WO2003046163A2

WO2003046163A2 - Multifunctional caffeic acid o-methyltransferase

Info

Publication number: WO2003046163A2
Application number: PCT/EP2002/013320
Authority: WO
Inventors: Wilfried Schwab; Ralf Kaldenhoff; Martina Wein
Original assignee: Bayerische Julius-Maximilians-Universität Würzburg
Priority date: 2001-11-26
Filing date: 2002-11-26
Publication date: 2003-06-05
Also published as: AU2002356735A1; AU2002356735A8; WO2003046163A3

Abstract

The present invention is related to an isolated polypeptide capable of methylating an ortho-dihydroxy substituted ring system, whereby the ring system preferably is an aromatic, or heteroaromatic ring system or furanone based ring, and mimetics of said ortho-dihydroxy substituted ring system, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 1 to 365 for the mature polypeptide of SEQ ID NO. 2; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) the open reading frame coding for SEQ ID No 2 within the nucleotide sequence SEQ ID No. 1, (ii) a subsequence of (i) of at least 100 nucleotides, preferably of at least 100 consecutive nucleotides, or (iii) a complementary strand of (i), or (ii); (c) a polypeptide which is encoded by a nucleic acid having a nucleic acid sequence according to SEQ. ID. No. 1 or SEQ. ID. No. 3; (d) an allelic variant of (a), (b), or (c) and (e) a fragment of (a), (b), (c) or (d) that has methyltransferase activity using as a substrate an ortho-dihydroxy substituted ring system or a mimetic thereof.

Description

Multifunctional Caffeic Acid O-methyltransferase

Field of the invention

The invention relates to the field of enzymes useful for the synthesis of naturally occurring substances. More specifically, the invention relates to certain O-methyltransferases and their use in the synthesis of aroma compounds.

Background of the invention

Natural compounds are of great interest for the food industry. Consequentially, the composition of flavor of natural fruits and vegetables is a subject of ongoing research. Among the most important flavors are strawberry and vanilla flavor.

Because of their attracting flavor, strawberries are widely used and their qualitative composition have been intensively studied. Strawberry flavor consists of a variety of volatile compounds, and up to now more than 360 substances have been identified. Not all volatile compounds emitted by strawberries are likely to contribute significantly to the flavor, as their organoleptic properties are different. For the food industry, the „aroma value" concept is of special importance, i.e., the ratio of concentration to odor threshold. Studies conducted with Strawberry fruits suggest that 2,5- dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 2,5-dimethyl-4-methoxy-3(2H)-furanone (mesifurane, methoxyDMHF, DMMF) are likely to contribute significantly to strawberry flavor. Of these, DMHF is most important due to its high concentration (up to 100 mg/kg strawberry fruit fresh weight) and low odor threshold (10 ppb). DMHF is also found in many other fruits together with its methylated derivative DMMF (Roscher et al., J. Agric. Food Chem, 45, 3202- 3205, 1997). DMHF was first isolated by Rodin et al. (J. Food Sci., 30, 280-285, 1965) from pineapples and in the same year by Willhalm et al. (Chem. Ind. (London) 38, 1629-1630, 1965) from strawberries. The methyl ether of DMHF (DMMF), first reported by Willhalm et al. (Chem. Ind. (London) 38, 1629-1630, 1965), has also been identified as an aroma component of different fruits such as overripe strawberries, mango fruits and arctic brambles (Roscher et al., J. Agric. Food Chem, 45, 3202-3205, 1997). Until now, the furanones have only been isolated from fruits but they have not been detected in roots, stems, leaves, flowers or other plant parts, see the above article by Roscher et al. The β-glucopyranoside of DMHF was identified as natural ingredient of strawberry and tomato fruits (Mayerl et al., Phytochemistry 28, 631-633, 1989) and the malonylated derivative of DMHF (Roscher et al., Phytochemistry 43, 155-160, 1996) was detected in strawberries. Their content in strawberries varies remarkably in the different cultivars and varieties. Enantiomeric analyses showed that DMHF and DMMF occur as racemates in the different fruits (Bruche et al., Z. Lebensm. Unters. Forsch. 201, 249-252, 1991). Zabetakis and Holden suggested in J Sci Food Agric, 74, 421-434, 1997, that the total amount and the ratio of DMHF and DMMF determines the different taste of wild strawberries in contrast to cultivated ones.

The synthesis of DMHF has not been elucidated clearly. Bruche et al., in Z. Lebensm. Unters. Forsch. 201, 249-252, 1995, determined the [¹³C]/[¹²C] ratio of naturally occurring DMHF and DMMF by isotope ratio mass spectrometry. The 6¹³C_PDB values of DMMF were constantly 9%o lower than those recorded for DMHF indicating a higher natural abundance of ¹²C in DMMF. A depletion in the methyl groups originating from the S-adenosyl-L-methionin (SAM) pool (5¹³CpDB < = -39%o ) has already been described for natural purine alkaloids (Weilacher et al., Phytochemistry, 41, 1073-1077, 1996) but this value does not account for the difference observed with DMHF and DMMF. Consequently, in the above-mentioned paper of Bruce et al. it is suggested two independent biogenetic pathways leading to DMMF and DMHF. However, the quantification of DMHF and DMMF during fruit ripening indicated a rapid conversion of DMHF into DMMF as well as DMHF-glucoside (Perez et al. (in Agric. Food Chem. 44, 3620-3624, 1996) and by in vivo feeding experiments the above-mentioned paper of Roscher et al, 1997, showed the incorporation of the ¹⁴C-label into DMMF after the application of S-[methyl-¹⁴C]- adenosyl-L-methionine (¹⁴C-SAM) and ¹⁴C-DMHF, respectively. The data supported the hypothesis that SAM is the natural source of the methyl group in the 4-methoxy compound DMMF.

Enzymatic O-methylation is catalyzed by O-methyltransferases (OMTs) [EC 2.1.1.x], and involves the transfer of the methyl group of SAM to the hydroxyl group of an acceptor molecule, with the formation of its methyl ether derivative and S-adenosyl-L-homocysteine as products. Before the present invention was made, no OMT capable of catalyzing 4-O-methylation of DMHF was known. Although the chemical mechanism of methyl transfer reactions are identical, OMTs differ in their selectivity with respect to the stereochemistry of the methyl acceptor molecules, as well as the substitution pattern of their phenolic hydroxyl groups (Gauthier et al., Arch. Biochem. Biophys. 351(2), 243-249, 1998). In spite of their particular specificity for phenolic substrates, flavonoids, alkaloids or m o-inositol, it is interesting to note that plant OMTs share high amino acid sequence similarity (Gauthier et al., Arch. Biochem. Biophys. 351(2), 243-249, 1998, Ibrahim et al., Plant Mol. Biol. 36, 1-10, 1998, Joshi and Chiang, Plant Mol Biol. 37, 663-674, 1998). To date, genes or cDNAs encoding at least ten distinct groups of SAM-OMTs that utilize SAM and a variety of substrates have been reported from higher plants. Three amino acid sequence motifs are conserved in most of these SAM-OMTs which are believed to be involved in SAM binding (see the above Joshi and Chiang and the above Gauthier et al., see also Kagan and Clarke, Arch. Biochem. Biophys. 310, 417-427, 1994). Plant OMTs accepting phenolic compounds such as o- diphenols, caffeic acid and caffeoyl-CoA as substrates have been studied intensively with regard to lignin production. However, until now no biochemical or molecular information was available concerning the methylation of 4-hydroxy-3(2H)-furanones.

Thus, until the present invention, there had been no knowledge on the mechanism responsible for the synthesis of 4-hydroxy-3(2H)-furanones in plants. The present invention for the first time provides an enzyme that is responsible for said turnover. The enzyme also catalyzes the formation of vanillin and is useful for the enhancement of flavor formation in Vanilla planifolia.

Summary of the invention

The invention provides an SAM-dependent OMT that is capable of forming 4-hydroxy-3(2H)- furanones. The invention also provides said Enzyme in isolated form, and variants and homologues thereof. The invention further provides an in vitro method for the production of said furanones. The invention also provides plants that have been modified to express such enzyme, and plants wherein expression of such enzyme as naturally occurs in said plants has been ^"modified, e.g., enhanced or diminished. Detailed description of the invention

Definitions:

DMHF, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol®)

DMMF, 2,5-dimethyl-4-methoxy-3(2H)-furanone (mesifurane, methoxyDMHF, Methoxy- furaneol).

O-methyltransferase, an enzyme capable of transforming a hydroxy group into a methoxy group by enzymatic action. In the context of this application, the enzyme is one that preferably uses S- adenosylmethionine as the source of the methyl group. The specific O-methyltransferase of the invention is characterized by being coded by a sequence substantially homologous to the coding sequence of SEQ ID No. 1 , or alternatively, by being able to use aromatic or furanone-derived dihydroxyphenolic compounds as a substrate, provided the hydroxy groups are in the ortho position. Most specifically, the O-methyltransferase of the invention is one that is capable of catalyzing the formation of DMMF from DMHF.

Allelic variant is an alternative form of a gene that can occupy a particular chromosomal locus.

It is an object of the invention to provide an enzyme capable of catalyzing the O-methylation of substrates having a ring structure wherein at least two hydroxy groups are in the ortho position. Preferred ring structures are furanones and phenols. It is a further object of the invention to provide an Enzyme capable of catalyzing the formation of 2,5-dimethyl-4-mefhoxy-3(2H)- furanone (DMMF) from 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF). A still further object of the invention is the provision of an enzyme that catalyzes the O-methylation of caffeic acid, caffeoyl-CoA, catechol, and protocatechuic aldehyde.

It is another object of the invention to provide nucleic acids encoding said enzyme. A further object of the invention is a process for the production of said enzyme. Still another object of the invention is an antibody binding to said enzyme, or to a part thereof. The invention thus provides an isolated polypeptide capable of conversion of DMHF into DMMF by O-methyltransferase activity, selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 1 to 364 for the mature polypeptide of SEQ ID NO. 2;

(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) the open reading frame coding for SEQ ID No 2 within the nucleotide sequence SEQ ID No. 1, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i), or (ii);

(c) an allelic variant of (a) or (b); and

(d) a fragment of (a), (b), or (c) that has methyltransferase activity capable of conversion of DMHF into DMMF by O-methylation,

provided that said peptide sequence is not SEQ ID No. 2 and said nucleotide sequence is not SEQ ID No. 1.

In a further aspect, the invention is related to a polypeptide capable of methylating an ortho- dihydroxy substituted ring system, whereby the ring system preferably is an aromatic, or heteroaromatic ring system or furanone based ring, and mimetics of said ortho-dihydroxy substituted ring system, selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 1 to 365 for the mature polypeptide of SEQ ID NO. 2;

(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) the open reading frame coding for SEQ ID No 2 within the nucleotide sequence SEQ ID No. 1, (ii) a subsequence of (i) of at least 100 nucleotides, preferably of at least 100 consecutive nucleotides, or (iii) a complementary strand of (i), or (ii); (c) a polypeptide which is encoded by a nucleic acid having a nucleic acid sequence according to SEQ. ED. No. 1 or SEQ. ID. No. 3;

(d) an allelic variant of (a), (b), or (c) and

(e) a fragment of (a), (b), (c) or (d) that has methyltransferase activity using as a substrate an ortho-dihydroxy substituted ring system or a mimetic thereof.

The invention further provides the said polypeptide, having an amino acid sequence which has at least 70% identity with amino acids 1 to 365 of SEQ ID NO. 2. The invention also provides the said polypeptide, having an amino acid sequence which has at least 80%, at least 90%, or at least 95 % identity with amino acids 1 to 365 of SEQ ID NO. 2.

The invention also provides the said polypeptide, which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) the open reading frame of SEQ ID NO. 1 that encodes SEQ ID No. 2, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i), or (ii).

An embodiment of the invention is an enzyme characterized by: (i) O-methylase activity, (ii) Conversion of DMHF into DMMF.

Another embodiment of the invention is the said enzyme, further characterized by: (i) A temperature optimum of about 37 degrees C, (ii) About 90% enzymatic activity at about 30 degrees C, (iii) Temperature-dependent irreversible inhibition at about 45 degrees C.

Further embodiments of the invention provides said enzyme is characterized by a pH optimum of about 8.5, and essentially no enzymatic activity at pH values significantly below about pH 6 or significantly above about pH 10. In a further embodiment of the invention, said enzyme is further characterized by having a molecular weight of about 80 kD, as measured by gel filtration, or of about 40 kD, as measured by SDS-polyacrylamide gel electrophoresis. In a still further embodiment of the invention, said enzyme is further characterized by being essentially independent of the presence of divalent metal ions, preferably with regard to the catalytic activity. Said divalent metal ions are preferably cations. Said cations are preferably selected from the group consisting of Ca, Mg, Co, Mn, Zn, and Fe ions. More preferably, the presence of said divalent metal ions in the reaction mixture inhibits the activity of said enzyme. The inhibition is preferably by a degree of about 10% with the reaction conditions preferably being 10 mM Ca, Mg, Co, Mn, Zn and/or Fe ions, pH 6-10 (optimum at pH 8.5), temperature 37°C, as also described in Wein et al., The Plant Journal 2002, 31 (6), 755-765. The concentration of said cations at which said inhibition can be observed, is preferably between about 1 mM and about 10 mM. More preferably, the divalent metal ions that inhibit the said enzyme are selected from the group consisting of Ca, Mg, Co, Mn, Zn, and Fe ions.

