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GENETIC MODIFICATION OF SECONDARY METABOLISM / Alkaloids 493

and supply of resources, will require deeper investigation particularly of how photosynthesis integrates with other whole-plant processes. Potential exists for modifying photosynthesis: the task is considerable as extensive modications (and hence large input of still uncertain technology, resources and expertise) may be required, but the gains may be large.
See also: Energy Crops: Biofuels from Crop Plants. Photosynthesis and Partitioning: C3 Plants; C4 Plants; CAM Plants; Photoinhibition; Photorespiration; Sources and Sinks. Plants and the Environment: Phytoremediation.

Further Reading
Atwell BJ, Kriedemann PE, and Turnbull CGN (1999) Plants in Action: Adaptation in Nature, Performance in Cultivation. South Yarra, Australia: Macmillan Education. Barber J (ed.) (1994) Advances in Molecular and Cell Biology, vol. 10, Molecular Processes of Photosynthesis. Greenwich: Jai Press. Caemmerer S von (2000) Techniques in Plant Science, vol. 2, Biochemical Models of Leaf Photosynthesis. Collingwood: Australia, CSIRO Publishing. Flugge U-I (1999) Phosphate translocators in plastids. Annual Review of Plant Physiology and Plant Molecular Biology 50: 2745. Fridlyand LE, Backhausen JE, and Scheibe R (1999) Homeostatic regulation of enzyme activities in the Calvin cycle as an example for general mechanisms of ux

control: what can we expect from transgenic plants? Photosynthetic Research 61: 227239. Huber SC and Huber JL (1996) Role and regulation of sucrose-phosphate synthase in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 47: 431444. Lawlor DW (2001) Photosynthesis, 3rd edn. Oxford: BIOS Scientic Publishers. Lawlor DW and Paul MJ (2000) Genetic modication of photosynthesis. Journal of Experimental Botany 51 (GMP Special Issue). Leegood RC, Sharkey TD, and von Caemmerer S (eds) (2000) Photosynthesis: Physiology and Metabolism. Dordrecht: Kluwer Academic. Parry MAJ, Loveland JE, and Andralojc PJ (1999) Regulation of rubisco. In: Bryant JA, Burrell MM, and Kruger NJ (eds) Plant Carbohydrate Biochemistry, pp. 1018. Oxford: BIOS Scientic Publishers. Paul MJ, Pellny T, and Goddijn O (2001) Enhancing photosynthesis with sugar signals. Trends in Plant Science 6: 197200. Poolman MG, Fell DA, and Thomas S (2000) Modelling photosynthesis and its control. Journal of Experimental Botany 51: 319328. Reynolds MP, van Ginkel M, and Ribaut J-M (2000) Avenues for genetic modication of radiation use efciency in wheat. Journal of Experimental Botany 51: 459473. Ruelland E and Miginiac-Maslow M (1999) Regulation of chloroplast enzyme activities by thioredoxin: activation or relief from inhibition? Trends in Plant Science 4: 136141. Schulze E-D and Caldwell MM (eds) (1994) Ecophysiology of Photosynthesis. Berlin: Springer-Verlag.

GENETIC MODIFICATION OF SECONDARY METABOLISM


Contents

Alkaloids Terpenoids Wood Quality

Alkaloids
R M Twyman, University of York, York, UK R Verpoorte, Leiden University, Leiden, The Netherlands J Memelink, Leiden University, Leiden, The Netherlands P Christou, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany
Copyright 2003, Elsevier Ltd. All Rights Reserved.

