Phytochemistry Reviews (2006) 5: 49–58 Ó Springer 2006 DOI 10.1007/s11101-005-3747-3

Metabolic engineering of terpenoid biosynthesis in plants

Asaph Aharoni1,*, , Maarten A. Jongsma2, Tok-Yong Kim2,4, Man-Bok Ri2,4, Ashok P. Giri2,5, Francel W. A. Verstappen2, Wilfried Schwab3 & Harro J. Bouwmeester2 1Weizmann Institute of Science, 26 Rehovot 76100 Israel; 2Plant Research International, 16 6700 AA Wageningen The Netherlands; 3Biomolecular Food Technology, TU Mu¨nchen, Lise-Meitner-Str. 3485354 Freising, Mu¨nchen Germany; 4Research Institute of Agrobiology, Academy of Agricultural Sciences, Ryongsong Pyongyang, Democratic People’s Republic of Korea; 5Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Dr Homi Bhabha Road, Pune 411008 India; *Author for correspondence (Tel: +0972-(0)8-9343643; Fax: +0972-(0)8-9344181; E-mail: asaph.aharoni@ weizmann.ac.il)

Key words: linalool, MEP pathway, mevalonate pathway, monoterpene, sesquiterpene

Abstract Metabolic engineering of terpenoids in plants is a fascinating research topic from two main perspectives. On the one hand, the various biological activities of these compounds make their engineering a new tool for improving a considerable number of traits in crops. These include for example enhanced disease resistance, weed control by producing allelopathic compounds, better pest management, production of medicinal compounds, increased value of ornamentals and fruit and improved pollination. On the other hand, the same plants altered in the profile of terpenoids and their precursor pools make a most important contri- bution to fundamental studies on terpenoid biosynthesis and its regulation. In this review we describe our recent results with terpenoid engineering, focusing on two terpenoid classes the monoterpenoids and ses- quiterpenoids. The emerging picture is that engineering of these compounds and their derivatives in plant cells is feasible, although with some requirements and limitations. For example, in terpenoid engineering experiments crucial factors are the subcellular localisation of both the precursor pool and the introduced enzymes, the activity of endogenous plant enzymes which modify the introduced terpenoid skeleton, the costs of engineering in terms of effects on other pathways sharing the same precursor pool and the phyto- toxicity of the introduced terpenoids. Finally, we will show that transgenic plants altered in their terpenoid profile exert novel biological activities on their environment, for example influencing insect behaviour.

Abbreviations: DMADP – dimethylallyl diphosphate; FDP – farnesyl diphosphate; GDP – geranyl diphosphate; GGDP – geranylgeranyl diphosphate; IDP – isopentenyl diphosphate; MEP – methylerythritol 4-phosphate; TPSs – terpene synthases.

Introduction agents, pharmaceuticals, perfumes, insecticides and anti-microbial agents (Martin et al., 2003). In Terpenoids are the most structurally varied class nature, they play significant roles in plant–envi- of plant natural products. They are commercially ronment interactions, plant–plant communication important due to their wide application in a vast and plant–insect and plant–animal interactions number of industrial products such as flavouring (Pichersky and Gershenzon, 2002). Although