In a preferred embodiment, the enzyme of the invention is characterized by having an Km value for DMHF of about 0,1 mM to about 10 mM, preferably of about 0,2 mM to about 7,5 mM, more preferably from about 0,4 mM to about 5 mM. In another preferred embodiment of the invention, said enzyme is characterized by having a Km value for S-Adenosylmethionin of about 1 to about 50 μM, preferably of about 3 to about 20 μM, more preferably of about 5 to about 10 μM, and most preferably of about 7,5 μM. In a further preferred embodiment said Km value does not vary greatly with different substrates. Preferably the Km value is about 50 to about 200 μM with the substrates preferably being protocatechnic aldehyde, caffeic acid, catechol, caffeoyl CoA and mimetics thereof. More preferably, that said Km value is about 7,5 μM for the different substrates DMHF, caffeic acid and catechol.

Further preferred embodiments of the invention include that said enzyme is characterized by having a Km value for catechol of about 120 μM. Another preferred embodiment of the invention includes that said enzyme is characterized by having a Km value for caffeic acid of about 145 μM. Still another preferred embodiment of the invention includes that said enzyme is characterized by not accepting monohyxdroxylated substrates, such as coumaric acid, ferulic acid, t-anol, and coniferyl alcohol. A preferred embodiment of the invention is an O-methyltransferase capable of accepting as a substrate a compound that has two hydroxyl groups attached to a ring system and being in ortho position to each other or a mimetic thereof. This kind of compound is referred to herein as the substrate(s) or the substrate(s) of or to the enzymatic activity of the polypeptide and enzyme, respectively, of the present invention. Preferably, said ring system is an aromatic or a hetero aromatic ring system. More preferably, said ring system is a phenolic ring system or a furanone ring system. Even more preferably, said compound is selected from the group consisting of caffeic acid, caffeoyl-CoA, DMHF, catechol, and protocatechuic aldehyde. The preferred Km values for said phenolic substrated are from about 1 μM to about 10 mM, preferably from about 10 μM to about 3 mM, more preferably from about 30 μM to about 0,8 mM, and most preferably from about 66 to 150 about μM. When expressed as a recombinant enzyme, the Km value for DMHF is preferably between 30 μM and about 8 mM, more preferably between 200 μM and about 5 mM, still more preferably between 300 μM and about 1,5 mM, and most preferably about 0,44 mM although this may vary depending on the particular host cell used for the expression of said polypeptide and enzyme, respectively, going along with the particularities of the expression system used as known to the one skilled in the art. As used herein a ring system is a simple ring or a group of rings, whereby the group of rings may be fully or partially condensed rings or rings connected through a or several linker moieties. Also, as used herein, a mimetic of an ortho-dihydroxy substituted ring system is a compound or a moiety of a compound which acts as a substrate to the O-methyltransferase described herein and which preferably has an electronic configuration similar or identical to the ortho-dihydroxy substituted ring system.

The specific methylation activity of the enzyme of the invention is preferably between about 0,01 and about 100 nmol^min^'^mg^"1 , more preferably between about 0,032 and about 20 nmol*min^" *mg^"1 , still more preferably between about 0,087 and about 10 nmol^min^'^mg^"1, more preferably between about 0,1 and about 5 nmol*min^"1*mg^"1 .

In another embodiment of the invention, the specific methylation activity in relation to the substrates of the enzymatic activity of the polypeptide and enzyme, respectively, of the present invention is between about 1,5 and about 2,5 nmo^min^'^mg^"1 . In a preferred embodiment of invention, the specific activity of the said enzyme for protocatechuic aldehyde is about 2.0 nmol*min^"I*mg^''1. In another embodiment of the invention, the specific activity for caffeic acid is about 1.6 nmol^min^'^mg^"1. In yet another embodiment of the invention, the specific activity for other substrates is between about 87 and about 32 pmol* rrm-^"1*mg^"1.

The invention further provides a process for the production of a peptide having an enzyme activity according to any of the above embodiments, comprising the steps of

(i) inserting a nucleotide sequence of the invention, or the region of SEQ ID No 1 coding for SEQ ID No 2, or for an enzyme of the invention, into an expression vector comprising, in operable sequence, a promoter, and optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers,

(ii) introducing said vector into a cell,

(iii) culturing said cell under conditions allowing for expression of said peptide or enzyme to occur within said cell,

(iv) isolating said peptide or enzyme from said cell, from the supernatant, or from a cell compartment, within or without the plasma membrane thereof.

The cell is preferably derived from a bacteria, insect, fungus, animal, or plant. The fungus cell is preferably derived from yeast. When the cell is derived from an insect, the said vector is preferably a baculovirus-derived vector. The insect cell is preferably SF9.

In another embodiment of the invention, the cell is a bacterial cell. The bacterial cell is preferably derived from E. coli. The E. coli cell is preferably BL 21.

In yet another preferred embodiment, the vector encodes a second peptide so as to express a fusion protein with the enzyme of the invention. The second peptide is preferably a signal peptide or a peptide for which a specifically binding compound is known. The signal peptide preferably directs the transportation of the fusion protein into intracellular and extracellular compartments such as the periplasmatic space. Such signal peptides are, among others, described in Chloroplast transit peptides: structure, function and evolution. Bruce, B. D., Trends in Cell Biology (2000), 10(10), 440-447, or Plastids and protein targeting. McFadden, Geoffrey I. Journal of Eukaryotic Microbiology (1999), 46(4), 339-346 the disclosure of which is hereby incorporated by reference. The specifically binding compound is preferably a substrate of the enzyme, an antibody or a metal chelate resin. The substrate is preferably Glutathion. In a further preferred embodiment, the invention provides a process for the production of the enzyme of the invention wherein the vector is pGEX, wherein said second coding region codes for Glutathion-S-transferase, and wherein the process of isolating the peptide comprises the use of GST-affinity chromatography and optionally, release of the O-methyltransferase activity containing peptide of the invention by thrombin cleavage.

The invention also provides a process for obtaining an antibody against a peptide of the invention, comprising the steps of

(i) providing said peptide or enzyme, or an immunogenic part thereof, in substantially purified form, (ii) introducing said peptide or enzyme, or said immunogenic part thereof, into a non- human animal capable of producing antibodies, (iii) optionally, introducing adjuvants into said non-human animal, (iv) optionally, repeating step (ii) and optionally, step (iii), (v) isolating antibodies or cells that produce said antibodies from said non-human animal.

(i) providing said peptide or enzyme, or a part thereof, in substantially purified form, (ii) bringing said peptide or enzyme, or said part thereof, into contact with a variety of cells, each of which containing a gene for the production of an antibody, and wherein each cell is producing said antibody, (iii) selecting cells that bind to said peptide or enzyme, or part thereof, (iv) using said cells to produce said antibody.

As used herein a peptide of the invention is the same as a polypeptide of the invention.

The invention further provides an antibody against the peptide of the invention. The invention also provides a transgenic plant comprising a genetically engineered DNA sequence encoding at least one polypeptide having an O-methyltransferase activity, wherein said polypeptide is encoded by a nucleotide sequence of the invention.

The invention also provides a transgenic plant characterized by enhanced flavor and/or taste, comprising the enzyme of the invention, produced therein by recombinant means.

Preferably, the nucleotide sequence that expresses the enzyme of the invention, is a heterologous nucleotide sequence.

Further preferably, the nucleotide sequence codes for SEQ ID No. 2, or a variant, homologue or functional part thereof. Most preferably, the nucleotide sequence codes for SEQ ID No. 2.

Preferably, the plant is selected from the group consisting of strawberry, mango, arctic brambles, raspberry, tomato, cucumber, soya, sweet potato, grape, rapeseed, sugar beet, cotton, tea, sunflower, rose, chrysanthemum, sweet pepper, potato and Vanilla planifolia or the yeast Zygosaccharomyces rouxii.

The invention also provides propagating material derived from the transgenic plant according to the invention.

Further, the invention provides a process for the production of a transgenic plant with enhanced flavor and/or taste, comprising:

(i) preparing a genetically engineered DNA sequence encoding at least one polypeptide having a O-methyltransferase activity, (ii) operably linking the DNA sequence to a promoter for said DNA; (iii) transforming a transformable, regenerable plant cell to contain the operably linked

DNA sequence of step(ii), and (iv) regenerating said plant cell to produce a transgenic plant.

As used herein a regenerating plant cell is a plant cell which may be used to regenerate a plant, whereby the regenerated plant has any of the following characteristics or combinations thereof, such as improved flavor and/or taste, intensive flavor, stabilized aroma also after defrosting and/or better storage stability)

In a preferred embodiment the DNA sequence is the DNA sequence according to the present invention. In a further preferred embodiment, the polypeptide is the polypeptide according to the present invention.

Preferably, said nucleotide sequence is contained in a vector under the control of a promoter allowing its expression in said transgenic plant.

The introduction of the nucleotide of the invention into the plant is preferably carried out by transfection using the Agrobacterium system.

The invention further provides a method for enhancing flavor and/or taste in a plant comprising:

DNA sequence of step (ii), (iv) transforming a transformable, regenerable plant cell to contain the operably linked

DNA sequence of step (ii).

In a preferred embodiment the DNA sequence is a DNA sequence according to the present invention. In a further preferred embodiment the polypeptide is the polypeptide according to the present invention.

^"Thus, in one aspect, the object of the present invention is a process for the production of plants which are capable of synthesizing the enzyme of the invention by recombinant means, characterized by introducing into plant cells a DNA sequence of the invention which comprises a region coding for a protein having the enzymatic activity of an O-methyltransferase of the invention linked to DNA sequences ensuring expression in plant cells and regeneration of whole plants from the transformed cells.

In addition, the present invention relates to a process for the production of plant cells and plants which are capable of O-methylating DMHF, comprising the following process steps:

(a) producing an expression cassette having the following partial sequences:

(i) a promoter being active in plants and ensuring formation of an RNA in the respective target tissue or target cells whereby the target tissue are preferably selected from the group comprising fruits, roots, stalks, blossoms, leaves and seeds and whereby the target cells are preferably selected from the group comprising cells of the dermal tissue, ground tissue, assimilation tissue and supporting tissue; dermal tissue includes, but is not limited to, epidermis, cork, bark; ground tissue includes, but is not limited to, parenchyma; assimilation tissue includes, but is not limited to palisade parenchyma and spongy parenchyma; supporting tissue includes, but is not limited to, collenchyma and sclerenchyma; vascular or conducting tissue includes, but is not limited to, vessels, vascular bundles, xylem and phloem.

(ii) at least one DNA sequence coding for a protein having the enzymatic activity of an O- methyltransferase of the invention and being operably linked to the promotor, preferably being fused to the promoter in sense orientation;

(iii) a signal being functional in plants for the transcription termination and polyadenylation of an RNA molecule;

(b) transferring the expression cassette into plant cells; and

(c) regenerating intact whole plants from the transformed plant cells.

Particularly useful promoters are those promoters that ensure a constitutive expression of the gene in all tissues of the plants such as the 35S promoter of the cauliflower mosaic virus (CaMV) as well as those that ensure expression only in certain organs or at certain times in the development of the plant. Promoters are known that ensure a specific expression in the tubers of potato plants, such as the B33 promoter (Liu et al, 1990, Mol. Gen. Genet. 223:401-406) or those that allow a specific expression in the roots of the sugar beet, tomato fruit (Krasnyanski et al., In Vitro Cell Biol: Plant 37(4), 427-433, 2001) or strawberry fruit (Marty et al., WO 2001051637, Conner, WO 9831812). Furthermore described are DNA sequences that allow a light-dependent and tissue-specific expression of DNA sequences downstream thereof in leaves (Orozco and Ogren, 1993, Plant Mol. Biol. 23:1129-1138).

The DNA sequence mentioned in process step (a) (ii) basically can be any DNA sequence comprising a coding region coding for a protein having the enzymatic activity of the invention. This is preferably an O-methyltransferase having the substrate specificities as described above. Useful DNA sequences are particularly DNA sequences derived from strawberries, especially from Fragaria ananassa. A preferred embodiment of the process of the invention contemplates the use of DNA sequences coding for a protein having the enzymatic activity of an O- methyltransferase, with the protein exhibiting the amino acid sequence depicted in Seq D No. 2 or an amino acid sequence that is substantially identical to that. It is preferred to use DNA sequences that exhibit a high degree of homology to the DNA sequence indicated under Seq ID No. 1 and that encode an O-methyltransferase. Also DNA sequences can be used that can be derived from said sequences by substitution, insertion or deletion, as long as their enzymatic activity is not impaired. A particularly preferred embodiment of the process relates to the use of a DNA sequence that exhibits the nucleotide sequence indicated under Seq ED No. 1 or parts thereof, with the parts being long enough to encode a protein having the enzymatic activity of an O-methyltransferase.

According to the invention, the DNA sequence coding for an O-methyltransferase is linked in sense orientation to the promoter (3' end of the promoter to the 5' end of the coding sequence). This sequence can be modified before or after linkage to the transcription control elements (promoter and termination signal) in order to vary, if necessary, the properties of the polypeptide or its localization as is described infra in more detail. The DNA sequence may contain a signal peptide. Secretion is then ensured by said signal sequence. Since such preferably procaryotic signal sequences normally lead to a secretion of the protein also in plant cells, the expressed protein is transported to the apoplast of the plant when using the DNA sequence indicated under Seq ED No. 1 in conjunction with a signal sequence. In order to express the enzyme in the cytosol of the plant cells, no signal sequence should be used. If the enzyme to be expressed is to be directed to certain subcellular compartments such as chloroplasts, amyloplasts, mitochondria or the vacuole, the signal sequence effecting secretion must be chosen to be a signal sequence or a sequence coding for a transit peptide which ensures the transport of the expressed protein to the respective compartment. Such sequences are known in the art such as described by Bruce, B. D. et al. (supra) or McFadden, G. (supra). For the transport into the plastids, e.g., the transit peptides of the precursor proteins of the small subunit of the ribulose bisphosphate carboxylase (RUBISCO) from potatoes (Wolter et al., 1988, Proc. Natl. Acad. Sci. USA 85:846-850) or of the acyl carrier protein (ACP) are useful. For the transport into the vacuole, e.g., the signal sequence of patatin can be used (Sonnewald et al., 1991, Plant J. 1:95-106). The sequences used must be fused in frame to the DNA sequence coding for the enzyme.