Alkaloids
Alkaloids are complex organic molecules containing a heterocyclic nitrogen ring, which have been widely exploited for their diverse pharmacological properties. Such compounds are produced by many different organisms, including animals and microbes, but a particularly diverse array of alkaloids is produced by plants. Alkaloids are often isolated

494 GENETIC MODIFICATION OF SECONDARY METABOLISM / Alkaloids

from plants and used as pure compounds, although they may also be administered as crude extracts. Approximately 10% of all plant species are thought to produce alkaloids as secondary metabolites, where they function predominantly in defense against herbivores and pathogens. Although such ecochemical functions are important in their own right, most of the interest in alkaloids stems from their potent and specic pharmacological effects in humans, and this is also the reason why metabolic engineering of alkaloid biosynthesis is an important area of research. The structures of over 16 000 different alkaloids have been elucidated, and some important examples are listed in Table 1. The chemical structures of selected alkaloids are illustrated in Figure 1.

the synthesis of a particular alkaloid and/or inhibiting the synthesis of related compounds to increase the ease of purication.

Complexities of Alkaloid Biosynthesis


Before considering engineering strategies to exploit alkaloid biosynthesis it is necessary to have a thorough understanding of the endogenous biosynthetic pathways. Despite the interest in engineering alkaloid biosynthesis, progress in this area has been slow primarily due to the lack of knowledge of such pathways and their regulation. Predominantly, this is because alkaloid biosynthesis involves very complex multistep pathways, which have been difcult to dissect. Traditional pathway analysis has involved feeding plants with radiolabeled metabolic intermediates and determining whether the label was incorporated into particular end products. This type of analysis is frustrated by the difculties involved in synthesizing some intermediates and the fact that, in many cases, there is a signicant degree of cross-talk between pathways, such that the label can be incorporated into various products via different routes. A major step towards elucidating a metabolic pathway is to dene the enzymes involved. Again, this can be a complex task due to the fact that some enzymes cannot be isolated in an active form or that intermediates cannot be synthesized, so no convenient assay for enzyme activity can be employed. Furthermore, it is possible that some biosynthetic steps occur as spontaneous chemical reactions without the use of enzymes at all, e.g., conversion of the intermediate neopine into codeinone in the morphine biosynthetic pathway. Also, some enzymes may

Reasons for Engineering Alkaloid Biosynthesis


Due mainly to their useful pharmacological properties, alkaloids are often isolated from plants and produced commercially as ne chemicals. The complexity of the molecules means that they are in most cases impossible to produce by total chemical synthesis, so extraction from the source plant remains the most economically viable strategy. However, plants often produce complex mixtures of alkaloids at low levels, with the result that commercially produced specic alkaloids are very expensive. As the genetic manipulation of plants becomes more sophisticated, research has focused on the engineering of alkaloid biosynthesis to generate transgenic plants or cell lines that overproduce specic alkaloids. This can be achieved by increasing

Table 1 Sources and pharmacological uses of selected plant-derived alkaloids Alkaloid Ajmaline Caffeine Camptothecin Cocaine Codeine Emetine Hyoscyamine Morphine Nicotine Pilocarpine Quinidine Quinine Reserpine Scopolamine Strychnine Taxol Vinblastine and vincristine Source Rauvola serpentina Coffea arabica Camptotheca acuminata Erythroxylon coca Papaver somniferum Uragoga ipecacuanha Atropa belladonna and others Papaver somniferum Nicotiana tabacum Pilocarpus jaborandi Cinchona spp. Cinchona spp. Rauvola serpentina Hyoscyamus niger and others Strychnos nux-vomica Taxus brevifolia Catharanthus roseus Properties Antiarrhythmic, antihypertensive Stimulant, insecticide Antineoplastic Analgesic, narcotic, local anesthetic Analgesic, antitussive Antiamoebic, expectorant, emetic Anticholinergic Analgesic, narcotic Stimulant Cholinergic Antiarrhythmic Antimalarial Tranquilizer Sedative, anticholinergic Stimulant, poison Antineoplastic Antineoplastic

GENETIC MODIFICATION OF SECONDARY METABOLISM / Alkaloids 495


Quinine N N H H OCO OCH3 OCH3 OCH3 OCH3 CH3O N H N H H H OCH3 H N H R O N H CH3O2C H NCH3 HO Morphine and codeine CH3O N H Emetine OH N N O H H O HO H N

