Flavonoids are the most widespread group of secondary metabolites found in plants, occurring in species ranging from mosses to angiosperms (Harborne, 1988). The generic term "flavonoid"describes a number of different polyphenolic compounds, all of which contain the same basic carbon skeleton.

The flavonoid skeleton is shown in Figure 1.

PRIOR ART Plants synthesize a range of flavonoids which may vary in respect of the degree of hydroxylation of, and subsequent modifications to, the basic flavonoid skeleton. Modifications to the flavonoid skeleton include methylation, glycosylation with a range of different sugar groups and acylation with aromatic or aliphatic acids.

The flavonoid biosynthetic pathway The biosynthetic pathway leading to the synthesis of anthocyanins (the main pigments in flowers), flavones and flavonols (co-pigments) and proanthocyanidins is well established in parts (reviewed by Heller and Forkmann, 1988; Stafford, 1990).

A basic pathway is shown in Figure 2.

The first step in flavonoid biosynthesis is catalysed by chalcone synthase (Ebel and Hahlbrock, 1982; Reimold et al., 1983). In most plants of commercial importance, chalcone synthase catalyses the condensation of three molecules of malonyl-CoA with one molecule of 4-coumaroyl-CoA (Ebel and Hahlbrock, 1982).

The product of this reaction, 4,2', 4', 6'-tetrahydroxychalcone is isomerised by the enzyme chalcone isomerase to produce (2S)- naringenin. The stereochemistry at C-2 of all flavonoid

compounds is established at this step (Heller et al., 1979; Ebel and Hahlbrock, 1982) [ (2S)-naringenin is generally considered to be the natural isomer of this compound, (2R)- naringenin is rarely encountered in nature].

(2S)-naringenin is a substrate for several different enzymes including the flavone synthase (Britsch, 1990b; Stotz and Forkmann, 1981), flavonoid 3'-hydroxylase (product is eriodictyol-see Hagmann et al., 1983), flavonoid 3', 5'- hydroxylase (product is 5,7,3', 4', 5'-pentahydroxyflavanone- see Tanaka et al., 1996) and flavanone 3ß-hydroxylase (Britsch and Grisebach, 1986). The flavonoid 3'-and 3', 5'-hydroxylases may also catalyse corresponding modifications using flavones, dihydroflavonols and flavonols as substrates. The action of these two flavonoid hydroxylases creates a biosynthetic grid system, rather than a linear pathway.

(2S)-naringenin is converted to apigenin by the enzyme flavone synthase (Britsch, 1990b). Flavones are the end products of a side-branch of the main flavonoid biosynthetic pathway. (2S)- naringenin is also converted into (2R, 3R)-dihydrokaempferol by the action of the enzyme, flavanone 3B-hydroxylase (Britsch and Grisebach, 1986; Britsch et al., 1992). The flavanones, eriodictyol and 5,7,3', 4', 5'-pentahydroxyflavanone, may also be substrates for the flavone synthase (products are luteolin and tricetin respectively) and the flavanone 3ß-hydroxylase [products are (2R, 3R)-dihydroquercetin and (2R, 3R)- dihydromyricetin respectively].

The dihydroflavonols, (2R, 3R)-dihydrokaempferol, -dihydroquercetin and-dihydromyricetin, can be reduced to their respective leucoanthocyanidins, 3,4-cis- leucopelargonidin,-leucocyanidin and-leucodelphinidin by the action of dihydroflavonol 4-reductase (Heller et al., 1985a, b) The leucoanthocyanidins are converted to the anthocyanidins, pelargonidin, cyanidin and delphinidin probably by the action of a single enzyme, the anthocyanidin synthase. Although putative clones of this enzyme exist, the activity encoded by these clones has never been demonstrated (Menssen et al., 1990) Anthocyanidins are unstable compounds which are stabilised within the cell by further modification eg. glycosylation (reviewed in Heller and Forkmann, 1988).

Dihydroflavonols are also substrates for the enzyme flavonol

synthase (Holton et al., 1993). (2R, 3R)-dihydrokaempferol,- dihydroquercetin,-dihydromyricetin are converted to the flavonols, kaempferol, quercetin and myricetin respectively.

Flavonols are also the end products of a side-branch of the main flavonoid biosynthetic pathway.

Proanthocyanidins are end-products of a third side-branch of the main flavonoid biosynthetic pathway.

Interestingly, whilst the majority of anthocyanins found in plants have 2,3-trans-stereochemistry, the commonest proanthocyanidin monomers found in plants, (-)-epicatechin and (-)-epigallocatechin, have 2,3-cis-stereochemistry (reviewed in Stafford, 1990). No enzyme has been identified which will either synthesize flavonoids with 2,3-cis-stereochemistry or will use such flavonoids as substrates.

