Transcript
Page 1: Molecular cloning and characterization of sesquiterpene

University of Calgary

PRISM: University of Calgary's Digital Repository

Graduate Studies The Vault: Electronic Theses and Dissertations

2012-07-19

Molecular cloning and characterization of

sesquiterpene synthases from valeriana officinalis

Pyle, Bryan Wilkinson

Pyle, B. W. (2012). Molecular cloning and characterization of sesquiterpene synthases from

valeriana officinalis (Unpublished master's thesis). University of Calgary, Calgary, AB.

doi:10.11575/PRISM/26983

http://hdl.handle.net/11023/129

master thesis

University of Calgary graduate students retain copyright ownership and moral rights for their

thesis. You may use this material in any way that is permitted by the Copyright Act or through

licensing that has been assigned to the document. For uses that are not allowable under

copyright legislation or licensing, you are required to seek permission.

Downloaded from PRISM: https://prism.ucalgary.ca

Page 2: Molecular cloning and characterization of sesquiterpene

UNIVERSITY OF CALGARY

Molecular Cloning and Characterization of Sesquiterpene Synthases from Valeriana officinalis

by

Bryan Wilkinson Pyle

B.Sc., The University of Calgary

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF BIOLOGICAL SCIENCES

FACULTY OF SCIENCE

CALGARY, ALBERTA

JULY, 2012

Bryan Wilkinson Pyle 2012

Page 3: Molecular cloning and characterization of sesquiterpene

ii

Abstract

Valeriana officinalis (valerian) is a popular medicinal plant in North America and

Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Valerenic acid, a

C15 sesquiterpenoid, has been suggested as the active ingredient responsible for the sedative

effect. Recently, medical uses of valerenic acid as anti-depressant and anti-inflammatory drugs

were suggested due to its affinity for the γ-aminobutyric acid type A (GABAA) receptor as an

agonist and its inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells

(NF-B) pathway, respectively. Despite its importance, biochemistry of valerenic acid in

valerian remains unknown. To identify the first committed enzymatic step in valerenic acid

biosynthesis, next-generation sequencing (Roche 454 titanium) was used to generate ~1 million

transcript reads from valerian root. Subsequently, three cDNAs for sesquiterpene synthases

(VoTPS1/2/3) were identified and their corresponding recombinant enzymes were purified.

Three recombinant enzymes efficiently catalyze the synthesis of valerena-4,7(11)-diene,

germacrene C/D, and drimenol, respectively, based on the spectral match in the mass

spectrometry library. Additional structural analyses using GC-MS and 13

C-NMR spectrometry

in comparison to a semi-synthesized standard confirmed the chemical identity of valerena-

4,7(11)-diene. This is the first report of valerena-4,7(11)-diene and drimenol synthases, and the

biosynthetic mechanisms of these two products from the substrate, farnesyl diphosphate, were

proposed.

Page 4: Molecular cloning and characterization of sesquiterpene

iii

Acknowledgements

I would like to thank Dr. Dae-Kyun Ro for introducing me to the world of plant

metabolites. Before I started this project I had little to no understanding of the vast complexity

and unfathomable quantity of compounds produced by plants. I can now say I comprehend that

number a little more. I must also thank Dr. Ro for pushing my own expectations of myself, for

that I owe you a great debt, the skills you have given me will help me in every decision I ever

make, from now on. To all members of the Ro lab, past and present, thank you for all your help.

Dr. Hue Tran, I thank you for teaching me the basics of protein purification. I must also thank

Dr. Benjamin Pickel for our extensive discussions on women, science and beer without which

very few men can survive science. I must also thank Dr. Pickel for valerenadiene purification

and NMR analysis. Thank you to Drs. John Vederas and Zhizeng Gao for valerenadiene

chemical synthesis. Thank you to Gillian MacNevin for the semi-quantitative PCR data. Finally

I would like to thank Dr. Paul O’Maille for the pH9GW vector, a generous gift.

Last and definitely not least I must thank my family and friends. All members of my

family have helped me in some way, shape or form throughout my life and that is priceless. To

my wife Lisa, thank you, for you have contributed so much emotionally to these past two years

and I will forever be indebted to you. Our daily walks with Bodie gave me an outlet to escape

from my second love, science. You are my best friend and I am sorry for being a “difficult” grad

student for the past two years.

Page 5: Molecular cloning and characterization of sesquiterpene

iv

Dedication

To my late grandfather, Byron W. Pyle, though our religious philosophies did not always agree

our educational philosophies did; everyone deserves an opportunity at education…wherever that

may go.

Page 6: Molecular cloning and characterization of sesquiterpene

v

Table of Contents

Abstract ....................................................................................................................................... ii

Acknowledgements .................................................................................................................... iii

Dedication .................................................................................................................................. iv

Table of Contents ........................................................................................................................ v

List of Tables ............................................................................................................................ vii

List of Figures .......................................................................................................................... viii

List of Abbreviations .................................................................................................................. x

CHAPTER 1: INTRODUCTION ................................................................................................ 1

1.1 Secondary Metabolites .......................................................................................................... 1

1.2 Terpenoids............................................................................................................................. 4

1.2.1 Ecological Functions of Isoprenoids and Terpenoids ................................................... 5

1.2.2 Terpenoid Biosynthesis in Plants ................................................................................... 8

1.3 Terpene Synthases .............................................................................................................. 15

1.3.1 Sesquiterpene Synthase Structure-Function Relationships ......................................... 16

1.3.2 Phylogenetic Relationships of Terpene Synthases ....................................................... 21

1.4 Metabolic Engineering of the MVA Pathway .................................................................... 22

1.5 Ligand-Receptor Binding.................................................................................................... 25

1.6 Sesquiterpene Biosynthesis in Valeriana officinalis ........................................................... 27

1.7 Objectives ........................................................................................................................... 30

CHAPTER 2: MATERIALS AND METHODS ...................................................................... 32

2.1 Plant Cultivation and Metabolite Preparations ................................................................... 32

2.2 RNA preparations ............................................................................................................... 32

2.3 cDNA Library Preparation from Total RNA ...................................................................... 33

2.4 Plasmid Construction for Yeast Expression ....................................................................... 33

2.5 Quantitative Transcript Analysis ........................................................................................ 34

2.6 Yeast Transformation.......................................................................................................... 35

2.7 In vivo Production of Terpenoids in Yeast ......................................................................... 36

2.8 Plasmid Construction for E. coli Expression ...................................................................... 36

2.9 Heterologous Expression Trials .......................................................................................... 39

2.10 Expression in E. coli and Protein Purification .................................................................. 39

2.11 Gas-chromatography and Mass Spectroscopy Analysis ................................................... 41

2.12 Purification and NMR of Valerena-4,7(11)-diene ............................................................ 42

2.14 NMR Analysis of Valerena-4,7(11)-diene Standard ........................................................ 42

2.15 Enzyme Activity Assays ................................................................................................... 43

2.16 Enzyme Characterization .................................................................................................. 43

2.17 Phylogenetic Analysis ....................................................................................................... 44

CHAPTER 3: RESULTS ........................................................................................................... 45

3.1 Metabolite Profiling of Valerian Root ................................................................................ 45

3.2 Transcript Sequencing and Candidate Gene Isolation ........................................................ 47

3.3 Functional Screening of VoTPS cDNAs in Engineered Yeast ........................................... 51

3.4 Characterization of the VoTPS2 Product ............................................................................ 57

Page 7: Molecular cloning and characterization of sesquiterpene

vi

3.5 In vitro Characterization of VoTPS1 and VoTPS2 ............................................................. 60

3.6 Cyclization Mechanism of Valerena-4,7(11)-diene ............................................................ 65

3.7 Identification and Characterization of an Additional Sesquiterpene Synthase, VoTPS3.... 68

3.8 Phylogenetic Analysis of VoTPS1/2/3 ............................................................................... 73

CHAPTER 4: DISCUSSION ..................................................................................................... 76

LITERATURE CITED .............................................................................................................. 80

Appendix I ................................................................................................................................ 89

Appendix II ............................................................................................................................... 93

Page 8: Molecular cloning and characterization of sesquiterpene

vii

List of Tables

Table 1. Table of primers used in cloning experiments. ............................................................. 38

Table 2. GC-MS analysis of terpenoids synthesized from VoTPS1, VoTPS2 and VoTPS3 ...... 54

Table 3. Comparison of the 13

C-NMR signals from the purified compound of peak 4 with the

published data. .............................................................................................................................. 58

Page 9: Molecular cloning and characterization of sesquiterpene

viii

List of Figures

Figure 1. Selected examples of common specialized metabolites. ............................................... 3

Figure 2. Chemical structures of isoprene and isopentenyl diphosphate. ..................................... 5

Figure 3. Schematic depiction of the MVA pathway. ................................................................. 13

Figure 4. Schematic depiction of the DXP pathway. .................................................................. 14

Figure 5. Schematic diagram representing the carbocation mechanism of tobacco epi-

aristolochene synthase (TEAS). .................................................................................................... 20

Figure 6. Proposed biosynthetic pathway for valerenic acid production in V. officinalis. ......... 31

Figure 7. GC-MS profile of volatile metabolites from valerian root. .......................................... 46

Figure 8. Sequence alignment of deduced amino acid sequences from VoTPS1 and VoTPS2. ... 49

Figure 9. Semi-quantitative RT-PCR analysis of the VoTPS1 and VoTPS2 transcripts in V.

officinalis root and leaf. ................................................................................................................ 50

Figure 10. Unique terpene compounds synthesized from the yeast expressing VoTPS1 or

VoTPS2. ........................................................................................................................................ 53

Figure 11. Chemical structures relating to numbers from text. .................................................. 55

Figure 12. GC-MS analysis of VoTPS1 products and the terpene standards synthesized by

tomato germacrene B/C synthase. ................................................................................................. 56

Figure 13. Validation of VoTPS2 enzyme product (peak 4) as valerena-4,7(11)-diene. ............ 59

Figure 14. Expression trials of his-tagged recombinant VoTPS1 and VoTPS2. ........................ 62

Figure 15. Purification of VoTPS1/2 by Ni-NTA column using a gradient elution. .................. 63

Figure 16. In vitro enzyme assays of VoTPS1 and VoTPS2 recombinant enzyme..................... 64

Figure 17. A proposed mechanism for valerena-4,7(11)-diene formation catalyzed by VoTPS2

(valerenadiene synthase). .............................................................................................................. 67

Page 10: Molecular cloning and characterization of sesquiterpene

ix

Figure 18. Expression trial of his-tagged recombinant VoTPS3 (67 kDa). ................................ 70

Figure 19. In vitro assays for VoTPS3. ....................................................................................... 71

Figure 20. A proposed mechanism of drimenol formation by VoTPS3 (drimenol synthase). ... 72

Figure 21. A phylogenetic tree representing the seven subfamilies (a-g) of terpene synthase

enzymes......................................................................................................................................... 74

Page 11: Molecular cloning and characterization of sesquiterpene

x

List of Abbreviations

3H

13C

CDP

DMAPP

DPP1

DXP

DXS

EI

ERG9

FOH

FPLC

FPP

G3P

GABAA

Gal

GC-MS

GPCR

HMG-CoA

HMGR

IPP

LSD

MEP

min

MSA

Hydrogen isotope (tritium)

Carbon-13 isotope

Cytidyl diphosphate

Dimethylallyl diphosphate

Diacylglycerol pyrophosphate phosphatase

Deoxyxylulose phosphate

Deoxyxylulose synthase

Electron impact

Squalene synthase

Farnesol

Fast-protein liquid chromatography

Farnesyl diphosphate

Glyceraldehyde-3-phosphate

γ-aminobutyric acid type A receptor

Galactose

Gas chromatography-mass spectrometry

G-protein coupled receptor

3-hydroxy-3-methyl glutaryl coenzyme A

HMG-CoA reductase

Isopentenyl diphosphate

Lysergic acid diethylamide

2-C-methyl-D-erythritol 4-phosphate

minutes

Microtubule stabilizing agent

Page 12: Molecular cloning and characterization of sesquiterpene

xi

MVA

NF-κB

NMR

PCR

RI

RT

sec

SERCA

TEAS

TPS(s)

UPC2

VLS

VoTPS

Mevalonic acid

Nuclear factor kappa-light-chain-enhancer

of activated B cells

Nuclear magnetic resonance

Polymerase chain reaction

Retention index

Reverse transcriptase

Seconds

Sarcoplasmic endoplasmic reticulum Ca2+

ATPase

Tobacco epi-aristolochene synthase

Terpene synthase(s)

Yeast transcription factor

Valerena-4,7(11)-diene synthase

Valeriana officinalis terpene

synthase

Page 13: Molecular cloning and characterization of sesquiterpene

1

CHAPTER 1: INTRODUCTION

1.1 Secondary Metabolites

Secondary (or specialized) metabolites encompass a vast number of low-molecular-

weight organic compounds naturally synthesized in plants and microbes. The canonical

definition describes secondary metabolites as any compound that contributes no apparent benefit

to the host organism’s growth and reproduction. However, the specialized metabolites enhance

the fitness of synthesizers in distinct ecological niches, and therefore play a central role in

evolutionary selection of plants and microbes. In contrast, primary metabolites are essential for

day-to-day function of all organisms and are normally present at higher levels. As mutations in

genes involved in primary metabolism cause fatal effects on the survival of organisms, variation

in primary metabolism is restricted and is highly conserved across the kingdoms. On the other

hand, specialized metabolism can tolerate alterations and thus display a great metabolic plasticity.

Four major classes of secondary metabolites are terpenoids, alkaloids, phenylpropanoids,

and polyketides (Figure 1). Although these compounds have a small finite metabolic role in an

individual species, many of these compounds collectively have important functions as pollinator

attractants, anti-feedants, repellents, toxins, and antibiotics. The extremely large structural

diversity of specialized metabolites makes them, in an anthropocentric view, useful to humans as

food additives, fibers, bio-polymers, pharmaceuticals, and nutraceuticals. For example, Papaver

somniferum, Valeriana officinalis, Cannabis sativa, Humulus lupulus, Atropa belladona have all

been used as medicinal plants for thousands of years. Ancient documents dating over 4,600

years old listed 1,000 plant species for possible medical uses, and most of these plants are still in

use today (Newman et al., 2000).

Page 14: Molecular cloning and characterization of sesquiterpene

2

The contemporary impact of specialized metabolites in our day-to-day lives may have a

much broader influence on our society than generally realized. For example, it has been

suggested that hydrocarbon terpenes and aromatic phenolics can be developed as alternative

fuels (Zhang et al., 2011). Although the estimates of oil reserves tend to vary depending on

various factors (e.g., source of information, production, consumption, and quality), undoubtedly

its quantity is finite (Owen et al., 2010). For example, synthetic rubber manufactured from

petroleum (~3.9 million tonnes per year), for example, will ultimately need to be replaced by

natural rubber, which currently accounts for 40% of total rubber production (Cornish, 2001).

This will make substantial impacts on many manufacturing industries, such as goods, medical

devices, research, and pharmaceuticals.

Medicinal plants also impact our lives as 63% of all new chemical entities from 1981-

2006 were specialized metabolites or their semi-synthetic derivatives (Newman and Cragg,

2007). For example, three generic anti-cancer drugs currently produced by partial chemical

synthesis are vinblastine, vincristine, and paclitaxel, which were first identified from the plant

species Catharanthus roseus and Taxus brevifolia. Consequently, these compounds are

produced at minute levels by their respective plants. The Pacific yew (T. brevifolia) produces

~30 mg taxol/kg of bark in one 100-year-old tree which is equivalent to a single dosage of

treatment (Horwitz, 1994; Kirby and Keasling, 2009). Currently, paclitaxel is produced either by

semi-synthesis from a naturally more abundant intermediate, 10-deacetyl baccatin III, or from

plant cell culture, significantly reducing the cost and also protecting the environment (Horwitz,

1994; Kirby and Keasling, 2009). Similar efforts have been attempted to produce vinblastine

and vincristine in C. roseus cell cultures, but it is still very challenging to meet the 3 kg/yr

worldwide demand (Verpoorte et al., 1993; Julsing et al., 2006).

Page 15: Molecular cloning and characterization of sesquiterpene

3

Figure 1. Selected examples of common specialized metabolites.

Selected examples of specialized products are: cocaine (alkaloid), tetrahydrocannabinolic acid

(terpene phenolic), proanthocyanidin (phenylpropanoid), lovastatin (polyketide).

Page 16: Molecular cloning and characterization of sesquiterpene

4

1.2 Terpenoids

Terpenoids have a long etymological and biosynthetic history. The word terpenoid,

sometimes called isoprenoid (historical term), was derived from the German word “terpentin”, or

more conspicuously known as turpentine. Turpentine refers to the essential oils of conifer

species used to investigate chemical structures in the 19th

century (Chappell, 1995). Turpentine

oils are composed of mono-, di-, and minor amounts of sesqui-terpenes which are believed to be

used as a chemical defense against pests and pathogens in conifer trees (Zulak and Bohlmann,

2010). Terpenoids contribute to primary metabolism as sterols, photosynthetic pigments, prenyl

modification of proteins, and various hormones, but they also play critical eco-physiological

roles in plant-plant, plant-pathogen, and plant-herbivore interactions. This is largely due to the

sessile nature of plants and relates to the complex evolution of specialized metabolism. In the

past 25 years, research in the field of terpene metabolism has exploded with chemical structure

estimates reaching 65,000 (Oldfield and Lin, 2012), making terpenoids, by far, the largest and

most structurally diverse class of natural products known.

The extreme chemical diversity of terpenoids attracted scientists to elucidate the structure

of camphor, a monoterpene. Otto Wallach was able to propose the structure of camphor by

proposing the isoprene rule. The ‘isoprene rule’ establishes the C5 isoprene as structural

building blocks, which are synthesized in a head-to-tail conjugation reaction. This simple

proposal could explain why many terpenes have carbon structures following the C5 x n (n = 2, 3,

4, 6, 8) rule (Ruzicka, 1953). Leopold Ruzicka further advanced the theoretical aspect of terpene

biogenesis by defining the ‘biogenetic isoprene rule’, which is based on the unique carbocation

mechanism involving the various allylic rearrangements, hydride- and methyl-shifts, and

Page 17: Molecular cloning and characterization of sesquiterpene

5

deprotonation reactions. The central biological precursor of all terpenoids was then proposed to

be isopentenyl diphosphate (IPP), and not isoprene (Figure 2) (Ruzicka, 1953).

Figure 2. Chemical structures of isoprene and isopentenyl diphosphate.