CH3O Reserpine

H CH3O2C

CH3O CH3O H NH H

Strychnine OCH3

N H OH OCOCH3

CO2CH3 R Vinblastine and vincristine

Figure 1 Structures of selected alkaloids. Note that the structures of morphine and codeine are based on the same skeleton, but are decorated with different functional groups in the position represented by R. In morphine, this group is OH, while in codeine it is CH2O. Similarly, vinblastine and vincristine are based on the same skeleton, but differ in the nature of the R-group, which for vinblastine is CH3 and for vincristine is CHO.

catalyze two or more separate reactions, e.g., hyoscyamine 6-hydroxylase, which carries out two consecutive steps in the scopolamine biosynthetic pathway. Another complication in alkaloid biosynthesis is compartmentalization. The biosynthesis of the indole alkaloids vinblastine and vincristine in Catharanthus roseus provides an example where different enzymatic steps are carried out in different cellular compartments (Figure 2). The nal steps in the pathway are also carried out in a different cell type to the early steps, which requires the intercellular transport of metabolic intermediates. Similarly, scopolamine biosynthesis requires two different cell types.

block competitive pathways, and the simultaneous modulation of multiple enzymatic steps. An alternative is to stimulate or inhibit the catabolism of the target compound, although to date this has not been used with respect to alkaloids.
Single-Step Engineering

Strategies for Engineering Alkaloid Biosynthesis


There are three basic goals of metabolic engineering: (1) producing more of a specic compound, (2) producing less of a specic compound, and (3) producing a novel compound (this may be a known compound that is heterologous in the expression system being used or a completely novel compound). Strategies for achieving these goals can involve the engineering of single steps in a pathway to increase or decrease metabolic ux to target compounds, or

The simplest strategy to increase the levels of a specic target alkaloid is to increase the metabolic ux towards that compound. As discussed above, this requires a thorough understanding of the endogenous pathway because it is necessary to identify the rate-limiting step in the pathway and to use this as the primary target for engineering. For example, consider the biosynthesis of terpenoid indole alkaloids such as vinblastine and vincristine in C. roseus (Figure 2). Although synthesized in whole plants, these valuable alkaloids are not synthesized in cell culture, probably due to the tissue-specic compartmentalization in whole plants discussed above. The universal terpene indole alkaloid precursor strictosidine is produced in culture, and is metabolized to form other alkaloids such as ajmalicine and catharanthine. For indole alkaloid biosynthesis, a rate-limiting step is the conversion of the amino acid tryptophan to tryptamine, catalyzed by the enzyme tryptophan decarboxylase (TDC).

496 GENETIC MODIFICATION OF SECONDARY METABOLISM / Alkaloids

Chorismate AS Anthranilate Plastid Tryptophan

Chloroplast
Desacetoxyvindoline

D4H Deacetylvindoline

NMT
16-Hydroxytabersonine

TDC Tryptamine

Geraniol G10H 10-Hydroxygeraniol Loganin DAT T16H Tabersonine Endoplasmic reticulum SGD Cathenamine Catharanthine Vindoline

STR

SLS Secologanin

Strictosidine Vacuole Vinblastine

Figure 2 Very simplied scheme showing the compartmentalization of terpenoid indole alkaloid biosynthesis in Catharanthus roseus. En route to vinblastine synthesis, different enzymes (shown as disks) are located in distinct compartments, including the plastid, vacuole, endoplasmic reticulum membrane, mature chloroplast, and cytosol (the location of some enzymes is not precisely known). Alkaloid biosynthesis therefore involves extensive trafcking of metabolic intermediates. Although not shown in this gure, certain reactions are also cell-type specic, so intermediates must also be transported between cells. Broken arrows represent multiple steps, while intact arrows represent single reactions. Abbreviations: AS, anthranilate synthase; TDC, tryptophan decarboxylase; G10H, geraniol-10-hydroxylase; SLS, secologanin synthase; STR, strictosidine synthase; SGD, strictosidine b-D-glucosidase; T16H, tabersonine-16-hydroxylase; NMT, S-adenosyl-L-methionine: 16-methoxy-2,3-dihydro-3-hydroxy-tabersonine-N-methyltransferase; D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline 4-O-acetyltransferase.