The diversity of flavonoid structures is reflected in their multi-functionality in plant biology as colouring agents in fruits, flowers and seeds; as signalling molecules in development, pathogenesis, symbiosis and reproduction and as defence compounds against both environmental and biotic stresses (reviewed in Shirley, 1996; Koes et al., 1993).

Published reports of flavonoid biosynthetic enzymes Genes encoding the following enzymes from the main flavonoid biosynthetic pathway have been cloned:- (i) chalcone synthase (Reimold et al., 1983), (ii) chalcone isomerase (Mehdy and Lamb, 1987), (iii) flavanone 3ß-hydroxylase (Britsch et al., 1992), (iv) dihydroflavonol 4-reductase (O'Reilly et al., 1985) (v) flavonol synthase (Holton et al., 1993).

Genes encoding flavanone 3B-hydroxylases have been isolated from a range of species (Britsch et al., 1992,1993). This enzyme catalyses the stereospecific addition of a hydroxyl group to the 35 position of C-3 (Britsch and Grisebach, 1986).

This enzyme introduces the 2,3-trans-stereochemistry found in the majority of anthocyanidins, leucoanthocyanidins and dihydroflavonols. The only flavanone 3ß-hydroxylase to have been studied in detail at the biochemical level is the enzyme from Petunia (Britsch and Grisebach, 1986; Britsch, 1990a;

Britsch et al., 1992; Lukacin and Britsch, 1997).

Genes thought to encode flavonol synthases have been isolated from several species (Holton et al., 1993; Pelletier et al., 1997; van Eldik et al., 1997). The only genes which have been shown unequivocally to encode a flavonol synthase are those isolated from Petunia and Chrysanthemum which catalysed the synthesis of quercetin from dihydroquercetin and kaempferol or quercetin from dihydrokaempferol or dihydroquercetin respectively when expressed as recombinant proteins in yeast (Holton et al., 1993; WO 94/03606). Pelletier et al., (1997) suggest that a number of Arabidopsis thaliana flavonol synthase isozymes, having different substrate specificities, control the amount and types of flavonols present in a given tissue.

Flavanone 3ß-hydroxylase and flavonol synthase are members of the superfamily of enzymes called 2-oxoglutarate-dependent dioxygenases (reviewed by Prescott, 1993 and Prescott and John, 1996). These enzymes require ferrous iron, ascorbate and 2- oxoglutarate for maximal rates of activity in vitro.

The 2-oxoglutarate-dependent dioxygenases are characterised at the protein level by the presence of a number of conserved motifs and amino acid residues (reviewed by Prescott, 1993; Prescott and John, 1996). Flavonol synthases and flavanone 3R- hydroxylases are typical members of the family, containing all of the expected conserved residues e. g. the two conserved histidine residues which are thought to be iron ligands and the conserved arginine residue which may be involved in binding 2- oxoglutarate (see Hegg and Que, 1997; Lukacin and Britsch, 1997).

The sequence of the gene thought to encode a flavonol synthase from the species Arabidopsis thaliana has been placed in the nucleic acid databases under the following accession numbers U72631 (genomic sequence from Landsberg erecta), U84258 (genomic from Columbia), U84259 (cDNA from Columbia), U84260 (cDNA from Landsberg erecta). The sequence has also been published in Pelletier et al., (1997).

Similarly a putative A. thaliana flavanone 3ß-hydroxylase sequence has been published by Pelletier & Shirley (1996) Plant Physiology 11: 339-345. These two enzymes from A. thaliana are however only 47E identical at the nucleic acid level, and 322

identical (58% similar) at the amino acid level to each other.

The enzymes encoded by these clones have not been shown to have any biological activity.

Uses of flavonoid biosynthetic enzymes A plant species may not contain all of the enzymes described previously, eg. Petunia flowers do not make flavones as they lack a flavone synthase. Similarly, roses lack a flavonoid 3', 5'-hydroxylase, causing an inability to make blue/purple pigments in this species (reviewed in Elomaa and Holton, 1994).

Whilst many species contain flavonoids with a 5,7-hydroxylation pattern of the A ring (the phloroglucinol type), some lack the hydroxyl group at C-5 (the resorcinol type found in Leguminosae and Anancardiaceae). These are the two commonest types of flavonoid skeleton, although others have been reported (see Harborne, 1988). Enzymes isolated from plants containing only phloroglucinol flavonoids are unable to use the analogous resorcinol flavonoids as substrates (reviewed by Heller and Forkmann, 1988).

Enzymes isolated from species lacking 5'-hydroxylated flavonoids eg (2R, 3R)-dihydromyricetin show little or no activity with 5'-hydroxylated substrates (reviewed in Elomaa and Holton, 1994). Conversely the presence of a particular flavonoid and an appropriate enzyme within a plant does not always mean that the flavonoid can be utilised as a substrate by the enzyme. For example, the dihydroflavonol 4-reductase from Petunia uses only (2R, 3R)-dihydroquercetin and- dihydromyricetin as substrates and is unable to use the (2R, 3R)-dihydrokaempferol which is also present in the cell (Meyer et al., 1987).

