WO2010106318A1

WO2010106318A1 - Screening plant glycosyltransferases for glycosylation of terpenoids in planta

Info

Publication number: WO2010106318A1
Application number: PCT/GB2010/000477
Authority: WO
Inventors: Kiat Lim Eng; Dianna Joy Bowles
Original assignee: The University Of York
Priority date: 2009-03-18
Filing date: 2010-03-17
Publication date: 2010-09-23
Also published as: GB0904639D0

Abstract

This disclosure provides a novel means to rapidly assay the utility of different GTs to optimise yields of specific small molecule products (e.g. terpenoids), in planta by means of agroinf iltration. The GTs can be used seperately and in combination with a diverse array of additional biosynthetic enzymes. We disclose that transient expression in plant leaves of a terpene synthase leads to elevated levels of the respective terpenoid product. By combining expression of the terpene synthase with that of a GT known to glucosylate terpenoid leads to a massive increase in terpenoid glucoside. This has utility in rapid screening to bypass need for plant transformation. Combinations of GTs and terpene synthases can be compared with respect to the production of selected terpenoids. The combinations of glycosyltransf erase and terpene synthase can be combined with additional enzyme activities to give still further versatility to the assay.

Description

SCREENING PLANT GLYCOSYLTRANSFERASES FOR GLYCOSYLATION OF TERPENOIDS IN PLANTA

The invention relates to an assay to monitor the glycosylation of terpenoids.

Terpenoids, also called isoprenoids, are products derived from a five carbon isoprene unit and have diverse activities. They are also used to flavour and/or scent a variety of commercial products. They also include pharmaceuticals e.g. taxol, artemisinin. Terpenoids are classified with reference to the number of isoprene units that comprise the particular terpenoid. For example a monoterpenoid comprises two isoprene units; a sesquiterpenoid comprises three isoprene units and a di-terpenoid four isoprene units. Polyterpnoids comprise multiple isoprene units. There are many thousands of examples of terpenoids. Artemisinin is a sesquiterpene lactone endoperoxide and is a natural product produced by the plant Artemesia annua. Artemisinin is typically used in combination with anti-malarial therapeutics, for example lumefantrine, mefloquine, amodiaquine, sulfadoxine, chloroquine, in artemisinin combination therapies (ACT). Endoperoxides like artemisinin, for example dihydroartemisinin, artemether, and sodium artesunate have been used in the treatment of malaria. Examples of monoterpenoids include linalool, citronellol, menthol, geraniol and terpineol. Linalool and citronellol are used as a scent in soap, detergents, shampoo and lotions. Linalool is also an intermediate in the synthesis of vitamin E. Menthol is isolated from peppermint or other mint oils and is known for its anaesthetic properties; it is often included sore throat medications and oral medications e.g. for the treatment of bad breath in toothpaste and mouth wash. Geraniol is known for its insect repellent properties and and is also used as a scent in perfumes. Terpineol is also used as an ingredient in perfumes and cosmetics and as flavouring. It is apparent that in addition to the pharmaceutical applications of monoterpenoids such as perillyl alcohol there are additional uses as scents, flavourings and as insect deterrents.

The synthesis of terpenoids involves a large number of enzymes with different activities. For example isoprene units are synthesized from monosaturated isoprene units by prenyltransferases into multples of 2, 3 or 4 isoprene units. These molecules serve as substrates for terpene synthase enzymes [also called "terpene cyclase"]. Plant terpene synthases are known in the art. For example WO00/17327 describes terpene synthases and characterisation of the synthase active site. WO2006/133013 discloses a method to generate variant terpene synthases and also variants with altered substrate specificity using a directed evolution technique. WO2007/024718 describes genetically modified eukaryotic cells expressing enzymes of the mevalonate pathway in particular phenyltransferases and squalene synthase enzymes. In WO2007/009958, two terpene synthases are characterized from Origanum vulgare [oregano] and their use in the improvement of terpenoid synthesis in the production of essential oils in transgenic plants. WO2008/039499 discloses genetically modified cells with a large number of terpene synthase nucleic acids encoding terpene synthases with different substrate specificities.

Glycosyltransferases (GTases) are enzymes that post-translationally transfer glycosyl residues from an activated nucleotide sugar to monomeric and polymeric acceptor molecules such as other sugars, proteins, lipids and other organic substrates. These glucosylated molecules take part in diverse metabolic pathways and processes. The transfer of a glucosyl moiety can alter the acceptor's bioactivity, solubility and transport properties within the cell and throughout the plant. One family of GTases in higher plants is defined by the presence of a C-terminal consensus sequence. The GTases of this family function in the cytosol of plant cells and catalyse the transfer of glucose to small molecular weight substrates, such as phenylpropanoid derivatives, coumarins, flavonoids, other secondary metabolites and molecules known to act as plant hormones. Available evidence indicates that GTases enzymes can be highly specific.