Tryptamine is then condensed with the terpenoid molecule secologanin to produce strictosidine. To alleviate the regulation of TDC, the C. roseus tdc gene was overexpressed under the control of a strong and constitutive promoter. The resulting transgenic cultures produced high levels of tryptamine, but not further-downstream alkaloids. Thus, overcoming the rate-limiting step merely revealed the next ratelimiting step, at the level of the enzyme strictosidine synthase (STR). This is likely to reect a limitation in the supply of the terpenoid precursor secologanin. Single-step engineering can also be used to extend a metabolic pathway in a heterologous plant. For example, the alkaloid scopolamine is produced in Hyoscyamus niger but not in Atropa belladonna, where the tropane alkaloid pathway stops at Lhyoscyamine. However, introduction of a cDNA from H. niger encoding hyoscyamine-6-hydroxylase into A. belladonna resulted in the production of scopolamine in this plant (Figure 3).
Multiple-Step Engineering

gene transgenics) or by simultaneous multiple gene transfer. For the most part, such experiments have involved marker genes rather than genes of agronomic interest, but examples of the latter are beginning to be reported, particularly involving the simultaneous transfer of multiple genes conferring resistance to different pests and pathogens. In terms of metabolic engineering, one way to overcome the limitations of the single-gene approach above would be to simultaneously transform plants with genes encoding enzymes that act at different steps of a biosynthetic pathway. For example, transgenic tobacco (Nicotiana tabacum) plants and C. roseus cell cultures have been generated simultaneously overexpressing both tdc and str transgenes, the latter showing high levels of strictosidine accumulation when fed with loganin. Particle bombardment has been used to transform plants simultaneously with 13 different genes, so it is theoretically possible to transfer genes encoding the enzymes of an entire pathway into a suitable expression host.
Engineering Regulatory Genes

Over the last few years it has become increasingly common for multiple genes to be introduced into plants either stepwise (by crossing independent single

In most organisms, transcription factors act as master regulators of complex pathways of

GENETIC MODIFICATION OF SECONDARY METABOLISM / Alkaloids 497


CH3 N O O H O Hyoscyamus niger
L-Hyoscyamine

CH3 N OH H6H O OH

H O

(intermediate)

Scopolamine (normal metabolic end product) Transfer h6h gene from H. niger to A. belladonna

CH3 O O H O OH

CH3 N

H O

OH

Atropa belladonna

L-Hyoscyamine (normal metabolic end product)

Scopolamine (end product in transgenic plants)

Figure 3 An example of single-gene metabolic engineering. The enzyme hyoscyamine-6-hydroxylase (H6H) is responsible for two reactions that convert L-hyoscyamine into scopolamine. Atropa belladonna accumulates L-hyoscyamine because the enzyme H6H is not produced in this species. However, by transferring the enzyme from another plant such as Hyoscyamus niger, which does produce scopolamine, the metabolic pathway in A. belladonna can be extended.

downstream genes, with important examples recognized in development, signal transduction and metabolism. The important feature of such regulatory systems is that a number of downstream genes are coordinately regulated, either in response to the developmental program of the organism or in response to external inductive signals. As well as responding to the developmental program in plants, a number of external stimuli are known to induce alkaloid biosynthesis, including UV light, fungal elicitors, auxin starvation, and the signaling molecule jasmonic acid. Cell cultures are valuable for testing such responses because signals can be applied to the cultured cells and the expression of different biosynthetic genes can be assayed directly by northern blot or reverse transcriptase polymerase chain reaction (RT-PCR), or using reporter genes linked to the endogenous gene promoters. In this way, it has been shown that the C. roseus genes for tryptophan decarboxylase (tdc), strictosidine synthase (str), geraniol-10-hydroxylase (g10h), NADPH:cytochrome P450 reductase (cpr), strictosidine b-D-glucosidase (sgd), deacetylvindoline 4-O-acetyltransferase (dat), and desacetoxyvindoline 4-hydroxylase (d4h) are all inducible by methyl jasmonate and/or fungal elicitor in cell culture. In addition, tdc and str are known to be UV-B-inducible in leaves.