The transfer of the expression cassette constructed in process step (a) in plant cells is preferably carried out using plasmids, for example, binary plasmids. It is preferred to use techniques that ensure that the expression cassette is stably integrated into the genome of the transformed plant cell.

The process of the invention can basically be applied to any plant species. Both monocotyledonous and dicotyledonous plants are of interest, however, especially fruit-bearing plants are of interest. Transformation techniques have already been described for various monocotyledonous and dicotyledonous plant species and are described in the art such as „Use of Ri-mediated transformation for production of transgenic plants". Christey, Mary C. Crop and Food Research, Christchurch, N. Z. Plant (2001), 37(6), 687-700; Transformation. Lopez-Meyer, Melina; Maldonado-Mendoza, Ignacio E.; Nessler, Craig L. Plant Tissue Culture Concepts and Laboratory Exercises (2nd Edition) (2000), 297-303; and Plant transformation and transgenic crops. Katsube-Tanaka, Tomoyuki; Utsumi, Shigeru. Food Science and Technology Research ^"(2000), 6(4), 241-247.) the disclosure of which is incorporated herein by reference.

The DNA sequences of the invention allow to modify plants such that they express proteins having the enzymatic activity of an O-methyltransferase of the invention, thereby allowing the synthesis or enhanced synthesis of certain compounds that contribute to the flavor and/or taste of a plant. Since such compounds that are O-methylated by the enzyme of the invention are also involved in the anthocyan pathway, also the colour of a plant may be influenced by using the methods of the invention and more particularly the steps and combination of steps disclosed in connection therewith.

Preferred subjects of the various methods of the present invention are fruit and vegetable plants such as strawberries, apples, plums, carrots or tomatoes, but also crop plants may be used, such as maize, rice, wheat, barley, sugar beet, sugar cane, tobacco, potatoes or cassava.

There are many cloning vectors available containing a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells which can be used to prepare the introduction of foreign genes into higher plants. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184, pGEX, etc. The desired sequence can be introduced into the vector at an appropriate restriction site. The plasmid obtained is used to transform E. coli cells. Transformed E. coli cells are cultivated in an appropriate medium and are then harvested and lysed. The plasmid is recovered. Methods of analysis generally used to characterize the obtained plasmid DNA are restriction analyses, gel electrophoresis, sequencing reactions and further methods known in biochemistry and molecular biology, as known to the person skilled in the art and as illustrated further below. After every manipulation the plasmid DNA can be cleaved and linked to other DNA sequences. Every plasmid DNA sequence can be cloned into the same or other plasmids. Many techniques are available for the introduction of DNA into a plant host cell. These techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, the fusion of protoplasts, injection, electroporation of DNA, introduction of DNA by the bioballistic method as well as other possible techniques. Depending on the method of introduction of the desired genes into the plant cells, further DNA sequences may be required. If, e.g., the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right border sequence, but often the right and left border sequence of the Ti and Ri plasmid T-DNA as flanking area must be linked with the genes to be introduced. If Agrobacteria are used for transformation, the DNA to be introduced must be cloned into special plasmids, either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated by homologous recombination into the Ti or Ri plasmid of the Agrobacteria due to sequences that are homologous to sequences in the T-DNA. Said plasmid contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors are not able to replicate in Agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens using a helper plasmid (conjugation). Binary vectors or shuttle vectors are able to replicate both in E. coli and in Agrobacteria. They contain a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border regions. They can be directly transformed into Agrobacteria (Holsters et al., 1978, Mol. Gen. Genet. 163:181-187). The Agrobacterium serving as host cell should contain a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA to the plant cell. Additional T-DNA may be present. The thus transformed Agrobacterium is used to transform plant cells. The use of T-DNA for the transformation of plant cells has been extensively examined and is sufficiently described in EP 120516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam, 1985, Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4:1-46 and An et al; 1985, EMBO J. 4:277-287. For the transfer of the DNA to the plant cells plant explants can expediently be cocultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g., pieces of leaves, stem segments, roots but also protoplasts or suspension- cultivated plant cells) whole plants can be regenerated on an appropriate medium which may contain antibiotics or biocides for the selection of transformed cells. The plants thus obtained can be screened for the presence of the introduced DNA. There are no specific requirements for the plasmids used for the injection and electroporation of DNA into plant cells. Simple plasmids such as pUC derivatives can be used. However, if it is intended to regenerate whole plants from the thus transformed cells, the presence of a selectable marker is required. Once the introduced DNA is integrated into the genome of the plant cell, it generally remains there stably and can also be found in the successor of the originally transformed cell. Normally it contains a selection marker which imparts to the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or gluphosinate etc. The individually selected marker should therefore allow for the selection of transformed cells over cells lacking the introduced DNA. The transformed cells grow within the cell as usual (cf., e.g., McCormick et al., 1986, Plant Cell Reports 5:81-84). These plants can be grown in the usual manner and can be cross-bred with plants possessing the same transformed genetic material or other genetic materials. The resulting hybrid individuals have the corresponding phenotypic properties. Two or more generations should be cultivated in order to make sure that the phenotypic features are stably retained and inherited. Furthermore, seeds should be harvested in order to make sure that the corresponding phenotype or other characteristics have been retained.

A further object of the invention are the modified plant cells and plants resulting from the above- mentioned process of the invention, particularly plant cells and plants containing a DNA sequence of the invention in combination with DNA sequences that allow the expression of the DNA sequence of the invention in plant cells. Said plant cells are characterized by expressing a protein of the invention having the enzymatic activity of an O-methyltransferase, thereby resulting in the synthesis of the products of the O-methylation reaction of the inventive enzyme, as described above, in the cells or the plants. The transgenic plant cells and plants are furthermore characterized in that they contain a recombinant DNA molecule stably integrated into their genome which comprises an expression cassette, said expression cassette containing a DNA sequence coding for the said O-methyltransferase.

The products of the enzymatic reaction of the enzyme of the invention, especially 2- methoxyphenol, vanillin, ferulic acid, feruoyl-CoA, and DMMF, formed in the transgenic plant cells and plants with the help of the recombinant O-methyltransferase of the invention, can be isolated from transgenic plant cells and plants in the same manner as the products which are normally formed, and which is illustrated below. They are likewise an object of the present invention.

The invention furthermore relates to the use of the DNA sequences of the invention or parts thereof for the expression of a polypeptide having O-methyltransferase activity, preferably in microorganisms having no O-methyltransferase activity of their own. In this application, microorganisms are to be understood as bacteria as well as all protists such as defined by, e.g., Schlegel "Allgemeine Mikrobiologie" (Georg Thieme Verlag, 1985, pages 1-2). Today, biotechnological research to a large extent uses microorganisms to synthesize and process the most varied substances. This has become possible by the provision of a multitude of various systems for the efficient expression of procaryotic and eucaryotic genes in microorganisms (for an overview see, e.g., Methods in Enzymology 153:385-516). Widely used are, e.g., the strains of the bacterial species Escherichia coli and Bacillus subtilis. By providing the DNA sequences of the invention, particularly the DNA sequence depicted in Seq ID No. 1, it is now possible to express a protein having O-methyltransferase activity in microorganisms for which the appropriate expression systems are available. Examples of such expression systems are provided further below.

The present invention concerns an O-methyltransferase that is capable of methylating a single hydroxy group in compounds that comprise a ring system and at least two hydroxy groups in ortho position which are also referred to herein as ortho-dihydroxy phenols, or in compounds which are substrates of or to the enzymatic activity of the polypeptide and enzyme, respectively, of the present invention. Preferably, said ring system is a furanone ring system, and further preferably, the compound is DMHF. In another embodiment of the invention, the ring system is a phenolic or diphenolic ring system, and more preferably, the compound is selected from the group comprising caffeic acid, caffeoyl CoA, catechol, and protocatechuic aldehyde. As the prototype enzyme of the invention is an O-methyltransferase (OMT) and has been isolated from Strawberry fruits, the enzyme of the invention is called herein STOMT.

The present invention also concerns the DNA sequence encoding a STOMT protein and the STOMT proteins encoded by the DNA sequences.

Moreover, the present invention further concerns the DNA sequences encoding biologically active analogs, fragments and derivatives of the STOMT protein, and the analogs, fragments and derivatives encoded thereby. The preparation of such analogs, fragments and derivatives is by standard procedure (see for example, Sambrook et al., 1989) in which in the DNA sequences encoding the STOMT protein, one or more codons may be deleted, added or substituted by another, to yield analogs having at least one amino acid residue change with respect to the native protein.

Of the above DNA sequences of the invention which encode a STOMT protein, isoform, analog, -fragment or derivative, there is also included, as an embodiment of the invention, DNA sequences capable of hybridizing with a cDNA sequence derived from the coding region of a native STOMT protein, in which such hybridization is performed under moderately stringent conditions and which hybridizable DNA sequences encode a biologically active STOMT protein. As used herein high stringency conditions comprise washing at 0.2 x SSC at 55°C or washing with 1 x SSC and subsequently with 0.1 x SSC at 55°C. This kind of stringent conditions are also those described in Wein et al. (supra). These hybridizable DNA sequences therefore include DNA sequences which have a relatively high homology either at the nucleic acid level or at the amino acid level. The homology may preferably be at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% or any integer describing a degree of homology of 65% or more to the native STOMT cDNA sequence and as such represent STOMT-like sequences which may be, for example, naturally-derived sequences encoding the various STOMT isoforms, or naturally- occurring sequences encoding proteins belonging to a group of STOMT-like sequences encoding a protein having the activity of STOMT. Further, these sequences may also, for example, include non-naturally occurring, synthetically produced sequences, that are similar to the native STOMT cDNA sequence but incorporate a number of desired modifications. Such synthetic sequences therefore include all of the possible sequences encoding analogs, fragments and derivatives of STOMT, all of which have the activity of STOMT.

To obtain the various above noted naturally occurring STOMT-like sequences, standard procedures of screening and isolation of naturally-derived DNA or RNA samples from various tissues of plants, preferably of fruits of strawberry plants, may be employed using the natural STOMT cDNA or portion thereof as probe (see for example standard procedures set forth in Sambrook et al., 1989).

Likewise, to prepare the above noted various synthetic STOMT-like sequences encoding analogs, fragments or derivatives of STOMT, a number of standard procedures may be used as are detailed herein below concerning the preparation of such analogs, fragments and derivatives.

A polypeptide or protein "substantially corresponding" to STOMT protein includes not only STOMT protein but also polypeptides or proteins that are analogs of STOMT.

Analogs that substantially correspond to STOMT protein are those polypeptides in which one or more amino acid of the STOMT protein's amino acid sequence has been replaced with another amino acid, deleted and/or inserted, provided that the resulting protein exhibits substantially the same or higher biological activity as the STOMT protein to which it corresponds. In order to substantially correspond to STOMT protein, the changes in the sequence of STOMT proteins, such as isoforms are generally relatively minor. Although the number of changes may be more than ten, preferably there are no more than ten changes, more preferably no more than five, and most preferably no more than three such changes. While any technique can be used to find potentially biologically active proteins which substantially correspond to STOMT proteins, one such technique is the use of conventional mutagenesis techniques on the DNA encoding the protein, resulting in a few modifications. The proteins expressed by such clones can then be screened for their O-methyltransferase activity, for instance by using the assays described hereinbelow, e.g., in section "Enzyme Assay" in the Materials and Methods part hereinbelow, and in Examples 2 and 3 hereinbelow. Such proteins may further be screened by their ability to induce the desired effects as described above and below, e.g., enhancing the formation of desired substances such as aroma substances, especially, vanilla, strawberry and the like aroma substances, upon expression of said clones in plant cell cultures or plants.

"Conservative" changes are those changes which would not be expected to change the activity of the protein and are usually the first to be screened as these would not be expected to substantially change the size, charge or configuration of the protein and thus would not be expected to change the biological properties thereof.

Conservative substitutions of STOMT proteins include an analog wherein at least one amino acid residue in the polypeptide has been conservatively replaced by a different amino acid. Such substitutions preferably are made in accordance with the following list as presented in Table A, which substitutions may be determined by routine experimentation to provide modified structural and functional properties of a synthesized polypeptide molecule while maintaining the biological activity characteristic of STOMT protein.

Table A

Original Exemplary

Residue Substitution

Ala Gly;Ser

Arg Lys

Asn Gln;His Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Ala;Pro

His Asn;Gln

He Leu;Val

Leu Ile;Val Lys • Arg;Gln;Glu

Met Leu;Tyr;Ile

Phe Met;Leu;Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp;Phe

Val Ile;Leu

Alternatively, another group of substitutions of STOMT protein are those in which at least one amino acid residue in the polypeptide has been removed and a different residue inserted in its place according to the following Table B. The types of substitutions which may be made in the polypeptide may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al., G.E., Principles of Protein Structure Springer- Verlag, New York, NY, 1798, and Figs. 3-9 of Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, CA 1983. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups:

TABLE B

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gin;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic nonpolar residues: Met, Leu, He, Val (Cys); and 5. Large aromatic residues: Phe, Tyr, Tip.

The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. This however tends to promote the formation of secondary structure other than a-helical. Pro, because of its unusual geometry, tightly constrains the chain and generally tends to promote β-turn-like structures, although in some cases Cys can be capable of participating in disulfide bond formation which is important in protein folding. Note that Schulz et al, supra, would merge Groups 1 and 2, above. Note also that Tyr, because of its hydrogen bonding potential, has significant kinship with Ser, and Thr, etc.

Conservative amino acid substitutions according to the present invention, e.g., as presented above, are known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or polypeptide molecule. "Characteristics" is defined in a non- inclusive manner to define both changes in secondary structure, e.g. a-helix or β-sheet, as well as changes in biological activity, e.g., O-methylation of DMHF or of a similar substrate.