A plant platform for the production of terpenoids, such as those used in flavours and fragrances, depends on a mechanism to ensure high yield and stability of the desired product. Glucosylation of a small molecule through the overexpression of a glucosyltransferase (GT) gene in a transgenic plant and the consequent increased enzyme activity is known to lead to high levels of the small molecule glucoside in plant tissue. Release from the glucoside is straight forward post-harvest, through the action of glucosidases, enabling the increased yield of desired product to be easily recovered.

In the context of volatile terpenoids, it is also known that their glucosylation increases their stability and prevents their loss from the plant as volatiles. In addition, it is possible that combining the overexpression of a GT gene with a gene encoding a specific terpene synthase could further enhance the yield of a specific terpene glucoside. However whilst the use of GTs potentially provides a valuable means for increasing yield and stability of a plant production platform, there are currently considerable time constraints involved to assay the effectiveness of different GTs in transformed plants. This disclosure provides a novel means to rapidly assay the utility of different GTs to optimise yields of specific small molecule products [e.g. terpenoids], in planta. The GTs can be used separately and in combination with a diverse array of additional biosynthetic enzymes. We disclose that transient expression in plant leaves of a terpene synthase leads to elevated levels of the respective terpenoid product. By combining expression of the terpene synthase with that of a GT known to glucosylate terpenoid leads to a massive increase in terpenoid glucoside. This has utility in rapid screening to bypass need for plant transformation. Combinations of GTs and terpene synthases can be compared with respect to the production of selected terpenoids. The combinations of glycosyltransferase and terpene synthase can be combined with additional enzyme activities to give still further versatility to the assay. The assay is an alternative to microbial fermentation and is inexpensive relative to fermentation with equivalent yields.

We provide a new high throughput combinatorial method in planta to evaluate the impact of GTs on yield of terpenoids and/or for the evaluation of GTs to increase yields of specific small molecules.

According to an aspect of the invention there is provided at least one bacterial cell wherein said cell is transformed with at least one nucleic acid molecule that encodes a terpene synthase and a glycosyltransferase for use in the glycosylation of at least one plant terpenoid.

In a preferred embodiment of the invention said bacterial cell[s] are used to transform a plant tissue and to in planta modify said terpenoid.

In a preferred embodiment of the invention said bacterial cell is of the genus Agrobacterium spp; preferably A.tumefaciens.

In a preferred embodiment of the invention said terpene synthase is encoded by a nucleic acid molecule as represented in Figure 1 , or a nucleic acid molecule that hybridizes under stringent hybridization and encodes a polypeptide with terpene synthase activity.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

In a further preferred embodiment of the invention said glycosyltransferase is a glucosyltransferase.

Glucosyltranferases are known in the art; for example see WO2004/106508 [US 10/558,220]; WO2008/065370 [US12/517,109] and WO2008/062165 [US12/515,940] the GT sequences of which are specifically incorporated by reference.

According to an aspect of the invention there is a provided a method to glucosylate a terpenoid wherein said method comprises: i) forming a preparation comprising at least first and second bacterial cells wherein one cell is transformed with a nucleic acid molecule that encodes a terpene synthase polypeptide and a second cell which is transformed with a nucleic acid molecule that encodes a glycosyltransferase polypeptide wherein said nucleic acid molecules are adapted for expression of said polypeptides, preferably over-expression of said polypeptides; ii) contacting plant tissue with said preparation to allow transient expression of said terpene synthase and said glycosyltransferase; and optionally iii) monitoring the accumulation of glucosylated terpenoids in said plant tissue.

In a preferred embodiment of the invention said plant is selected from the group consisting of: In a preferred embodiment of the invention said plant is selected from: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals e.g. rose, geranium.

Preferably, plants of the present invention are crop plants for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea, and other root, tuber or seed crops and including peppermint and spearmint.

Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, cajuput, pine, petitgrain and sorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, celery and cauliflower, and carnations and geraniums. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, cherry, sunflower, tomato, pepper, and chrysanthemum, coriander. Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava been, lentils, chick pea.

In a preferred embodiment of the invention said plant is of the genus Artemesia; preferably Artemesia annua.

In a preferred embodiment of the invention said plant is selected from the group consisting of; Artemisia annua, A.serrata, A.ludovicana, A.suksdorfii, A. tillisii, A, verlotiorum, A.vulgaris, A.arborescens, A. absinthium, A. canareinsis, A. granatensis, A.spendens, A. umbelliformis, A.lucentica, A. reptans, A. rupestήs, A.filifolia, A.californica, A. abrotanum, A. molineiri, A. chamaemelifolia, A. afra, A. tournefortiana, A. campestris, A. crithimifolia, A. scoparia, A.monosperma, A. dracunculus.