1.2.1 Ecological Functions of Isoprenoids and Terpenoids

Plants are sessile organisms. This inherent stationary nature results in complex

interactions on many different trophic levels as plants must deal with many biotic and abiotic

stressors, often concurrently. Emission of mixtures of volatile compounds from floral organs

and vegetative parts after herbivore damage, and from roots into the soil are examples of

evolutionary mechanisms that plants have developed to deal with such stressors. Plant volatiles

consist mostly of terpenoids, phenylpropanoids, benzenoids, fatty acids, and amino acid

derivatives, but terpenoids are the most diverse (Dudareva et al., 2004). Normally, volatiles are

lipophilic with high vapor pressures, and hence are able to cross membranes and diffuse through

the atmosphere or soil. Consequently, these compounds are important for plant defense and

reproduction. The simplest example is C5 isoprene synthesized by enzymatic dephosphorylation

of IPP (precursor to all terpenoids) in certain plant species. In isoprene synthesizing plants, up to

Page 18: Molecular cloning and characterization of sesquiterpene

6

1-2% of the carbon fixed by photosynthesis is released to the atmosphere as a volatile gas

(Vickers et al., 2009) and most of this is in the form of isoprene ~500 Tg C/yr globally (Sharkey

and Yeh, 2001; Sasaki et al., 2007). The biological implication of such massive isoprene release

is still being debated, but some physiological experiments suggest that plants release a large

amount of isoprene in response to thermal stresses (Vickers et al., 2009). Interestingly, some

plants that have lost the ability to synthesize and emit isoprene have replaced isoprene with

mono-terpenes (Harley et al., 1997). Therefore, evolutionarily it may be reasonable to assume

that plants lacking the ability to synthesize isoprene for protection against thermal stress have

replaced isoprene with mono- and sesqui-terpenes (Vickers et al., 2009), implying a significant

evolutionary consequence for ecological function of isoprene or a terpene replacement.

Strictly speaking, research into the ecological function of isoprene (C5 units) with respect

to plants has been limited to mostly abiotic and oxidative stress (Dudareva et al., 2006; Vickers

et al., 2009). However, terpenoids (C10) are much more diverse in their ecological functions

and are implicated in many plant defense, plant-plant, and reproductive interactions. Hybrid

poplar under herbivore attack by forest tent caterpillars showed local (wound site) and systemic

emission of E--ocimene (monoterpene) in addition to several other mono- and sesquiterpenes

(Arimura et al., 2004). Other examples exist wherein plants damaged by an herbivore may

induce expression of pathways involved in production of plant defense compounds such as

jasmonic acid or ethylene (Arimura et al., 2000; Arimura et al., 2002). Plants also use terpenoids

to influence the life cycle of adjacent plants (referred to as allelopathy), as it has been shown that

emission of the monoterpene 1,8-cineole from a root can inhibit germination and growth of

competing plants (Romagni et al., 2000). Recently, belowground interactions involving the

sesquiterpene E--caryophyllene in maize was identified as the first root insect-induced

Page 19: Molecular cloning and characterization of sesquiterpene

7

belowground plant signal recorded in controlled conditions. This attraction involves a parasitic

nematode Heterorhabditis megidis, which infects a herbivorous beetle Diabrotica virgifera

virgifera, but only after herbivory induced emission of E--caryophyllene by maize (Rasmann et

al., 2005). In a similar interaction, transgenic Arabidopsis thaliana engineered to produce the

sesquiterpene E--farensene prevented attack of a common aphid pest (Aharoni et al., 2003) by

mimicking the common aphid alarm pheromone (Beale et al., 2006). Examples of tobacco

species attracting herbivore predators in the wild by volatile terpenoids has also been

documented (Kessler and Baldwin, 2001).

The terpenoids and -pinene, -mycrene, and -phellandrene have been implicated in

plant reproductive fitness experiments in an orchid species, Epipactis ventrifolia (Stokl et al.,

2011). Herbivorous aphids known to feed on E. ventrifolia emit a similar mixture of terpenoids

as alarm pheromones in times of distress. Consequently, the orchid species has evolved to emit

these terpenoids from its flower as a ‘generalized mimicry’, which means that the volatile

compounds emitted do not exactly mimic the aphid alarm pheromone proportions, but mimic

only the compounds present. This generalized mimicry by the orchid attracts hoverfly females

for oviposition on the orchid. Afterward, the larvae predate herbivorous aphids, grow into

adults, and become pollinators.

Consequently, as plants have developed mechanisms to deal with herbivore and pathogen

attack, herbivores have also evolved to acquire counter solutions. For example, emission of a

volatile with the intent of attracting carnivores or perhaps as a warning signal to other plants

could inadvertently attract herbivores. Therefore, many plants have evolved to emit volatiles in a

rhythmic pattern. For example, some plants may emit volatiles to attract specific carnivores

Page 20: Molecular cloning and characterization of sesquiterpene

8

which are only diurnally active, whereas certain herbivores have evolved to feed nocturnally to

avoid diurnal predators (Shiojiri et al., 2006). Finally, an example of simultaneous herbivory of

aerial and root tissues results in systemic reduction in volatile emission and can cause increased

attack by herbivorous insects on adjacent unharmed plants (Rasmann and Turlings, 2007; Soler

et al., 2007). All of these cases exemplify the dynamic nature of life and the constant

evolutionary pressures that result in specialized metabolite profiles in plants.

1.2.2 Terpenoid Biosynthesis in Plants

The discovery of IPP, a biologically active precursor of terpenoids, influenced the works

of Lynen, Bloch, Cornforth, and Popjak in establishing the metabolism of cholesterol. By

combining genetic and biochemical studies, they elucidated that the mevalonic acid (MVA)

pathway is responsible for IPP biosynthesis in both human and yeast (Bloch, 1965, 1987).

However, stable-isotope labeling patterns of IPP in bacteria did not fit the accepted prediction,

suggesting that an independent IPP pathway could be present in bacteria. Further studies

identified mevalonate-independent pathways operating in bacteria, which use pyruvate and

glyceraldehyde 3-phosphate as starting precursors. Definitive evidence for the 1-deoxy-D-

xylulose 5-phosphate (DXP) pathway was obtained from the NMR analysis of hopanoids

(cholesterol equivalent in bacteria) (Rohmer et al., 1993; Rohmer, 1999). The DXP pathway was

only fully understood in 2000, and is perhaps the last hidden metabolic pathway conserved

across various kingdoms. The eponymous DXP pathway has also been termed the non-

mevalonate, MEP (methyl erythritolphosphate pathway), or Rohmer pathway.

Through decades of work, it is now firmly established that the biosynthesis of terpenoids

occurs in almost all living organisms via two distinct metabolic pathways, the MVA and DXP

Page 21: Molecular cloning and characterization of sesquiterpene

9

pathways. The MVA pathway is present in the cytosol of most eukaryotes and some

archaebacteria, but most prokaryotes do not have the MVA pathway (Rohmer, 1999; Estevez et

al., 2001). Therefore, most bacteria utilize the DXP pathway to synthesize IPP. However, plants

are the only organisms that possess both MVA and DXP pathways. The DXP pathway is present

in the plastid of the plant, and the MVA pathway in the cytosol. Since it is generally accepted

that plastids originated from bacteria by an ancient symbiotic event, presence of the DXP

pathway in plastid is not surprising. Both pathways, independent of starting materials, produce

isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are key

precursors of all terpenoids. DMAPP and IPP are structural isomers of each other, and are

interchangeable by IPP isomerase. IPP isomerase converts IPP to DMAPP which acts as a

fundamental primer molecule in the synthesis of longer prenyl diphosphates, such as C10 geranyl

diphosphate, C15 farnesyl diphosphate, and C20 geranyl geranyl diphosphate. These prenyl

diphosphates are the direct biosynthetic precursors of C10 monoterpenes, C15 sesquiterpenes,

and C20 diterpenes respectively (Figures 3 and 4). Additionally, C5 hemiterpenes, C30

triterpenes, and C40 tetraterpenes can be synthesized from IPP and DMAPP.

The MVA pathway uses acetyl-CoA in the cytosol as a precursor to synthesize

cholesterol or the corresponding equivalent compounds, depending on the organisms (Figure 3).

Two carbon-carbon bonds are formed in the first two reactions of the MVA pathway by

acetoacetyl-CoA thiolase and HMG-CoA synthase, which convert two acetyl-CoA molecules to

3-hydroxy-3-methylglutaryl CoA (HMG-CoA). HMG-CoA is subsequently reduced to

mevalonate by the highly regulated HMG-CoA reductase (HMGR). Mevalonate is then

phosphorylated by two kinases and finally decarboxylated to produce IPP from mevalonate

diphosphate. IPP and its isomer DMAPP are condensed to produce various prenyl diphosphates

Page 22: Molecular cloning and characterization of sesquiterpene

10

described above (Miziorko, 2011). One major metabolic fate of IPP synthesized from the MVA

pathway is cholesterol and its derivatives in animals.

The DXP pathway utilizes pyruvate and glyceraldehyde-3-phosphate (G3P) from

glycolysis (Figure 4). In the first step of the DXP pathway, pyruvate and G3P are

decarboxylated followed by two reductions and a skeletal rearrangement, catalyzing the

formation of methylerythritol phosphate (MEP). Subsequently, a cytidyl phosphate moiety is

transferred to DXP followed by phosphorylation. A unique 8-membered ring is then formed

which is facilitated by the cleavage of the nucleoside group. Ring opening and reduction are

followed by a last reduction step yielding either IPP or DMAPP. Both of the last enzymatic

steps are thought to employ a carbocation reaction (Graewert et al., 2011).

The rate-limiting enzyme for the MVA pathway is HMGR, which is competitively

inhibited by lovastatin, whereas the slowest enzyme in the DXP pathway is the reductoisomerase

(DXR), which is inhibited by fosmidomycin. Therefore, these two enzymes are the central

targets to regulate the MVA and DXP pathways. However, detailed regulation of MVA and DXP

pathways has yet to be fully understood in plants, and recent research has revealed a much more

complex feedback regulation system with multiple bottlenecks regulated at transcriptional and

post-transcriptional levels, depending on environmental and developmental cues (Rodriguez-

Concepcion, 2006).

What also seems to be unclear is the level at which metabolic crosstalk exists between the

two pathways. Based on the metabolic compartmentalization, sesquiterpenes (C15) are

synthesized in the cytosol from FPP (C15) derived from the MVA pathway, whereas

monoterpenes (C10) and diterpenes (C20) are synthesized in the plastid from GPP (C10) and

Page 23: Molecular cloning and characterization of sesquiterpene

11

GGPP (C20), respectively, derived from the DXP pathway. However, some experimental

evidence suggests that the precursors for terpene synthases (TPS; prenyl diphosphates such as

FPP, GPP, and GGPP) can be transported to and from the plastid. In addition, some terpene

synthases can efficiently use physiologically non-relevant substrates. For example, Aharoni et

al. have found a cytosolic sesquiterpene synthase (FaNES1) from Fragaria ananassa (garden

strawberry) capable of synthesizing both linalool (monoterpene) and nerolidol (sesquiterpene)

from GPP and FPP, respectively (Aharoni et al., 2004). In their experiment, overexpression of

FaNES1 in the plastid surprisingly resulted in production of relatively high quantities of linalool

as well as small amounts of nerolidol (Aharoni et al., 2004). Therefore, this cytosolic enzyme has

the capability to synthesize a monoterpene from a physiologically non-relevant substrate, GPP.

Other literature evidence further implies a plastidal proton symporter which could transport

plastidic prenyl diphosphates to the cytosol, although there is no additional biochemical or

genetic data to support this report (Bick and Lange, 2003). In snapdragon, sesquiterpene

volatiles were shown to be synthesized from IPP, originating from the plastid, by labeling and

inhibitor studies (Bick and Lange, 2003). However, it is not certain if this metabolic crosstalk is

a specific case in one species or if it is a widespread phenomenon in the plant kingdom.

Nonetheless, it is evident that prenyl diphosphates can be transported from plastid to cytosol

efficiently (Dudareva et al., 2005). Similarly, cytosol to plastid transport has also been proposed

to occur in some plants (Rodriguez-Concepcion, 2006). Whether the activities of TPS enzymes

can truly catalyze these two distinctly separated reactions or if it happens inadvertently due to

promiscuous activities developed during the evolution of homologous TPSs remains to be seen

and implies a deeply complex system of subcellular regulation (Nagegowda et al., 2008).

Page 24: Molecular cloning and characterization of sesquiterpene

12

Advancement in reverse genetics (e.g., RNAi and virus-induced gene silencing) has

allowed researchers the ability to investigate complex transcriptional regulation by silencing

specific transcripts, thus allowing further elucidation of crosstalk between the DXP and MVA

pathways. Additional support for the crosstalk was observed by RNAi-silencing of DXS in the

DXP pathway. Knock-down of DXS unexpectedly increased the level of sesquiterpenes in the

cytosol; subsequently, precursors from the MVA pathway were incorporated into monoterpenes

in the plastid by isotope-labeling studies (Paetzold et al., 2010). Therefore, a body of direct and

indirect experimental data strongly suggests that metabolic pools originating from both the MVA

and DXP pathways are interchangeable in plants.

The condensation of IPP to the priming molecule (DMAPP) occurs in a trans-

configuration, and until recently it has been believed that trans-GPP, FPP, and GGPP (or E,E-

prenyl diphosphates) are the only natural substrates for terpene synthases. However, a novel

pathway in wild tomato capable of producing sesquiterpenes from a cis-configured FPP (or Z,Z-

FPP) was identified and shown to be localized in the plastid (Sallaud et al., 2009). This is a

good example that TPS function is highly versatile and cannot be predicted by sequence

information alone.

Page 25: Molecular cloning and characterization of sesquiterpene

13

Figure 3. Schematic depiction of the MVA pathway.

Grey text indicates continuation of pathway to primary metabolite production. The MVA

pathway’s subcellular location is within the cytosol.

Page 26: Molecular cloning and characterization of sesquiterpene

14

Figure 4. Schematic depiction of the DXP pathway.

Grey text indicates continuation of pathway to production of primary metabolites. The DXP

pathway’s subcellular location is contained within chloroplasts.

Page 27: Molecular cloning and characterization of sesquiterpene

15

1.3 Terpene Synthases

The exceptionally large diversity of terpenoids can be attributed to the catalytic plasticity

of the terpene synthase (TPS) enzyme family. TPSs catalyze acyclic and cyclic rearrangements

of their linear prenyl diphosphate and squalene precursors into a plethora of different terpenoids.

TPSs have a great degree of specificity towards their respective prenyl diphosphate precursors

but exhibit large variation in their catalytic mechanisms, resulting in enzymes producing single

and multiple terpene products. For example, two multi-product TPSs from Abies grandis were

shown to produce 34 and 52 different terpenes, respectively, whereas a third synthase from the

same species produces only -bisabolene (Steele et al., 1998). Evolution of the TPS family is

proposed to have occurred by duplication of general or specific metabolic genes, and subsequent

adaptive radiation of duplicated TPS genes (i.e., mutations), leading to enzymes that synthesize a

distinct product from the same substrate (Pichersky and Gang, 2000). To date, roughly 300

specific terpene skeletal structures have been reported, which most likely arose from the diverse

activities originating from gene duplications and neo-functionalization of TPSs (Bohlmann et al.,

1998).

Currently, the crystal structures for several TPSs from plant, bacteria, and fungi have

been determined. Several plant TPS structures have been described so far, including the (+)-

bornyl diphosphate synthase from Salvia officinalis (Whittington et al., 2002), -bisabolene

synthase from Abies grandis (McAndrew et al., 2011), epi-aristolochene synthase from

Nicotiana tabacum (Starks et al., 1997), (+)--cadinene synthase from Gossypium arboreum

(Gennadios et al., 2009), and ent-copalyl synthase from A. thaliana (Koeksal et al., 2011), as

well as taxadiene synthase from Taxus brevifolia (Koeksal et al., 2011). In general, the study of

TPS-mediated reactions involves either ionization-dependent or protonation-dependent

Page 28: Molecular cloning and characterization of sesquiterpene

16

carbocation formation. This is quite similar to the prenyl transferase enzymes from which the

terpene synthases are believed to have evolved. For example, the ionization-dependent terpene

synthases have an -helical fold termed the class I TPS-fold, whereas the protonation-dependent

synthases possess an unrelated -barrel fold, class II fold. However, exceptions to this rule exist

throughout the terpene synthase family. Abietadiene synthase, a diterpene synthase, possesses

both class I and II folds, in a single polypeptide and hence can catalyze both ionization- and

protonation-dependent reactions. Tobacco epi-aristolochene synthase (TEAS) also has both

structural elements, but only the class I fold is active and located in the C-terminal domain

(Christianson, 2006). The sesquiterpene synthase -bisabolene synthase from A. grandis is an

exceptional enzyme in that it has a vestigial -domain normally present in diterpene synthases

(McAndrew et al., 2011). This has interesting implications in terpene synthase evolution, as the

current theory reasons that sesquiterpene synthases evolved from diterpene synthases (Trapp and

Croteau, 2001), which could point to -bisabolene synthase from A. grandis as the most recently

diverged sesquiterpene synthase known.

1.3.1 Sesquiterpene Synthase Structure-Function Relationships

Many TPSs, from bacteria, fungi, and plant, lack similarity in primary structure but share

distinct structural domains, such as the N-terminal domain and the catalytic C-terminal domain

(Starks et al., 1997; Bohlmann et al., 1998). The structural characterization of the first TPS from

plant, TEAS, revealed that it completely consists of an -helical structure with short connecting

loops forming an -helical barrel active site (Starks et al., 1997), which is now known to be

conserved throughout bacteria, fungi, and plants termed the ‘terpene synthase fold’ (Bohlmann et

al., 1998). Other specific structural elements of terpene synthases include a conserved DDXXD

Page 29: Molecular cloning and characterization of sesquiterpene

17

motif involved in binding divalent metal ions for stabilization of the diphosphate moiety upon

ionization, and variations or duplications of this ‘aspartate rich’ motif result in reduced activity.

The highly hydrophobic aromatic-rich active site in TPS accommodates the long olefin chain of

the prenyl diphosphate substrate while the two Mg2+

ions are complexed with the aspartate-rich

motif (DDXXD), stabilizing the ionized diphosphate group. A third Mg2+

is complexed by a

(L,V)(V,L,A)-(N,D)D(L,I,V)X(S,T)XXXE (metal-binding ligands in bold) motif and a water

molecule (Christianson, 2006). The N-terminal domain contains two flexible regions termed the

A-C and J-K loops which help to prevent solvent-access to the hydrophobic active site when

bound to a substrate. The A-C loop contains two generally conserved arginine residues. One

helps to stabilize the lid forming action of the J-K loop when a substrate binds to the TPS and the

other helps to stabilize the negatively charged diphosphate. This action is presumably important

to prevent the regeneration of the initial FPP or its tertiary allylic isomer, nerolidyl diphosphate.

Further stabilization of the carbocation intermediates occur through conserved aromatic residues

via -cation interaction. Whether these and other aliphatic residues have active or passive

functions within the carbocation reaction mechanism is currently a topic of debate (Miller and

Allemann, 2012). For example, recent studies of patchouli alcohol synthase from the plant

species, Pogostemon cablin, putatively implicated a single leucine in active reorientation during

catalysis, effectively creating a second active site pocket (Faraldos et al., 2010). Conversely, the

skeletal structure of the terpene may rely more on the initial orientation of the substrate upon

binding the active site, implying a more passive role for the active-site residues as chaperones to

a product. For example, TEAS and Hysocyamus premnaspirodiene synthase are two

evolutionarily related enzymes that have been shown to share a carbocation intermediate but

yield different products. Studies in which various amino acids were mutated, independent of the

Page 30: Molecular cloning and characterization of sesquiterpene

18

active sites and in increasing radii from the active sites, resulted in switching of their respective

products (Greenhagen et al., 2006).