Examples of production of amino acid substitutions in proteins which can be used for obtaining analogs of STOMT proteins for use in the present invention include any known method steps, such as presented in U.S. patent RE 33,653, 4,959,314, 4,588,585 and 4,737,462, to Mark et al.; 5,116,943 to Koths et al., 4,965,195 to Namen et al; 4,879,111 to Chong et al.; and 5,017,691 to Lee et al.; and lysine substituted proteins presented in U.S. patent No. 4,904,584 (Shaw et al.).

Besides conservative substitutions discussed above which would not significantly change the activity of STOMT protein, either conservative substitutions or less conservative and more random changes, which lead to an increase in biological activity of the analogs of STOMT proteins, are intended to be within the scope of the invention.

When the exact effect of the substitution or deletion is to be confirmed, one skilled in the art will appreciate that the effect of the substitution(s), deletion(s), etc., will be evaluated by routine binding and cell death assays. Screening using such a standard test does not involve undue experimentation.

Acceptable STOMT analogs are those which retain at least the capability of O-methylating an aromatic or furanone-based ring system comprising two hydroxy groups in ortho position, and thereby, as noted above, catalyze the formation of, e.g., 2-methoxyphenol, vanillin, ferulic acid, feruoyl-CoA, and/or DMMF, in plants where the unmethylated precursors of such compounds exist.

At the genetic level, these analogs are generally prepared by site-directed mutagenesis of nucleotides in the DNA encoding the STOMT protein, thereby producing DNA encoding the analog, and thereafter synthesizing the DNA and expressing the polypeptide in recombinant cell culture. The analogs typically exhibit the same or increased qualitative biological activity as the naturally occurring protein, Ausubel et al, Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

Preparation of a STOMT protein in accordance herewith, or an alternative nucleotide sequence encoding the same polypeptide but differing from the natural sequence due to changes permitted by the known degeneracy of the genetic code, can be achieved by site-specific mutagenesis of DNA that encodes an earlier prepared analog or a native version of a STOMT protein. Site- specific mutagenesis allows the production of analogs through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 complementing nucleotides on each side of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by publications such as Adelman et al, ^'DNA 2: 183 (1983), the disclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site- directed mutagenesis include vectors such as the Ml 3 phage, for example, as disclosed by Messing et al, Third Cleveland Symposium on Macromolecules and Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), the disclosure of which is incorporated herein by reference. These phages are readily available commercially and their use is generally well known to those skilled in the art. Alternatively, plasmid vectors that contain a single-stranded phage origin of replication (Veira et al, Meth. Enzymol. 153:3, 1987) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant polypeptide. An oligonucleotide primer bearing the desired mutated sequence is prepared synthetically by automated DNA oligonucleotide synthesis. This primer is then annealed with the single-stranded protein-sequence-containing vector, and subjected to DNA- polymerizing- enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli JM101 cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

After such a clone is selected, the mutated STOMT protein sequence may be removed and placed in an appropriate vector, generally a transfer or expression vector of the type that may be employed for transfection of an appropriate host.

Accordingly, gene or nucleic acid encoding for a STOMT protein can also be detected, obtained and/or modified, in vitro, in situ and/or in vivo, by the use of known DNA or RNA amplification techniques, such as PCR and chemical oligonucleotide synthesis. PCR allows for the amplification of specific DNA sequences by repeated DNA polymerase reactions. This reaction can be used as a replacement for cloning; all that is required is a knowledge of the nucleic acid ^"sequence. In order to carry out PCR, primers are designed which are complementary to the sequence of interest. The primers are then generated by automated DNA synthesis. Because primers can be designed to hybridize to any part of the gene, conditions can be created such that mismatches in complementary base pairing can be tolerated. Amplification of these mismatched regions can lead to the synthesis of a mutagenized product resulting in the generation of a peptide with new properties (i.e., site directed mutagenesis). See also, e.g., Ausubel, supra, Ch. 16. Also, by coupling complementary DNA (cDNA) synthesis, using reverse transcriptase, with PCR, RNA can be used as the starting material for the synthesis of the STOMT, or of a preferably functional part thereof, without cloning.

Furthermore, PCR primers can be designed to incorporate new restriction sites or other features such as termination codons at the ends of the gene segment to be amplified. This placement of restriction sites at the 5' and 3' ends of the amplified gene sequence allows for gene segments encoding STOMT protein or a fragment thereof to be custom designed for ligation other sequences and/or cloning sites in vectors.

PCR and other methods of amplification of RNA and/or DNA are well known in the art and can be used according to the present invention without undue experimentation, based on the teaching and guidance presented herein. Known methods of DNA or RNA amplification include, but are not limited to polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis et al; 4,795,699 and 4,921,794 to Tabor et al; 5,142,033 to Innis; 5,122,464 to Wilson et al; 5,091,310 to Innis; 5,066,584 to Gyllensten et al; 4,889,818 to Gelfand et al; 4,994,370 to Silver et al; 4,766,067 to Biswas; 4,656,134 to Ringold; and Innis et al, eds., PCR Protocols: A Guide to Method and Applications) and RNA mediated amplification which uses anti-sense RNA to the target sequence as a template for double stranded DNA synthesis (U.S. patent No. 5,130,238 to Malek et al, with the tradename NASBA); and immuno-PCR which combines the use of DNA amplification with antibody labeling (Ruzicka et al, Science 260:487 (1993); Sano et al, Science 258:120 (1992); Sano et al, Biotechniques 9:1378 (1991)), the entire contents of which patents and reference are entirely incorporated herein by reference.

In an analogous fashion, biologically active fragments of STOMT proteins (e.g. those of any of ^"the STOMT or its homologs) may be prepared as noted above with respect to the analogs of STOMT proteins. Suitable fragments of STOMT proteins are those which retain the STOMT capability and which can mediate the biological activity of STOMT. Accordingly, STOMT protein fragments can be prepared which have dominant-positive effect, i.e., will result in enhanced production of methylated products according to the specificity of STOMT in a plant or cell where they are introduced and where the corresponding precursors exist.

Similarly, derivatives may be prepared by standard modifications of the side groups of one or more amino acid residues of the STOMT protein, its analogs or fragments, or by conjugation of the STOMT protein, its analogs or fragments, to another molecule e.g. an antibody, enzyme, receptor, etc., as are well known in the art. Accordingly, "derivatives" as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention. Derivatives may have chemical moieties such as carbohydrate or phosphate residues, provided such a fraction has the same or higher biological activity as STOMT proteins.

For example, derivatives may include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed with acyl moieties.

The term "derivatives" is intended to include only those derivatives that do not change one amino acid to another of the twenty commonly occurring natural amino acids.

STOMT is a protein or polypeptide, i.e. a sequence of amino acid residues. A polypeptide consisting of a larger sequence which includes the entire sequence of a STOMT protein, in accordance with the definitions herein, is intended to be included within the scope of such a polypeptide as long as the additions do not affect the basic and novel characteristics of the invention, i.e., if they either retain or increase the biological activity of STOMT protein or can be cleaved to leave a protein or polypeptide having the biological activity of STOMT protein. Thus, for example, the present invention is intended to include fusion proteins of STOMT protein with ^"other amino acids or peptides.

The STOMT protein, their analogs, fragments and derivatives thereof, have a number of possible uses, for example: STOMT protein, its analogs, fragments and derivatives thereof, may be used to enhance the function of naturally-occurring STOMT in plants or tissue cultured cells. For example, ii STOMT can enhance the production of flavor-enhancing substances such as vanillin or DMMF, then such a STOMT effect would be desirable in the production of plant with enhanced flavor/taste characteristics, or in the in-vitro production of flavor substances. In this case the STOMT protein, its analogs, fragments or derivatives thereof, which have the desired enzymatic effect, may be introduced to the cells by standard procedures known per se. It is possible to introduce the STOMT gene as described further above, or as illustrated in the examples below. Another possibility is to introduce the sequences of the STOMT protein (e.g., any one of the STOMT or its isoforms) in the form of oligonucleotides which can be absorbed by the cells and expressed therein.

Another possibility is to use antibodies specific for the STOMT protein to inhibit its enzymatic effects. This may be desirable where it is found that the substances whose production STOMT is catalyzing, have an undesirable effect on flavor or taste. Likewise, it is possible that the effect of STOMT in the pathway of production of pigments, e.g., anthocyanins, leads to a colour change when the activity of STOMT is inhibited.

One way of inhibiting the STOMT enzyme is by the recently developed ribozyme approach. Ribozymes are catalytic RNA molecules that specifically cleave RNAs. Ribozymes may be engineered to cleave target RNAs of choice, e.g., the mRNAs encoding the STOMT protein of the invention. Such ribozymes would have a sequence specific for the STOMT protein mRNA and would be capable of interacting therewith (complementary binding) followed by cleavage of the mRNA, resulting in a decrease (or complete loss) in the expression of the STOMT protein, the level of decreased expression being dependent upon the level of ribozyme expression in the target cell. To introduce ribozymes into the cells of choice, any suitable vector may be used, e.g., plasmid, bacterial (e.g. agrobacterium) vectors, that are usually used for this purpose (see also (i) above, where the vector has, as second sequence, a cDNA encoding the ribozyme sequence of choice). (For reviews, methods etc. concerning ribozymes see Chen et al., 1992; Zhao and Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama et al., 1993; Cantor et al., 1993; Barinaga, 1993; Crisell et al., 1993 and Koizumi et al., 1993). This approach is suitable when the STOMT activity enhances formation of undesirable substances or when it is desired to change the colour of a plant or part thereof where STOMT activity leads to the formation of pigments or enhances such formation.

Further nucleic acids which have a similar function and may thus also be used in a similar manner in connection with the present invention are antisense oligonucleotides and siRNA.

The activity and design of antisense oligonucleotides for the manufacture of an agrochemical and as a diagnostic agent, respectively, is based on a mode of action similar to the one of ribozymes. Basically, antisense oligonucleotides hybridise based on base complementarity, with a target RNA, preferably with a mRNA, thereby activate RNase H. RNase H is activated by both phosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupled DNA, however, is rapidly degraded by cellular nucleases with the exception of phosphorothioate-coupled DNA. These resistant, non-naturally occurring DNA derivatives do not inhibit RNase H. upon hybridisation with RNA. In other words, antisense polynucleotides are only effective as DNA RNA hybride complexes. Examples for this kind of antisense oligonucleotides are described, among others, in US-patent US 5,849,902 and US 5,989,912. In other words, based on the nucleic acid sequence of the target molecule which in the present case are the nucleic acid molecules for the polypeptide and/or enzyme according to the present invention, either from the target protein from which a respective nucleic acid sequence may in principle be deduced, or by knowing the nucleic acid sequence as such, particularly the mRNA, suitable antisense oligonucleotides may be designed base on the principle of base complementarity.

Particularly preferred are antisense-oligonucleotides which have a short stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNA bases is required for activation of bacterial RNase H and a minimum of 5 bases is required for mammalian RNase H activation. In these chimeric oligonucleotides there is a central region that forms a substrate for RNase H that is flanked by hybridising "arms" comprised of modified nucleotides that do not form substrates for RNase H. The hybridising arms of the chimeric oligonucleotides may be modified such as by 2'-O-methyl or 2 '-fluoro. Alternative approaches used methylphosphonate or phosphoramidate linkages in said arms. Further embodiments of the antisense oligonucleotide useful in the practice of the present invention are P-methoxyoligonucleo tides, partial P- methoxyoligodeoxyribonucleotides or P-methoxyoligonucleotides. Of particular relevance and usefulness for the present invention are those antisense oligonucleotides as more particularly described in the above two mentioned US patents. These oligonucleotides contain no naturally occurring 5'- 3'-linked nucleotides. Rather the oligonucleotides have two types of nucleotides: 2 '-deoxyphosphorothioate, which activate RNase H, and 2 '-modified nucleotides, which do not. The linkages between the 2 '-modified nucleotides can be phosphodiesters, phosphorothioate or P-ethoxyphosphodiester. Activation of RNase H is accomplished by a contiguous RNase H-activating region, which contains between 3 and 5 2'- deoxyphosphorothioate nucleotides to activate bacterial RNase H and between 5 and 10 2'- deoxyphosphorothioate nucleotides to activate eucaryotic and, particularly, mammalian RNase H. Protection from degradation is accomplished by making the 5' and 3' terminal bases highly nuclease resistant and, optionally, by placing a 3' terminal blocking group.

More particularly, the antisense oligonucleotide comprises a 5' terminus and a 3' terminus; and from 11 to 59 5'- 3'-linked nucleotides independently selected from the group consisting of 2'- modified phosphodiester nucleotides and 2 '-modified P-alkyloxyphosphotriester nucleotides; and wherein the 5 '-terminal nucleoside is attached to an RNase H-activating region of between three and ten contiguous phosphorothioate-linked deoxyribonucleo tides, and wherein the 3 '-terminus of said oligonucleotide is selected from the group consisting of an inverted deoxyribonucleotide, a contiguous stretch of one to three phosphorothioate 2 '-modified ribonucleo tides, a biotin group and a P-alkyloxyphosphotriester nucleotide.

Also an antisense oligonucleotide may be used wherein not the 5 ' terminal nucleoside is attached to an RNase H-activating region but the 3' terminal nucleoside as specified above. Also, the 5' terminus is selected from the particular group rather than the 3' terminus of said oligonucleotide.

siRNA may, in principle, be used for the same purposes as the afore-mentioned antisense oligonucleotides. siRNA is a double stranded RNA having typically a length of about 21 to about 23 nucleotides. The sequence of one of the two RNA strands corresponds to the sequence of the target nucleic acid such as the nucleic acid coding for the polypeptide and the enzyme, respectively, according to the present invention which are also referred to as the target molecule, to be degraded. In other words, knowing the nucleic acid sequence of the target molecule, preferably the mRNA sequence, a double stranded RNA may be designed with one of the two strands being complementary to said, e. g. mRNA of the target molecule and, upon application of said siRNA to a system containing the gene, genomic DNA, hnRNA or mRNA coding for the target molecule, the respective target nucleic acid will be degraded and thus the level of the respective polypeptide and enzyme, respectively, be reduced. The basic principles of designing, constructing and using said siRNA as agrochemicals and diagnostic agent, respectively, is, among others, described in international patent applications WO 00/44895 and WO 01/75164.

