. novogranatense, we were able to determine a large abundance of the EcPKS2 gene in L1 tissue in comparison with L3 tissue in both tested organisms. These findings correlate with previous studies that indicate that tropane alkaloid production in Erythroxylum species is more abundant in young leaf tissue than in other plant tissue132, 187. Enzyme isolation and characterization experiments are now under process. The results of these studies indicate that the three putative enzymes presented here show the hypothesized activity. Future experiments will be aimed at profiling transcript levels in other plant tissues and other species of the Erythroxylaceae family.

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CHAPTER IV

IDENTIFICATION OF A SALYCILIC ACID METHYL TRANSFERASE (SAMT) IN E. COCA AND E. NOVOGRANATENSE

4.1 Introduction

Methyl salicylate is among the most abundant components in floral scent of several species191. This compound is believed to play an important role in plant defense and reproduction192-194. The presence of this compounds in certain edible plants produces a pleasant minty taste that is today used in the flavoring of medicines and candies195. Humans have positively selected for plants that are able to produce methyl salicylate in abundance to fulfill their own benefits. The biochemical reaction that produces methyl salicylate from salicylic acid in plants is catalyzed by an S-adenosyl-methionine dependent methyl transferase and is often denoted as SAM:salicylate carboxyl methyltransferase or SAMT192, 196. SAMT enzymes correspond to a structurally related group of methyltransferases known as the SABATH family of methyltransferases. The term SABATH was incorporated into the literature after three of the first five identified genes of enzymes belonging to this group197. The first two enzymes from the SABATH family to be isolated and characterized were an SAMT from Clarkia breweri and a SAM;benzoic acid carboxyl methyltransferase from snapdragon flowers196, 198. Additionally, three novel methyltransferases involved in caffeine biosynthesis were elucidated contemporary to these two enzymes, also corresponding to the SABATH family199-201. As previously mentioned, human selection for plants that make methyl salicylate in abundance has affected many plant species. For instance, the two most cultivated Erythroxylum species for their high content of cocaine in leaf material, E. novogranatense and E. coca are fundamentally different in their methyl salicylate content. E. novogranatense has a higher content of methyl salicylate than E. coca72. This can be reasoned by the cultural value it was given since ancient times in the Incan

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Texas Tech University, Olga P. Estrada, May 2017 empire. The leaves of E. novogranatense were exclusively used by important priests and were considered to be able to render divine powers to the consumer73. Here, we provide a focus in the intrinsic investigation of transcriptomic and proteomic differences between E. coca and E. novogranatense production of methyl salicylate. A putative SAMT in Erythroxylum was tested for transcript profiling in E. coca and E. novogranatense and heterologous expression optimization of the enzyme was attempted. Methylecgonone, a precursor of cocaine, has a methyl ester group that is responsible for the narcotic properties of cocaine (please refer to Chapter II). We can not discard the possibility that the protection of the ester group by methylation in methylecgonone is catalyzed by a methyltransferase belonging to the SABATH family. This will be subject to further investigations.

4.2 Materials and Methods

4.2.1 qPCR experiments All qPCR materials and methods were performed as described in Chapter III.

4.2.2 Construct EcSAMTpHGW construct was used throughout these experiments. The clone was sequenced and analyzed to confirm the presence of the full open reading frame.

4.2.3 Enzymological experiments Bacterial Work

Cultures were grown in LB medium 25g/L (Fisher, USA). Agar plates contained LB medium 25g/L supplemented with 7g/L of agar (Fisher, USA). SOC medium used for transformation of E. coli. Antibiotic concentrations were 50 μg/mL of kanamycin and 20 μg/mL gentamicyn for the corresponding liquid and agar mediums. Liquid cultures were shaken at 250 rpm.

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Transformation

Three types of E. coli cell lines were used throughout these experiments, E. coli BL21 (DE3), BL21 Rosetta Blue, and BL21 Arctic cell lines. All three were stored at - 80° C until required. Approximately 100 ng to 150 ng of EcSAMTpH9 GW plasmid were used for transformation. After the addition of plasmid, the mixture was incubated for 10 minutes on ice and heat-shocked at 42° C for 42 seconds and finally incubated on ice for an additional 2 minutes. The mixture was then supplemented with 200 μl of SOC medium and incubated at 37° C shaking for 1 hour. The cells were then plated in agar containing the corresponding antibiotic.