In an alternative preferred method of the invention said plant is of the genus Seriphidium spp.

In a preferred embodiment of the invention said plant is selected from the group consisting of: Seriphidium palmeri, S. arbusculum, S. canum, S. novum, S.tripartitum, S. longilobum, S. rothrockii, S.tridentatum, S. pygmaeam, S. bigelowii, S. rigidum, S. araxinum, S.fragans, S.barrelieri, S. caerulescens, S. sublessingianum, S.herba-album, S. seiberi, S. incultum.

In an alternative embodiment of the invention said plant is selected from the group consisting of: Sphaeromeria diversifolia, Neopallasia pectinata, Crossostephuim chinense, Filifolium sibericum.

In an alternative preferred embodiment said plant is Nicotiana benthamiana.

According to an aspect of the invention there is provided a transgenic plant wherein the genome of said plant is transfected with a nucleic acid that encodes a terpene synthase polypeptide and a nucleic acid molecule that encodes a glucosyltransferase polypeptide. In a preferred embodiment of the invention said nucleic acid moleculefs] are adapted to over express said polypeptide^].

In a preferred embodiment of the invention said nucleic acid molecule as hereindisclosed is part of a vector and is operably linked to a promoter element.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter. In a preferred aspect, the promoter is an inducible promoter or a developmentally regulated promoter. Of particular of interest in the present context are nucleic acid constructs which operate as plant vectors. For example those described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148. Suitable vectors may include plant viral-derived vectors (see e.g. EP194809). By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-Ia promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) MoI. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792- 803; Hansen et al. (1997) MoI. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341 ; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant MoI. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20): 9586- 9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.

Vectors may also include a selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following materials, methods and figures: Figure 1 is analysis of geranyl linalool glucoside production in plants infiltrated with geranyl linalool synthase and glycosyltransferase;

Figure 2 is the nucleotide sequence of geranyl linalool synthase from Arabidopsis; Figure 3 is the nucleotide sequence of glucosyltransferase of GT 73C5;

Figure 4 is the nucleotide sequence of glucosyltransferase GT 76E11 ;

Figure 5 is the amino acid sequence of geranyl linalool synthase;

Figure 6 is the amino acid sequence of glucosyltransferase of GT 73C5; and Figure 7 is the amino acid sequence of glucosyltransferase GT 76E11.

Materials and Methods

DNA constructs

The cDNA of geranyl linalool synthase was amplified from arabidopsis genomic DNA by PCR using the primers listed in Table 1. The cDNA was cloned into pBin61 binary vector using the BamH I site.

Two arabidopsis glycosyltransferases were selected in this work. GTs 73C5 and 76E11 have previously been cloned in our laboratory. The cDNAs corresponding to these two genes were amplified by PCR and ligated into the BamH I site of the vector pBin61.

Transient expression in tabocco For the agrobacterium-mediated transient expression in tobacco, the binary vector containing either the geranyl linalool synthase cDNA, the glycosyltransferase GT 73C5 or GT 76E11 was transferred into Agrobacterium tumefaciens by electroporation. The agrobacterium cultures were grown to OD₆O₀ 0.6. The cultures were then washed by the infiltration buffer (50 mM MES pH 5.6, 2 mM Na₃PO₄, 0.5% glucose [w/v], 100 DM acetosyringone). The agrobacterium culture containing the geranyl linalool synthase cDNA was either diluted to OD₆₀₀ 0.3 using the infiltration buffer, or mixed with the culture containing GT 73C5 or GT 76E11 at 1 :1 ratio. The cultures were injected into young tobacco (Nicotiana benthamiana) leaves. The leaves were harvested 48 h after agrobacterium infiltration for metabolite extraction. Metabolite extraction

The infiltrated leaves were harvested, weighted and frozen in liquid nitrogen. The leaves (1 g) were incubated for 1 h at room temperature with 80% methanol. After filtration through glass wool, the supernatant was collected and concentrated to approximately 1 ml on a rotary evaporator. The samples were cleaned up on an activated Strata-X solid phase extraction column (Phenomenex, UK). The elution was carried out with 1 ml methanol. An aliquote of 50 μl was used for LC-MS analysis.