Initial insight into the structure-function relationships between sesquiterpene synthases

and their substrate (FPP) came from the TEAS crystal structure. This evidence led to the

synthetic carbocation mechanism of TEAS and became the template for which most TPS

catalyzed reactions proceed. Stabilization and positioning of the electrophilic carbon (C1)

facilitates attack by the C10-C11 pi-bond creating a cyclic carbocation (Figure 5). Termination

of this carbocation, which produces a germacrene A intermediate, was initially proposed to occur

by an acidic tyrosine. However, this has also become a contentious observation. Site-directed

mutagenesis of other Tyr residues from sesquiterpene synthases from two bacterial species

Penicillium roquefortii (Felicetti and Cane, 2004) and Fusarium sporotrichioides resulted in no

change of product. This result caused researchers to conclude that the diphosphate ion may be

involved with acid/base catalysis (Shishova et al., 2007). Substrate docking of the bisabolyl

cation in modeling simulations, with two sesquiterpene synthases from Sorghum bicolor,

indicates the diphosphate ion as a proton acceptor (Garms et al., 2012). Subsequently, the

reaction from the germacrene A intermediate in TEAS proceeds by addition of a proton to C6 via

a Asp-Tyr-Asp catalytic triad where the last two residues are contained within the J-K loop

(Figure 5). Consequently, a second ring closure at C2 and C7 would occur creating the

eudesmane carbocation intermediate. Final termination of the carbocation cascade by

deprotonation of the eudesmane intermediate by the indole ring of a tryptophan would be

facilitated by the formation of an arenium cation (Figure 5). Fundamentally, the termination of

the carbocation cascade can occur by capture of water, which creates a terpene alcohol or by

proton abstraction and different TPSs apply different quenching methods.

Page 31: Molecular cloning and characterization of sesquiterpene

19

Relationships between reaction mechanism and enzyme kinetics have yet to be

scientifically explored. Roughly 100 sesquiterpene synthases have been characterized as of 2008

(Degenhardt et al., 2009), and most sesquiterpene synthases have an apparent Km ranging from

0.4-10 M (Picaud et al., 2005) with the exception of -bisbolene synthase which exhibits a Km

of 49.5 M (McAndrew et al., 2011). Slower rates of catalysis are observed with enzymes

involved in sesquiterpene biosynthesis, in general, and sesquiterpene synthases show relatively

low kcat values ranging from 0.033 - 4.0x10-3

s-1

(Chen et al., 1995; Shen et al., 2007).

Page 32: Molecular cloning and characterization of sesquiterpene

20

Figure 5. Schematic diagram representing the carbocation mechanism of tobacco epi-

aristolochene synthase (TEAS).

Page 33: Molecular cloning and characterization of sesquiterpene

21

1.3.2 Phylogenetic Relationships of Terpene Synthases

TPSs are believed to have originated from prenyl transferases (e.g., GPP and FPP

synthase). However, little empirical evidence exists for such conclusions. Elucidation of TEAS

3D structure revealed convincing evidence as the C-terminal backbone of TEASs tertiary

structure aligns with avian FPP synthase, despite apparent lack of primary sequence similarity

(Starks et al., 1997). Further convincing evidence from TPS phylogenetic alignments of amino

acid sequences (>40% similarity) revealed that gymnosperm monoterpene, sesquiterpene, and

diterpene synthases are more closely related to each other than to their counterparts in

angiosperm (Bohlmann et al., 1997; Bohlmann et al., 1998). This indicates convergent evolution

of specialized TPSs after the angiosperm and gymnosperm bifurcation (Bohlmann et al., 1997;

Bohlmann et al., 1998). Classification of TPSs based on the phylogenetic analysis showed that

seven TPS clades or sub-families are present in nature and fit into the following nomenclature,

TPS-a to -g (Bohlmann et al., 1998; Aubourg et al., 2002; Dudareva et al., 2003). The TPS-a

subfamily consists of casbene synthase, a diterpene synthase, and sesquiterpene synthases from

various angiosperms. TPS-b consists of monoterpene synthases from angiosperm but is distinct

from TPS-a. TPS-c and TPS-e contain diterpene synthases from primary metabolism, and

therefore have fewer representative members. The subfamily of TPS-f contains only one

presumably ancient linalool synthase, and the TPS-d subfamily contains gymnosperm TPSs.

Recently, three monoterpene synthases from Antirrhinum majus, one monoterpene synthase from

A. thaliana, and a sesquiterpene synthase, nerolidol synthase, from Fragaria ananassa comprise

a new subfamily, TPS-g, characterized by a lack of an RRx8W motif, which is present in all

characterized monoterpene synthases from angiosperm TPS-b and gymnosperm TPS-d

subfamilies (Bohlmann et al., 1997; Aubourg et al., 2002; Dudareva et al., 2003; Jones et al.,

Page 34: Molecular cloning and characterization of sesquiterpene

22

2011). Function of this motif is thought to be involved in cyclization of prenyl diphosphates as

all synthases lacking this motif produce acyclic products (Dudareva et al., 2003).

1.4 Metabolic Engineering of the MVA Pathway

Many plant terpenoids have been traditionally used as aromas, flavors, pharmaceuticals,

and nutraceuticals, but the natural abundance of terpenoids is minute. Furthermore, the structural

complexity of terpenoids has prevented their chemical synthesis on a commercial scale.

Therefore, biotechnological efforts have focused on the over-production of rare but valuable

terpenoids in fast growing heterologous microbial hosts such as E. coli and yeast. E. coli and

yeast provide genetically amenable platforms for reconstitution and manipulation of complex

metabolic pathways, such as the MVA and DXP pathways for improved terpenoid production.

Reconstitution and manipulation of the MVA or DXP pathways have been attempted and

proven to be successful in E. coli and yeast. Prokaryotes, such as E. coli, do not possess the

MVA pathway, and thus reconstruction of the pathway in E. coli could create an organism

implemented with an entirely synthetic metabolic pathway. The synthetic MVA pathway in E.

coli is expected to be free from any endogenous regulatory mechanisms and hence avoids

complicated feedback regulation (Dudareva et al., 2003). Although manipulation of the

endogenous DXP pathway in E. coli has been proven to increase the level of terpenoids, the

endogenous regulatory mechanisms controlling the DXP pathway in E. coli are highly complex

and not fully understood and hence the scalable production of terpenes was not achieved

(Kajiwara et al., 1997; Farmer and Liao, 2001; Kim and Keasling, 2001). Recently, complete

reconstitution of the MVA pathway in tobacco chloroplasts was successful in producing higher

Page 35: Molecular cloning and characterization of sesquiterpene

23

than normal amounts of FPP derivatives, indicating plant metabolic engineering is also feasible

(Kumar et al., 2012).

Metabolic engineering of yeast relies heavily on modifications of the endogenous MVA

pathway, and the best example for increased C15 sesquiterpene production involved increasing

FPP abundance (Ro et al., 2006; Shiba et al., 2007; Ro et al., 2008). Four central points of

importance in achieving enhanced carbon flux for de novo terpene synthesis are: i) to increase

the pool of acetyl-CoA that serves as a precursor to the MVA pathway, ii) to increase cellular

activity of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase

(HMGR) and deregulate it from feedback inhibition, iii) to re-route FPP from ergosterol (yeast

sterol) to sesquiterpene biosynthesis, and iv) to overexpress the transcription factor activating

the steroid (i.e., MVA) biosynthetic pathway.

Firstly, implementing the pyruvate dehydrogenase bypass in yeast can alleviate the

bottleneck created by pathway precursor supply of acetyl-CoA to the MVA pathway. The

pyruvate dehydrogenase bypass converts pyruvate into acetyl-CoA in three steps by pyruvate

decarboxylase, acetaldehyde dehydrogenase, and acetyl-CoA synthetase. By overexpressing the

endogenous acetaldehyde dehydrogenase and heterologously expressing a Salmonella enterica

acetyl-CoA synthetase variant, Shiba et al. were able to increase acetate production in

engineered Saccharomyces cerevisiae (Shiba et al., 2007). Secondly, the major metabolic

bottleneck of the MVA pathway is caused by the rate-limiting enzyme HMGR, and thus

overexpression of a deregulated version (N-terminal truncated) of HMGR could significantly

enhance the flux. HMGR is regulated by several intermediate products of the MVA pathway

including FPP, and its membrane bound N-terminal domain appears to mediate the feedback

Page 36: Molecular cloning and characterization of sesquiterpene

24

inhibitory effect. N-terminal truncation of tHMGR was shown to abolish inhibitory activity and

increase squalene production in yeast (Donald et al., 1997; Polakowski et al., 1998). Thirdly,

squalene synthase condenses two C15 FPP molecules to synthesize C30 squalene, however this

synthase can be down-regulated to increase the availability of FPP. Sterol biosynthesis in yeast

is an essential pathway, and the biosynthesis of sterol in S. cerevisiae involves over 20 distinct

reactions from the precursor acetyl-CoA, proceeding through FPP, which is a branch point for

sterol and sesquiterpene production (Shiba et al., 2007). Squalene synthase is the first committed

step in sterol biosynthesis. Therefore, down-regulating the expression of squalene synthase

(ERG9) can have a marked impact on increasing FPP (Ro et al., 2006). Lastly, a point-mutant

version of UPC2 transcription factor (upc2-1) can constitutively up-regulate several genes in the

MVA pathway.

Additional studies have employed more drastic methods to block squalene synthesis from

FPP by the complete knockout of squalene synthase. Complete aberrant removal of this gene

would result in a lethal mutant (sue) (Takahashi et al., 2007), but the phenotype can be rescued

by an external supply of ergosterol (yeast cholesterol), producing an abundant level of FPP.

However, cytotoxicity becomes a significant problem with engineering overproduction of FPP,

and consequently the yeast dephosphorylate FPP by diacylglycerol pyrophosphate phosphatase

(DPP1) to form farnesol (FOH), which is less toxic. Therefore, knocking out dpp1 is a rational

step in committing carbon to the production of sesquiterpenes (Faulkner et al., 1999). Similar

efforts to improve flux of FPP towards terpene hydrocarbon production, such as overexpression

of the FPP synthase, have been used but have little additive effect (Jackson et al., 2003; Ro et al.,

2006).

Page 37: Molecular cloning and characterization of sesquiterpene

25

Another potentially significant problem exists with the consequence of high-level

production of terpenoids as there may be innate toxicity related to the terpene being produced

(Ro et al., 2008). Consequences of such toxicity have been observed in yeast engineered to

produce high levels of arteminisic acid. Ro et al. found that yeast engineered to produce large

quantities of artemisinic acid, an anti-malarial drug precursor to artemisinin, resulted in the

induction of multiple pleiotropic drug resistance genes (Ro et al., 2008). Global transcription

analysis by yeast microarray, as well as quantitative PCR, identified genes from the major

facilitator superfamily, in addition to ATP-binding cassette transporters, in response to the

overproduction of the weak acid, artemisinic acid.

1.5 Ligand-Receptor Binding

Several terpenoids have been shown to bind pharmacologically important receptors with

high specificity, and therefore have relevance as anti-cancer anti-psychotic and anti-malarial

drugs (Eckstein-Ludwig et al., 2003; Jordan and Wilson, 2004; Yan et al., 2005; Winther et al.,

2010). For example, salvinorin A is a lipophilic neutral small molecule, which selectively binds

a G-protein coupled receptor (GPCR) (Yan et al., 2005). Another diterpene anti-cancer drug,

paclitaxel, has been shown to be a potent mitotic inhibitor (Jordan et al., 1996). Other examples

of terpenoids that have selective biological targets are artemisinin and thapsigargin which both

attenuate activity of Ca2+

ion pumps in Plasmodium falciparum ATP6 and its mammalian

homolog sarcoplasmic endoplasmic reticulum Ca2+

ATPase (SERCA), respectively (Eckstein-

Ludwig et al., 2003; Winther et al., 2010).

Salvinorin A is a hallucinogenic diterpene produced by the sage Salvia divinorum and has

historically been used by the Mazatec people of Oaxaca, Mexico in shamanic rituals. The

Page 38: Molecular cloning and characterization of sesquiterpene

26

hallucinogenic properties of salvinorin A lie in its ability to selectively bind the -opioid receptor

(Yan et al., 2005). Salvinorin A was the first non-alkaloid opioid subtype-selective drug and

exhibits no affinity for the traditional target of most natural hallucinogenic compounds, such as

N,N-dimethyltryptamine, psilocybin, and mescaline, and it rivals the potency of synthetic

hallucinogens, such as lysergic acid diethylamide (LSD) (Roth et al., 2002). Stabilization of the

compound in the binding pocket is through unusual and generally unconserved binding residues

isoleucine, glutamate and tyrosine (Yan et al., 2005).

Inhibition of mitosis represents a powerful approach in controlling cancer cell

proliferation. Microtubules play an extremely important role in the proliferation of metastatic

tumors as these generally advance through mitosis rapidly. For example, during prometaphase,

microtubules must rapidly extend and retract in an effort to adhere to the kinetochores. This

highly dynamic nature of microtubule formation is the basis for ‘microtubule binding agents’.

Paclitaxel is a microtubule stabilizing agent (MSA) which promotes polymerization whereas

vinblastine or vincristine bind and inhibit polymerization. The consequence to the cell is loss of

the dynamic nature needed for advancement to anaphase, and consequently the cell enters

apoptosis. Paclitaxel binds the -subunit of tubulin and is located on the inner surface of

microtubule structures (Nogales et al., 1995). Elucidation of the actual binding site has

identified an arginine residue as the specific amino acid involved (Rao et al., 1999). The

mechanism by which paclitaxel promotes polymerization is unknown, and there is only one

binding site on every molecule of tubulin (heterodimer of and subunits). Initially it was

believed that taxanes and other MSAs diffused through fenestrations in the microtubule wall.

However, kinetic studies have revealed that binding of paclitaxel is too fast to occur in this

manner, leading scientists to propose a second mechanism (Diaz et al., 2003). Recent modeling

Page 39: Molecular cloning and characterization of sesquiterpene

27

evidence supports a proposed second binding site whereby taxanes bind first to the outer-

microtubule surface before moving to the -subunit binding site on the inner surface of the

tubulin structure (Magnani et al., 2009).

Calcium balance within the endoplasmic reticulum is an important process as Ca2+

is an

important second messenger in cell signaling processes. Consequently, Ca2+

flux is tightly

governed by Ca2+

ion channels, and disruption of such processes can lead to pro-apoptotic

cascades, indirectly inducing cytochrome c release, caspases, and finally cell death (Scorrano et

al., 2003; Deng et al., 2009). Therefore, inhibition of normal SERCA function by thapsigargin

regardless of the proliferative state of the cell could make this sesquiterpene lactone a potent

anti-cancer drug (Winther et al., 2010). The lipophilic nature of thapsigargin allows it to

selectively bind the E2 form of SERCA (Toyoshima and Nomura, 2002). Unfortunately,

selective targeting of non-proliferative cells is not feasible with most anti-cancer drugs, and

hence a pro-drug mechanism has been designed for thapsigargin where a short H-S-S-L-Q-L

amino acid sequence attached to a short linker at O-8 allows for specific recognition by a

prostate-specific antigen protease (Denmeade et al., 2003). In a similar mechanism, artemisinin

inhibits PfATP6 Ca2+

levels in P. falciparum, and mutagenesis studies have identified a single

amino acid which can abolish the inhibitory activity of artemisinin (Uhlemann et al., 2005). The

impetus for which malarial parasites develop resistance to artemisinin may impinge on

elucidation of this mutation in natural settings (Krishna et al., 2010).

1.6 Sesquiterpene Biosynthesis in Valeriana officinalis

Valeriana officinalis is a medicinal plant native to Asia and Europe where it has been

used for centuries as a potent sedative, although contemporary uses are more common to Europe

Page 40: Molecular cloning and characterization of sesquiterpene

28

and the United States. In fact, valerian made the top-ten list of top selling herbal remedies in the

United States in 2002 (Anderson et al., 2005). The first biological activity relating to valerian

root extract was observed over 50 years ago (Stoll et al., 1957). Various metabolites of the

essential oil extracts from dried root show hundreds of specialized metabolites that include but

are not limited to chlorogenic acid, monoterpene alkaloids, terpenoids, valepotriates, furanofuran

lignans, and phenylpropanoids (Torssell and Wahlberg, 1966, 1967; Houghton, 1999; Navarrete

et al., 2006). Major terpene compounds identified from valerian root extracts are

sesquiterpenoids, such as valeranone, valerenal, valerenic acid, and several valerenic acid

derivatives (Stoll et al., 1957; Houghton, 1988, 1999). The precise compound exhibiting activity

has been contentious, but recent studies implicated the sesquiterpene, valerenic acid, as an

inhibitor of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) pathway

(i.e., anti-inflammatory) and agonist of the -aminobutyric acid type A (GABAA) receptor (i.e.,

sedative) (Figure 7) (Jacobo-Herrera et al., 2006; Benke et al., 2009).

Mediation of neuronal excitability in the human brain is highly reliant on -aminobutyric

acid, which potently inhibits GABAA receptors (Khom et al., 2010). GABAA receptors are the

targets of the drug class benzodiazepines due to the important role they play in mediating the

balance between excitation and inhibition of the central nervous system. Consequently,

benzodiazepines have potentially serious side-effects. Therefore, discovery of drugs with similar

efficacy but benign manifestation of side-effects has led to extracts from plants, such as valerenic

acid from V. officinalis. Similarly, in vivo and in vitro experiments have reported valerenic acid

to be an allosteric inhibitor of the GABAA receptor, constituting it as a potential anxiolytic drug

with little toxicity (Benke et al., 2009; Khom et al., 2010). The hydrocarbon precursor to

valerenic acid, valerena-4,7(11)-diene, has also been implicated as an anxiolytic compound

Page 41: Molecular cloning and characterization of sesquiterpene

29

(Takemoto et al., 2009). Ligand-receptor binding and mutagenesis studies of valerenic acid and

valerenic acid derivatives with GABAA receptors implicates Gln265 on the -3 subunit as

absolutely necessary for interaction (Benke et al., 2009).

Valerenic acid itself has been shown to have nM level binding constants with respect to

GABAA receptors (Benke et al., 2009). The unique structure of valerenic acid may be important

for activity as other derivatives have been shown to have similar and sometimes higher potency

(Khom et al., 2010; Kopp et al., 2010). Interestingly, because valerena-4,7(11)-diene and

valerenal (possible aldehyde precursor to valerenic acid) have also been determined to potentiate

GABAA receptors, therefore several of the possible pathway intermediates in valerenic acid

metabolism may have significance as sedatives and there may be a synergistic effect occuring.

Page 42: Molecular cloning and characterization of sesquiterpene

30

1.7 Objectives

The goal of this project is to identify a novel sesquiterpene synthase, which catalyzes the

first committed step to valerenic acid from a medicinal plant Valeriana officinalis. Structural

analysis of valerenic acid suggests that valerena-4,7(11)-diene is the product from the

unidentified sesquiterpene synthase (Figure 6). Importantly, this sesquiterpene skeleton is

unique, and its synthase from FPP substrate has yet to be identified. Ultimately, I aim to

demonstrate the enzymatic synthesis of the medically important terpene, valerena-4,7(11)-diene.

Integrative approaches involving genomics, chemistry, and metabolic engineering tools will be

included in this project.