Protein Expression and Harvest in E. coli BL21 (DE3) and BL21 Rosetta Blue cell lines

Starter cultures were made by the inoculation of 2 ml of LB supplemented with kanamycin, grown overnight at 37° C shaking. Expression cultures were made by inoculation of 50 ml of LB medium supplemented with kanamycin with 500 μl of starter culture. Expression cultures were grown to OD .4 to .5, let to rest on ice for 30 minutes, and induced with IPTG to a final concentration of 1mM, grown at 37° C shaking at 200 rpm for 3 hours and harvested by centrifugation 4000 xg for 20 minutes at 4° C in pre- cooled equipment. The supernatant was discarded and the pellet was re-suspended in HisTag biding buffer composed of 20mM potassium phosphate, .5M NaCl, 20mM imidazole, 10% glycerol in a pH of 7.4 at 4° C. Cell lysis was achieved by sonication four times in intervals of 30 seconds at power 60%. Cell lysate was centrifuged at 15,000 xg for 20 minutes at 4° C in pre-cooled equipment. Supernatant was prepared for purification. Sample of crude was taken for SDS-PAGE analysis.

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Texas Tech University, Olga P. Estrada, May 2017 Protein Expression and Harvest in BL21 Rosetta Blue using Overnight Express Medium

Starter cultures were made as previously described. LB Overnight Express (Novagen, Germany) medium was prepared according to manufacturer’s instructions. 2.25 g of medium was added to 30 ml of water, .5 ml of glycerol were added to this solution. pH was adjusted to 6.9 using HCL or NaOH. Medium was sterilized in microwave to avoid caramelization. When the medium reached room remperature it was supplemented with kanamycin and inoculated with 500 ul of starter culture. Culture was incubated at 18° C for 48 hours, shaking. Cells were harvested by centrifugation at 4000 xg for 20 minutes at 4° C. The following steps were performed as previously described.

Protein Expression and Harvest in BL21 Arctic cell line

Starter cultures were made by the inoculation of 2 ml of LB supplemented with kanamycin and gentamycin, grown overnight at 37° C shaking. Expression cultures were made by inoculation of 50 ml of LB medium supplemented with kanamycin and gentamycin with 500 μl of starter culture. Expression cultures were grown to OD .4 to .5, left to rest on ice for 30 minutes, and induced with IPTG to a final concentration of 1mM, grown at 13° C shaking at 200 rpm for 24 hours and harvested by centrifugation 4000 xg for 20 minutes at 4° C in pre-cooled equipment. The supernatant was discarded and the pellet was re-suspended in HisTag biding buffer composed of 20mM potassium phosphate, .5M NaCl, 20mM imidazole, 10% glycerol in a pH of 7.4 at 4° C. Cell lysis was achieved by sonication four times in intervals of 30 seconds at power 60%. Cell lysate was centrifuged at 15,000 xg for 20 minutes at 4° C in pre-cooled equipment. Supernatant was prepared for purification. Sample of crude was taken for SDS-PAGE analysis.

Protein Purification

Protein purification was conducted in a GE Healthcare AKTA purifier (Munchen,Germany). Columns and calibration kits for protein purification were

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Texas Tech University, Olga P. Estrada, May 2017 purchased from the same manufacturer. After purification, fractions were collected, supplemented with 10mM DTT, aliquoted for analysis and stored at -20° C.

SDS-PAGE

Purified samples of protein were ran in 12% acrylamide gels in a Bio-Rad gel chamber connected to a Bio-Rad Power Pack Basic (Bio-Rad, USA). 20 μl of sample were mixed with 5 μl of protein loading buffer containing 50 mM Tris-HCl in pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, .02% bromophenol blue and 12.5 mM EDTA. The mixture was boiled at 95° C for 7 minutes. 20 μl of each sample was loaded to the gel. Gels were run at 100 V until loading buffer dye reached the bottom of the gel. Benchmark Protein Ladder (Invitrogen, Germany) was run simultaneously to all samples. SDS-PAGE running buffer consists of 25 mM Tris, 192 mM glycine, .1% (w/v) SDS, at a pH of 8.3.