A. Aharoni is an Incumbent of the Adolfo and Evelyn Blum Career Development chair. 50 many of them are associated with primary terpenoid structures (Lucker et al., 2001). Terpe- metabolism (e.g. the phytol side chain of chloro- noid biosynthesis occurs in the cytosol and the phyll, carotenoid pigments, and the plant hormone plastids (Figure 1). IDP and DMADP are synthe- gibberellin) others are typical plant secondary sized through the 2-methylerythritol 4-phosphate metabolites. pathway (MEP) via deoxy-D-xylulose 5-phosphate All terpenoids are synthesized through the in plastids. However, IDP is also synthesized in the condensation of isopentenyl diphosphate (IDP) cytosol via the mevalonate pathway (Bick and and its allylic isomer dimethyl allyl diphos- Lange, 2003). It is generally accepted that GDP phate (DMADP) (Carretero-Paulet et al., 2002). and GGDP in the plastids are used as substrate for The sequential head-to-tail addition of IDP units monoterpene and diterpene biosynthesis, respec- to DMADP yields the prenyl diphosphates geranyl tively whereas FDP in the cytosol is used for ses- diphosphate (GDP), farnesyl diphosphate quiterpene biosynthesis (Figure 1). (FDP) and geranylgeranyl diphosphate (GGDP) Why would we like to engineer terpenoid pro- (Figure 1). These three components serve as pre- duction in plants? Primarily, plants engineered for cursors for the monoterpenes, sesquiterpenes and their terpenoid profile could serve as a tool for diterpenes, respectively. Terpenoid synthases or improving a large number of traits in different cyclases catalyze the reactions in which the pri- crop species. Examples for such traits are mary terpene skeletons are formed from these enhanced disease resistance, weed control by pro- substrates. The parent skeletal type of mono-, ducing allelopathic compounds, improved pest sesqui- and diterpenes is normally further modified control, increased value of ornamentals and fruit by the activity of an array of different enzymes (fragrance and flavour) and improved pollination (e.g. hydroxylases, dehydrogenases, reductases and by altering scent profiles. In addition, large-scale glycosyl, methyl and acyl transferases) which to- production of terpenoids in plants, either for gether generate the many thousands of different medicinal uses or for other industries such as

Figure 1. The mevalonate and MEP pathways producing different terpenoid classes in the cytosol and plastids, respectively. GA3P, D-glyceraldehyde-3-phosphate; TPSs, terpene synthases; IDP, isopentenyl diphosphate; DMADP, dimethylallyl diphosphate; FDP, farnesyl diphosphate; MEP, methylerythritol 4-phosphate; GDP, geranyl diphosphate; GGDP, geranylgeranyl diphosphate. Broken arrows represent multiple enzymatic steps. 51 cosmetics and food would be attractive. A second, The FaNES1 protein as a ‘sensor’ for both but not less important reason is that plants altered mono- and sesquiterpene precursors in the profile of terpenoids (and pool of precur- sors) make an important contribution to funda- We chose the strawberry FaNES1 (Fragaria anan- mental studies on their biosynthesis and assa Nerolidol Synthase 1) gene for performing regulation. For example, metabolic engineering metabolic engineering experiments in several plant experiments often reveal undiscovered branches to species (Aharoni et al., 2004). The recombinant an already known metabolic pathway or point to FaNES1 protein was previously shown to catalyze feedback loops within a pathway or between the conversion of GDP and FDP to (S)-linalool pathways. and (3S)-(E)-nerolidol, respectively with equal In recent years attempts to produce high levels efficiency. The ability of other recombinant terpene of monoterpenes in transgenic plants have been synthases to generate both sesquiterpenes and successful. Several different plant species were monoterpenes was already observed earlier (Crock engineered, mainly by overexpressing terpene et al., 1997; Steele et al., 1998). However, in these synthases under constitutive promoters. Petunia, cases the recombinant enzyme could generate the tomato, carnation, potato and Arabidopsis plants sesquiterpene with high efficiency while a combi- were generated that over-expressed genes encoding nation of monoterpenes would be formed from linalool synthases. Such plants produced and GDP but with low efficiency. FaNES1 could emitted the monoterpene linalool and its glycosy- therefore serve as an excellent ‘sensor’ for levels of lated or hydroxylated derivatives (Lewinson et al., both monoterpene and sesquiterpene precursors in 2001; Lucker et al., 2001; Lavy et al., 2002; the cell or even, as will be described later in this Aharoni et al., 2003). Mint and tobacco plants report, in a specific sub-cellular compartment. expressing limonene, c-terpinene and a-pinene synthases were also altered in their terpenoid Engineering monoterpenoids in Arabidopsis plants profile (Diemer et al., 2001; Lucker et al., 2004b). Levels of terpenoid precursors could also be ele- Recent research in Arabidopsis revealed that what vated by overexpressing genes encoding enzymes at first seemed a metabolically simple plant species from various steps of the MEP pathway (DXR and is in reality a reasonable producer of secondary HDR) (Mahmoud and Croteau, 2001; Botella- metabolites (D’Auria and Gershenzon, 2005). For Pavia et al., 2004). In addition, genes encoding example, nearly two dozen monoterpenes and ses- enzymes which modify monoterpene structures quiterpenes are emitted from its flowers (Aharoni have been successfully over-expressed or knocked et al., 2003; Chen et al., 2003; Tholl et al., 2005). down in tobacco and mint (Mahmoud and Leaves of Arabidopsis on the other hand emit only Croteau, 2001; Wang et al., 2001; Lucker et al., traces of one monoterpene, limonene. Transgenic 2004a; Mahmoud et al., 2004). In conclusion, in Arabidopsis plants were raised which expressed the many studies it was demonstrated that it is feasible FaNES1 gene driven by the CaMV 35S promoter. to engineer several steps of the monoterpenoid The FaNES1 protein was targeted to the plastids pathway. However, attempts to engineer sesquit- by fusing the wild strawberry FvNES1 (Fragaria erpenes in plants using terpene synthases resulted vesca Nerolidol Synthase 1) plastid targeting region in only low level production (Hohn and Ohlrogge, to the N-terminal of FaNES1. Various headspace 1991; Wallaart et al., 2001). analyses methods revealed that the volatile profile In this report we will describe our recent work of rosette leaves derived from transgenic lines was on engineering mono- and sesquiterpenes in Ara- altered compared to control plants. High levels of bidopsis and potato. Several issues important for linalool were produced by transgenic lines and this field of research will be discussed, including: levels of linalool released from the highest pro- (a) metabolic fate of engineered terpenoids, (b) ducing lines reached 7.2 to 13.3 lgday)1 plant)1 ease of engineering monoterpenes versus sesquit- (Aharoni et al., 2003). Multidimensional GC-MS erpenes, (c) availability of precursors (in different analysis revealed that transgenic Arabidopsis lines sub-cellular compartments) (d) cost of engineering produced exclusively (S)-linalool. This result mat- terpenoids (e) biological activity of transgenic ches with the linalool enantiomer produced by ripe plants altered in their terpenoid profile. fruit of cultivated strawberry and the recombinant 52