In a further aspect the present invention relates to functional nucleic acids interacting with any of the polypeptides according to the present invention, and a method for the manufacture of such functional nucleic acids whereby the method is characterized by the use of the polypeptides according to the present invention and the basic steps are known to the one skilled in the art. The functional nucleic acids are preferably aptamers and spiegelmers.

Aptamers are D-nucleic acids which are either single stranded or double stranded and which specifically interact with a target molecule which is in the present case the polypeptide and/or the enzyme according to the present invention. The manufacture or selection of aptamers is, e. g., described in European patent EP 0 533 838. Basically the following steps are realized. First, a mixture of nucleic acids, i. e. potential aptamers, is provided whereby each nucleic acid typically comprises a segment of several, preferably at least eight subsequent randomised nucleotides. This mixture is subsequently contacted with the target molecule whereby the nucleic acid(s) bind to the target molecule, such as based on an increased affinity towards the target or with a bigger force thereto, compared to the candidate mixture. The binding nucleic acid(s) are/is subsequently separated from the remainder of the mixture. Optionally, the thus obtained nucleic acid(s) is amplified using, e. g. polymerase chain reaction. These steps may be repeated several times giving at the end a mixture having an increased ratio of nucleic acids specifically binding to the target from which the final binding nucleic acid is then optionally selected. These specifically binding nucleic acid(s) are referred to aptamers. It is obvious that at any stage of the method for the generation or identification of the aptamers samples of the mixture of individual nucleic acids may be taken to determine the sequence thereof using standard techniques. It is within the present invention that the aptamers may be stabilized by such as, e. g., introducing defined chemical groups which are known to the one skilled in the art of generating aptamers. Such modification may for example reside in the introduction of an amino group at the 2 '-position of the sugar moiety of the nucleotides. However, it is also within the present invention that the thus selected or generated aptamers may be used for target validation and/or as lead substance for the development of agrochemicals, preferably of agrochemicals based on small molecules. This is actually done by a competition assay whereby the specific interaction between the target molecule and the aptamer is inhibited by a candidate agrochemical whereby upon replacement of the aptamer from the complex of target and aptamer it may be assumed that the respective agrochemical candidate allows a specific inhibition of the interaction between target and aptamer, and if the interaction is specific, said candidate agrochemical will, at least in principle, be suitable to block the target and thus decrease its biological availability or activity in a respective system comprising such target. The thus obtained small molecule may then be subject to further derivatisation and modification to optimise its physical, chemical, biological and/or agrochemical characteristics such as toxicity, specificity, biodegradability and bioavailability.

Spiegelmers and their generation or manufacture is based on a similar principle. The manufacture of spiegelmers is described in international patent application WO 98/08856. Spiegelmers are L- nucleic acids, which means that they are composed of L-nucleotides rather than D-nucleotides as aptamers are. Spiegelmers are characterized by the fact that they have a very high stability in biological system and, comparable to aptamers, specifically interact with the target molecule against which they are directed. In the process of generating spiegelmers, a heterogonous population of D-nucleic acids is created and this population is contacted with the optical antipode of the target molecule, in the present case for example with the D-enantiomer of the naturally occurring L-enantiomer of the polypeptide or the enzyme according to the present invention. Subsequently, those D-nucleic acids are separated which do not interact with the optical antipode of the target molecule. But those D-nucleic acids interacting with the optical antipode of the target molecule are separated, optionally determined and/or sequenced and subsequently the corresponding L-nucleic acids are synthesized based on the nucleic acid sequence information obtained from the D-nucleic acids. These L-nucleic acids which are identical in terms of sequence with the aforementioned D-nucleic acids interacting with the optical antipode of the target molecule, will specifically interact with the naturally occurring target molecule rather than with the optical antipode thereof. Similar to the method for the generation of aptamers it is also possible to repeat the various steps several times and thus to enrich those nucleic acids specifically interacting with the optical antipode of the target molecule.

Because of the mode of action any of the afore-mentioned compounds may be used for the inhibition of the polypeptide and enzyme, respectively, subject to the present invention in its various terms and in its various uses. In accordance therewith they may be used for the inhibition of a methylation of ortho-dihydroxy phenols and mimetics thereof and any enzyme catalyzing this or any of the reactions as disclosed herein.

As noted hereinabove and hereinbelow, the STOMT protein, or its analogs, fragments or derivatives thereof, of the invention may also be used as immunogens (antigens) to produce specific antibodies thereto. These antibodies may also be used for the purposes of purification of the STOMT protein (e.g., STOMT or any of its isoforms) either from cell extracts or from transformed cell lines producing STOMT protein, or its analogs or fragments. Further, these antibodies or functional nucleic acids may be used for diagnostic purposes for identifying plants having particular properties, e.g., particular abilities with respect to the synthesis of the products that STOMT may synthesize, e.g., caffeic acid, caffeoyl-CoA, DMHF, catechol and the like.

It should also be noted that the isolation, identification and characterization of the STOMT protein of the invention may be performed using any of the well known standard screening procedures. As noted above and below, procedures may be employed such as affinity chromatography, DNA hybridization procedures, etc. as are well known in the art, to isolate, identify and characterize the STOMT protein of the invention or to isolate, identify and characterize additional proteins, factors, etc. which are capable of catalyzing the reaction that STOMT is capable of catalyzing.

As set forth hereinabove, the STOMT protein may be used to generate antibodies specific to STOMT proteins, e.g., STOMT and its isoforms. These antibodies or fragments thereof may be used as set forth hereinbelow in detail, it being understood that in these applications the antibodies or fragments thereof are those specific for STOMT proteins. Since it may be advantageous to design peptide inhibitors that selectively inhibit STOMT enzymatic activity without interfering with other physiological processes in which other enzymes are involved, the pool of peptides binding to STOMT in an assay such as the one described by Geysen (Geysen, 1985; Geysen et al., 1987) can be further synthesized as a fluorogenic substrate peptide to test for selective binding to such other proteins to select only those specific for STOMT. Peptides which are determined to be specific can then be modified to enhance cell permeability and inhibit the enzymatic activity of STOMT either reversibly or irreversibly. Accordingly, peptides that selectively bind to STOMT can be modified with, for example, an aldehyde group, chloromethylketone,(acyloxy) methyl ketone or a CH OC (O)-DCB group to create a peptide inhibitor of STOMT activity. Further, to improve permeability, peptides can be, for example, chemically modified or derivatized to enhance their permeability across the cell membrane and facilitate the transport of such peptides through the membrane and into the cytoplasm. Muranishi et al. (1991) reported derivatizing thyrotropin-releasing hormone with lauric acid to form a lipophilic lauroyl derivative with good penetration characteristics across cell membranes. Zacharia et al. (1991) also reported the oxidation of methionine to sulfoxide and the replacement of the peptide bond with its ketomethylene isoester (COCH ) to facilitate transport of peptides through the cell membrane. These are just some of the known modifications and derivatives that are well within the skill of those in the art.

U.S. Patent 5,149,782 discloses conjugating a molecule to be transported across the cell membrane with a membrane blending agent such as fusogenic polypeptides, ion-channel forming polypeptides, other membrane polypeptides, and long chain fatty acids, e.g. myristic acid, palmitic acid. These membrane blending agents insert the molecular conjugates into the lipid bilayer of cellular membranes and facilitate their entry into the cytoplasm.

Low et al., U.S. Patent 5, 108,921, reviews available methods for transmembrane delivery of molecules such as, but not limited to, proteins and nucleic acids by the mechanism of receptor mediated endocytotic activity. These receptor systems include those recognizing galactose, mannose, mannose 6-phosphate, transferrin, asialoglycoprotein, transcobalamin (vitamin B₁₂), α- 2 macroglobulins, insulin and other peptide growth factors such as epidermal growth factor (EGF). Low et al. teaches that nutrient receptors, such as receptors for biotin and folate, can be advantageously used to enhance transport across the cell membrane due to the location and multiplicity of biotin and folate receptors on the membrane surfaces of most cells and the associated receptor mediated transmembrane transport processes. Thus, a complex formed between a compound to be delivered into the cytoplasm and a ligand, such as biotin or folate, is contacted with a cell membrane bearing biotin or folate receptors to initiate the receptor mediated trans-membrane transport mechanism and thereby permit entry of the desired compound into the cell.

As will be appreciated by those of skill in the art of peptides, the peptide inhibitors of the STOMT interaction according to the present invention is meant to include peptidomimetic drugs or inhibitors, which can also be rapidly screened for binding to STOMT enzyme to design perhaps more stable inhibitors.

It will also be appreciated that the same means for facilitating or enhancing the transport of peptide inhibitors across cell membranes as discussed above are also applicable to the STOMT or its isoforms themselves as well as other peptides and proteins which exert their effects intracellularly.

As regards the antibodies mentioned herein throughout, the term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti- Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques, and especially single-chain (sc) antibodies, which have the advantage that they are coded for by a single chain of nucleic acids and may therefore easily be introduced into and expressed in cells.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which populations contains substantially similar epitope binding sites. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature, 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., eds., Harlow and Lane ANTIBODIES : A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience N.Y., (1992-1996), the contents of which references are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GELD and any subclass thereof. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.

Chimeric antibodies are molecules of which different portions are derived from different animal species, such as those having the variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc. Natl Acad. Sci. USA 81:3273-3277 (1984); Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published November 14, 1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171496 (published February 19, 1985); Morrison et al., European Patent Application 173494 (published March 5, 1986); Neuberger et al., PCT Application WO 8601533, (published March 13, 1986); Kudo et al., European Patent Application 184187 (published June 11, 1986); Sahagan et al., J. Immunol 137:1066-1074 (1986); Robinson et al., International Patent Application No. WO8702671 (published May 7, 1987); Liu et al., Proc. Natl Acad. Sci USA 84:3439-3443 (1987); Sun et al., Proc. Natl Acad. Sci USA 84:214-218 (1987); Better et al., Science 240:1041- 1043 (1988); and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra. These references are entirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Patent No. 4,699,880, which is herein entirely incorporated by reference.

The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the STOMT proteins, analogs, fragments or derivatives thereof, of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for an epitope of the above STOMT protein, or analogs, fragments and derivatives thereof.

The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein-a.

The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of the STOMT protein according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The antibodies, including fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the STOMT protein in a sample or to detect presence of cells which express the STOMT protein of the present invention. This can be accomplished by immunofiuorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.

The antibodies (or fragments thereof) and/or the functional nucleic acids useful in the present invention may be employed histologically, as in immunofiuorescence or immunoelectron microscopy, for in situ detection of the STOMT protein of the present invention. In situ detection may be accomplished by removing a histological specimen from a plant, and providing the labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the STOMT protein, but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. Such assays for the STOMT protein of the present invention typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, such as an extract prepared from root, petiole, leaf, flower, green fruit, white fruit, turning fruit, or ripe fruit tissue, preferably an extract prepared from white fruit, turning fruit, or ripe fruit tissue, most preferably an extract prepared from turning fruit or ripe fruit tissue, an extract prepared from freshly harvested cells, such as freshly harvested cells derived from said tissues, or from cells which have been incubated in tissue culture, such as cultured cells derived from the said tissues, in the presence of a detectably labeled antibody or functional nucleic acid capable of identifying the STOMT protein, and detecting the antibody by any of a number of techniques well known in the art.

The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with a detectably labeled antibody or functional nucleic acid in accordance with the present invention, as noted above. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means.

"Solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" as used herein is any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen, antibody or any of the functional nucleic acids. Thus, the support or carrier configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody, antigen or any functional nucleic acid, or will be able to ascertain the same by use of routine experimentation. The binding activity of a given lot of antibody, of the invention as noted above, may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.

One of the ways in which an antibody in accordance with the present invention can be detectably labeled is by linking the same to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spec trophotome trie, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomeras, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholin-esterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactive labeling the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label an antibody in accordance with the present invention with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can be then detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

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The antibody can also be detectably labeled using fluorescence emitting metals such as E, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

It is within the present invention that any of the functional nucleic acids as described herein are labeled and/or detected in an manner similar to the one described for antibodies.

An antibody molecule of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody. Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.

The antibody of the invention may further be used to identify such plants, parts thereof, tissues, or cells, that express STOMT, preferably such plants, parts thereof, tissues, or cells, that express STOMT in high amounts. Such plants, parts thereof, tissues, or cells may be useful in the production of aroma substances, such as the aroma substances of vanillin or Strawberry, as mentioned herein. The so identified plants, parts thereof, tissues, or cells, may also be useful in the creation of other cells, tissues, or plants, by way of fusion of cells derived from said plants, parts thereof, tissues, or by way of fusion of said cells, with said other cells, that may have other desirable properties but lack the expression, preferably the high expression, of STOMT, and thus lack the desirable properties conferred by STOMT, in particular, production of aroma substances as described herein.