Gel Staining

After SDS-PAGE were run, they were left to stain overnight in Colloidal Coomassie staining solution consisting of .1% Coomassie Blue, 40% ethanol and 10% acetic acid. After staining, the gels were put to de-stain for 1 hour in de-staining solution consisting of 40% ethanol and 10% acetic acid.

Gel imaging

Images of gels were taken using a white/dual UV Transilluminator photoimager (VWR, US).

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Texas Tech University, Olga P. Estrada, May 2017 4.3 Results

4.3.1 Relative Quantification of EcSAMT Transcript Levels Primer Design and Optimization

qPCR grade primers were designed for the putative EcSAMT sequence, and the reference genes, see tables 5 and 6. Single bands corresponding to our genes of interest were present in the 4% Agarose gel analysis for both, E. coca and E. novogranatense (please refer to chapter III figure 11). The results were indicative of the presence of a single amplicon corresponding to our gene of interest.

Melting Curve Analysis

Melting curve analyses showed the presence of specific amplicons for reference genes and the gene of interest in both organisms, E. coca and E. novogranatense. In figure 25 we are able to observe the presence of a single peak, indicative of a single amplicon product. For melting curve analysis of reference genes, please refer to figure 12 in chapter III.

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A)

B)

Figure 25. Melting curve analysis of EcSAMT amplicons. A) Melting curve of EcSAMT in E. coca. B) Melting curve of EcSAMT in E. novogranatense.

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Texas Tech University, Olga P. Estrada, May 2017 Primer Efficiencies

The primer set specific to the gene of interest was tested for amplification efficiency in both, E. coca and E. novogranatense. Standard curves were created for the gene of interest in both organisms of interest, see figure 26. Primer efficiencies were calculated from standard curve linear regression plot by the qBase analysis program, all efficiencies were close to 2 (Tables 5 and 6) and used for normalization in relative quantification analysis. The coefficients of correlation (R2) values were also calculated by the qBase software and listed in Tables 5 and 6. These values are indicative of linearity within the range of tested parameters, necessary for relative quantification of the genes of interest. For standard curves of reference genes please refer to figures 18 and 21 in chapter III.

Table 5. Primer Efficiency Values in E. coca

Table 6. Primer Efficiency Values in E. novogranatense

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A)

B

Figure 26. Standard curves of EcSAMT primers A) Standard curve of EcSAMT primers in E. coca. B) Standard curve of EcSAMT primers in E. novogranatense. Each point represents a dilution of template. 81

Texas Tech University, Olga P. Estrada, May 2017 Relative Quantification of EcSAMT in E. coca and E. novogranatense

To determine significance among Ct values obtained from qPCR analysis, t-tests were performed for all comparisons made. P values greater than .05 were determined to have no significance. Relative quantification analyses are presented in figure 27. Significant differences were determined for EcSAMT in E. coca between tissues L1 and L2 and between tissues L1 and L3. The relative expression levels of EcSAMT in E. coca were about 50 times greater in L1 than in L2 and L3. Significant differences were determined for EcSAMT relative expression in all tissues of E. novogranatense. We found a relative expression 10 times larger in L1 tissue than in L3 tissue, and about 5 times larger in L1 tissue than in L2 tissue. No significant differences in relative expression were determined between L2 tissue and L3 tissue.

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A)

B)

Figure 27. Relative quantification analysis of EcSAMT gene. A) Relative quantification analysis in E. novogranatense. B) Relative quantification analysis in E. coca. Error bars represent standard deviation.

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Texas Tech University, Olga P. Estrada, May 2017 4.3.2 Expression optimization of EcSAMTpH9GW Several experiments were performed to optimize the expression of EcSAMT in a heterologous organism.

Expression in E. coli BL21 (DE3) cell line

Expression of EcSAMTpH9 in E. coli BL21 (DE3) cells was not favorable. The SDS-PAGE analysis of the crude and purified fractions (please see figure 28) showed no band corresponding to the 41 kDa putative SAMT protein. The analysis of the pellet showed a band corresponding to our protein of interest. These results indicate that the putative SAMT could be present as an insoluble sample in the harvested pellet.

Figure 28. SDS-PAGE of EcSAMTpH9 expression in E. coli BL21 (DE3) cell line.