FaNES1 enzyme in vitro. Interestingly, emission of that low levels of glycosidically bound E-8-hydroxy the newly produced linalool showed a diurnal linalool could also be detected in leaves of wild- rhythm in the same way as other mono- and ses- type Arabidopsis, and, therefore, active endoge- quiterpenes naturally emitted by flowers of wild- nous enzymes apparently were present that could type plants (Aharoni et al., 2003). also act efficiently on the newly produced linalool. Plants overexpressing FaNES1 not only produced and emitted linalool, but also three Engineering monoterpenoids in potato plants linalool derivatives including E-8-hydroxy linalool, Z-8-hydroxy linalool and E-8-hydroxy-6,7-di- To engineer monoterpenes in potato plants we hydrolinalool. E-8-hydroxy linalool and E-8-hy- expressed the FaNES1 gene using the same con- droxy-6,7-dihydrolinalool were identified as both struct as the one used for transforming Arabid- glycosidically bound and as aglycons and Z-8-hy- opsis (see above). Leaves of wild-type potato droxy linalool only as glycoside (Aharoni et al., plants already emit linalool but transgenic lines 2003). Formation of these derivatives is most emitted linalool to much higher levels (Figure 2). probably a result of endogenous enzyme activities As described earlier (Aharoni et al., 2004), the mediating hydroxylation, double bond reduction, FaNES1 recombinant protein catalyzes the bio- and glycosylation (e.g. cytochrome P450s, reduc- transformation of GDP to S-linalool. We per- tases and glycosyl transferases). E-8-hydroxy formed chiral analyses on linalool produced by linalool and its glycoside were detected to the transgenic and wild-type plants using multidi- highest levels. This might be explained by the fact mensional gas chromatography mass spectrometry