Four specific objectives to achieve this goal are as follows.

Specific Objectives

1. Utilize genomics resources to identify TPS genes from Valeriana officinalis.

2. Functional activity evaluation of the encoded recombinant enzymes in yeast and E. coli

systems.

3. Structural elucidation of the sesquiterpene to be valerena-4,7(11)-diene.

4. Propose the mechanism for valerena-4,7(11)-diene synthesis based on TPS product profile.

Page 43: Molecular cloning and characterization of sesquiterpene

31

Figure 6. Proposed biosynthetic pathway for valerenic acid production in V. officinalis.

Page 44: Molecular cloning and characterization of sesquiterpene

32

CHAPTER 2: MATERIALS AND METHODS

2.1 Plant Cultivation and Metabolite Preparations

V. officinalis seeds were obtained from B & T world seeds (France). Seeds were

germinated at 20 °C, and seedlings were grown in the University of Calgary greenhouse.

Valerian root was ground by mortar and pestle with liquid N2, and 100 mg of the ground tissue

was extracted using 1 mL ethyl acetate. The organic layer was partitioned by centrifugation,

diluted 10 times, and analyzed by GC-MS under the conditions described below.

2.2 RNA preparations

Total RNAs were isolated according to a modified version of the published protocol

(Meisel et al., 2005). Valerian root and leaf were ground under liquid N2 and 1.5-2 g were

extracted with 5 mL/g tissue of extraction buffer (1% (w/v) Cetyl Trimethyl Ammonium

Bromide (CTAB), 0.5 M TRIS HCl pH 8.0, 0.25 M EDTA pH 8.0, 4% (w/v) NaCl, 0.5% (w/v)

polyvinylpyrrolidone (PVP) in diethyl pyrocarbonate (DEPC) treated H2O preheated to 65ºC.

100 L of fresh -mercaptoethanol and 50 L spermidine trihydrochloride (SPD) were added to

5 mL extraction buffer. After the extraction slurry was vortexed for 30 sec. an equivalent

volume of 24:1 (chloroform:isoamyl alcohol) was added followed by vortexing and

centrifugation at 12,000 x g for 20 min. at room temperature. The resulting aqueous phase was

decanted and extracted a second time with 24:1 (chloroform:isoamyl alcohol). The aqueous

phase was then decanted and LiCl was added to a concentration of 2 M. After an overnight

incubation at 4ºC the solution was centrifuged at 12,000 x g for 35 min at room temperature and

the supernatant removed and the pellet dried but avoiding complete dessication. The pellet was

then resuspended in 0.5 mL of DEPC-treated H2O and extracted once more with 24:1

(choloroform:isoamyl alcohol). After vortexing, the centrifugation step was performed at 14,000

Page 45: Molecular cloning and characterization of sesquiterpene

33

x g for 30 min at 4ºC. Aqueous phase was extracted, 1 mL of 100% ethanol was added and the

solution incubated on ice before vortexing and precipitating for 30 min at -80ºC. The ethanol

solution was then centrifuged at 14,000 x g for 20 min at 4ºC. The supernatant was removed and

the pellet dried avoiding complete desiccation. The resulting pellet was washed with 75%

ethanol, centrifuged at 14,000 x g for 10 min at 4ºC and dried once more. Total RNA was

dissolved in 50 L DEPC-treated H2O followed by concentration and purity measurements by a

Nanodrop 1000.

2.3 cDNA Library Preparation from Total RNA

7 µg of double-stranded cDNA from root tissue were prepared by the supplier’s protocol

(Invitrogen) using Superscript II Reverse Transcriptase (Invitrogen). The 454 GS FLX Titanium

was used to sequence valerian cDNA, and the raw reads were assembled by the University of

Calgary Bioinformatics Center through the Magpie informatics platform.

2.4 Plasmid Construction for Yeast Expression

Full length sesqui-TPS cDNAs (VoTPS1/2/3) were obtained by in silico analysis of the V.

officinalis database from the PhytoMetaSyn project at the University of Calgary. VoTPS1/2/3

were amplified from valerian root cDNA by a forward primer and a reverse primer with a

restriction enzyme digestion site integrated into the primer (Table 1). General PCR conditions

were as follows: 1 cycle of 30 sec at 98C; 29 cycles of 10 sec at 98C, 30 sec. at 60C (Table

1), 1 min 45 sec at 72C; followed by 1 cycle for 10 min. at 72C. The amplified PCR product

was ligated into a pGEM vector using a TA-cloning kit (Promega). The resulting pGEM clone

harbouring one of VoTPS1/2/3 was then transformed into Top10 cells and grown overnight at

37C on plates containing 100 g/mL ampicillin. Colony PCR was then performed to confirm

Page 46: Molecular cloning and characterization of sesquiterpene

34

the presence of inserts and a single positive colony was selected for growth overnight at 37C in

3 mL LB broth containing 100 g/mL ampicillin. Isolation of pGEM clones harbouring one of

VoTPS1/2/3 was done by kits (Gene-All, Korea) and subsequently, restriction mapped and

sequenced. pGEM clones containing one of VoTPS1/2/3 were then digested with their respective

restriction enzymes (Table 1) and ligated into a linearized pESC-Leu2d vector and transformed

into Top10 cells. Colonies from plates were then verified to contain the insert by colony PCR.

Positive colonies were grown at 37C in 3 mL LB broth containing 100 g/mL ampicillin and

clones isolated using a kit (Gene-All, Korea). Clones were resequenced to confirm the insert

was present in the desired vector as ampicillin was the selection marker for both pGEM and

pESC-Leu2d cloning.

2.5 Quantitative Transcript Analysis

Semi-quantitative RT-PCR analyses for VoTPS1/2 were performed using 250 ng cDNA

from V. officinalis root or aerial tissue for 30 cycles with an annealing temperature of 55°C. For

VoTPS1, the primers used were a forward primer, 5’-CTGTTTACGAACAAGACAAGTCATG

CAAC-3’, and a reverse primer, 5’-AAGTCACAAAGCGCACCAAATTCAGAACT-3’. For

VoTPS2, the primers used were a forward primer, 5’-TATCGTCGAACGATACATTATTAGC

ATCAG-3’, and a reverse primer, 5’- CTTTGTAGAATACATTCATAAAGCATG-3’. The

restriction enzyme mapping of the resulting 921-bp (VoTPS1) and 1032-bp (VoTPS2) amplicons

were performed using EcoRV and HindIII separately to confirm their sequence identities.

Identical conditions and primers were used with 250 ng of RNA from V. officinalis root or aerial

tissues as a negative control to rule out possible genomic DNA contamination. Elongation factor

1α (EF1) was used as an internal control with a forward primer, 5’-GACTGTCACACTTCTCA

CATTGCC-3’, and a reverse primer, 5’-TCTCGACCACCATAGGTTTGGT-3’, using 5 ng of

Page 47: Molecular cloning and characterization of sesquiterpene

35

cDNA from V. officinalis root or aerial tissues by the same PCR conditions mentioned above.

Amplified VoTPS1/2 and EF1 fragments were mixed and run in the same lane for visualization.

Quantitative PCR of VoTPS1 was performed with a forward primer, 5’- TGGTCAAAGCATC

AACAATTATCGCT-3’, and a reverse primer, 5’-CTTCTTCTTTTGTGGCACCATGTTGT-3’.

Ten ng of cDNA from V. officinalis root or aerial tissues were used with an annealing

temperature of 58°C. The above mentioned EF1 primers were also used as the reference gene.

2.6 Yeast Transformation

All yeast transformations were done with the EPY300 strain according to the protocol

described by (Gietz and Schiestl, 2007). A single colony was selected for growth overnight at

30C in 2 mL SC (500 mL of media containing 0.695 g of a mixture of amino acids containing

various amounts of the following: L-Ala, L-Arg, L-Asn, L-Asp, L-Lys, L-Glu, L-Ile, L-Lys, L-

Phe, L-Pro, L-Ser, L-Thr, L-Tyr, L-Val, L-Trp, Gly, uracil and adenine; 3.35 g yeast nitrogen

base) media omitting His and Met with 2% (v/v) glucose and shaken at 200 rpm. The overnight

culture was diluted 25-fold to a 50 mL SC medium of the same components, at the same

concentrations and grown at 30C for 4-6 hrs shaking at 200 rpm, followed by two wash steps

with sterile ddH2O, pelleted for 5 min. at 4,150 rpm. This was followed by an additional two

wash steps with sterile ddH2O, centrifuged at 14,000 rpm for 30 sec. After the last wash the cells

were resuspended in 50% polyethyleneglycol, 1 M lithium acetate and single-stranded salmon

testes DNA (Sigma Aldrich). 0.5-1.0 g plasmid DNA was used for each respective

transformation and incubated at 42C for 40 min. After incubation transformations were left on

ice for 2-5 min. before plating on SC-agar media omitting His, Met and Leu supplemented with

2% (v/v) glucose and grown for 3 days at 30C..

Page 48: Molecular cloning and characterization of sesquiterpene

36

2.7 In vivo Production of Terpenoids in Yeast

Transgenic yeasts were inoculated in 2 mL Synthetic Complete (SC) media omitting the

amino acids His, Met and Leu with 2% glucose, and the sub-cultures were cultivated overnight at

30C and 200 rpm. The overnight culture was diluted 25-fold to a 50 mL SC media omitting His

and Leu with 2% (v/v) galactose, 0.2% (v/v) glucose, and 2 mM Met. Five mL of dodecane

(10% of the culture volume) was overlaid to the culture medium to trap volatile terpenoids

released during culture. The 50 mL yeast was cultured at 30C for 200 rpm for 3 days. The

yeast cultures were then centrifuged at 4,000 rpm for 5 min, and 1 mL of dodecane was extracted

and diluted in hexane (100-fold dilution) for GC-MS analysis.

2.8 Plasmid Construction for E. coli Expression

The Gateway Cloning (Invitrogen) system was used for construction of the bacterial

expression clone. VoTPS1/2/3 genes were initially cloned into the pDONR207 vector using gene

specific primers with attB1 specific 5’ tails (Table 1). According to the Gateway manual a PCR

reaction was performed using the following conditions: 1 cycle for 2 min. at 95°C ; 10 cycles for

15 sec. at 94 °C, 30 sec. at 60°C; 1 min. 45 sec. at 68°C. 10 L of the previous reaction were

immediately added to 40 L of a second reaction using the following conditions: 1 cycle for 1

min. at 95°C; followed by 5 cycles for 15 sec. at 94°C, 30 sec. at 45°C, 1 min. 45 sec. at 68°C;

followed by 15 cycles for 15 sec. at 94°C, 30 sec. at 55°C, and 1 min. 45 sec. at 68°C using

primers with 3’ tails specific to the respective genes and their 5’ portions specific to attB1 sites

(Table 1). Homologous recombination of the PCR product and the pDONR207 vector were

done using conditions suggested in the Gateway manual and resulted in an entry clone

harbouring one of the genes VoTPS1/2/3. The entry clone was then transformed into Top10 cells

and grown overnight on a plate containing 30 g/mL gentamicin. After colony PCR a positive

Page 49: Molecular cloning and characterization of sesquiterpene

37

single colony was selected and grown in 3 mL LB broth containing 30 g/mL gentamicin and

subsequently isolated using a kit (Gene-All, South Korea). Isolated entry clone was then

restriction mapped to confirm integration of VoTPS1/2/3. Similarly, a second recombination

reaction was performed using the entry clone harbouring VoTPS1/2/3 with the expression vector

pH9GW (provided by Dr. Paul O’Maille, John-Innes Centre, UK) according to the Gateway

manual. 1 L of the reaction product was then used to transform Top10 cells and grown

overnight on plates containing 50 g/mL kanamycin. Colony PCR was performed to verify

integration of VoTPS1/2/3 into pH9GW. A single positive colony was then selected for growth

overnight at 37C in 3 mL LB broth containing 50 g/mL kanamycin and the resulting

expression clone was isolated by a kit (Gene-All, Korea). Purified expression clones were then

restriction mapped and sequenced.

Page 50: Molecular cloning and characterization of sesquiterpene

38

Table 1. Table of primers used in cloning experiments.

Sequences in bold indicate Gateway homologous recombination sites. Underlined sequences are relevant to integrated restriction sites.

Amplicon Cloning

System

Primers Integrated

Sites

VoTPS1

pGEM/

pESC-Leu2d

5’-AAGTGGATCCGCCATGGAGAGTTGCCTTAGTTTTTC-3’F BamHI

5’-TCCAGCTAGCTTAATACGGAACACTTTCTACTAG-3’R NheI

Gateway 5'-AAAAAAGCAGGCTTCATGGAGAGCTGCCTTAGTGTATC-3' F attB1

5'-CAAGAAAGCTGGGTTTAACTCGGGATGCTCTCTACTAG-3'R attB2

VoTPS2

pGEM/

pESC-Leu2d

5’-TAATGGATCCGCCATGGAGAGCTGCCTTAGTGTATC-3’F BamHI

5’-AATTGCTAGCTTAACTCGGGATGCTCTCTACTAG-3’R NheI

Gateway 5'-AAAAAAGCAGGCTTCATGGAGAGTTGCCTTAGTTTTTC-3'F attB1

5'-CAAGAAAGCTGGGTATTAATACGGAACACTTTCTACTA-3'R attB2

VoTPS3

pGEM/

pESC-Leu2d

5’-CTCGAGGATCCAACATGTCTACTGCATTAAACAGTGAGC-3’F BamHI

5’-CGATACGGGGCCCTATATTAGAAAATAAACAGACAACAGTCCGTAGA-

3’R ApaI

Gateway 5'-AAAAAAGCAGGCTTCATGTCTACTGCATTAAACAGT-3'F attB1

5'-CAAGAAAGCTGGGTAGAAACTGTGGCTCCCTTCTATAT-3'R attB2

Adapter Gateway 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′F attB1

5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′R attB2

38

Page 51: Molecular cloning and characterization of sesquiterpene

39

2.9 Heterologous Expression Trials

VoTPS1/2/3 genes were cloned into the expression vector pH9GW (provided by Dr. Paul

O’Maille, John-Innes Centre, UK), which contains an in-frame N-terminal 9x hisitidine tag. E.

coli (BL21AI) with 50 g/mL kanamycin expressing either VoTPS1 or VoTPS2 were cultured at

37 °C until an A600 of 0.3-0.6 was reached and subsequently induced. For each clone an

uninduced and induced culture were grown at temperatures of 15 and 37°C and time points of 2,

4, and 6 hrs were sampled for protein expression by pelleting followed by lysis and visualized on

an SDS-PAGE gel. Expression of VoTPS3 was tested in a similar way but at a single

temperature of 37°C and using the Rosetta cell line.

2.10 Expression in E. coli and Protein Purification

VoTPS1 or VoTPS2 were cultured at 37 °C until an A600 of 0.3-0.6 was reached,

incubated for 30 min at 4°C, and induced for 24 hr at 15°C with 0.2% (v/v) arabinose. The

cultures were centrifuged (4,000 rpm for 30 min at 4°C), and pellets were resuspended in 25 mL

of extraction buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM imidazole, 10% (v/v)

glycerol, 1 mM PMSF, and 1 mM DTT), frozen in liquid N2, and stored at -80°C. After thawing

cells harbouring VoTPS2 in a 42°C water bath cells were lysed by sonication, the total lysate

centrifuged (30 min, 10,000 rpm at 4°C) and the supernatant incubated for 1 hr end-over-end at

4°C with 1 ml Ni-NTA affinity resin (Novagen). The sample was then loaded into an empty 10

mL BioRad Econo column. The column was washed with 35 column volumes of 50 mM Tris-

HCl pH 7.5, 750 mM KCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM DTT, and 0.1 % (v/v)

Triton X-100, and then washed with 5 column volumes of the same buffer without Triton X-100.

The column was eluted with 10 column volumes of 50 mM Tris-HCl pH 7.5, 100 mM KCl, 500

Page 52: Molecular cloning and characterization of sesquiterpene

40

mM imidazole, 10% (v/v) glycerol, and 1 mM DTT, and 1 mL fractions were collected.

Fractions containing VoTPS2 were pooled and concentrated to 250 µL with an Amicon Ultra-4

centrifugation filter unit (10-kDa cutoff). Alternatively, the cleared lysate after centrifugation

was filtered by a 0.2 m filter, and the recombinant VoTPS1 or VoTPS2 enzymes were purified

through a Bio-scale Mini Profinity IMAC cartridge (1 mL bed volume; Bio-Rad) installed on a

Bio-Rad FPLC. Before loading the protein extract, the Ni-NTA column was equilibrated with

extraction buffer (50 mM TRIS-HCl, 1 mM PMSF, 1 mM DTT, pH 7.5). A single wash step (10

mL of 50 mM TRIS-HCl, 750 mM KCl, 10% glycerol (v/v), 40 mM imidazole) followed

equilibration. Sample loading was performed at a 1 mL min-1

rate, while wash and elution steps

were performed at 2 mL min-1

. VoTPS1 was eluted by 5 mL of 275 mM imidazole, followed by

a gradient to 500 mM imidazole (50 mM TRIS-HCl, 100 mM KCl, 10% glycerol, 500 mM

imidazole, 1 mM DTT; against the same buffer without imidazole) over a volume of 5 mL. An

additional 5 mL of buffer containing 500 mM imidazole were passed through the column to elute

any residual protein. VoTPS2 was also eluted by a linear gradient over a 10 mL volume from 0-

500 mM imidazole with the same buffers as above. Fractions of 1 mL were collected over the

entire elution and run on a 10% SDS-PAGE gel. Fractions containing either VoTPS1 or

VoTPS2 were pooled and concentrated on an Amicon concentrator (>30 kDa exclusion size).

Protein was subsequently quantified by the Bradford method (Bio-Rad).

Similarly, VoTPS3 was cloned into the expression vector pH9GW. Constructs carrying

VoTPS3 were expressed in E. coli Rosetta (DE3) pLysS cells (Novagen) and cultured with LB

broth during sub-culture stages. All cultures were incubated with 30 g/mL chloramphenicol

and 15 g/mL kanamycin. Rosetta cells carrying the VoTPS3 construct were grown in 2

Page 53: Molecular cloning and characterization of sesquiterpene

41

Fernbach flasks containing 1L of rich media (TB) at 37C until an OD600 of 0.6-0.8 was reached.

After cooling cultures for 20 min. at 4C 1 mM IPTG (Inalco, Italy) was added for production of

recombinant VoTPS3 by growth at 15C for 20 hrs. Cultures were pelleted at 4,000 rpm for 30

min. at 4C. Cell pellets were weighed and resuspended in 1.5 mL/gpellet in extraction buffer (50

mM TRIS-HCl, 300 mM NaCl, 10 mM imidazole, 10% glycerol). The resuspended cells were

frozen at -80C until the day of purification.

Thawed cells were lysed by sonication (see above) and centrifuged at 10,000 rpm for 40

min at 4C. followed by decanting the supernatant and a second centrifugation step of 10,000

rpm for 30 min at 4C. The cleared lysate was then incubated at 4C overnight end-over-end

with 200 L Ni-NTA resin (Novagen). The slurry was then loaded onto a 1 mL Econo-column

(Bio-Rad) and washed with 50 mM TRIS-HCl, 500 mM KCl, 10% glycerol (v/v), 20 mM

imidazole. The column was eluted with 10 column volumes of 50 mM Tris-HCl pH 7.5, 100

mM KCl, 500 mM imidazole, and 10% (v/v) glycerol.