The antibody of the invention may not only bind the STOMT protein, but also inhibit its enzymatic activity. This may be tested easily by methods known to the person of skill in the art. For instance, adding various amounts of the antibody into an enzyme will result in a lower enzymatc activity observed if that antibody is capable of inhibiting the enzyme reaction, as compared to irrelevant control antibody. Thus, antibodies can be identified that specifically inhibit STOMT activity. Such antibodies are useful in control assays for enzymatic activity. Such antibodies are also useful in inhibiting endogenous or exogenous STOMT activity in plants, parts thereof, tissues, or cells. Basically, the same applies to the functional nucleic acids described herein, preferably any aptamers or spiegelmer. To this end, the antibodies, or parts thereof that exhibit theirs functional, i.e., enzyme-inhibiting properties, may be introduced into the cells of said plants, parts thereof, tissues, or directly into said cells, by methods well known in the art. When it is desired to introduced proteins into cells, it is generally desired to use proteins of low molecular weight. Therefore, in the said process a single-chain antibody may advantageously used. Methods for introducing proteins into cells are described, e.g., in EP1127133 and WO0026379, which publications are included herein in their entirety by reference.

The STOMT proteins of the invention may be produced by any standard recombinant DNA procedure (see for example, Sambrook, et al., 1989 and Ansabel et al., 1987-1995, supra) in which suitable eukaryotic or prokaryotic host cells well known in the art are transformed by appropriate eukaryotic or prokaryotic vectors containing the sequences encoding for the proteins. Accordingly, the present invention also concerns such expression vectors and transformed hosts for the production of the proteins of the invention. As mentioned above, these proteins also include their biologically active analogs, fragments and derivatives, and thus the vectors encoding them also include vectors encoding analogs and fragments of these proteins, and the transformed hosts include those producing such analogs and fragments. The derivatives of these proteins, produced by the transformed hosts, are the derivatives produced by standard modification of the proteins or their analogs or fragments. The polypeptide and enzyme, respectively, of the invention may also be used in the production of a transgenic plant or a method for enhancing flavor and/or taste in a plant as described in more detail in the claims.

In a further aspect the invention relates to a transgenic plant with enhanced flavor comprising a genetically engineered DNA sequence encoding at least one polypeptide having an O- methyltransferase activity, wherein said polypeptide is encoded by a nucleotide sequence as described in any of claims 1-10, or by SEQ DD No. 1 or SEQ. DD. No. 3, and a propagating material derived from the transgenic plant of the present invention.

In a still further aspect the present invention is related to a method for the methylation of an ortho-dihydroxy substituted ring system and/or mimetics of such ortho-dihydroxy substituted ring system, whereby preferably the ring system is an aromatic ring system or a heteroaromatic ring system or a furanone based ring system, comprising the following steps:

a) providing an ortho-dihydroxy substituted ring system and/or a mimetic of such ortho-dihydroxy substituted ring system b) providing the enzyme according to any of claims 11 to 22 or a polypeptide according to any of claims 1 to 10, and c) reacting the ortho-dihydroxy substituted ring system and/or the mimetic of such ortho-dihydroxy substituted ring system and the enzyme or the polypeptide in the presence of a methyl group donor.

In an embodiment of the method according to the invention the methyl group donor is S- adenosyl-L-methionine.

In a further embodiment the ortho-dihydroxy substituted ring system and/or mimetics thereof is selected from the group comprising catechol, caffeic acid, protocatechuic aldehyde and pyrogallol.

In an alternative embodiment the mimetic of the ortho-dihydroxy substituted ring system is a 4- hydroxy-3 (H)-furanone.

In a preferred embodiment of the method of the invention the methylation is selected from the group comprising

- methylation of protocatechuic aldehyde to vanillin

- methylation of caffeic acid to ferulic acid

- methylation of catechol to guaiacol

- methylation of caffeoyl-CoA to feruloyl CoA, and

- methylation of DMHF to DMMF.

In a further preferred embodiment of the method of the invention the methylation is carried out in an aqueous solution, preferably in an aqueous buffer.

In a still further preferred embodiment of the method of the invention the reaction is carried out with the enzyme or the peptide being immobilized onto a surface.

The invention is now more fully described and illustrated by the figures, examples and the sequence listing as set out below from which further features, embodiments and advantages of the present invention may be taken. It is understood that the examples are not limiting the scope of the invention, and that e.g. other enzyme sequences may be cloned according to the teaching of the invention, and other substrates may be used by the enzymes of the invention.

Fig. 1 shows the structures of the furanone DMHF and its methylation by STOMT to DMMF, whereby on the lower panel the methylation of dihydroxyl-carrying phenolic compounds are shown.

Fig. 2 shows the substrate specificity of OMT from strawberry fruits with the assays being performed as described in Materials and Methods. p-Coumaric acid, (E)-cinnamic acid, (E)-ferulic acid, vanillic acid, p-anol, chavicol, coniferyl alcohol, phenol, hydroquinone, and o-cresol were ineffective substrates for the OMT activity.

Fig. 3 shows the OMT activity towards caffeic acid (white bars), catechol (grey bars) and DMHF (black bars) in three strawberry varieties during fruit ripening.

Fig. 4 shows the expression pattern of STOMT in different strawberry tissues: root (R), petiole (P), leaf (L), flower (F), green fruit (G), white fruit (W), turning fruit (T), and ripe fruit (RF). Northern blot analysis of strawberry total RNA (10 μg/lane) probed with ³²P- labeled STOMT (A). Equal amounts of RNA were run in each lane as indicated by the intensities of rRNA bands prior to transfer (B).

Fig. 5 shows the SDS PAGE analysis of GST-fusion protein expressed in E. coli after purification with GST affinity column (A) (El to E3: fraction 1 to 3, M: marker proteins) and purified STOMT after cleavage of the fusion protein with thrombin (B) (M: marker proteins, P: STOMT released by thrombin). Band were visualized by silver straining.

Fig. 6 shows the kinetic analysis of the methylation of DMHF to DMMF. Material and methods

The following materials and methods may be used for the practice of the present invention and were used in connection with the examples described herein.

Plant material:

Plant tissue was collected from strawberry plants (Fragaria x ananassa ) cv. Elsanta, in the Botanical Garden, Wurzburg, Germany, frozen in liquid nitrogen and stored at -80°C until use. Fresh or frozen fruits of different varieties of Fragaria x ananassa var. Tamar, Yael, Malach were obtained from the Volcani Institute, Israel.

Extraction of volatiles:

Fresh strawberry fruits (30 g) were homogenized in a food processor (Braun) and extracted with 50 ml methyl tert-butyl ether containing 10 ug internal standard (isobutylbenzene) by shaking for 2 hours. The organic phase was dried on anhydrous Na₂SO₄ and evaporated under nitrogen to 1 ml.

GC-MS analysis:

Volatile compounds were analyzed on a HP-GCD apparatus equipped with an HP-5 (30 m x 0.25 mm) fused-silica capillary column (Hewlett-Packard, USA). Helium (1 ml/min) was used as a carrier gas. The injector temperature was 250 °C, set for splitless injection. The oven was set to 50 °C for 1 min, then the temperature was increased to 200 °C at a rate of 4 °C/min. The detector temperature was 280 °C. Mass range was recorded from 45 to 450 m/z, with electron energy of 70 eV. Identification of the main components was done by comparison of mass spectra and retention time data with those of authentic samples and supplemented with a Wiley GC-MS library. The quantitative analyses were determined using isobutylbenzene as an internal standard.

Chemicals and radiochemicals:

S-adenosyl-L-methionine was purchased from Sigma Chemical Co. S-adenosyl-L-methyl H- methionine (s.a. 15 Ci/mmol) and S-adenosyl-L-methyl ¹⁴C-methionine (s.a. 55 mCi/mmol) were from Amersham. Preparation of crude cell-free extracts:

Fresh or frozen strawberry fruits were cut into slices (4-5 gr), weighed and placed in a chilled mortar. The fruits were then ground with a pestle in the presence of 0.5 gr PVPP to spell-out phenolic materials, and 15 ml extraction buffer A (100 mM Tris-HCl pH 8.5 containing 10% glycerol, 5 mM Na₂S₂O₅, 10 mM 2-mercaptoethanol, 1% PVP-10) were added. The slurry was centrifuged at 20,000 g for 10 min at 4°C. The supernatant (crude extract) was used for further purification steps and enzymatic assays.

Partial purification:

All procedures were performed at 0-4°C. Proteins were purified as follows:

Four ml of the crude extracts were loaded onto XAD-2 columns (1x15 cm). Column equilibration and protein elution were done with buffer B (buffer A without PVP). Fractions were tested for enzyme activities and active fractions were combined.

Three ml of the XAD-active fractions were loaded to Bio-gel P-6 column (1x10 cm). Column equilibration and protein elution were done with buffer B. Fractions were tested for enzyme activity and active fractions were combined.

Three ml of the P-6-active fractions were loaded onto DE52 column (1x3 cm). Column equilibration was done with buffer B. Protein elution was done with 10 ml of buffer B in gradient of 0 to 1 M KC1. Fractions were tested for enzyme activity and active fractions were combined and used for further tests.

Assays of enzyme activity in strawberry protein extracts:

The standard assay mixture consisted of 30 μl buffer B (100 mM Tris-HCl pH 8.5 containing 10% glycerol, 5 mM Na₂S₂O₅, 10 mM 2-mercaptoethanol), 50 μl enzyme solution, 10 mM DMHF, and 10 μl ³H-SAM, in a total volume of 100 μl. The mixture was incubated at 30°C for 1 to 2 h. The reaction was stopped by adding 10 μl 2N HC1 and stirring. Then 1 ml ethyl-acetate was added to each tube, vigorously vortexed and spun for 1 min at 20,000 g to separate the phases. The upper ethyl-acetate phase layers containing the radioactive labeled enzyme products were transferred to scintillation tubes containing 3 ml of scintillation fluid (4 gr/L 2,5- diphenyloxazol (PPO) and 0.05 gr/L 2,2'-p-phenylen-bis(5-phenyloxazol) (POPOP) in toluene. The radioactivity was quantified using a liquid scintillation counter.

To confirm the identity of the biosynthetic products, similar incubations were performed, except that ¹⁴C-labeled SAM was used. In this case the ethyl-acetate layer was evaporated to a volume of 20 μl using a gentle stream of N₂, and analyzed by TLC-autoradiography using Silica gel 60 F₂₅ plates developed with pentane: diethyl ether (5:1). Spots were visualized by UV light and radioactive spots detected by autoradiography on Kodak X-OMAT paper.

Gel permeation chromatography:

The molecular mass of the native enzyme was determined by gel filtration chromatography through a Superdex 75 Hiload Prep 16/60 (FPLC, Amersham Pharmacia Biotech), using buffer B at flow rate of 1 ml/min, and compared to the molecular mass of known proteins.

Protein determination:

Protein was measured according to the method described by Bradford (Anal. Biochem. 72, 248-

254, 1976), using the Bio-Rad protein reagent (Bio-Rad) and bovine serum albumin (Sigma) as standard.

cDNA library:

Poly(A)⁺ RNA isolated from turning strawberries was used to construct a cDNA library in the Uni-ZAP^® XR vector (Stratagene, La Jolla, CA, USA) and it was amplified according to the manufacture's instructions.

Isolation of STOMT:

Two degenerate primers (forward primer 5'-GTI-GAC/T-GTI-GGI-GGI-GGI-ACI-GGI-GC-3', MTsI; reverse primer 5 '-GGI-GCA/G-TCC/T-TCI- ATI-AC A/G-TGI-GG-3' MTasϋ with I = Inosin) based on two highly conserved regions of plant O-methyltransferases (Frick and Kutchan, Plant J., 17, 329-339, 1999) were synthesized and used for PCR cloning. One microliter of the amplified λ-ZAP phage suspension was used as templates for PCR. The amplification program consisted of 35 cycles of denaturation (94°C, 30 sec), annealing (50°C, 30 sec), and primer extension (72°C, 20 sec). The resulting PCR product of 106 bp was cloned into the pCR^®2.1 vector (Invitrogen). This fragment was used as probe for an initial screening of 3xl0⁵ PFU from the amplified cDNA library (manual of the cDNA Synthesis Kit, Stratagene, La Jolla, CA, USA) resulting in 44 positive clones. After two further rounds of plaque purification 24 clones were obtained and sequenced. All clones revealed an identical sequence but differed in size. The longest clone, designated STOMT (Strawberry O-methyltransferase) was chosen for further examinations.

Cloning of STOMT:

PCR was used to introduce a BamHl site at the 5' end and a Xhoϊ site at the 3' end of the open reading frame of STOMT. The PCR product was ligated into the multiple cloning site (MCS) of the predigested pGEX-4T-2 fusion vector (Pharmacia, Freiburg, Germany). This glutathione S- transferase (GST) fusion vector contains the coding region of the GST gene and a thrombin recognition site located between the GST gene and the MCS.

The resulting plasmid was transferred into E. coli BL21(DE3) cells (Stratagene, La Jolla, CA, USA)

Expression of STOMT

An overnight culture of the transformed E. coli was diluted 1:60 with fresh LB medium containing 50 mg/1 ampicillin. The culture was grown at 37°C until an OD₆oo of 0.6 - 0.8 was reached. The expression of the fusion protein was induced by addition of 0.3 mM isopropyl-1- thio-β-D-galactopyranoside (D?TG). The culture was grown at 16°C for 16 h.

Cells were harvested at 3000xg for 10 min. The pellet was resuspended in 1/20 vol. prechilled extraction buffer (EB) consisting of PBS buffer (140 mM NaCl, 2.7 mM KC1, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄, pH 7.2-7.4), 5 mM β-mercaptoethanol and 5 % glycerol. All following steps were performed on ice. Lysis of the cells was achieved by sonification (5x6sec) and the cell debris were separated by centrifugation at 15000xg for 15 min. Preequilibrated GST-affinity resins (Stratagene, La Jolla, CA, USA) were added to the supernatant (lOOμl resin/20 ml supernatant). After 1.5 h of incubation at 4°C the resins were spun down at 500 g for 5 min. They were washed 5 times with EB. The beads were finally resuspended in EB including thrombin (200μl buffer + 0,2NIH Units thrombin/ lOOμl resin). After incubation overnight at 4°C with gentle agitation the suspension was centrifuged at 500xg for 5 min. The supernatant was collected and directly used for SDS PAGE and enzyme activity assays. Protein concentration was determined by the Bradford method (Bradford, Anal. Biochem. 72, 248-254, 1976) using BSA as a standard.