(a) E-8-hydroxy- 4.0 linalool 3.5 Z-8-hydroxy- 3.0 linalool E-8-hydroxy-6,7- 2.5 dihydro-linalool 2.0

fresh weight nerolidol -1 1.5 linalool 1.0 mg kg 0.5 0.0 WT-2 WT-3 WT-5 WT-6 TM-1 TM-9 TM-13 TM-24 TM-29 TM-31

(b) 20 E-8-hydroxy- 18 linalool 16 Z-8-hydroxy- 14 linalool 12 E-8-hydroxy-6,7- dihydro-linalool 10 fresh weight nerolidol -1 8 6 linalool mg kg 4 2 0 WT-2 WT-3 WT-5 WT-6 TM-1 TM-9 TM-13 TM-24 TM-29 TM-31 Plant line

Figure 2. Concentration of linalool, nerolidol, E-8-hydroxy-6,7-dihydro-linalool, Z-8-hydroxy-linalool, and E-8-hydroxy-linalool in wild-type (WT) and transgenic potato (S. tuberosum) leaves expressing FaNES1 with a plastidic targeting signal (TM) (a). Concen- tration of the metabolites released from their glycosides by enzymatic hydrolysis (b). Detection limit was 0.01 mg kg)1 with phenol as internal standard and assuming a response factor of 1. 53

(MDGC-MS). The results showed that wild-type analyzed in more detail. Wild-type leaves already potato leaves contain linalool with an average produce linalool and E-8-hydroxylinalool (Fig- enantiomeric ratio of 98:2 (R:S) while in trans- ure 2a). However, in leaves of FaNES1 transgenic genic potato plants overexpressing FaNES1 the plants, levels of linalool and E-8-hydroxylinalool enantiomeric ratio of linalool is completely were considerably higher than in the wild-type the opposite with an average ratio of 8:92 (R:S) leaves and Z-8-hydroxylinalool as well as E-8-hy- (Figure 3). Transgenic potato plants emitting high droxy-6,7-dihydrolinalool were also detected levels of linalool had a very distinct smell com- (Figure 2a). Glycosidically bound terpenoids were pared to wild-type plants. While leaves of wild- determined after enzymatic hydrolysis of glyco- type plants had the typical green odor, leaves of sides (Figure 2b). GC-MS analysis of liberated transgenic lines had a sweet, flowery, citrus fruity alcohols showed that glycosylated linalool and impression which is characteristic of S-linalool. E-8-hydroxylinalool were present in wild-type Volatiles and glycosidically bound terpenoids plants but that their levels were considerably produced by young potato leaf tissue were also higher in leaves of transgenic plants. Transgenic