Thus the level of different flavonoids synthesised by any species is dependent on which individual biosynthetic enzymes are present within a particular cell, in what amounts they are present, and what the catalytic specificity, substrate specificity, and specific activity of the enzymes are.

Thus a number of genes encoding enzymes involved in flavonoid biosynthesis have been used in transgenic plants to manipulate the levels of endogenous flavonoid compounds or to cause the synthesis of flavonoids not native to the species in question (reviewed by Elomaa and Holton, 1994).

Publication WO 94/03606 (International Flower Developments Pty. Ltd.) disclosed a flavonol synthase enzyme from Petunia hybrida and discussed the use of flavonol synthases (FLS) to modulate the metabolism of dihydroflavonols (dihydrokaempferol, dihydroquercetin and dihydromyricetin-see page 3, lines 8- 16). The aim of such modulation was to manipulate flower colour.

It can be seen from the forgoing that the characterisation of flavonoid biosynthesis enzymes (and/or the nucleic acids encoding them) which demonstrate novel catalytic activities or specificities would provide a contribution to the art.

DISCLOSURE OF THE INVENTION The present inventors have demonstrated that a recombinantly expressed, plant-derived, flavonoid biosynthetic enzyme, previously described only as a flavonol synthase (FLS), is capable of exhibiting catalytic specificities in addition to flavonol synthase activity. Some of these catalytic specificities are not found in any existing cloned enzymes.

Briefly, the inventors used a recombinant protein expressed from a cDNA clone thought to encode the flavonol synthase from Arabidopsis thaliana. The recombinant protein was produced in a native form in Escherichia coli. The purified recombinant enzyme exhibited multiple catalytic specificities when assayed in a reaction containing a flavanone as a substrate (together with 2-oxoglutarate as a cosubstrate and ferrous iron and ascorbate as cofactors). These catalytic specificities were: (i) flavone synthase activity, (ii) flavanone 3a-hydroxylase activity (iii) flavanone 3ß-hydroxylase activity (iv) flavonol synthase activity This is the first time a flavonoid biosynthetic enzyme has been shown to possess multiple catalytic specificities. Individual characterised genes which appear in the literature have in all cases been ascribed only a single catalytic specificity, albeit in some cases using a variety of substrates.

Thus the demonstration has implications for the use of flavonol synthases, and other enzymes which share motifs with these enzymes and may therefore share the demonstrated properties, in manipulating flavonoid biosynthesis. It is particularly of

interest in respect of the manipulation of reactions other than those previously ascribed to any cloned enzyme, or in cases where it may be desirable to manipulate multiple catalytic specificities. In particular: This is the first enzyme with flavone synthase activity for which the gene has been cloned. Thus it opens up the possibility synthesising flavones in species which do not normally synthesize these flavonoids.

It is also the first enzyme with flavanone 3a-hydroxylase activity to be described and thus it may provide a means to manipulate the levels of (-) -epicatechin and (-)- epigallocatechin in plants.

Interestingly, the enzyme has been shown to utilise substrates which are not found in its plant of origin specifically those based on a resorcinol skeleton [(2S)-liquiritigenin, (2R, 3R)- dihydrofisetin, (2R, 3R)-dihydrorobinetin]; 5'-hydroxylated dihydroflavonols [(2R, 3R)-dihydromyricetin, (2R, 3R)- dihydrorobinetin)], 4'-hydroxyflavanone and the rare isomer, (2R)-naringenin. Thus it is possible to use this enzyme to manipulate the flavonoid pathway in transgenic plants which contain a different range of flavonoids to those found in Arabidopsis. Indeed it may also be possible to use this enzyme to make a wide range of flavonoid compounds in vitro, including a number which have not been found in nature.

The FLS enzyme shares a number of conserved motifs with other, characterised, FLS enzymes. Thus, in the light of the present disclosure, it is expected that other enzymes having these motifs will also exhibit multiple catalytic specificities.

These and other aspects of the present invention will now be discussed in more detail.

Generally the invention provides various methods of influencing a flavonoid biosynthetic catalytic activity in a cell (preferably a plant cell) which methods comprise the step of modifying in that cell the activity (e. g. nature or concentration) of an enzyme as discussed above e. g. an enzyme exhibiting one or both of (i) flavone synthase activity; (ii) flavanone 3a-hydroxylase activity. Such methods will usually form a part of, possibly one step in, a method of producing a flavonoid, or modifying the production of a flavonoid, in a

plant.