Enzyme Assay

Enzyme activity was tested in EB buffer (140 mM NaCl, 2,7 mM KC1, 10 mM Na₂HPO₄, 1,8 mM KH₂PO₄,), 5 mM β-mercaptoethanol and 5 % glycerol, pH 7,2-7,4) containing 1 μM - 1 mM substrate, 9,1 μM adenosyl-L-[methyl-¹⁴C]-methionine (S.A. 55mCi/mmol oder 1.85 GBq/mmol) and 1.8 - 3.7 μg protein in a final volume of 100 μl. After incubation at 30°C for 15 min to 30 min the reaction was stopped by adding 6 μl of 6 N HC1. If caffeoyl-CoA was used as substrate, the assays were treated as described in Meng and Campbell (Plant Mol. Biol., 38, 513- 520, 1998). The reaction mixture were extracted twice with 600 μl of ethyl acetate respectively. The organic layers were combined and analyzed by liquid scintillation counting LSC (Packard Tri-Carb Liquid Scintillation Analyzer, Meriden, CT, USA). Control assays were performed using eluates obtained by induction of E. coli cells containing the empty pGEX vector.

Identification of methylated products by HPLC

Enzyme assays were stopped as described above and extracted twice with 700 μl of diethyl ether. The organic layers were pooled, dried, concentrated and transferred into 100 μl of water which were analyzed by high performance liquid chromatography (HPLC). HPLC separations were carried out on an Eurospher 100 C-18 column (25 cm x 4.0 mm i.d., particle size 5μm, Knauer, Berlin, Germany) using a linear gradient with a flow rate of 1 ml/min. The gradient proceeded from 95% water acidified with 0.05% formic acid and 5 % acetonitrile to 100% acetonitrile in 30 min. Fractions of 1 ml each were collected, scintillation cocktail Emulsifier-Safe™ (Packard BioScience, Groningen, The Netherlands) was added and analyzed by LSC (LKB Rackbeta 1214, Pharmacia).

Isolation of RNA and Northern-blot hybridization

For northern analysis, RNA was isolated from different strawberry tissues as described by

Manning (Anal. Biochem., 195, 45-50, 1991). Total RNA (10 μg) was separated on a formaldehyde gel, followed by capillary transfer with lOxSSC to a nylon membrane (Hybond N, Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany). The STOMT cDNA probe was made by random labeling oligonucleotide priming (Feinberg and Vogelstein, Anal. Biochem. 137, 266-267, 1984). Hybridization was carried out at 68°C overnight in Roti-Hybri-Quick solution (Roth Chemikalien, Karlsruhe, Germany). The membrane was washed consecutively in lxSSC, 0.1% SDS and O.lxSSC, 0.1 SDS at 68°C and exposed to X-ray retina film (XBD) at - 80°C.

Example 1: Determination of SAM:DMHF O-methyltransferase activity from strawberry fruits

Incubation of strawberry cell-free extracts with ¹⁴C- or ³H-labeled S-adenosyl-L-methionine (SAM) and DMHF, resulted in the accumulation of ethyl-acetate-soluble radiolabeled product(s), absent when the cell-free extracts were previously boiled for 5 min or without a protein extract. This suggested that an O-methyltransferase enzymatic activity is involved in the conversion of DMHF into DMMF. To confirm the identity of the radiolabeled compounds formed, the ethyl- acetate extracts were evaporated and analyzed by TLC-autoradiography. Only one radioactive substance originating in DMHF and ¹⁴C-SAM fed extracts was detected and its R_f co-incided with that of authentic DMHF. These results indicate that a methyltransferase activity, present in strawberry fruits is able to O-methylate DMHF and release DMMF.

Example 2: Extraction and properties of the SAM:DMHF O-methyltransferase activity from strawberry fruits

Inclusion of PVP and PVPP during the extraction was crucial for stabilizing the enzymatic activity, which could be kept for more than three months at -20 °C without an apparent loss of activity. However, control assays conducted without added DMHF also contained soluble radiolabeled product (10-30% in crude cell-free extracts), indicating the presence of some endogenous substrates. In order to remove these internal substrates, partial purification was conducted using adsorbance-, gel filtration- and ion exchange chromatography. This effectively removed almost all the contaminants, and the activity was stable after the purification. The activity levels linearly increased with incubation time and were linearly dependent on protein concentration up to 25 μg protein. Optimum temperature for activity was found around 37 °C, being the levels at 30 °C about 90% of that at 37 °C. Activity was irreversible lost at incubation temperature over 45 °C, probably due to protein denaturation.

The pH optimum was found to be at about 8.5, using Tris-HCl buffer and after employing several other buffers. Below pH 6 activity sharply decreased, loosing all activity at pH values below 6 or above 10.

Some methyltransferases require the presence of metal cofactors for activity. To test whether the DMHF O-methyltransferase from strawberry requires any metal cof actor for activity, 1 or 10 mM of either CaCl₂, MgCl₂, MnCl₂, CoCl₂, ZnSO₄ or FeSO₄ were added to the assays. All the additions at 1 mM caused diminution of the activity by 10% as compared to controls without any further addition. This indicated that the DMHF dependent O-methyl transferase activity from strawberry apparently does not require a metal cofactor to be active.

A native molecular weight of approx. 80 kD was determined for DMHF-OMT by gel permeation chromatography on a calibrated Superdex HR 75 column (Pharmacia FPLC). This is within the same order found for OMT's from other sources (Bugos et al., Plant Mol. Biol., 17, 1203-1215, 1991; Ibrahim et al., Plant Mol. Biol., 36, 1-10, 1998).

A constant 15 μM SAM level was used to calculate an apparent Km for DMHF of 5 mM both utilizing Lineweaver-Burk and Eadie-Hofstee equations. This is not within the range of Km's obtained for substrates of O-methyltransferases from other sources, that normally display Km values lower by orders of magnitude (Ibrahim et al., Plant Mol. Biol,. 36, 1-10, 1998). Conversely, keeping a constant 10 mM of the acceptor substrate DMHF, a Km for SAM was found to be 7.5 μM. Example 3: Strawberry O-methyltransferase specificity

As shown above, in strawberry, DMHF can be enzymatically O-methylated, to generate methyl DMHF by S-adenosyl-L-methionine dependent O-methyltransferase activities. Nevertheless, the Km for this activity (5 mM) is very high in comparison with usual values of OMT's (Km = 5-500 μM. Ibrahim et al., Plant Mol. Biol., 36, 1-10, 1998).

To test substrate-specificity of this/these enzyme(s), several natural alcohols were chosen, in 2 concentrations (1 and 10 mM) (Fig. 2). Only ortho-dihydroxyphenols (catechol, caffeic acid) or similar ortho-dihydroxy compounds (DMHF, DTT) were found to react with this enzyme, resulting in transferring a methyl group from SAM and create methoxy compounds. This enzyme preparation had much lower Km towards catechol (120 μM) and caffeic acid (145 μM) than towards DMHF (5 mM). Substrates that contained only one OH group, such as coumaric- and ferulic acid, t-anol and coniferyl alcohol, were apparently not accepted by this activity. A Km for SAM was found to be 7.5 μM, the same for DMHF, caffeic acid and catechol.

Example 4: DMHF, DMMF and OMT activity in different varieties and maturation stages

Three strawberry varieties differing in their aroma properties were analyzed. Content of total volatiles, DMHF, DMMF, and OMT activity in three strawberry varieties was assessed during ripening. Content of these compounds sharply increased along fruit ripening with maximum values at the ripe stage. This is in agreement with Sanz et al. (In: Fruit flavors, biogenesis, characterisation, and authentication (Rouseff, R.L. and Leahy, M.M., eds). ACS Symposium Series 596, ACS, Washington, pp. 268-275, 1995) who found that in most cases content of these compounds sharply increased along fruit ripening with maximum values at the overripe stage. 'Malach' is an aromatic variety, and accumulates relatively high levels of total volatiles, including both DMHF and DMMF. This variety also contains the highest values of DMHF- specific OMT at the ripe stage (Fig. 3). Cell-free extracts from 'Malach' variety also displayed significant OMT activity when catechol and caffeic acid were used as substrates (Fig. 3). Interestingly, at the unripe green stage, no OMT activity was detected when using either DMHF, catechol or caffeic acid as acceptor substrates. 'Tamar' and 'Yael' varieties are much less aromatic, and this is reflected in the lower levels of total volatiles (about 1/10 of the levels found in 'Malach'). Still, cell-free extracts from ripe fruits of these two varieties displayed significant OMT activities towards DMHF, catechol and caffeic acid (Fig. 3). Interestingly, OMT activities were found only in red fruits, and cell-free extracts from unripe fruits of any of the three varieties analyzed were devoid of any OMT activity towards any of the three substrates analyzed. We also observed that the ratio of OMT activity towards caffeic acid, catechol and DMHF was the same in the enzyme extracts obtained from the cultivars Malach, Tamar und Yael (Fig 3). This implies that the ratios of three OMTs in three different varieties are the same or only one enzyme is capable to perform the methylation of the different substrates.

Caffeic acid is involved in anthocyanin pigmentation biosynthesis, that takes place concomitantly with ripening and DMMF formation in strawberry. Ripening-related gene sequences that code for proteins involved in key metabolic events including anthocyanins biosynthesis were isolated from strawberry (Manning, Planta, 205, 622-31, 1998), and were not found active in green fruits. Cyanidin, an anthocyanin precursor in strawberry contains the same ortho-di-hydroxyphenol structure typical for this OMT activity. Peonidin-glucoside, the o-methyl derivative of cyanidin glucoside, has been found in strawberry cell suspensions and strawberry fruits (Nakamura et al., Enzyme and Microbial Technology, 22, 404-408, 1998). It is assumed that DMHF methylation occurs as a side-effect action of caffeic acid, catechol and anthocyanidin methylation, that sharply increase during fruit ripening.

Example 5: Identification of OMT partial cDNAs from strawberry fruits

Poly(A)⁺ RNA was isolated from Fragaria x ananassa fruits in the pink ripening stage according to Manning (Anal. Biochem., 195, 45-50, 1991). Isolated poly(A)⁺ RNA was used to construct a cDNA library in the Uni-ZAP^® XR vector and it was amplified according to the manufacturer's instructions. SAM-OMT specific fragments were isolated by PCR using the undiluted phage suspension of the lambda library as a DNA template. The amplification primers were based on highly conserved sequences in plant OMTs representing the SAM binding site (primer pair MTsI/MTasD) (Frick and Kutchan (Plant J., 17, 329-339, 1999). In this manner, a PCR product of 106 bp in length was obtained which was used as a probe to screen 3xl0⁵ PFU of the cDNA library. The obtained positive plaques were purified twice and 24 clones were isolated after in vivo excision of the phagemid. Sequencing of the cDNAs revealed that all clones were full length clones with identical sequences in the 5' untranslated region (5'UTR) and the coding region. The majority of the cDNA inserts ranged from 1.3 to 1.6 kb whereas few cDNAs exhibited a prolonged 3'UTR resulting in a total length of approx. 1.9 kb. Northern analysis revealed that only the 1.9 kb messenger is originally present in turning/ripe strawberry tissue. The shorter clones were therefore judged to be artifacts, formed during the first-strand cDNA synthesis. For that reason, further investigations were focused on one of the longer cDNAs, which was designated STOMT (strawberry O-methyltransferase) This cDNA was 1876 bp in length with an open reading frame of 1098 bp, encoding a putative polypeptide of 366 amino acids and a predicted molecular weight of 39.817 kDa. A partial sequence of STOMT was already published by Manning, (Planta 205, 622-31, 1998) and was described as a ripening-related gene.

Example 6: Developmental expression of STOMT

Northern analysis was performed on eight different strawberry tissues: root, petiole, leaf, flower, green fruit, white fruit, turning fruit (from white to red) and ripe fruit to demonstrate the specific expression of STOMT (Fig. 4). The STOMT cDNA probe was random-prime-labeled and used to examine the expression pattern in the different tissues. STOMT is barely expressed in root, petiole, leaf and flower. Increasing expression was observed in the different developmental stages of the fruit. As expected, green and white fruits showed weak expression level whereas maximum level was reached in turning. Thus, STOMT is fruit-specific and ripening-related as the highest expression was observed in the maturing and ripe fruit.

Example 7: Heterologous expression of STOMT

The entire coding region of the STOMT cDNA was cloned in frame into the expression vector pGEX to produce a GST-fusion protein (gluthation S-transferase) in E. coli. The recombinant protein was isolated by GST-affinity chromatography and the OMT was released by thrombin cleavage. The successful expression of the fusion protein was monitored by SDS-PAGE confirming the calculated molecular weights of both the fusion protein (66 kDa) and the free O- methyltransferase (40 kDa) (Fig. 5). Concentrations of 50 μg/ml were obtained.

Example 8: Purification and characterization of the recombinant STOMT

For functional characterization of recombinant STOMT aliquots of the thrombin eluate were assayed for their methylation activity. The tested substrates included compounds which are natural constituents of strawberry fruits such as caffeic acid, caffeoyl-CoA and DMHF, as well as those which have not been detected in strawberry fruits until now e.g. catechol and protocatechuic aldehyde. Catechol, caffeic acid and caffeoyl-CoA were used as substrates that represented the major classes of compounds transformed by OMTs. All substrates share an aromatic system substituted by two adjacent hydroxy groups. DMHF may also be considered a heterocyclic aromatic compound as one of its tautomeric structures is formally a dienolic furan (Rodin et al., J. Food Sci. 30, 280-285, 1965) (Fig. 1) resembling a o-diphenol structure. The kinetic constants were measured at a saturated and constant concentration of SAM and evaluated according to Hanes (Cornish-Bowden, A., 1995 Fundamentals of enzyme kinetics, Portland Press, London). All tested compounds were accepted as substrate by STOMT. The apparent K_m values of the phenolic substrates ranged from 66 to 150 μM comparable with the results obtained with the crude strawberry enzyme whereas DMHF exhibited an K_m of 440 μM which is one order of magnitude lower than for the native enzyme isolated from strawberry as depicted in table 1.