Figure 3. Enantiomeric distribution of linalool in transgenic potato plants compared to wild-type plants. Enantio-selective analysis of endogenous linalool in wild-type potato leaves and linalool extracted from leaves of transgenic potato plants expressing the FaNES1 gene. Analysis was conducted by using Multidimensional Gas Chromatography-Mass Spectrometry (MDGC-MS; for methods see Aharoni et al., 2004). 54 plants also produced glycosylated E-8-hydroxy- very recent reports described the formation of 6,7-dihydrolinalool and Z-8-hydroxylinalool, higher levels of sesquiterpenes in transgenic which were not detected in wild-type plants. Arabidopsis plants. Kappers et al. (2005) could HPLC-MS analysis confirmed the presence of generate higher levels of the sesquiterpene nerol- linalyl-b-D-glucopyranoside in wild-type and idol by altering the localization of the strawberry transgenic potato leaves and we assume that the FaNES1 protein to the mitochondria. Emission of carbohydrate moiety in the glycosylated 8-hydroxy 4,8-dimethyl-1,3(E),7-nonatriene [(E)-DMNT] the linalool derivatives are also attached to the tertiary homoterpene derivative of nerolidol, which plays a hydroxyl group (Figure 4). Thus, the glycosylation crucial role in insect predator attraction in tri- patterns of identical linalool derivatives are trophic interactions (Bouwmeester et al., 1999) different in potato and Arabidopsis. was also detected in the same transgenic plants. Also in transgenic Arabidopsis, Degenhardt et al. Attempts to engineer sesquiterpenes in Arabidopsis (2005) overexpressed the maize TPS8 and TPS10 genes and could generate a range of sesquiterpenes Transgenic Arabidopsis plants overexpressing the in planta. FaNES1 gene emitted also the sesquiterpene ner- olidol, albeit at much lower levels (levels of ner- Cost of engineering terpenoids in plants olidol emitted were 100- to 300-fold lower than of and biological activity of transgenics linalool) (Aharoni et al., 2003). As sesquiterpenes are known to be produced in the cytosol, and these Up to now most experiments aiming at engineer- transgenic lines produced the FaNES1 protein in ing terpenoids in plants utilized the constitutive the plastids, this observation indicates that FDP is CaMV35S promoter for driving the expression of also present in the plastids. In another attempt to a gene of interest. Although most of these plants engineer sesquiterpenes, Arabidopsis plants pro- produced and emitted the expected terpenoid (in ducing a cytosolic germacrene A synthase from the case of monoterpenes) it was not without chicory were generated. GC-MS analysis of vola- paying a price in terms of plant vitality. In most tiles emitted from transgenic vs. control plants did cases, if transgenic plants were affected in their show the presence of the thermal rearrangement growth, the strength of the phenotype correlated product of germacrene A, b-elemene, but only in with the production level of the new terpenoid. trace amounts (Aharoni et al., 2003). Thus, engi- For example, Arabidopsis plants expressing neering sesquiterpene production in plants is more FaNES1 were retarded in growth compared to difficult as compared to the engineering of mon- their wild-type counterparts (Aharoni et al., 2003). oterpenoids, most probably due to shortage in the An even stronger phenotype was observed in sesquiterpene precursor pool. Future work on potato plants when expressing the FaNES1 gene metabolic engineering of sesquiterpene production under the Rubisco small subunit promoter from in plants might prove to be more successful as two chrysanthemum (Figure 5). This promoter is

Figure 4. Structures of linalool and derivatives formed in transgenic potato plants over-expressing the strawberry FaNES1 gene. 55

Figure 5. Phenotypic effect in potato plants with very strong expression of the strawberry FaNES1 gene under the control of the chrysanthemum rubisco small subunit promoter. Upon transfer from in vitro to the greenhouse leaves of plants with the highest levels of expression turned white and were retarded in growth.

approximately 10-fold stronger and transgenic transgenic plants have higher aphidicidal activity, potato lines with such high levels of linalool pro- in vivo assays with these plants showed greatly duction were strongly reduced in growth, but also reduced aphid colonisation. Aphid behaviour was showed heavy bleaching in leaves upon transfer also altered in transgenic Arabidopsis plants from in vitro to the greenhouse (Figure 5). Again, producing linalool (Aharoni et al., 2003). In plants with the highest levels of linalool showed dual-choice assays with Myzus persicae, transgenic the most severe phenotype. It is still not clear lines producing linalool significantly repelled the whether these effects are due to terpene toxicity or aphids. Similar results were recently obtained with the reduction in the availability of precursors for transgenic chrysanthemum (Chrysanthemum  isoprenoid primary metabolite pathways such as grandiflorum) and the western flower thrips, chlorophyll and carotenoid biosynthesis. Frankliniella occidentalis (M.A. Jongsma, 2004. The biological effects and possible practical Novel genes for control and deterrence of sucking application of these compositional changes in the insect pests. http://www.isb.vt.edu/news/2004/ terpenoid profiles of engineered plants have so far Nov04.pdf). The transgenic Arabidopsis plants hardly been tested. The first indications of the engineered to emit (3S))E-nerolidol and (E)- possibility of altering insect behaviour were DMNT, as described above, attracted carnivorous provided by transgenic tobacco plants producing predatory mites (Phytoseiulus persimilis), the higher levels of the diterpene cembratriene-ol natural enemies of spider mites (Kappers et al., (Wang et al., 2001). Not only did exudates from 2005). 56