The common regulation of many of the genes involved in terpenoid indole alkaloid biosynthesis in C. roseus suggests that a useful strategy for increasing the levels of alkaloids in this system would be to identify and manipulate transcription factors that control the expression of these genes. Such transcription factors have been sought in a number of ways. The yeast one-hybrid system (Figure 4A) has been very successful, yielding two AP2/ERF-type transcription factors called ORCA1 and ORCA2 (octadecanoid-responsive Catharanthus AP2) that bind to the jasmonate/elicitor response element (JERE) in the str gene promoter. While ORCA1 appears not to be involved in jasmonate/elicitor induction, ORCA2 has been directly shown to activate the str gene by binding to the JERE, and the orca2 gene is itself induced by jasmonate and elicitor stimulation. A further transcription factor called ORCA3 has been identied using an alternative insertional mutagenesis strategy called activation tagging (Figure 4B). Briey, Agrobacterium tumefaciens was used to transform C. roseus with a T-DNA construct containing the strong and constitutive CaMV35S promoter adjacent to the right border. The rationale behind this strategy was that the promoter would activate genes adjacent to the site of integration. Occasionally, the construct might integrate near a gene regulating alkaloid biosynthesis, resulting in the

498 GENETIC MODIFICATION OF SECONDARY METABOLISM / Alkaloids


(A) (1) P GAL4 (act) ORCA (B) (1) T-DNA Pmax

(2) (2) GAL4 ORCA

Pmax

ORCA

(3)
GAL4 GAL4 GAL4 GAL4 ORCA ORCA ORCA ORCA

ORCA ORCA ORCA ORCA ORCA

(3)

JERE JERE JERE JERE Pmin HIS3

(4) P

ORCA JERE ORCA P


JERE

tdc str (others)

(4) P

ORCA JERE

Figure 4 Approaches that have been used to isolate regulatory genes controlling alkaloid biosynthesis in Catharanthus roseus. (A) The yeast one-hybrid system. A C. roseus cDNA expression library is generated in yeast. Each cDNA is expressed as a fusion protein with the activation domain of the yeast transcription factor GAL4 as shown in (1). If the C. roseus cDNA encodes a transcription factor such as ORCA2, which regulates alkaloid biosynthesis, the fusion protein (2) will activate a HIS3 reporter gene via a minimal promoter (Pmin) linked to four jasmonate/elicitor response elements (JERE) as shown in (3). Yeast cells in which the construct is active are able to grow on minimal medium lacking histidine (4). These cells are likely to contain fusion proteins in which the C. roseus component is a transcription factor that binds to the JERE. (B) Activation tagging. An Agrobacterium T-DNA construct contains a strong promoter (Pmax) adjacent to the right border as shown in (1). If this integrates adjacent to a gene that encodes a relevant transcription factor such as ORCA3 (2), its expression will be stimulated (3) resulting in the upregulation of genes such as tdc and str. Cells with elevated TDC activity can be selected using 4-methyltryptophan, and the expression levels of tdc, str and other genes can be analyzed.

upregulation of one or more structural genes in the pathway. Populations of C. roseus cells transformed in this manner were selected in a medium containing the tryptophan analog 4-methyltryptophan (4-mT). The levels of 4-mT used in the experiment would be toxic to normal cells, but not those in which the activity of the tdc gene was stimulated, since TDC converts 4-mT into a harmless derivative, 4-methyltryptamine. Of course this selection strategy identies not only global regulators of alkaloid biosynthesis, but also the tdc gene itself. To specically identify regulatory genes, surviving cell lines with elevated levels of tdc mRNA were screened for overexpression of other biosynthetic genes, such as str, and this resulted in the isolation of ORCA3. The ORCA2 and ORCA3 transcription factors are both induced by jasmonate/elicitor with similar kinetics and they both bind to the tdc and str promoters. The two proteins are highly conserved in the AP2/ERF DNA-binding domain but are otherwise dissimilar in sequence. Cells that overexpress ORCA3 show increased expression of tdc, str, sgd, cpr, and d4h, although