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Texas Tech University, Olga P. Estrada, May 2017 Expression in E. coli BL21 Rosetta cell Blue line

Expression of EcSAMTpH9 in E. coli BL21 Rosetta Blue cells was not favorable. Similarly to the E. coli BL21 (DE3) expression analysis, the SDS-PAGE analysis of the crude and purified fractions (please see figure 29) showed no band corresponding to the 41 kDa putative SAMT protein being expressed in E. coli BL21 Rosetta Blue cell line. The analysis of the pellet showed a band corresponding to our protein of interest. These results indicate that the putative SAMT could be present as an insoluble sample in the harvested pellet.

Figure 29. SDS-PAGE of EcSAMTpH9 expression in E. coli BL21 Rosetta Blue cell line.

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Texas Tech University, Olga P. Estrada, May 2017 Expression in E. coli BL21 Rosetta Blue cell line using Overnight Express medium

Expression of EcSAMTpH9 in E. coli BL21 Rosetta Blue cells using the Overnight Express autoinducible expression system was not favorable. The SDS-PAGE analysis of the crude and purified fractions (please see figure 30) showed several 40 kDa bands in three purified fractions, but also many other bands all over the purified fractions. The analysis of the pellet showed a band at around 38 kDa, which does not correspond to our protein of interest. These results can not be used to determine if the putative SAMT was present in the expression sample.

Figure 30. SDS-PAGE of EcSAMTpH9 expression in E. coli BL21 Rosetta cell line using the OvernightExpress autoinducible expression medium.

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Texas Tech University, Olga P. Estrada, May 2017 Expression in E. coli ArcticExpress cell line

Expression of EcSAMTpH9 in E. coli BL21 ArcticExpress cells was not favorable. SDS-PAGE analysis of the crude and purified fractions (please see figure 31) showed no band corresponding to the 41 kDa putative SAMT protein being expressed in E. coli BL21 ArcticExpress cell line, but showed a band corresponding to about 60 kDa in two purified fractions. It was thought that these bands corresponded to the chaperonins encoded in the ArcticExpress cell line; Cpn10 and Cpn60(10 and 60 kDa correspondingly) from the psychrophile Oleispira Antarctica. The size of Cnp60 corresponds to the purified band size. The analysis of the pellet showed an abundant band corresponding to the size of our protein of interest. These results indicate that the putative SAMT could be present as an insoluble sample in the harvested pellet and that there could be an error in the construct.

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Figure 31. SDS-PAGE of EcSAMTpH9 affinity purification of expression in E. coli BL21 ArcticExpress cell line.

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Texas Tech University, Olga P. Estrada, May 2017 4.4 Discussion

Here we present the data required for the proper report of relative expression levels of EcSAMT in three leaf stage tissues of E. coca and E. novogranatense with the use of qPCR techniques. The relative transcript levels of EcSAMT in E. coca and E. novogranatense are more abundant in young leaf tissue in comparison to mature leaf tissue. As previously described, the production of tropane alkaloids is more abundant in young leaf tissue. It is hypothesized that in correlation to these findings, production of secondary metabolites such as methyl salicylate, is carried out in younger leaf stages. Based on the abundance of methyl salicylate in E. coca and E. novograntense, we have predicted that the relative transcript abundance of the gene encoding for a salicylic acid methyltransferase (SAMT) would be higher in E. novogranatense, which has a higher methyl salicylate content72. The relative transcript abundance analysis here presented indicates that the studied SAMT gene is present in comparative levels in E. coca and E. novogranatense. It is suspected that the enzyme responsible for the methylation of the thioester present in the ecgonone-CoA ester is a methyltransferase belonging to the SABATH family. Taking this into consideration, it is possible that the gene studied is, in fact, the methyltransferase responsible for the formation of methylecgonone rather than the salicylic acid methyltransferase responsible for the formation of methyl salicylate. Future experiments will aim to identify a novel putative methyltransferase gene in E. coca and E. novogranatense to be able to express and analyze the activity of the enzymes. Attempts to optimize the expression of a putative EcSAMT enzyme were carried out in several cell lines. Unfortunately, the optimization was not achieved in any of these experiments. The attempted expression in the ArcticExpress cell line yielded what is suspected to be the expression of the chaperonins present in this cell line. These results could be indicative of an erroneous construct. Further analyses will be aimed to determine the errors present in the construct to enable us to express this enzyme and carry out enzyme assays.

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