Concluding remarks erpenes in the other), were shown to lack a plastidic targeting signal. This was identified by in silico The recent reports on metabolic engineering of analysis as well as by testing the targeting capacity terpenoids in plants have clearly shown that these of the N-termini of these proteins in vivo (Aharoni type of approaches could be used successfully to et al., 2004). These two proteins were active in two generate substantial levels of terpenoids. Engi- different strawberry species, wild (Fragaria vesca) neering of some classes of terpenoids is apparently and cultivated (Fragaria  ananassa). Thus, intra- more difficult than others as the pool of terpenoid cellular biosynthesis of the different terpene classes precursors may not be sufficient for the production and the presence of their precursors is not as strict of substantial amounts of the required terpenoid. as believed earlier when the MEP pathway was In the case of monoterpenes, the availability of discovered. GDP is apparently high in both leaf tissues (as we Engineering experiments to alter terpenoid observed in both potato and Arabidopsis and biosynthesis can take advantage of these two others in additional plant species such as tobacco points (intracellular transport and biosynthesis of and mint) as well as in fruit (Lewinson et al., precursors) by targeting proteins to different sub- 2001), tuber (Aharoni and Jongsma, unpublished) cellular compartments. For example, Arabidopsis and flower tissues (Lucker et al., 2004b). Newly plants targeting the FaNES1 protein to the plast- introduced monoterpene skeletons, but most likely ids unexpectedly also produced low levels of the also those of other terpene classes, will be trans- sesquiterpene nerolidol (Aharoni et al., 2003). formed by endogenous modifying enzymes such as Targeting of the same protein to the mitochondria cytochrome P450s, reductases and glycosyl trans- resulted in even higher level of nerolidol produc- ferases. Interestingly, in Arabidopsis and potato tion (Kappers et al., 2005). Ohara et al. (2003) we detected the same 8-hydroxy derivatives targeted the limonene synthase (LS) cDNA of (E-8-hydroxy linalool, Z-8-hydroxy linalool and Perilla frutescens to the plastid, the cytosol and the E-8-hydroxy 6,7-dihydrolinalool) but their glyco- endoplasmic reticulum (ER) in transgenic tobacco. sylation pattern was different. In both plant Limonene formation was detected in leaf extracts species E-8-hydroxy linalool was also present at of both plastid- and cytosol-localized limonene low levels in the wild-type plants, and, thus, synthase transgenic plants but not in the ER. The endogenous enzymes already active in the wild- amount of limonene in plastid-localized LS trans- type plants could act on the introduced monoter- genic plants was more than three times higher pene and produce substantially higher levels of this compared to transgenic plants with cytosol local- particular derivative. ized LS. Thus, targeting different cell compart- Engineering of sesquiterpene production in the ments when engineering terpenoids might be a cytosol compared to the plastidic production of valuable tool for obtaining higher levels of terpe- monoterpenes seems more difficult. At this point it noids and producing novel ones. In addition, fur- is not clear why only such a small amount of FDP, ther modification of the introduced terpenoid the sesquiterpene precursor is available although it might be different in each cell compartment. was earlier speculated that this is due to tight Since the isoprenoid pathway in both the metabolic regulation directing precursors to sterol plastid and the cytosol supplies precursor not only biosynthesis (Chappell et al., 1995). In recent years for mono- and sesquiterpene biosynthesis but also several studies showed that transport of terpenoid for an array of other compounds (some of them precursors occurs between subcellular compart- crucial to plant growth and fitness such as sterols, ments (McCaskill and Croteau, 1998; Bick and gibberellins, carotenoids and chlorophyll), strong Lange, 2003; Hemmerlin et al., 2003; Laule et al., and constitutive expression of introduced genes 2003; Schuhr et al., 2003; Dudareva et al., 2005). could be most harmful. It is therefore highly rec- We discovered, however, that in ripe strawberry ommended in future metabolic engineering exper- fruit monoterpene synthase proteins are localized iments to direct gene expression to a specific tissue to the cytosol and not as expected to the plastids. or organ or use an inducible system. One example Two independent strawberry monoterpene syn- for an inducible approach was recently reported thase proteins, which generate unrelated products for the production of the diterpene taxadiene, (linalool in one case and several olefinic monot- which was engineered using the glucocorticoid 57

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