2.11 Gas-chromatography and Mass Spectroscopy Analysis

Organic extracts of EPY300 yeast expressing TPS1 or TPS2 were analyzed by total ion

scan and single ion mode (m/z 204) for product identification. Analysis was conducted on an

Agilent 6890N gas chromatography system coupled to an Agilent 5975B mass spectrometer.

Peaks pertaining to the expected parental mass of sesquiterpenes (m/z 204), specifically

germacrene D, valerenic acid and valerena-4,7(11)-diene, were analyzed by authentic standard.

All other sesquiterpenes identified were by the NIST5/Wiley7 mass spectra library, Massfinder

4, and/or by literature. Retention indices were calculated by using alkane standard (C10-C40)

and compared with the values in the literature and Massfinder 4 database. One L samples were

Page 54: Molecular cloning and characterization of sesquiterpene

42

injected at an inlet temperature of 250C with a flow rate of 1 mL min-1

helium on a DB1-UI-MS

and DB-Wax column (30 m X 250 m i.d. X 0.25 m film thickness). The initial temperature of

the program was set to 40C followed by a linear increase of 10 C min-1

to a temperature of

220C. The Cyclodex B chiral column (30 m x 250 µm inner diameter x 0.25 µm film thickness)

was also used to compare retention behavior of authentic valerena-4,7(11)-diene and the terpene

product from VoTPS2. The program used for chiral analysis was: initially at 50 °C (hold for 5

min) followed by 5 °C min-1

linear increase to 70 °C and final ramp of 2.5 °C min-1

to 200 °C.

2.12 Purification and NMR of Valerena-4,7(11)-diene

100 mL of SC medium without His, Met, and Leu supplemented with 1.8 % (v/v)

galactose and 0.2 % (v/v) glucose was inoculated with 1.5 mL overnight culture of the EPY300

expressing VoTPS2. After 6 h, the culture was supplied with 1 mM methionine and 5 mg of

Amberlite™ XAD-4 (Sigma-Aldrich), which was washed with MeOH prior to use. After

cultivating 3 days at 30 °C and 180 rpm, the Amberlite resin™ was recovered by filtration,

washed with distilled water and submerged in MeOH. The suspension was extracted three times

with 10 mL hexane, and the combined supernatants were dried over Na2SO4 and evaporated by a

gentle N2 stream to 0.2 ml. The concentrate was separated by silica column chromatography in a

Pasteur pipette and eluted with 10 ml of n-hexane. The collected 0.5 ml fractions were analyzed

by GC-MS and valerena-4,7(11)-diene-containing fractions were pooled and evaporated to

dryness by a gentle N2 stream. For NMR analysis, the dried residue (app. 0.7 mg) was dissolved

in CDCl3, and spectra were recorded on an UltrashieldPlus 600 MHz spectrometer (Bruker) in

CDCl3 at -20°C. Chemical shifts were reported as parts per million relative to CDCl3.

2.14 NMR Analysis of Valerena-4,7(11)-diene Standard

Page 55: Molecular cloning and characterization of sesquiterpene

43

The natural product, valerenic acid, was purchased from Extrasynthese (France).

Valerena-4,7(11)-diene was synthesized from valerenic acid by John Vederas’ laboratory

(University of Alberta), and a detailed synthesis procedure is given in the Appendix I. Nuclear

magnetic resonance (NMR) spectra were obtained on Varian Inova 500 MHz and 600 MHz

spectrometers. 1H NMR chemical shifts are reported in parts per million (ppm) using the residual

proton resonance of solvents as reference: CDCl3 δ 7.26, and CD2Cl2, δ 5.32. 13

C NMR chemical

shifts are reported relative to CDCl3 δ 77.0, and CD2Cl2 δ 53.8. Infrared spectra (IR) were

recorded on a Nicolet Magna 750 or a 20SX FT-IR spectrometer. Film Cast refers to the

evaporation of a solution on a NaCl plate. Mass spectra were recorded on a Kratos IMS-50 (high

resolution, electron impact ionization (EI)), or a ZabSpec IsoMass VG (high resolution

Electrospray (ES)).

2.15 Enzyme Activity Assays

Verification of enzyme activity was done according to a modified protocol originally

described by (O'Maille et al., 2004). In 1.5 mL glass GC vials; 50 mM TRIS HCl pH 7.5, 10

mM MgCl2, 100 M FPP and 50 g protein in 500 L ddH2O were gently overlaid with 500 L

pentane and incubated at 30°C for 1 hr. The reaction was terminated by vortexing for 1 min. and

centrifuging at 4,150 rpm. Initially 300 L of pentane was extracted and concentrated with a

gentle N2 (g) stream to a volume of ~50 L. A second volume of 500 L pentane was added to

the assay vial, vortexed and centrifuged. An additional 300 L was extracted and concentrated.

Negative controls of a boiled enzyme, enzyme with 100 mM EDTA and no enzyme in addition

to the above buffer system were run in parallel with each enzyme assay.

2.16 Enzyme Characterization

Page 56: Molecular cloning and characterization of sesquiterpene

44

Appropriate assay incubation time and enzyme amount were determined to ensure that

the initial velocity of the reaction was linear in the given conditions. 100 M FPP substrate was

spiked with [1-3H]-FPP (Perkin Elmer, Boston, USA, 23 Ci mmol

-1) and used as a stock solution

for serial dilution. Biochemical properties were determined in the substrate concentrations

ranging from 0.25 to 50 M in triplicates for each concentration. Assays were carried out in 100

L volumes with 1.5 g of purified protein in each assay. The reactions were overlaid with 900

L hexane and incubated for 15 min at 30C. Reactions were terminated by adding 100 L of

0.5 M EDTA and 4 M NaOH, followed by 1 min of vortexing. Reactions were then centrifuged,

and 500 L of hexane was mixed with 3.5 mL of scintillation cocktail. Total activity of the

radioisotope labeled product was analyzed by liquid scintillation counting (LS 6500 Multi-

Purpose Scintillation Counter, Beckman Coulter). Apparent Vmax and Km values were calculated

using the Enzyme Kinetics Module Sigmaplot 12.0.

2.17 Phylogenetic Analysis

All TPS sequences were extracted from the public domain. Any mono- or di-TPSs were

analyzed for a chloroplast targeting peptide by ChloroP (Expasy), which was subsequently

removed due to lack of homology of such sequences between species. All sequences were

aligned by CLC Main Workbench, saved as clustal (aln) files and analyzed by the website

www.phylogeny.fr with the following settings in á la carte mode: bootstrap value 100, neighbor

joining method and treedyn for tree rendering (Dereeper et al., 2008; Dereeper et al., 2010).

Page 57: Molecular cloning and characterization of sesquiterpene

45

CHAPTER 3: RESULTS

3.1 Metabolite Profiling of Valerian Root

Valerenic acid is known to accumulate in the root of the valerian plant (Valeriana

officinalis) (Bos et al., 1997). To ensure the presence of valerenic acid in the V. officinalis root

prior to 454 pyrosequencing, the volatile metabolites from V. officinalis root were analyzed by

gas-chromatography mass-spectrometry (GC-MS). The metabolites from root were identified by

spectral match to the mass spectra library and to an authentic valerenic acid standard. The root

sample presented a complex mixture of volatile compounds, but the two most abundant volatiles

were identified as bornyl acetate and valerenal (Figure 7A, D). Although the metabolite

composition of valerian varies depending on their ecotypes, these two compounds have been

reported as major constituents in valerian root (Bos et al., 1997; Letchamo et al., 2004). In our

initial analysis, valerenic acid was not detected, but the valerenic acid standard also could not be

measured at concentrations lower than 100 M likely due to its low volatility. To increase the

volatility of valerenic acid, the valerian root extract and valerenic acid standard were derivatized

(i.e., methylated) and re-analyzed by GC-MS. After derivatization, several new peaks appeared,

and the retention index (RI) and mass fragmentation of one later-eluting compound coincided

with the methylated valerenic acid (Figure 7A, B, C). The valerenic acid content from

greenhouse grown valerian was quantified to be 0.56 ± 0.02 mg (n=4) per g fresh weight, but no

valerenic acid was detected in aerial parts (stem and leaves) of the plant. Metabolite analysis

therefore confirmed that valerenal and valerenic acid are major terpenoid constituents of V.

officinalis root. This result also suggested valerena-4,7(11)-diene sesqui-TPS transcripts are

specific to root and likely to be abundant.

Page 58: Molecular cloning and characterization of sesquiterpene

46

Figure 7. GC-MS profile of volatile

metabolites from valerian root.

A) Valerenic acid from valerian root can

be detected after methylation, and it

shows an identical retention index and

mass fragmentation pattern to those of

the authentic standard (B and C). D)

Mass fragmentation of valerenal is

shown (Dae-Kyun Ro).

Page 59: Molecular cloning and characterization of sesquiterpene

47

3.2 Transcript Sequencing and Candidate Gene Isolation

From the same valerian root sample analyzed by GC-MS, cDNA was prepared and

subjected to 454 pyrosequencing. This deep transcript sequencing yielded a total of 949,214

reads with an average read length of 347-bp. After removing repetitive, AT-rich, and low quality

sequences, 759,335 high quality reads were collected and assembled via the Magpie

bioinformatics platform (The Bioinformatics Center, University of Calgary) using the MIRA

algorithm (Chevreux et al., 2004; Meisel et al., 2005). The MIRA assembly of the 454 reads

generated 55,093 unigenes, which covers 42.3 M-bp of the total transcripts. From this sequence

data set, transcripts homologous to the previously reported sesqui-TPS (e.g., amorpha-4,11-

diene, 5-epi-aristolochene, and germacrene A synthases) were retrieved by BLASTX homology

search (Wallaart et al., 2001; Ro et al., 2008).

Two full-length valerian sesqui-TPS cDNAs were distinctly identified owing to their

abundance in the database, and they are referred to as VoTPS1 and VoTPS2. The read numbers

for VoTPS1 and VoTPS2 transcripts constitute 0.03% (ranked 200th) and 0.04% (ranked 259th)

of all reads, respectively. The amino acid sequences deduced from the ORFs of VoTPS1 and

VoTPS2 share 75% identity, encoding 563 and 562 amino acids, respectively (Figure 8). These

two sesqui-TPS clones appear to be unique to valerian because the BLAST analysis shows that

the closest terpene synthase to VoTPS1 and VoTPS2 was germacrene D synthase from Vitis

vinifera with only 53% amino acid identity. The proteins encoded by VoTPS1 and VoTPS2 did

not possess any motif for plastid targeting, implying that they are not di-terpene or mono-terpene

synthases, which are known to be localized to the plastid. Semi-quantitative RT-PCR analyses

of these two transcripts in valerian root and aerial tissues (stem and leaves) showed predominant

expression patterns of VoTPS1/2 in valerian root (Figure 9). Although a marginal level of

Page 60: Molecular cloning and characterization of sesquiterpene

48

VoTPS1 expression was detected in aerial tissues, the level of VoTPS1 transcripts in aerial tissues

was quantified to be 178 ± 6 fold (n=4) lower than that from root by quantitative PCR.

Accordingly, we decided to focus on these two cDNAs because of their transcript abundance and

specific expression pattern in root.

Page 61: Molecular cloning and characterization of sesquiterpene

49

Figure 8. Sequence alignment of deduced amino acid sequences from VoTPS1 and VoTPS2.

Sequence highlighted in black indicates identical and grey indicates similar residues. Conserved

DDXXD motif is boxed.

Page 62: Molecular cloning and characterization of sesquiterpene

50

Figure 9. Semi-quantitative RT-PCR analysis of the VoTPS1 and VoTPS2 transcripts in V.

officinalis root and leaf.

Total RNA was used as a RT (reverse transcriptase) minus control to ensure the absence of

genomic DNA contamination in the template. The sizes of the DNA fragments were 921 and

1032-bp for VoTPS1 and VoTPS2, respectively (Gillian MacNevin).

Page 63: Molecular cloning and characterization of sesquiterpene

51

3.3 Functional Screening of VoTPS cDNAs in Engineered Yeast

We have previously shown that the in vivo assessment of sesqui-TPS clones in yeast

allowed technically reliable and cost-effective means for the characterization of sesqui-TPS

(Gopfert et al., 2009). To evaluate the biochemical activities of the two cDNAs, their ORFs

were individually cloned under the Gal10 promoter in pESC-Leu2d plasmid (Ro et al., 2008).

VoTPS1 and VoTPS2 cDNAs were then expressed in the EPY300 yeast strain, which was

previously engineered to synthesize abundant FPP, an immediate precursor of sesquiterpene

synthase (Ro et al., 2006; Ro et al., 2008). The volatile metabolites synthesized from the

transgenic EPY300 were sequestered by dodecane overlaid in the culture. Accordingly, the

dodecane fraction from the culture medium was analyzed by GC-MS, and newly synthesized

terpenoids were analyzed in comparison to the electron impact (EI)-mass fragmentation pattern

and RIs of standards or MS library data.

As results, six terpenoids unique to VoTPS1 or VoTPS2-expressing yeast were identified

(Figure 10A, Table 2). The yeast expressing VoTPS1 produced predominantly -elemene and

germacrene D (peak 1 and 2, respectively) with a minor amount of germacrene B (peak 3)

(Figure 10A; Figure 11). It was previously reported that germacrene C is unstable and thermally

converted to -elemene in GC-MS analysis (Colby et al., 1998), and therefore the appearance of

-elemene in GC was further assessed at different GC-inlet temperatures. Upon injection of the

sample at an inlet temperature of 300 C, a dominant peak of -elemene appeared, but this peak

completely disappeared when injected at 150 C inlet temperature with a noticeable increase of

the baseline (Figure 10B). Germacrene C could not be detected as it is continuously converted to

the fast eluting -elemene during its migration on the GC column and thus increased baseline.

Published tomato recombinant germacrene B/C synthase (Colby et al., 1998) and germacrene D

Page 64: Molecular cloning and characterization of sesquiterpene

52

standard were used to unambiguously determine the chemical identities of the VoTPS1 products

(Figure 12A/B). Therefore, the VoTPS1 clone encodes a multi-product sesqui-TPS synthesizing

germacrene C/D as major terpenes. On the other hand, the yeast expressing VoTPS2 produced a

major terpene (peak 4) (Figure 10A; Figure 11) whose EI-fragmentation and RI matched with

valerena-4,7(11)-diene in the MS library (MassFinder 4). As minor products, bicyclogermacrene

and alloaromadendrene (peaks 5 and 6, respectively) were also detected (Figure 10A; Figure 11).

Page 65: Molecular cloning and characterization of sesquiterpene

53

Figure 10. Unique terpene compounds synthesized from the yeast expressing VoTPS1 or

VoTPS2.

A) Dashed lines indicate the metabolites present in the vector-transformed control. Numbers are

unique metabolites identified from VoTPS1- or VoTPS2-expressing yeast. In comparison to the

mass fragmentation data from the mass spectrometry library and retention indices, these were

Page 66: Molecular cloning and characterization of sesquiterpene

54

identified as: peak 1, -elemene; 2, germacrene D; 3, germacrene B; 4, valerena-4,7(11)-diene; 5.

bicyclogermacrene; 6, alloaromadendrene. Chemical structures for compounds 1-6 are depicted

in Figure 19. B) The terpenes from VoTPS1-expressing yeast were analyzed at different inlet

temperatures by GC-MS.

Table 2. GC-MS analysis of terpenoids synthesized from VoTPS1, VoTPS2 and VoTPS3

Peak Compound RIexp RIstd

1 -elemene 1338 c1338

2 Germacrene D 1480 b1480

3 Germacrene B 1556 c1556

4 Valerena-4,7(11)-diene 1454 a1454

5 Bicyclogermacrene 1495 d1494

6 Alloaromadendrene 1463 b1463

7 Unknown1 1618 N.A.

8 Unknown2 1492 N.A.

9 Drimenol 1754 e1758

RIexp : experimentally determined retention index values; RIstd :

standard retention index values

aSynthesized,

bcommercial, or

cenzymatically prepared standards by

recombinant germacrene B/C synthase (AF035630) were used to

identify the terpenes produced from VoTPS1/2. dMassFinder 4 MS

library. eLiterature value (Samaneh et al., 2010).

Page 67: Molecular cloning and characterization of sesquiterpene

55

Figure 11. Numbered chemical structures.

1) δ-elemene; 2) germacrene D; 3) germacrene B; 4) valerena-4,7(11)-diene; 5)

bicyclogermacrene; 6) alloaromadendrene; 9) drimenol

Page 68: Molecular cloning and characterization of sesquiterpene

56

Figure 12. GC-MS analysis of VoTPS1 products and the terpene standards synthesized by

tomato germacrene B/C synthase.

Terpenes sequestered in dodecane were fractionated through a silica column, and the three

fractions (2, 4, and 6) enriched for the terpenes were analyzed by GC-MS in comparison to the

standards. A) The bottom chromatograms are a commercial germacrene D standard (left) and

the -elemene (germacrene C) and germacrene B (right) enzymatically synthesized by published

tomato germacrene B/C synthase (AF035630)(Colby et al., 1998). The numbers indicated are

identical to those in Fig. 10. 1, δ-elemene; 2, germacrene D; 3, germacrene B. Retention times

are labeled on the side of the peaks. B) EI-Mass fragmentations of three VoTPS1-products and

standards are shown.

Page 69: Molecular cloning and characterization of sesquiterpene

57

3.4 Characterization of the VoTPS2 Product

To ensure that the compound at peak 4 is valerena-4,7(11)-diene, we attempted to purify

the compound from the dodecane layer of the culture; however, it was difficult to separate the

compound at peak 4 from dodecane due to their similar chemical properties. Therefore, instead

of dodecane, a hydrophobic resin (AmberliteTM

) was added to the yeast culture, and ~0.7 mg of

the peak 4 was purified from the resin. When the purified product was analyzed by NMR, the

chemical shifts from the 13

C-NMR perfectly matched to those of the published NMR signals of

valerena-4,7(11)-diene (Paul et al., 2001; Kitayama et al., 2010) (Table 3). However, 1H-NMR

data overlapped with other contaminants, making accurate integration and signal assignments

very difficult. As an alternative approach, commercially available natural product, valerenic

acid, was used to synthesize valerena-4,7(11)-diene by sequential reductions of the C-12

carboxylic acid. The chemically synthesized valerena-4,7(11)-diene standard and the purified

compound were then analyzed by three different GC columns including one chiral selective

column (DB1, DB-wax, and cyclodex B). The retention time of the synthesized standard and the

compound at peak 4 were identical in all three columns (Figure 13A), and they showed identical

EI-fragmentation patterns (Figure 13B). When the valerena-4,7(11)-diene standard was spiked

with the purified compound and the mixture was analyzed by GC, perfectly symmetrical single

peaks were obtained from all three columns. Therefore, the 13

C-NMR and GC-MS analyses (i.e.,

EI-fragmentation and retention time) confirmed that the enzymatically synthesized compound at

peak 4 is valerena-4,7(11)-diene.