other genes are not induced, even though they are normally responsive to jasmonate/elicitor stimulation. This suggests that although the structural genes of the pathway may be coordinately induced, this may be under combinatorial control by ORCA2, ORCA3 and perhaps other regulators. ORCA3overexpressing cells accumulate tryptamine but no downstream alkaloids, suggesting the incoming terpenoid branch of the pathway is regulated independently of ORCA3.

Future Perspectives
The valuable pharmacological properties of alkaloids together with their low level of production by natural sources means that active research into novel strategies for metabolic engineering will continue to be of great importance in the future. The approaches discussed above single-step engineering, multiplestep engineering, and the engineering of regulatory genes are likely to be used in combination with new high-throughput strategies for dening cellular biochemistry at a global level. This metabolomics

GENETIC MODIFICATION OF SECONDARY METABOLISM / Alkaloids 499

approach will become more applicable as the number of plants with completed genome sequences increases, and it becomes possible to analyze the expression of all genes simultaneously using microarrays and oligonucleotide chips. In the future, it may be possible to identify the most suitable route to metabolic engineering simply by carrying out a small number of array-based hybridization experiments to identify genes and external conditions that upregulate appropriate sets of biosynthetic genes correlating with increased metabolite biosynthesis.

Microarray

A collection of cDNA sequences arranged on a miniature solid support allowing the simultaneous analysis of many gene expression proles. A collection of oligonucleotides printed onto a miniature solid support allowing the simultaneous analysis of many gene expression proles. A yeast-based expression cloning system in which DNA-binding proteins are preferentially isolated. Gene transfer achieved by ring DNAcoated microprojectiles into a plant cell. A bottleneck in a metabolic pathway, where conversion from one intermediate to another is slowest. A DNA sequence in a genes promoter that responds to external stimuli by binding transcription factors whose activity is dependent on signal transduction pathways stimulated by external molecules binding to receptors. The sum of metabolic pathways producing molecules with specialized functions, not involved in general homeostasis. Metabolic engineering through the modication of one step in a metabolic pathway. A protein that controls gene expression by binding to regulatory elements and controlling transcription.

Oligonucleotide chip

One-hybrid system Particle bombardment Rate-limiting step Response element

List of Technical Nomenclature


Activation tagging The isolation of genes through the integration of a DNA element containing a strong promoter that can activate genes adjacent to the insertion site. Any of the complex nitrogen-containing heterocyclic organic compounds, mainly of plant origin, with potent pharmacological properties in humans. The situation in which different steps in a metabolic pathway are carried out by enzymes localized in different compartments within the cell. The situation in which one transcription factor controls several genes in the same manner, so that they are activated as a unit. Molecules produced by microorganisms, which are recognized by receptors on the plant cell and induce a defense response in the plant. A gene whose product confers an easily recognizable property on a cell, such as the ability to grow in the presence of antibiotics or the production of a visible product. The deliberate alteration of metabolism by gene manipulation, in order to modify the levels of a particular metabolite. A series of enzymatic steps that converts a particular precursor into a given end product through a number of intermediates. The global study of metabolism. The simultaneous analysis of metabolites produced by a cell or organism. An intermediate in elicitor-stimulated signaling which can also induce defense responses when applied to plant cells.

Alkaloid

Compartmentalization

Secondary metabolism

Coordinate regulation

Single-step engineering Transcription factor

Elicitor

Marker gene

See also: Genetic Modication, Applications: Cell Factories. Genetic Modication of Secondary Metabolism: Terpenoids. Tissue Culture: Secondary Metabolism in Plant Cell Cultures.