HPLC-MS analysis of glucoside The analyses of the extracts were performed with a SpectraSYSTEM HPLC system (ThermoQuest) coupled with a LCQ ion trap mass spectrometer equipped with an APCI source (Finnigan MAT). The chromatographic separation was performed using a Columbus 5μ C18 (150'4.6 mm, Phenomenex) column, at a flow rate of 0.5 ml/min with a linear gradient of solvent A (methanol, 10-50%) against solvent B (10 mM ammonium acetate) over 10 min, followed by a linear gradient A (50-100%) against B over 20 min. The column was then washed with A (100%) for 5 min and re-equilibrated for 5 min. The mass spectrometry analysis was performed in positive mode (source voltage, 5.09 kV; source temperature, 450°C; nebulizing sheath gas flow rate 63.48; auxiliary gas flow rate 29.27; capillary voltage 10.24 V; capillary temperature 514.6⁰C). The instrument was operated at Unit resolution in full scan MS-MS mode, scanning the product ion spectrum from m/z 50-800. The LCQ was interfaced to a computer workstation running Xcalibur 2.0 software.

Before injection the samples were centrifuged at 13000 rpm for 5 min and the supernatants filtered using 0.2 mm PET filters. 30 ml of sample were injected each time.

Example

An arabidopsis geranyl linalool synthase was cloned into the binary vector pBin61. The construct was then transferred into Agrobacterium tumefaciens. The agrobacterium carrying the bionary vector was injected into tobacco (Nicotiana benthamiana) leaves. In a separate experiment, the tobacco leaves were infiltrated with a mixture of agrobacterium consisting of a bacterial culture carrying the geranyl linalool synthase and a bacterial culture carrying glycosyltransferases. The infiltrated leaves were detached from the plants two days later for metabolite analysis. This is illustrated in Figure 1.

Claims

1 Use of at least one bacterial cell wherein said cell is transformed with at least one nucleic acid molecule that encodes a terpene synthase and a glycosyltransferase in the glycosylation of at least one plant terpenoid in planta.

2. The use according to claim 1 wherein said at least one bacterial cell is transformed with a nucleic acid molecule encoding a terpene synthase and a second bacterial cell is transformed with a nucleic acid molecule that encodes a glycosyltransferase.

3. The use according to claim 1 or 2 wherein said terpene synthase is encoded by a nucleotide sequence as represented in Figure 2, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleotide sequence in Figure 1 and encodes a terpene synthase.

4. The use according to claim 3 wherein said terpene synthase is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 2.

5. The use according to any of claims 1-4 wherein said glycosyltransdferase is encoded by a nucleotide sequence as represented in Figure 3, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleotide sequence in Figure 3 and encodes a glycosyltransferase.

6. The use according to any of claims 1-4 wherein said glycosyltransdferase is encoded by a nucleotide sequence as represented in Figure 4, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleotide sequence in Figure 4 and encodes a glycosyltransferase.

7. A method to glucosylate a terpenoid in planta wherein said method comprises: i) forming a preparation comprising at least first and second bacterial cells wherein one cell is transformed with a nucleic acid molecule that encodes a terpene synthase polypeptide and a second cell which is transformed with a nucleic acid molecule that encodes a glycosyltransferase polypeptide wherein said nucleic acid molecules are adapted for expression of said polypeptides, ii) contacting plant tissue with said preparation to allow transient expression of said terpene synthase and said glycosyltransferase; and optionally iii) monitoring the accumulation of glycosylated terpenoids in said plant tissue.

8. The method according to claim 7 wherein said terpene synthase is encoded by a nucleotide sequence as represented in Figure 2, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleotide sequence in Figure 2 and encodes a terpene synthase

9. The method according to claim 8 wherein said terpene synthase is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 2.

10. A transgenic plant wherein the genome of said plant is transfected with a nucleic acid that encodes a terpene synthase polypeptide and a nucleic acid molecule that encodes a glucosyltransferase polypeptide.

11. A plant according to claim 10 wherein said nucleic acid molecule[s] are adapted to over express said polypeptidefs].

12. A bacterial cell wherein said cell is transformed with at least one nucleic acid molecule that encodes a terpene synthase and a glycosyltransferase for use in the glycosylation of at least one plant terpenoid.

13. A cell according to claim 12 wherein said bacterial cell is of the genus Agrobacterium spp; preferably A.tumefaciens.

14. A cell according to claim 12 or 13 wherein said terpene synthase is encoded by a nucleic acid molecule as represented in Figure 1 , or a nucleic acid molecule that hybridizes under stringent hybridization and encodes a polypeptide with terpene synthase activity.

15. A cell according to any of claims 12-14 wherein said glycosyltransdferase is encoded by a nucleotide sequence as represented in Figure 3, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleotide sequence in Figure 3 and encodes a glycosyltransferase.

16. A cell according to any of claims 12-14 wherein said glycosyltransdferase is encoded by a nucleotide sequence as represented in Figure 4, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleotide sequence in Figure 4 and encodes a glycosyltransferase.