Page 70: Molecular cloning and characterization of sesquiterpene

58

Table 3. Comparison of the 13

C-NMR signals from the purified compound of peak 4 with

the published data.

1Carbon

number

This work 2(Paul et al.,

2001)

2(Kitayama et al.,

2010)

C-14 12.06 12.07 12.1

C-15 13.39 13.39 13.4

C-12 17.78 17.78 17.8

C-2 24.61 24.60 24.6

C-13 26.12 26.11 26.1

C-8 26.55 26.56 26.6

C-9 28.67 28.69 28.7

C-10 33.56 33.57 33.6

C-6 33.63 33.65 33.6

C-3 37.53 37.55 37.5

C-1 47.38 47.40 47.4

C-7 126.18 126.21 126.2

C-4 128.38 128.38 128.4

C-11 129.77 129.77 129.8

C-5 136.06 136.04 136.0

1The carbon numbers are according to (Paul et al., 2001) and are

also depicted in Figure 6.

2Full citation information is given in the literature cited.

Page 71: Molecular cloning and characterization of sesquiterpene

59

Figure 13. Validation of VoTPS2 enzyme product (peak 4) as valerena-4,7(11)-diene.

A) Chiral column (Cyclodex B) was used as described in the method to separate the compound at

peak 4 and valerena-4,7(11)-diene standard. B) The EI-fragmentation of peak 4 is identical to

that of the synthesized standard, valerena-4,7(11)-diene.

Page 72: Molecular cloning and characterization of sesquiterpene

60

3.5 In vitro Characterization of VoTPS1 and VoTPS2

To examine the catalytic properties of recombinant VoTPS1/2, enzymes were expressed

in E. coli as N-terminal His-tags. Expression trials were performed to identify optimal growth

conditions for recombinant enzyme purification. Trials were conducted in the presence and

absence of arabinose at 37C. Clearly, large bands relating to induced cultures indicate

overexpression of 68 kDa proteins VoTPS1 (65.4 kDa without N-term 9X-His tag) and VoTPS2

(65.8 kDa without N-term 9X-His tag) (Figure 14). However, a large majority of the expressed

enzymes were present in the insoluble fraction of the lysate indicating that enzymes were

produced as inclusion bodies. Theoretically, it was believed that if the enzymes were expressed

regardless of solubility, they were most likely expressed in a soluble form but at levels low

enough to be masked by the large amount of soluble endogenous protein, as visualized by SDS-

PAGE (Figure 14). Therefore, cultures were scaled up to 1 L and grown at 15C. Yields for

VoTPS1 and VoTPS2 were between 0.6-1.2 mg for 2 L of culture indicating a relatively low

expression level. VoTPS1 and VoTPS2 were purified by Ni-affinity chromatography using a

gradient elution to >90% purity (Figure 15A/B).

When purified VoTPS1 or VoTPS2 enzyme was incubated with FPP in vitro, the major

terpenes synthesized were essentially the same as those from transgenic yeast (Figure 16A/C,

peaks 1-6; Figure 11). However, other minor unknown terpenes (Figure 16A/C, peaks 7 and 8)

were additionally identified from the in vitro assays. When the kinetic properties of VoTPS1 and

VoTPS2 were measured using 3H-FPP, hyperbolic FPP saturation kinetics were obtained for

both VoTPS1 and VoTPS2 enzymes (Figure 16B/D). The apparent Km and kcat values for

VoTPS1 were determined to be 13.7 ± 2.5 µM and 1.0 (± 0.1) x 10-2

s-1

(n=3), and for VoTPS2

were 9.5 ± 1.6 µM and 1.3 (± 0.1) x 10-2

s-1

(n=3). These values are similar to those reported for

Page 73: Molecular cloning and characterization of sesquiterpene

61

other sesqui-TPS enzymes (Colby et al., 1998; Picaud et al., 2005; Picaud et al., 2005; Picaud et

al., 2006).

Page 74: Molecular cloning and characterization of sesquiterpene

62

Figure 14. Expression trials of his-tagged recombinant VoTPS1 and VoTPS2.

Expression was conducted at 37C in BL21(AI) cells with (+) arabinose or without (-) arabinose

for induction. A Coomassie stain of a 10% SDS-PAGE was used to visualize protein bands. A)

Expression of VoTPS1 (65 kDa) was 6 hrs. S, supernatant; and P, pellet. B) Expression of

VoTPS2 (65 kDa) was 2 hr.

Page 75: Molecular cloning and characterization of sesquiterpene

63

Figure 15. Purification of VoTPS1/2 by Ni-NTA column using a gradient elution.

10% SDS-PAGE stained with Coomassie to visualize protein bands. TL, total lysate; FT, flow

through; W, wash; E, elution. A) VoTPS1 (65 kDa). B) VoTPS2 (65 kDa).

Page 76: Molecular cloning and characterization of sesquiterpene

64

Figure 16. In vitro enzyme assays of VoTPS1 and VoTPS2 recombinant enzyme.

A) Purified recombinant VoTPS1 and VoTPS2 were incubated with FPP to synthesize terpenes

in vitro with GC-MS profiles shown. An additional two compounds identified from the in vitro

assays were shown in peaks 7 and 8. The identities of these minor compounds are unknown.

Plots of VoTPS1 (B) and VoTPS2 (D) reaction rates versus FPP concentration are shown. FPP

saturation curves were determined at pH 7.5 and fitted to a single catalytic site Michaelis-Menten

model. Calculated Km and kcat values are given in the figures.

Page 77: Molecular cloning and characterization of sesquiterpene

65

3.6 Cyclization Mechanism of Valerena-4,7(11)-diene

The carbon cores of most sesquiterpenes are formed by ten carbons. However, valerena-

4,7(11)-diene has a unique structural core constituted with nine carbons (Figure 17). This nine-

carbon core requires ring-contraction by formation of a new C-C bond. This unusual reaction is

catalyzed by VoTPS2 from Valeriana and possibly other closely related genera such as

Nardostachys. Interestingly, its closest homolog VoTPS1, exhibiting 75% amino acid identity to

VoTPS2, catalyzes the synthesis of germacrene B/C/D, which are commonly found in multiple

plant species outside of Valeriana and Nardostachys. We postulated that VoTPS2 recently

diverged from VoTPS1 by gene duplication and neo-functionalization in one evolutionary

lineage of the Valerianaceae family, and therefore comparative mechanistic study of VoTPS1

and VoTPS2 may provide an insight into the appearance of the unique valerena-4,7(11)-diene

from more commonly found terpenes. In addition, structural observations of other minor

terpenes (i.e., bicyclogermacrene and alloaromadendrene) released from VoTPS2 may help infer

the VoTPS2 mechanism for valerena-4,7(11)-diene synthesis. Taking these into consideration,

one possible mechanism for the synthesis of valerena-4,7(11)-diene by VoTPS2 is proposed in

Figure 17. In this scheme, the germacrene bearing a C6 carbocation is the central precursor for

all four major terpenes produced from VoTPS1/2 (i.e., germacrene C/D, bicyclogermacrene, and

valerena-4,7(11)-diene). Other minor products (i.e., germacrene B and alloaromadendrene) can

be coupled to the main reaction framework as depicted in Figure 17. The formation of

germacrene B/C/D, bicyclogermacrene, and alloaromadendrene can be explained by standard

carbocation reaction mechanisms, such as hydride shift (indicated as an arrow in b in Figure 17),

deprotonation (a, c, e, g, and i), double-bond migration (d), and protonation (h). The simplest

way to link the valerena-4,7(11)-diene synthesis to this reaction scheme is to involve the new C-

Page 78: Molecular cloning and characterization of sesquiterpene

66

C bond formation between C-6 and C-8. This reaction will evoke a unique ring-contraction

resulting in a nine-carbon core (reaction f). Subsequently, a cascade of deprotonation and

reprotonation reactions (j, k, and l) will lead to the formation of valerena-4,7(11)-diene guided

by VoTPS2. Further studies are necessary to understand how VoTPS2 promotes the new -bond

formation between C-6 and C-8 from the germacrene C6 carbocation while it suppresses all other

apparently simpler reactions, such as deprotonation and allylic rearrangement.

Page 79: Molecular cloning and characterization of sesquiterpene

67

Figure 17. A proposed mechanism for valerena-4,7(11)-diene formation catalyzed by

VoTPS2 (valerenadiene synthase).

The dashed rectangles indicate the VoTPS1 products, and the dashed circles indicate the

VoTPS2 products. Letters (a - l) designate distinct carbocation reactions proposed to be

catalyzed by VoTPS1 and VoTPS2. The rectangle with a solid line displays the valerena-4,7(11)-

diene biosynthetic mechanism proposed to have evolved in the genus of Valeriana.

Page 80: Molecular cloning and characterization of sesquiterpene

68

3.7 Identification and Characterization of an Additional Sesquiterpene Synthase, VoTPS3

An additional full-length TPS transcript lacking a chloroplast transit peptide, VoTPS3,

and a deduced amino acid length of 556, was also identified from a BLAST search against the V.

officinalis assembly database. However, the transcript abundance of VoTPS3 was ~10-fold

lower than VoTPS1/2, constituting 0.004% of the total transcript reads. Deduced amino acid

sequences of VoTPS3 showed only 41% and 39% identity to VoTPS1 and VoTPS2, respectively.

Initially, the project priority was given to VoTPS1/2 due to their transcript abundance; however,

after completing VoTPS1/2 characterization and considering many unique terpenoids found in V.

officinalis root, it was worth investigating the function of VoTPS3.

To examine the biochemical activity encoded in VoTPS3, His-tagged VoTPS3 was

expressed in BL21AI E. coli cells, and cells were induced by arabinose. However, no expression

was detected (data not shown). The expression of eukaryotic genes in E. coli may be impeded

by certain rare tRNAs in E. coli. Therefore, the cell line Rosetta (DE3) pLysS was chosen as it

carries the pRARE plasmid, which codes for six rare tRNAs normally not present in E. coli.

Expression trials with and without the inducer resulted in a large protein band of ~67 kDa (65.8

kDa without the 9X His-tag) present in the soluble fraction (Figure 18), and subsequently the

identical method for VoTPS1/2 purification was used to purify His-tagged VoTPS3.

Unfortunately, pure VoTPS3 was difficult to obtain as the VoTPS3 enzyme was unable to bind

to the Ni-NTA resin with comparable affinity to VoTPS1/2. In vitro enzyme assays using the

flow-through fraction, however, showed an efficient conversion of (FPP) substrate to a terpene,

of which the mass fragmentation pattern and RI value matched perfectly to drimenol, found in

the MS database (Figure 19B/C and Table 2). As an independent approach, VoTPS3 was also

Page 81: Molecular cloning and characterization of sesquiterpene

69

cloned in a yeast expression vector and expressed in an engineered yeast strain (EPY300) as

described previously. The GC-MS analysis of the induced yeast culture clearly showed the

synthesis of a new terpene (peak 9 in Figure 18), and its mass fragmentation pattern was also

identical to drimenol from the MS database. From these data, it was concluded that VoTPS3 is

able to synthesize drimenol from FPP, although this enzyme and its sesquiterpene product

require additional purification for proper characterization.

Attempts to infer the drimenol synthetic mechanism by VoTPS3 using the ionization-

initiated carbocation reaction as depicted in Figure 16 could not lead to any plausible proposal.

However, when the protonation-initiated reaction was applied, drimenol synthesis could be

easily explained through a concerted double-bond migration, followed by a sequential

deprotonation, and quenching by hydroxyl ion (Figure 20). Despite the fact that VoTPS3 shares

general primary structure with other sesquiterpene synthases which use ionization-initiated

mechanisms, VoTPS3 from V. officinalis appears to have developed a unique synthetic

mechanism which can be initiated by the protonation of a double-bond.

Page 82: Molecular cloning and characterization of sesquiterpene

70

Figure 18. Expression trial of his-tagged recombinant VoTPS3 (67 kDa).

Expression was conducted at 37C in Rosetta (DE3) pLysS cells using 1mM IPTG as an inducer.

10% SDS-PAGE stained by Coomassie was used to visualize protein bands. S, supernatant; P,

pellet; I, induced and U, uninduced.

Page 83: Molecular cloning and characterization of sesquiterpene

71

Figure 19. In vitro assays for VoTPS3.

For chemical structure of 9 see Figure 19. A) Unique terpene from expression of VoTPS3 in

engineered yeast. B) Partially purified recombinant VoTPS3 was incubated with FPP to

synthesize terpenes in vitro, with GC-MS chromatogram shown. C) Compound 9 spectrum is a

close match to the library.

Page 84: Molecular cloning and characterization of sesquiterpene

72

Figure 20. A proposed mechanism of drimenol formation by VoTPS3 (drimenol synthase).

Page 85: Molecular cloning and characterization of sesquiterpene

73

3.8 Phylogenetic Analysis of VoTPS1/2/3

Phylogenetic analysis showed that VoTPS1/2/3 are part of the subfamily TPS-a, which

encompasses angiosperm sesquiterpene synthases and a diterpene synthase, casbene synthase.

Casbene synthase’s relatedness to angiosperm sesquiterpene synthases is based on reaction

mechanism. Phylogenetic reconstruction of selected sesquiterpene synthases indicates that

VoTPS1 and VoTPS2 are closely clustered (Figure 21), supporting VoTPS2’s evolutionary

origin through gene duplication of VoTPS1 and justifying the proposed similarity in reaction

mechanisms between the two enzymes. The third more distantly related VoTPS3 (~40% identity)

is more related to other sesquiterpene synthases, such as vetisperadiene synthase and tobacco

epi-aristolochene synthases, than to VoTPS1/2 (Figure 21). However, as shown in section 3.7,

VoTPS3 catalyzes the synthesis of an entirely different product (drimenol), and the ionization-

initiated reaction typically found in sesquiterpene synthases is not likely to drive the reaction for

drimenol. VoTPS3 appeared to accumulate minor but distinct mutations, allowing it to be

grouped together with other sesquiterpene synthases from TPS-a, but acquired a novel function

not found in other sesquiterpene synthases. Further structure-function analysis will help

elucidate the underlying mechanisms of these three enzymes.

Page 86: Molecular cloning and characterization of sesquiterpene

74

Figure 21. A phylogenetic tree representing the seven subfamilies (a-g) of terpene synthase

enzymes.

VoTPS1/2/3 all belong to the Tps-a subfamily of angiosperm sesquiterpene synthases. Tree was

generated using a bootstrap value of 100, neighbor joining method, and rendered by Treedyn.

Artemisia annua, (Aa); Abies grandis, (Ag); Arabidopsis thaliana, (At); Antirrhinum majus,

(Am); Clarkia breweri, (Cb); Curcubita maxima, (Cm); Helianthus annuus, (Ha); Lactuca

sativa, (LS); Nicotiana attenuate, (Na); Pisum sativum, (Ps); Picea abies, (Pa); Taxus brevifolia,

Page 87: Molecular cloning and characterization of sesquiterpene

75

(Tb); Santalum album, (Sa); Santalum austrocaledonicum, (Saus); Solanum lycopersicum, (Sl);

Santalum spicatum, (Ss); Solanum tuberosum, (St); Valeriana officinalis, (Vo); and Zingiber

officinale, (Zo).

Page 88: Molecular cloning and characterization of sesquiterpene

76

CHAPTER 4: DISCUSSION

Recent progress in next-generation sequencing (NGS) has facilitated efficient gene-

mining from various non-model and under-studied medicinal plants. In this work,

transcriptomics generated by 454 sequencing and metabolically engineered yeast were used to

identify three sesquiterpene synthases, of which one synthase was found to synthesize valerena-

4,7(11)-diene, a precursor of valerenic acid. The additional synthases, one being a germacrene

C/D synthase and a potentially novel synthase that tentatively produces drimenol, were also

identified. The work presented here overcame traditional difficulties in investigating plant

natural products. For example, cost-effective NGS was used to easily identify full-length

candidate cDNAs for sesquiterpene synthesis; metabolically engineered yeast served as a simple

in vivo platform to evaluate gene function; costly substrate (i.e., FPP) was replaced by de novo

synthesized FPP in yeast; milligram levels of sesquiterpenes, were produced microbially and

subsequently purified in adequate amounts for NMR analysis. Such integrative approaches

involving genomics and metabolic engineering tools, as well as, analytical chemistry and

biochemistry, allowed us to functionally identify the targeted gene in a short time-frame. This

will showcase further studies of specialized metabolism in other medicinal or non-model plants.

Valerena-4,7(11)-diene is not a metabolic end-product in V. officinalis, and it is further

oxidized to valerenic acid. The metabolic profiling data from this work (Figure 7) and the

literature (Baranauskiene, 2007) showed that C12-oxidized forms of valerena-4,7(11)-diene are

also synthesized in V. officinalis root, and in particular valerenal is one of the major products

accumulated in root. These metabolic intermediates (i.e., valerenol and valerenal) were reported

to possess comparable levels of sedative properties (Kopp et al., 2010). Therefore, it would be

Page 89: Molecular cloning and characterization of sesquiterpene

77

worth investigating additional oxidations of valerena-4,7(11)-diene by yet uncharacterized

oxidase enzymes in V. officinalis. In pursuing this C12 oxidation biochemistry, similar genomics,

chemical approaches, gene-mining and expression can be utilized. Overall, acquiring and

understanding more enzymes in terpenoid metabolism will help us understand how enzymes

have evolved and developed new functions in different plant lineages.

The key discovery from this work is the identification of valerena-4,7(11)-diene synthase

from V. officinalis. Synthesis of an unusual nine-carbon core (Figure 6) by this enzyme implies

that a distinctive ring-contraction mechanism is encoded in valerena-4,7(11)-diene synthase, and

one possible mechanism was proposed (Figure 16). It should be noted that the nine-carbon

valerenadiene core structure is unique to the Valerianaceae family. Considering that VoTPS1

and VoTPS2 share very high sequence homology (~75% amino acid identity) and VoTPS1

showed rather common terpene synthesizing activities (i.e., germacrene C and D), it is

reasonable to postulate that the unique valerena-4,7(11)-diene synthase (VoTPS2) diverged

recently from the common germacrene C/D synthase (VoTPS1). The proposed reaction

mechanism of valerena-4,7(11)-diene synthase additionally suggests that both VoTPS1 and

VoTPS2 share a carbocation reaction intermediate (Figure 16). It is thought that the

temporospatial selection pressure, imposed on a certain lineage in the Valerianaceae family, may

have evoked the occurrence of valerena-4,7(11)-diene synthase from germacrene C/D synthase.

One particularly interesting aspect of VoTPS1/2 research is the structure-function

relationships coded in VoTPS1 and VoTPS2. Using homology modeling and site-directed

mutagenesis, critical residues determining the terpene product specificity could be identified.

From the proposal shown in Figure 16, it can be predicted that certain residues in valerena-

Page 90: Molecular cloning and characterization of sesquiterpene

78

4,7(11)-diene synthase (VoTPS2) may suppress the formation of simple terpene structures such

as germacrene C/D and bicyclogermacrene while other residues may accelerate the formation of

the unique nine-carbon ring by shrinking the germacrene skeleton. Such information could help

us describe TPS evolution more clearly as the current theory is that sesquiterpene synthases

evolved from diterpene synthases, but little is known about the evolution within sesquiterpene

synthases.