Metabolic engineering

Further Reading
De Luca V and St Pierre B (2000) The cell and developmental biology of alkaloid biosynthesis. Trends in Plant Science 5: 168173. Facchini PJ, Huber-Allanach KL, and Tari LW (2000) Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications. Phytochemistry 54: 121138. Kutchan TM (1995) Alkaloid biosynthesis: the basis of metabolic engineering of medicinal plants. Plant Cell 7: 10591070. Kutchan TM (1998) Molecular genetics of plant alkaloid biosynthesis. In: Cordell GA (ed.) The Alkaloids, vol. 50, pp. 257316. San Diego: Academic Press.

Metabolic pathway

Metabolomics

(Methyl)jasmonic acid

500 GENETIC MODIFICATION OF SECONDARY METABOLISM / Terpenoids Memelink J, Verpoorte R, and Kijne JW (2001) ORCaniation of jasmonate-responsive gene expression in alkaloid metabolism. Trends in Plant Science 6: 212219. Primrose SB, Twyman RM, and Old RW (2001) Principles of Gene Manipulation, 6th edn. Oxford: Blackwell Science. Verpoorte R and Alfermann AW (eds) (2000) Metabolic Engineering of Plant Secondary Metabolism. Dordrecht: Kluwer Academic Publishers. Verpoorte R, van der Heijden R, and Memelink J (1998) Plant biotechnology and the production of alkaloids: prospects of metabolic engineering. In: Cordell GA (ed.) The Alkaloids, vol. 50, pp. 453508. San Diego: Academic Press. Verpoorte R, van der Heijden R, ten Hoopen HJG, and Memelink J (1999) Metabolic engineering of plant secondary metabolic pathways for the production of ne chemicals. Biotechnology Letters 21: 467479. Verpoorte R, van der Heijden R, and Memelink J (2000) Engineering the plant cell factory for secondary metabolite production. Transgenic Research 9: 323343.

Terpenoids
J Degenhardt and J Gershenzon, Max Planck Institute for Chemical Ecology, Jena, Germany
Copyright 2003, Elsevier Ltd. All Rights Reserved.

industry, serving a world market of several billion dollars annually. In medicine, terpenes are generally used as antiseptics, antioxidants, or anti-inammatory agents, as well as for treatment of diseases, such as cancer and malaria. For example, a high demand exists for paclitaxel (Figure 1), a highly substituted diterpene with potent anticancer activity. Since paclitaxel can only be isolated from plants in low yields, it is currently produced by semisynthesis from a more widely available plant product of related structure. After decades of conventional breeding, researchers have recently begun to employ genetic engineering methods for the manipulation of terpene metabolism, in order to improve the overall yield and composition of terpenes from plants with high economic importance. These efforts have often been hampered by the complexity of terpene biosynthetic pathways and their regulation, but a few notable successes have already been achieved. This article will discuss the different phases of terpene biosynthesis and the strategies that have been pursued for altering each phase to modify the quantity and quality of terpenes in plants.

OH

Introduction
Terpenes, also known as terpenoids or isoprenoids, form the largest group of plant natural products, comprising about 30 000 substances, with new compounds being discovered at the rate of about 1000 per year. The large structural diversity of terpenes in plants is mirrored by their extremely high functional diversity. They play a myriad of roles in both primary metabolism, as pigments, membrane components and hormones, and in secondary metabolism, as defenses against herbivores and pathogens, and attractants for pollinators and herbivore enemies. In human society also, terpenes play many important roles. They are indispensable in nutrition (vitamins A, D, and E) and in providing certain industrial raw materials (rubber, resins, and turpentine). They also impart avors and fragrances to foods, beverages, perfumes, soaps, toothpaste, tobacco products, and many other items of daily commerce. Because the majority of customers usually prefer natural odors and tastes, rather than synthetic or semisynthetic substitutes, the cultivation of crops for avor and fragrance production is an economically important sector of the agricultural
OH

Linalool

Limonene

Menthol

Menthofuran

O O O O (E)--Farnesene (E )--Caryophyllene AcO O Artemisinin O OH

O N H OH Paclitaxel

O O OH O AcO O

Figure 1 Examples of terpenes of economic importance.