Table 1. Substrate specificity of partially purified native OMT isolated from strawberry fruits and recombinant enzyme STOMT.

Substrate K_m (μM) K_m (μM) V_max (pkat*mg^_1) product native recombinant recombinant

Protocatechuic aldehyde nd 99.5 32.57 Vanillin Caffeic acid 145 146 19.21 Ferulic acid

Catechol 120 169.5 1.33 Guaiacol Caffeoyl CoA nd 66 0.71 Feruloyl CoA

DMHF 5000 440 0.53 DMMF

SAM 7.5 nd nd SAH

Enzyme assays were carried out using 1.7 - 3.8 μg of the purified recombinant protein or 25 μg of the strawberry protein, 1 μM-1 mM phenolic substrates or 0.1-10 mM DMHF and 0.05 μCi ¹⁴C-SAM. Kinetic parameters were determined according Hanes (Cornish-Bowden, 1995) Fundamentals of enzyme kinetics, Portland Press, London), nd, not determined

If we assume that the dienolic tautomer of DMHF (Fig 1) exhibits the correct conformation for successful conversion by STOMT, it is obvious that the K_m-value of DMHF has to be much higher in comparison to other o-diphenolic substrates. Moreover, the o-diphenolic compounds showed the typical course of a Michealis-Menten kinetic while a different behavior was observed for DMHF (Fig. 6). It seems that high amounts of DMHF inhibit STOMT as maximum enzyme activity was observed at 10 mM DMHF whereas at higher concentrations (> 15 mM) the activity decreased.

STOMT methylates rapidly protocatechuic aldehyde and caffeic acid, which is demonstrated by the specific activities of 2.0 and 1.6 nmo^min^'^mg^"1 respectively. The other substrates were converted less efficiently to their methylated counteφarts, recognizable in specific activities of 87 to 32 pmol* min^'^rng^"1. Catechol seems to be rather well accepted, while caffeoyl-CoA and DMHF are methylated to a lesser extend. Radio-HPLC analysis of the formed radioactively labeled compounds showed that only mono-methylated products were produced. The broad substrate specificity was very suφrising and was observed for the first time for an enzyme involved in phenylpropanoid formation and strawberry fruit flavor biosynthesis.

The features, aspects and embodiments of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

Claims

1. An isolated polypeptide capable of methylating an ortho-dihydroxy substituted ring system, whereby the ring system preferably is an aromatic, or heteroaromatic ring system or furanone based ring system, and mimetics of said ortho-dihydroxy substituted ring system, selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 1 to 365 for the mature polypeptide of SEQ ED NO. 2;

(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) the open reading frame coding for SEQ JJD No 2 within the nucleotide sequence SEQ DD No. 1, (ii) a subsequence of (i) of at least 100 nucleotides, preferably of at least 100 consecutive nucleotides, or (iii) a complementary strand of (i), or (ii);

(c) a polypeptide which is encoded by a nucleic acid having a nucleic acid sequence according to SEQ. ID. No. 1 or SEQ. ED. No. 3;

(d) an allelic variant of (a), (b), or (c) and

2. The polypeptide of claim 1, having an amino acid sequence which has at least 70% identity with amino acids 1 to 365 of SEQ ED NO. 2.

3. The polypeptide of claim 2, having an amino acid sequence which has at least 80% identity with amino acids 1 to 365 of SEQ ED NO. 2.

4. The polypeptide of claim 3, having an amino acid sequence which has at least 90% identity with amino acids 1 to 365 of SEQ ID NO. 2.

5. The polypeptide of claim 4, having an amino acid sequence which has at least 95% identity with amino acids 1 to 365 of SEQ ED NO. 2.

6. The polypeptide of claim 1, which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) the open reading frame of SEQ ED NO. 1, that encodes SEQ ED No. 2, (ii) the nucleic acid having a nucleic acid sequence according to SEQ. DD. No. 3, (iii) a subsequence of (i) or of (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii) or (iii).

7. The polypeptide of any of claims 1 to 6, wherein the methylating is a methylation of 4- hydroxy-3(2H)-furanones substrates.

8. The polypeptide of any of claim 1 to 6, wherein the ortho-dihydroxy substituted ring system is selected from the group comprising catechol, caffeic acid, protocatechuic aldehyde and pyrogallol.

9. The polypeptide of any of claims 1 to 7, wherein the mimetics of ortho-dihydroxy substituted ring system is selected from the group comprising DMHF and DTT.

10. The polypeptide of any of claims 1 to 9, which is capable of converting DMHF into DMMF by O-methyltransferase activity.

11. An enzyme comprising a polypeptide according to any of claims 1 to 10.

12. An enzyme, preferably an isolated enzyme, derived from Strawberries, characterized by:

(i) O-methyltransferase activity,

(ii) Conversion of DMHF into DMMF

(iii) Conversion of protocatechuic aldehyde to vanillin.

13. The enzyme of claim Ϊ2, further characterized by:

(i) A temperature optimum of about 37°C, (ii) about 90% enzymatic activity at about 30°C,

(iii) Temperature-dependent irreversible inhibition at about 45°C.

14. The enzyme of claims 12 or 13, further characterized by:

(i) A pH optimum of about 8.5,

(ii) essentially no enzymatic activity at pH values significantly below about pH 6 or significantly above about pH 10.

15. The enzyme according to any of claims 12 to 14, further characterized by a molecular weight of about 80 kDa when measured by gel filtration, or about 40 kDa when measured by SDS-PAGE.

16. The enzyme according to any of claims 12 to 15, further characterized by being essentially independent of the presence of divalent metal ions.

17. The enzyme according to any of claims 12 to 16, further characterized by being essentially independent of the presence of divalent Ca, Mg, Co, Mn, Zn, or Fe ions.

18. The enzyme according to claim 17 , further characterized by its enzymatic activity being about 10% inhibited in the presence of about ImM to about 10 mM of said cations.

19. The enzyme according to any of claims 12 to 18, further characterized by having an apparent Km value for DMHF of about 5mM.

20. The enzyme according to any of claims 12 to 19, further characterized by having an apparent Km value for catechol of about 120 μM.

21. The enzyme according to any of claims 12 to 20 , further characterized by having an apparent Km value for caffeic acid of about 145 μM.

22. The enzyme according to any of the proceeding claims, characterized by comprising a polypeptide according to any of claims 1 to 11.

23. A process for the production of a peptide according to any one of claims 1 to 10 or of an enzyme according to any one of claims 11 to 22, comprising the steps of

(i) inserting the region of SEQ ID No 1 coding for SEQ ED No 2, or a nucleotide sequence coding for the peptide of any one of claims 1 to 10 or for the enzyme of any one of claims 11 to 22, into an expression vector comprising, in operable sequence, a promoter, optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers,

(ii) introducing said vector into a cell,

24. The process of claim 23, wherein the cell is derived from a bacteria, insect, fungus, animal, or plant.

25. The process of claim 24 wherein said cell is derived from yeast.

26. The process of claim 25 wherein said cell is a cell selected from a cell derived from the strain Pichia pastoris X-33, Pichia pastoris GS115, or Pichia pastoris KM71H, or is a cell of the strain Pichia pastoris X-33, Pichia pastoris GS115, or Pichia pastoris KM71H.

27. The process of claim 24 wherein said cell is an insect cell and said vector is a baculovirus-derived vector.

28. The process of claim 27 wherein said cell is SF9, or HI5.

29. The process of claim 24 wherein said cell is derived from bacteria.

30. The process of claim 29 wherein said cell is derived from E.coli.

31. The process of claim 30 wherein the cell is a BL21 cell.

32. The process according to any of claims 23 to 31 wherein said vector comprises a second coding region for a second peptide which is fused to the coding region coding for SEQ ID No. 2, or to a coding region coding for the peptide according to any one of claims 1 to 10 or to the coding region coding for the enzyme of any one of claims 11 to 22.

33. The process of claim 32, wherein said second coding region codes for a signal peptide.

34. The process of claim 33, wherein said signal peptide causes the protein or peptide which it is fused to, to be transported into an intracellular or extracellular compartment, preferably into the periplasmatic space.

35. The process of claim 32, wherein said second coding region codes for a peptide for which a specific binding partner exists.

36. The process of claim 35, wherein said specific binding partner is an antibody.

37. The process of claim 36, wherein said vector is pGEX, wherein said second coding regions codes for Glutathion-S-transferase, and the process of isolating the peptide comprises the use of GST-affinity chromatography and release of the O-methyltransferase activity containing peptide by thrombin cleavage.

38. The process of claim 36 wherein said vector is pGEX, wherein said second coding regions codes for a Histidine-tag, and the process of isolating the peptide comprises the use of Histidine affinity chromatography.

39. A process for obtaining an antibody against a polypeptide of any of claims 1-10 or against an enzyme of claims 11-22, comprising

(i) providing said polypeptide or enzyme, or an immunogenic part thereof, in substantially purified form, (ii) introducing said polypeptide or enzyme, or said immunogenic part thereof, into a non-human animal capable of producing antibodies, (iii) optionally, introducing adjuvants into said non-human animal, (iv) optionally, repeating step (ii) and optionally, step (iii), (v) isolating antibodies or cells that produce said antibodies from said non- human animal.

40. A process for obtaining an antibody against a polypeptide of any of claims 1-11 or against an enzyme of claims 11-22, comprising

(i) providing said polypeptide or enzyme, or a part thereof, in substantially purified form,

(ii) bringing said polypeptide or enzyme, or said part thereof into contact with a variety of cells, each of which containing a gene for the production of an antibody, and wherein each cell is producing said antibody,

(iii) selecting cells that bind to said polypeptide or enzyme, or part thereof,

(iv) using said cells to produce said antibody.

41. An antibody against the peptide of any of claims 1-10 or against the enzyme of any of claims 11-22.

42. A transgenic plant with enhanced flavor comprising a genetically engineered DNA sequence encoding at least one polypeptide having an O-methyltransferase activity, wherein said polypeptide is encoded by a nucleotide sequence as described in any of claims 1-10, or by SEQ ID No. 1 or SEQ. ID. No. 3.

43. The transgenic plant according to claim 42, wherein said nucleotide sequence is a heterologous nucleotide sequence.

44. The transgenic plant according to claim 42, wherein the nucleotide sequence codes for SEQ ED No. 2, or a variant, homologue or functional part thereof.

45. The transgenic plant of claim 44 wherein the nucleotide sequence codes for SEQ ED No.

2.

46. The transgenic plant according to any of claims 42-45, wherein the plant is selected from the group consisting of strawberry, mango, arctic brambles, tomato, cucumber, soya, sweet potato, grape, rapeseed, sugar beet, cotton, tea, sunflower, rose, chrysanthemum, sweet pepper, potato, raspberry, Vanilla planifolia, tobacco, and Arabidopsis thaliana and wherein preferably a substrate for the O-methyltransferase is present.

47. A propagating material derived from the transgenic plant according to any of claims 42- 46.

48. A process for the production of a transgenic plant with enhanced flavor and/or taste, comprising:

(i) preparing a genetically engineered DNA sequence encoding at least one polypeptide having an O-methyltransferase activity,

(ii) operably linking the DNA sequence to a promoter for said DNA;

(iii) transforming a transformable, regenerable plant cell to contain the operably linked DNA sequence of step(ii), and

(iv) regenerating said plant cell to produce a transgenic plant.

49. The process according to claim 48, wherein said DNA sequence is contained in a vector under the control of a promoter allowing its expression in said transgenic plant.

50. The process according to claims 48 and 49, wherein said introduction is carried out by transfection using the Agrobacterium system.

51. The process according to any of claims 48-50, wherein the polypeptide is the polypeptide according to any of claims 1 to 10 or the enzyme according to any of claims 11 to 22.

52. A method for enhancing flavor and/or taste in a plant comprising:

(i) preparing a genetically engineered DNA sequence encoding at least one polypeptide having a O-methyltransferase activity, (ii) operably linking the DNA sequence to a promoter for said DNA; (iii) transforming a transformable, regenerable plant cell to contain the operably linked DNA sequence of step (ii).

53. The process according to claim 52, wherein the polypeptide is the polypeptide according to any of claims 1 to 10 or the enzyme according to any of claims 11 to 22.

54. A method for the methylation of an ortho-dihydroxy substituted ring system and/or mimetics of such ortho-dihydroxy substituted ring system, whereby preferably the ring system is an aromatic ring system or a heteroaromatic ring system or a furanone based ring system, comprising the following steps:

55. The method according to claim 54, wherein the methyl group donor is S-adenosyl-L- methionine.

56. The method according to claim 54 or 55, wherein the ortho-dihydroxy substituted ring system is selected from the group comprising catechol, caffeic acid, protocatechuic aldehyde and pyrogallol.

57. The method according to claim 54 or 55, wherein the mimetic of the ortho-dihydroxy substituted ring system is a 4-hydroxy-3(H)-furanone.

58. The method according to any of claims 54 to 57, wherein the methylation is selected from the group comprising

- methylation of protocatechuic aldehyde to vanillin

- methylation of caffeic acid to ferulic acid

- methylation of catechol to guaiacol

- methylation of caffeoyl-CoA to feruloyl CoA, and

- methylation of DMHF to DMMF.

59. The method according to any of claims 54 to 58, wherein the methylation is carried out in an aqueous solution, preferably in an aqueous buffer.

60. The method according to any of claims 54 to 59, wherein the reaction is carried out with the enzyme or the peptide being immobilized onto a surface.