Similar experiments could be used to characterize VoTPS3 and its putative drimenol

sesquiterpene product. Evidence for the presence of drimenol in valerian root extracts exists

(Baranauskiene, 2007) as well as in other plants, such as the liverwort Diplophyllum serrulatum

and wild cinnamon, Canella winterana (Toyota et al., 1994; Ying et al., 1995). It is intriguing to

observe that ionization-initiated reaction by dephosphorylation, typical for many sesquiterpene

synthases including VoTPS1/2, could not be applied to explain the synthesis of drimenol by

VoTPS3. To the best of our knowledge, the sesquiterpene synthase catalyzing the protonation-

initiated reaction has not been reported to date. However, such protonation-initiated reactions

are common in many diterpene cyclases, and many possess the highly conserved DXDD motif

(Prisic et al., 2007). Sequence analysis of VoTPS3, however, could not locate the DXDD motif

known to play a catalytic role in the protonation-initiated reaction, however recently an EDXXD

motif was identified to be involved in class II diterpene protonation-initiated reactions (Cao et al.,

2010) and a similar motif (EDXD) was found in the amino acid sequence of VoTPS3.

Additionally, VoTPS3 also has the class I motif (DDXXD) motif responsible for the ionization

reaction. Therefore, we believe that this DDXXD motif in VoTPS3 is still required for the

dephosphorylation and hydroxyl group addition in drimenol synthesis as shown in Figure 19.

Page 91: Molecular cloning and characterization of sesquiterpene

79

In the last decade, significant effort has been directed to uncover the novel terpene

synthesizing activities from various TPS enzymes, and currently countless numbers of TPS

genes are being deposited to the sequence database without knowing their functions. However,

with an extensive number of biochemically characterized TPSs and an expanding quantity of

crystal structures, it would be ultimately feasible to predict the functions of uncharacterized TPS

in the genome and also to design TPS of improved or altered activities. The new TPS enzymes

uncovered from V. officinalis transcriptomics, in this work, advance our knowledge of the TPS

enzyme family and also provide a foundation for the possible enzymatic synthesis of sedatives

and anti-depressants.

Page 92: Molecular cloning and characterization of sesquiterpene

80

LITERATURE CITED

Aharoni A, Giri AP, Deuerlein S, Griepink F, de Kogel WJ, Verstappen FWA, Verhoeven

HA, Jongsma MA, Schwab W, Bouwmeester HJ (2003) Terpenoid metabolism in

wild-type and transgenic Arabidopsis plants. Plant Cell 15: 2866-2884

Aharoni A, Giri AP, Verstappen FWA, Bertea CM, Sevenier R, Sun ZK, Jongsma MA,

Schwab W, Bouwmeester HJ (2004) Gain and loss of fruit flavor compounds produced

by wild and cultivated strawberry species. Plant Cell 16: 3110-3131

Anderson GD, Elmer GW, Kantor ED, Templeton IE, Vitiello MV (2005) Pharmacokinetics

of valerenic acid after administration of valerian in healthy subjects. Phytotherapy

Research 19: 801-803

Arimura G, Huber DPW, Bohlmann J (2004) Forest tent caterpillars (Malacosoma disstria)

induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar

(Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and

patterns of gene expression of (-)-germacrene D synthase, PtdTPS1. Plant Journal 37:

603-616

Arimura G, Ozawa R, Nishioka T, Boland W, Koch T, Kuhnemann F, Takabayashi J (2002) Herbivore-induced volatiles induce the emission of ethylene in neighboring lima

bean plants. Plant Journal 29: 87-98

Arimura G, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J (2000) Herbivory-

induced volatiles elicit defence genes in lima bean leaves. Nature 406: 512-515

Aubourg S, Lecharny A, Bohlmann J (2002) Genomic analysis of the terpenoid synthase

(AtTPS) gene family of Arabidopsis thaliana. Molecular Genetics and Genomics 267:

730-745

Baranauskiene R (2007) Essential oil composition of Valeriana officinalis ssp officinalis grown

in Lithuania. Chemistry of Natural Compounds 43: 331-333

Beale MH, Birkett MA, Bruce TJA, Chamberlain K, Field LM, Huttly AK, Martin JL,

Parker R, Phillips AL, Pickett JA, Prosser IM, Shewry PR, Smart LE, Wadhams

LJ, Woodcock CM, Zhang Y (2006) Aphid alarm pheromone produced by transgenic

plants affects aphid and parasitoid behavior. Proceedings of the National Academy of

Sciences of the United States of America 103: 10509-10513

Benke D, Barberis A, Kopp S, Altmann KH, Schubiger M, Vogt KE, Rudolph U, Mohler H (2009) GABA(A) receptors as in vivo substrate for the anxiolytic action of valerenic acid,

a major constituent of valerian root extracts. Neuropharmacology 56: 174-181

Bick JA, Lange BM (2003) Metabolic cross talk between cytosolic and plastidial pathways of

isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast

envelope membrane. Archives of Biochemistry and Biophysics 415: 146-154

Bloch K (1965) BIOLOGICAL SYNTHESIS OF CHOLESTEROL. Science 150: 19-&

Bloch K (1987) SUMMING-UP. Annual Review of Biochemistry 56: 1-19

Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: Molecular biology

and phylogenetic analysis. Proceedings of the National Academy of Sciences of the

United States of America 95: 4126-4133

Bohlmann J, Steele CL, Croteau R (1997) Monoterpene synthases from Grand fir (Abies

grandis) - cDNA isolation, characterization, and functional expression of myrcene

Page 93: Molecular cloning and characterization of sesquiterpene

81

synthase, (-)(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase. Journal of

Biological Chemistry 272: 21784-21792

Bos R, Woerdenbag HJ, Hendriks H, Scheffer JJC (1997) Composition of the essential oils

from underground parts of Valeriana officinalis L. s.l. and several closely related taxa.

Flavour and Fragrance Journal 12: 359-370

Cao R, Zhang Y, Mann FM, Huang C, Mukkamala D, Hudock MP, Mead ME, Prisic S,

Wang K, Lin F-Y, Chang T-K, Peters RJ, Odfield E (2010) Diterpene cyclases and the

nature of the isoprene fold. Proteins-Structure Function and Bioinformatics 78: 2417-

2432

Chappell J (1995) THE BIOCHEMISTRY AND MOLECULAR-BIOLOGY OF ISOPRENOID

METABOLISM. Plant Physiology 107: 1-6

Chen XY, Chen Y, Heinstein P, Davisson VJ (1995) Cloning, expression, and characterization

of (+)-delta-cadinene synthase: A catalyst for cotton phytoalexin biosynthesis. Archives

of Biochemistry and Biophysics 324: 255-266

Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Muller WEG, Wetter T, Suhai S (2004)

Using the miraEST assembler for reliable and automated mRNA transcript assembly and

SNP detection in sequenced ESTs. Genome Research 14: 1147-1159

Christianson DW (2006) Structural biology and chemistry of the terpenoid cyclases. Chemical

Reviews 106: 3412-3442

Colby SM, Crock J, Dowdle-Rizzo B, Lemaux PG, Croteau R (1998) Germacrene C synthase

from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation,

characterization, and bacterial expression of the multiple product sesquiterpene cyclase.

Proceedings of the National Academy of Sciences of the United States of America 95:

2216-2221

Cornish K (2001) Similarities and differences in rubber biochemistry among plant species.

Phytochemistry 57: 1123-1134

Degenhardt J, Koellner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases

and the origin of terpene skeletal diversity in plants. Phytochemistry 70: 1621-1637

Deng X, Wang Y, Zhou Y, Soboloff J, Gill DL (2009) STIM and Orai: Dynamic

Intermembrane Coupling to Control Cellular Calcium Signals. Journal of Biological

Chemistry 284: 22501-22505

Denmeade SR, Jakobsen CM, Janssen S, Khan SR, Garrett ES, Lilja H, Christensen SB,

Isaacs JT (2003) Prostate-specific antigen-activated thapsigargin prodrug as targeted

therapy for prostate cancer. Journal of the National Cancer Institute 95: 990-1000

Dereeper A, Audic S, Claverie J-M, Blanc G (2010) BLAST-EXPLORER helps you building

datasets for phylogenetic analysis. Bmc Evolutionary Biology 10

Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S,

Lefort V, Lescot M, Claverie JM, Gascuel O (2008) Phylogeny.fr: robust phylogenetic

analysis for the non-specialist. Nucleic Acids Research 36: W465-W469

Diaz JF, Barasoain I, Andreu JM (2003) Fast kinetics of Taxol binding to microtubules.

Effects of solution variables and microtubule-associated proteins. Journal of Biological

Chemistry 278: 8407-8419

Donald KAG, Hampton RY, Fritz IB (1997) Effects of overproduction of the catalytic domain

of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in

Saccharomyces cerevisiae. Applied and Environmental Microbiology 63: 3341-3344

Page 94: Molecular cloning and characterization of sesquiterpene

82

Dudareva N, Andersson S, Orlova I, Gatto N, Reichelt M, Rhodes D, Boland W,

Gershenzon J (2005) The nonmevalonate pathway supports both monoterpene and

sesquiterpene formation in snapdragon flowers. Proceedings of the National Academy of

Sciences of the United States of America 102: 933-938

Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt J, Miller B, Bohlmann

J (2003) (E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in

snapdragon: Function and expression of three terpene synthase genes of a new terpene

synthase subfamily. Plant Cell 15: 1227-1241

Dudareva N, Negre F, Nagegowda DA, Orlova I (2006) Plant volatiles: Recent advances and

future perspectives. Critical Reviews in Plant Sciences 25: 417-440

Dudareva N, Pichersky E, Gershenzon J (2004) Biochemistry of plant volatiles. Plant

Physiology 135: 1893-1902

Eckstein-Ludwig U, Webb RJ, van Goethem IDA, East JM, Lee AG, Kimura M, O'Neill

PM, Bray PG, Ward SA, Krishna S (2003) Artemisinins target the SERCA of

Plasmodium falciparum. Nature 424: 957-961

Estevez JM, Cantero A, Reindl A, Reichler S, Leon P (2001) 1-deoxy-D-xylulose-5-

phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants.

Journal of Biological Chemistry 276: 22901-22909

Faraldos JA, Wu S, Chappell J, Coates RM (2010) Doubly Deuterium-Labeled Patchouli

Alcohol from Cyclization of Singly Labeled 2-(2)H(1) Farnesyl Diphosphate Catalyzed

by Recombinant Patchoulol Synthase. Journal of the American Chemical Society 132:

2998-3008

Farmer WR, Liao JC (2001) Precursor balancing for metabolic engineering of lycopene

production in Escherichia coli. Biotechnology Progress 17: 57-61

Faulkner A, Chen XM, Rush J, Horazdovsky B, Waechter CJ, Carman GM, Sternweis PC (1999) The LPP1 and DPP1 gene products account for most of the isoprenoid phosphate

phosphatase activities in Saccharomyces cerevisiae. Journal of Biological Chemistry 274:

14831-14837

Felicetti B, Cane DE (2004) Aristolochene synthase: Mechanistic analysis of active site residues

by site-directed mutagenesis. Journal of the American Chemical Society 126: 7212-7221

Garms S, Chen F, Boland W, Gershenzon J, Kollner TG (2012) A single amino acid

determines the site of deprotonation in the active center of sesquiterpene synthases

SbTPS1 and SbTPS2 from Sorghum bicolor. Phytochemistry 75: 6-13

Gennadios HA, Gonzalez V, Di Costanzo L, Li A, Yu F, Miller DJ, Allemann RK,

Christianson DW (2009) Crystal Structure of (+)-delta-Cadinene Synthase from

Gossypium arboreum and Evolutionary Divergence of Metal Binding Motifs for

Catalysis. Biochemistry 48: 6175-6183

Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier

DNA/PEG method. Nature Protocols 2: 31-34

Gopfert JC, MacNevin G, Ro DK, Spring O (2009) Identification, functional characterization

and developmental regulation of sesquiterpene synthases from sunflower capitate

glandular trichomes. Bmc Plant Biology 9

Graewert T, Groll M, Rohdich F, Bacher A, Eisenreich W (2011) Biochemistry of the non-

mevalonate isoprenoid pathway. Cellular and Molecular Life Sciences 68: 3797-3814

Page 95: Molecular cloning and characterization of sesquiterpene

83

Greenhagen BT, O'Maille PE, Noel JP, Chappell J (2006) Identifying and manipulating

structural determinates linking catalytic specificities in terpene synthases. Proceedings of

the National Academy of Sciences of the United States of America 103: 9826-9831

Harley P, Guenther A, Zimmerman P (1997) Environmental controls over isoprene emission

in deciduous oak canopies. Tree Physiology 17: 705-714

Horwitz SB (1994) HOW TO MAKE TAXOL FROM SCRATCH. Nature 367: 593-594

Houghton PJ (1988) THE BIOLOGICAL-ACTIVITY OF VALERIAN AND RELATED

PLANTS. Journal of Ethnopharmacology 22: 121-142

Houghton PJ (1999) The scientific basis for the reputed activity of Valerian. Journal of

Pharmacy and Pharmacology 51: 505-512

Jackson BE, Hart-Wells EA, Matsuda SPT (2003) Metabolic engineering to produce

sesquiterpenes in yeast. Organic Letters 5: 1629-1632

Jacobo-Herrera NJ, Vartiainen N, Bremner P, Gibbons S, Koistinaho J, Heinrich M (2006)

NF-kappa B modulators from Valeriana officinalis. Phytotherapy Research 20: 917-919

Jones CG, Moniodis J, Zulak KG, Scaffidi A, Plummer JA, Ghisalberti EL, Barbour EL,

Bohlmann J (2011) Sandalwood Fragrance Biosynthesis Involves Sesquiterpene

Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including

Santalene Synthases. Journal of Biological Chemistry 286: 17445-17454

Jordan MA, Wendell K, Gardiner S, Derry WB, Copp H, Wilson L (1996) Mitotic block

induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal

mitotic exit and apoptotic cell death. Cancer Research 56: 816-825

Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nature Reviews

Cancer 4: 253-265

Julsing MK, Koulman A, Woerdenbag HJ, Quax WJ, Kayser O (2006) Combinatorial

biosynthesis of medicinal plant secondary metabolites. Biomolecular Engineering 23:

265-279

Kajiwara S, Fraser PD, Kondo K, Misawa N (1997) Expression of an exogenous isopentenyl

diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli.

Biochemical Journal 324: 421-426

Kessler A, Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions

in nature. Science (Washington D C) 291: 2141-2144

Khom S, Strommer B, Ramharter J, Schwarz T, Schwarzer C, Erker T, Ecker GF, Mulzer

J, Hering S (2010) Valerenic acid derivatives as novel subunit-selective GABA(A)

receptor ligands - in vitro and in vivo characterization. British Journal of Pharmacology

161: 65-78

Kim SW, Keasling JD (2001) Metabolic engineering of the nonmevalonate isopentenyl

diphosphate synthesis pathway in Escherichia coli enhances lycopene production.

Biotechnology and Bioengineering 72: 408-415

Kirby J, Keasling JD (2009) Biosynthesis of Plant Isoprenoids: Perspectives for Microbial

Engineering. Annual Review of Plant Biology 60: 335-355

Kitayama T, Kawabata G, Ito M (2010) Concise Synthesis of Valerena-4,7(11)-diene, a

Highly Active Sedative, from Valerenic Acid. Bioscience Biotechnology and

Biochemistry 74: 1963-1964

Koeksal M, Hu H, Coates RM, Peters RJ, Christianson DW (2011) Structure and mechanism

of the diterpene cyclase ent-copalyl diphosphate synthase. Nature Chemical Biology 7:

431-433

Page 96: Molecular cloning and characterization of sesquiterpene

84

Koeksal M, Jin Y, Coates RM, Croteau R, Christianson DW (2011) Taxadiene synthase

structure and evolution of modular architecture in terpene biosynthesis. Nature 469: 116-

U138

Kopp S, Baur R, Sigel E, Moehler H, Altmann K-H (2010) Highly Potent Modulation of

GABA(A) Receptors by Valerenic Acid Derivatives. Chemmedchem 5: 678-681

Krishna S, Pulcini S, Fatih F, Staines H (2010) Artemisinins and the biological basis for the

PfATP6/SERCA hypothesis. Trends in Parasitology 26: 517-523

Kumar S, Hahn FM, Baidoo E, Kahlon TS, Wood DF, McMahan CM, Cornish K, Keasling

JD, Daniell H, Whalen MC (2012) Remodeling the isoprenoid pathway in tobacco by

expressing the cytoplasmic mevalonate pathway in chloroplasts. Metabolic Engineering

14: 19-28

Letchamo W, Ward W, Heard B, Heard D (2004) Essential oil of Valeriana officinalis L.

cultivars and their antimicrobial activity as influenced by harvesting time under

commercial organic cultivation. Journal of Agricultural and Food Chemistry 52: 3915-

3919

Magnani M, Maccari G, Andreu JM, Diaz JF, Botta M (2009) Possible binding site for

paclitaxel at microtubule pores. Febs Journal 276: 2701-2712

McAndrew RP, Peralta-Yahya PP, DeGiovanni A, Pereira JH, Hadi MZ, Keesling JD,

Adams PD (2011) Structure of a Three-Domain Sesquiterpene Synthase: A Prospective

Target for Advanced Biofuels Production. Structure 19: 1876-1884

Meisel L, Fonseca B, Gonzalez S, Baeza-Yates R, Cambiazo V, Campos R, Gonzalez M,

Orellana A, Retamales J, Silva H (2005) A rapid and efficient method for purifying

high quality total RNA from peaches (Prunus persica) for functional genomics analyses.

Biological Research 38: 83-88

Miller DJ, Allemann RK (2012) Sesquiterpene synthases: Passive catalysts or active players?

Natural Product Reports 29: 60-71

Miziorko HM (2011) Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Archives

of Biochemistry and Biophysics 505: 131-143

Nagegowda DA, Gutensohn M, Wilkerson CG, Dudareva N (2008) Two nearly identical

terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers.

Plant Journal 55: 224-239

Navarrete A, Avula B, Choi YW, Khan IA (2006) Chemical fingerprinting of Valeriana

species: Simultaneous determination of valerenic acids, flavonoids, and

phenylpropanoids using liquid chromatography with ultraviolet detection. Journal of

Aoac International 89: 8-15

Newman DJ, Cragg GM (2007) Natural products as sources of new drugs over the last 25

years. Journal of Natural Products 70: 461-477

Newman DJ, Cragg GM, Snader KM (2000) The influence of natural products upon drug

discovery. Natural Product Reports 17: 215-234

Nogales E, Wolf SG, Khan IA, Luduena RF, Downing KH (1995) STRUCTURE OF

TUBULIN AT 6.5 ANGSTROM AND LOCATION OF THE TAXOL-BINDING SITE.

Nature 375: 424-427

O'Maille PE, Chappell J, Noel JP (2004) A single-vial analytical and quantitative gas

chromatography-mass spectrometry assay for terpene synthases. Analytical Biochemistry

335: 210-217

Page 97: Molecular cloning and characterization of sesquiterpene

85

Oldfield E, Lin F-Y (2012) Terpene biosynthesis: modularity rules. Angewandte Chemie

(International ed. in English) 51: 1124-1137

Owen NA, Inderwildi OR, King DA (2010) The status of conventional world oil reserves-Hype

or cause for concern? Energy Policy 38: 4743-4749

Paetzold H, Garms S, Bartram S, Wieczorek J, Uros-Gracia E-M, Rodriguez-Concepcion

M, Boland W, Strack D, Hause B, Walter MH (2010) The Isogene 1-Deoxy-D-

Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway

Allocation, and Density of Tomato Trichomes. Molecular Plant 3: 904-916

Paul C, Konig WA, Muhle H (2001) Pacifigorgianes and tamariscene as constituents of

Frullania tamarisci and Valeriana officinalis. Phytochemistry 57: 307-313

Picaud S, Brodelius M, Brodelius PE (2005) Expression, purification and characterization of

recombinant (E)-beta-farnesene synthase from Artemisia annua. Phytochemistry 66: 961-

967

Picaud S, Olofsson L, Brodelius M, Brodelius PE (2005) Expression, purification, and

characterization of recombinant amorpha-4, 11-diene synthase from Artemisia annua L.

Archives of Biochemistry and Biophysics 436: 215-226

Picaud S, Olsson ME, Brodelius M, Brodelius PE (2006) Cloning, expression, purification and

characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.

Archives of Biochemistry and Biophysics 452: 17-28

Pichersky E, Gang DR (2000) Genetics and biochemistry of secondary metabolites in plants: an

evolutionary perspective. Trends in Plant Science 5: 439-445

Polakowski T, Stahl U, Lang C (1998) Overexpression of a cytosolic hydroxymethylglutaryl-

CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and

Biotechnology 49: 66-71

Prisic S, Xu J, Coates RM, Peters RJ (2007) Probing the role of the DXDD motif in class II

diterpene cyclases. Chembiochem 8: 869-874

Rao S, He LF, Chakravarty S, Ojima I, Orr GA, Horwitz SB (1999) Characterization of the

taxol binding site on the microtubule - Identification of Arg(282) in beta-tubulin as the

site of photoincorporation of a 7-benzophenone analogue of taxol. Journal of Biological

Chemistry 274: 37990-37994

Rasmann S, Kollner TG, Degenhardt J, Hiltpold I, Toepfer S, Kuhlmann U, Gershenzon J,

Turlings TCJ (2005) Recruitment of entomopathogenic nematodes by insect-damaged

maize roots. Nature 434: 732-737

Rasmann S, Turlings TCJ (2007) Simultaneous feeding by aboveground and belowground

herbivores attenuates plant-mediated attraction of their respective natural enemies.

Ecology Letters 10: 926-936

Ro D-K, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus

RA, Ham TS, Kirby J, Chang MCY, Withers ST, Shiba Y, Sarpong R, Keasling JD (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Nature (London) 440: 940-943

Ro DK, Ouellet M, Paradise EM, Burd H, Eng D, Paddon CJ, Newman JD, Keasling JD (2008) Induction of multiple pleiotropic drug resistance genes in yeast engineered to

produce an increased level of anti-malarial drug precursor, artemisinic acid. Bmc

Biotechnology 8

Rodriguez-Concepcion M (2006) Early steps in isoprenoid biosynthesis: Multilevel regulation

of the supply of common precursors in plant cells. Phytochemistry Reviews 5: 1-15

Page 98: Molecular cloning and characterization of sesquiterpene

86

Rohmer M (1999) The discovery of a mevalonate-independent pathway for isoprenoid

biosynthesis in bacteria, algae and higher plants. Natural Product Reports 16: 565-574

Rohmer M, Knani M, Simonin P, Sutter B, Sahm H (1993) ISOPRENOID BIOSYNTHESIS

IN BACTERIA - A NOVEL PATHWAY FOR THE EARLY STEPS LEADING TO

ISOPENTENYL DIPHOSPHATE. Biochemical Journal 295: 517-524

Romagni JG, Allen SN, Dayan FE (2000) Allelopathic effects of volatile cineoles on two

weedy plant species. Journal of Chemical Ecology 26: 303-313

Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P,

Rothman RB (2002) Salvinorin A: A potent naturally occurring nonnitrogenous kappa

opioid selective agonist. Proceedings of the National Academy of Sciences of the United

States of America 99: 11934-11939

Ruzicka L (1953) THE ISOPRENE RULE AND THE BIOGENESIS OF TERPENIC

COMPOUNDS. Experientia 9: 357-367

Sallaud C, Rontein D, Onillon S, Jabes F, Duffe P, Giacalone C, Thoraval S, Escoffier C,

Herbette G, Leonhardt N, Causse M, Tissier A (2009) A Novel Pathway for

Sesquiterpene Biosynthesis from Z,Z-Farnesyl Pyrophosphate in the Wild Tomato

Solanum habrochaites. Plant Cell 21: 301-317

Samaneh ET, Tayebeh R, Hassan E, Vahid N (2010) Composition of essential oils in

subterranean organs of three species of Valeriana L. Natural Product Research 24: 1834-

1842

Sasaki K, Saito T, Laemsae M, Oksman-Caldentey K-M, Suzuki M, Ohyama K, Muranaka

T, Ohara K, Yazaki K (2007) Plants utilize isoprene emission as a thermotolerance

mechanism. Plant and Cell Physiology 48: 1254-1262

Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ (2003) BAX and BAK regulation of endoplasmic reticulum Ca2+: A control point for

apoptosis. Science 300: 135-139

Sharkey TD, Yeh SS (2001) Isoprene emission from plants. Annual Review of Plant Physiology

and Plant Molecular Biology 52: 407-436

Shen H-Y, Li Z-Q, Wang H, Ma L-Q, Liu B-Y, Yan F, Li G-F, Ye H-C (2007) Advances in

sesquiterpene synthases cyclases of Artemisia annua. Sheng wu gong cheng xue bao =

Chinese journal of biotechnology 23: 976-981

Shiba Y, Paradise EM, Kirby J, Ro DK, Keasing JD (2007) Engineering of the pyruvate

dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of

isoprenoids. Metabolic Engineering 9: 160-168

Shiojiri K, Ozawa R, Takabayashi J (2006) Plant volatiles, rather than light, determine the

nocturnal behavior of a caterpillar. Plos Biology 4: 1044-1047

Shishova EY, Di Costanzo L, Cane DE, Christianson DW (2007) X-ray crystal structure of

aristolochene synthase from Aspergillus terreus and evolution of templates for the

cyclization of farnesyl diphosphate. Biochemistry 46: 1941-1951

Soler R, Harvey JA, Kamp AFD, Vet LEM, Van der Putten WH, Van Dam NM, Stuefer

JF, Gols R, Hordijk CA, Bezemer TM (2007) Root herbivores influence the behaviour

of an aboveground parasitoid through changes in plant-volatile signals. Oikos 116: 367-

376

Starks CM, Back KW, Chappell J, Noel JP (1997) Structural basis for cyclic terpene

biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277: 1815-1820

Page 99: Molecular cloning and characterization of sesquiterpene

87

Steele CL, Crock J, Bohlmann J, Croteau R (1998) Sesquiterpene synthases from grand fir

(Abies grandis) - Comparison of constitutive and wound-induced activities, and cdna

isolation, characterization and bacterial expression of delta-selinene synthase and

gamma-humulene synthase. Journal of Biological Chemistry 273: 2078-2089

Stoll A, Seebeck E, Stauffacher D (1957) New investigations on Valerian. Schweizerische

Apotheker-Zeitung 95: 115-120

Takahashi S, Yeo Y, Greenhagen BT, McMullin T, Song L, Maurina-Brunker J, Rosson R,

Noel JP, Chappell J (2007) Metabolic engineering of sesquiterpene metabolism in yeast.

Biotechnology and Bioengineering 97: 170-181

Takemoto H, Yagura T, Ito M (2009) Evaluation of volatile components from spikenard:

valerena-4,7(11)-diene is a highly active sedative compound. Journal of Natural

Medicines 63: 380-385

Torssell K, Wahlberg K (1966) STRUCTURE OF PRINCIPAL ALKALOID FROM

VALERIANA OFFICINALIS(L). Tetrahedron Letters: 445-&

Torssell K, Wahlberg K (1967) ISOLATION STRUCTURE AND SYNTHESIS OF

ALKALOIDS FROM VALERIANA OFFICINALIS L. Acta Chemica Scandinavica 21:

53-&

Toyoshima C, Nomura H (2002) Structural changes in the calcium pump accompanying the

dissociation of calcium. Nature 418: 605-611

Toyota M, Ooiso Y, Kusuyama T, Asakawa Y (1994) DRIMANE-TYPE

SESQUITERPENOIDS FROM THE LIVERWORT DIPLOPHYLLUM-

SERRULATUM. Phytochemistry 35: 1263-1265

Trapp SC, Croteau RB (2001) Genomic organization of plant terpene synthases and molecular

evolutionary implications. Genetics 158: 811-832

Uhlemann AC, Cameron A, Eckstein-Ludwig U, Fischbarg J, Iserovich P, Zuniga FA, East

M, Lee A, Brady L, Haynes RK, Krishna S (2005) A single amino acid residue can

determine the sensitivity of SERCAs to artemisinins. Nature Structural & Molecular

Biology 12: 628-629

Verpoorte R, Vanderheijden R, Schripsema J, Hoge JHC, Tenhoopen HJG (1993) PLANT-

CELL BIOTECHNOLOGY FOR THE PRODUCTION OF ALKALOIDS - PRESENT

STATUS AND PROSPECTS. Journal of Natural Products 56: 186-207

Vickers CE, Gershenzon J, Lerdau MT, Loreto F (2009) A unified mechanism of action for

volatile isoprenoids in plant abiotic stress. Nature Chemical Biology 5: 283-291

Wallaart TE, Bouwmeester HJ, Hille J, Poppinga L, Maijers NCA (2001) Amorpha-4,11-

diene synthase: cloning and functional expression of a key enzyme in the biosynthetic

pathway of the novel antimalarial drug artemisinin. Planta 212: 460-465

Whittington DA, Wise ML, Urbansky M, Coates RM, Croteau RB, Christianson DW (2002) Bornyl diphosphate synthase: Structure and strategy for carbocation manipulation

by a terpenoid cyclase. Proceedings of the National Academy of Sciences of the United

States of America 99: 15375-15380

Winther A-ML, Liu H, Sonntag Y, Olesen C, le Maire M, Soehoel H, Olsen C-E,

Christensen SB, Nissen P, Moller JV (2010) Critical Roles of Hydrophobicity and

Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca(2+)-ATPase

with Thapsigargin and Thapsigargin Analogs. Journal of Biological Chemistry 285:

28883-28892

Page 100: Molecular cloning and characterization of sesquiterpene

88

Yan F, Mosier PD, Westkaemper RB, Stewart J, Zjawiony JK, Vortherms TA, Sheffler DJ,

Roth BL (2005) Identification of the molecular mechanisms by which the diterpenoid

salvinorin A binds to kappa-opioid receptors. Biochemistry 44: 8643-8651

Ying BP, Peiser G, Ji YY, Mathias K, Tutko D, Hwang YS (1995) PHYTOTOXIC

SESQUITERPENOIDS FROM CANELLA-WINTERANA. Phytochemistry 38: 909-915

Zhang F, Rodriguez S, Keasling JD (2011) Metabolic engineering of microbial pathways for

advanced biofuels production. Current Opinion in Biotechnology 22: 775-783

Zulak KG, Bohlmann J (2010) Terpenoid Biosynthesis and Specialized Vascular Cells of

Conifer Defense. Journal of Integrative Plant Biology 52: 86-97

Page 101: Molecular cloning and characterization of sesquiterpene

89

Appendix I

Chemical synthesis of valerena-4,7(11)-diene from valerenic acid (data provided by

Zhizeng Gao).

General Synthetic Procedures. All reactions involving air or moisture sensitive reactants were

conducted under a positive pressure of dry argon. All solvents and chemicals were reagent grade

and used as supplied unless otherwise stated. For anhydrous reactions, solvents were dried

according to the procedures detailed in Perrin and Armarego.1

Removal of solvent was

performed under reduced pressure, below 40 °C, using a Büchi rotary evaporator. Chemical

reagents were purchased from Sigma-Aldrich Chemical Company. Valerenic acid was purchased

from Extrasynthese Chemical Company. All reactions and fractions from column

chromatography were monitored by thin layer chromatography (TLC). Analytical TLC was done

on glass plates (5 × 1.5 cm) precoated (0.25 mm) with silica gel (SiO2, Merck 60 F254).

Compounds were visualized by exposure to UV light and by dipping the plates in 10 g

phosphomolybdic acid in 100 mL EtOH followed by heating on a hot plate. Flash

chromatography was performed on silica gel (EM Science, 60Å, 230-400 mesh). GC was done

on a Varian Aerograph 1400 using 1/4" x 6 ft. column (15% SE-30 on Chromosorb W).

Spectroscopic Analyses. Nuclear magnetic resonance (NMR) spectra were obtained on Varian

Inova 500 MHz and 600 MHz spectrometers. 1H NMR chemical shifts are reported in parts per

million (ppm) using the residual proton resonance of solvents as reference: CDCl3 δ 7.26, and

CD2Cl2, δ 5.32. 13

C NMR chemical shifts are reported relative to CDCl3 δ 77.0, and CD2Cl2 δ

53.8. Infrared spectra (IR) were recorded on a Nicolet Magna 750 or a 20SX FT-IR spectrometer.

Film Cast refers to the evaporation of a solution on a NaCl plate. Mass spectra were recorded on

Page 102: Molecular cloning and characterization of sesquiterpene

90

a Kratos IMS-50 (high resolution, electron impact ionization (EI)), or a ZabSpec IsoMass VG

(high resolution Electrospray (ES)).

Synthesis of Compounds 2 and 3 :

Scheme 1: Synthesis of 3

2: The known compound 2 was synthesized according to a literature procedure.2 To a stirred

solution of LiAlH4 (11.0 mg, 289 µmol) in dry THF (1 mL) was added valerenic acid (10.0 mg,

40.3 µmol) in dry THF (1 mL) at 0 °C under Ar. The reaction mixture was stirred at 0 °C for 5 h.

The reaction was quenched with 1 mL cold water. The layers were separated and the aqueous

CO2H

H H

HO

H

LiAlH4, THF

94%

1. PPh3, CBr4, CH2Cl2

2. LiEt3BH, THF

20% for two steps

1 2 3

H

HO

1

2

3

4

56

15

7

89

1112

13

14

10

2

Page 103: Molecular cloning and characterization of sesquiterpene

91

layer was extracted with EtOAc (3x1 mL). The combined organic layers were washed with brine

(2 mL) and dried over Na2SO4. The solvent was removed in vacuo and the residue was purified

using flash column chromatography (1:8 EtOAc/hexanes) to give 2 (8.9 mg, 94% yield) as a

colorless oil. IR (CH2Cl2, cast film) 3311, 2960, 2924, 2878, 2840, 1440, 1378 cm-1

; 1H NMR

(500 MHz, CDCl3): 5.75 (1H, m, H-11), 4.00 (2H, s, H-14), 3.45 (1H, m, H-9), 2.95–2.89 (1H,

m, H-5), 2.19 (2H, t, J = 7.60 Hz, H-3), 2.00–1.94 (1H, m, H-6), 1.89–1.76 (3H, m, H-4, H-7, H-

8), 1.72 (3H, s, H-13), 1.64 (3H, s, H-1), 1.58-1.49 (1H, m, H-4), 1.40–1.27 (2H, m, H-7, H-8),

0.77 (3H, d, J = 7.00 Hz, H-15); 13

C NMR (125 MHz, CDCl3): 135.2, 133.1, 129.1, 127.7,69.3,

47.4, 37.5, 33.4, 33.2, 28.7, 26.2, 24.5, 13.7, 13.4, 12.0;

D

25

= - 24.7 (c = 0.300, CH2Cl2); HREI

calcd for C15H24O 220.18271, found 220.18287 (M+), 202.17175 (M-H2O), 189.16430 (M-

CH3O), 187.14856 (M-CH5O).

3: The known compound 32 was synthesized according to a modified procedure. To a stirred

solution of PPh3 (16.0 mg, 61.0 µmol) in 0.2 mL dry CH2Cl2 was added CBr4 (18.0 mg, 54.3

µmol) at -10 °C (ice-NaCl bath). A solution of 2 (3.00 mg, 12.8 µmol) in 0.2 mL dry CH2Cl2 was

added. The resulting solution was stirred for another 30 min at -10 °C. Pentane was added and

H

1

2

3

4

56

14

7

89

1112

13

13

10

3

Page 104: Molecular cloning and characterization of sesquiterpene

92

the resulting mixture was stirred for 5 min. The precipitate was removed by filtration, and the

filtrate was concentrated by passing of a stream of Ar. The resulting residue was used directly in

the next step without purification.

To the residue in 1 mL of THF was added LiBHEt3 (100 µL, 100 µmol). The resulting solution

was stirred at 0 °C for 1h. Cold water was added to the solution and the mixture was extracted

with pentane (3x1 mL). The combined organic layers were washed with brine (2 mL) and dried

over Na2SO4. The solvent was evaporated at 1 atm and the resulting resudue was purified by GC

to give 3 (0.500 mg, 20 % yield for two steps) as a colorless oil. IR (CH2Cl2, cast film) 2962,

2925, 2879, 2856, 1449, 1378 cm-1

; 1H NMR (600 MHz, CD2Cl2) 5.46 (1H, dqq, J = 9.26,

1.43, 1.43 Hz, H-12), 3.40 (1H, m, H-9), 2.93 – 2.89 (1H, m, H-5), 2.18 (2H, m, H-3), 1.94 (1H,

m, H-6), 1.87 - 1.82 (1H, m, H-7), 1.82 – 1.77 (1H, m, H-4), 1.71 – 1.65 (1H, m, H-4), 1.68 (3H,

d, J = 1.32 Hz, H-13), 1.66 (3H, d, J = 1.32 Hz, H-13), 1.63 (3H, m, H-1), 1.57 – 1.48 (1H, m,

H-4), 1.37 – 1.30 (1H, m, H-7), 1.30 – 1.26 (1H, m, H-8), 0.75 (3H, d, J = 6.94 Hz, H-14); 13

C

NMR (125 MHz, CDCl3): 136.3, 130.0, 128.6, 126.5, 47.8, 37.8, 34.0, 34.0, 29.0, 26.8, 26.1,

24.9, 17.8, 13.4, 12.2;

D

25

= - 1.43 (c = 0.07, CH2Cl2); HREI calcd for C15H24 204.18781, found

204.18793 (M+), 189.16405 (M-CH3).

References:

1. D. D. Perrin and W. L. F. Armarego, Purification of Laboratory Chemicals 3rd

Edition ,

Pergamon Press.

2. T. Kitayama, G. Kawabata, M, Ito. Biosci. Biotechnol. Biochem. 2010, 74, 1963-1964

Page 105: Molecular cloning and characterization of sesquiterpene

93

Appendix II

Figure A1. Alignment of VoTPS1/2/3 nucleotide sequence.

Black indicates absolute conservation.

Page 106: Molecular cloning and characterization of sesquiterpene

94

Figure A2. Alignment of the amino acid sequences deduced from VoTPS1/2/3.

Residues identified by black highlights indicate complete conservation between VoTPS1/2/3.


Recommended