Thus in one aspect of the present invention there is disclosed a method of producing a flavonoid, or modifying the production of a flavonoid, said method comprising the step of using an enzyme exhibiting multiple catalytic specificities such as to manipulate a plurality of catalytically distinct reactions involved in flavonoid biosynthesis.

The methods of the present invention embrace both the in vitro and in vivo production, or manipulation, of one or more flavonoids.

As discussed in more detail below, in this and other aspects of the invention, when used in vitro the enzyme will generally be in isolated, purified, or semi-purified form. Optionally it will be the product of expression of a recombinant nucleic acid molecule.

Likewise the in vivo methods will generally involve the step of causing or allowing the transcription of, and then translation from, of a recombinant nucleic acid molecule encoding the enzyme.

Generally, in manipulating flavonoid production, the enzyme will be used in conjunction with other enzymes. The substrates (including co-substrates) which are appropriate for the plurality of catalytic reactions may be supplied directly to the enzyme, or may themselves be the products of other enzymes, or other reactions catalysed by the same enzyme. The methods of the present invention are carried out in the presence of any co-factors required by the enzyme to catalyse one or more of the plurality of catalytic reactions. In particular ferrous iron and 2-oxoglutarate may be supplied directly or as the products of other reactions.

The B rings of the flavonone substrates which are the substrates or products of the plurality of catalytic reactions will preferably have a single hydroxyl group at the C-4' position. The B rings of dihydroflavonol substrates may have one or more hydroxyl groups positioned at, for example, C-3', C-4'or C-5'.

By"multiple catalytic specificity"is meant that the enzyme is capable of catalysing several quite different reactions,

optionally with the same substrate. This should be contrasted with a"broad specificity"enzyme which catalyses a single reaction with a number of substrates, to yield a number of corresponding products, although it is particularly envisaged that the enzyme used in the methods of the present invention may optionally have both of these qualities. The several different (catalytically distinct) reactions will generally correspond to those already ascribed to single enzymes in the flavonoid biosynthesis pathway: Thus, preferably, the encoded enzyme will exhibit 2 or more, preferably 3 or 4 of: (i) flavone synthase activity, (ii) flavanone 3a-hydroxylase activity (iii) flavanone 3ß-hydroxylase activity, (iv) flavonol synthase activity. and will be used to manipulate 2 or more, preferably 3 or 4 of the corresponding catalytic activities.

In a further aspect of the present invention there is disclosed use of the flavonol synthase as any one or more of: (i) a flavone synthase, (ii) a flavanone 3a-hydroxylase (iii) a flavanone 3ß-hydroxylase In further aspects of the present invention there are disclosed: A method of producing a flavonoid, or modifying the production of a flavonoid, said method comprising use of a nucleic acid molecule encoding an enzyme having flavone synthase activity.

A method of producing a flavonoid, or modifying the production of a flavonoid, said method comprising use of an enzyme as a flavanone 3a-hydroxylase (optionally via use of a nucleic acid molecule encoding the same). Preferably the enzyme is used to manipulate the levels of (-) -epicatechin and (-)- epigallocatechin in plants.

In the methods described above, the enzyme may use either resorcinol or phorglucinol flavonoids. The substrates used in the reaction may be selected from those that are not used naturally'by the enzyme e. g. which do not occur in its

organism of origin, particularly as regards stereochemistry of isomers. These artificial'substrates may be useful in producing novel flavonoids not found in nature.

In particular, in the methods described above, the enzyme may use any one or more of the following as substrates: (i) (2S)-liquiritigenin, (ii) (2R, 3R)-dihydrofisetin, (iii) (2R, 3R)-dihydrorobinetin, (iv) (2R, 3R)-dihydromyricetin, (v) (2R, 3R)-dihydrorobinetin, (vi) 4'-hydroxyflavanone, (vii) (2R)-naringenin, (viii) (2S)-naringenin, (ix) (2R, 3R)-dihydrokaempferol, (x) (2R, 3R)-dihydroquercetin, (xi) 3,4', 7-trihydroxyflavone Preferably the amino acid sequence of the enzyme used in the methods above is a flavonoid dioxygenase containing 1,2,3, or preferably all of the following motifs: i) IP (X) E (X) IR (X) E (X) EQPA (X) TT ii) AS (XX) WG (X) FQ (XX) NHGIP (X) E iii) WPPS (XX) NY (XX) WPK (X) P (XX) YR (XX) NEEY iv) SNG (X) YK (X) V (X) H (XX) TV (X) K (XX) TR (X) SWP wherein X'can be any amino acid, or may optionally represent the site of insertion or deletion of an amino acid. These motifs appear to be present in a number of enzymes designated as flavonol synthases. Preferably the enzyme is itself a flavonol synthase, and most preferably it is a flavonol synthase from Arabidopsis thaliana.

As mentioned above, the methods of the present invention may include the step producing the enzyme recombinantly i. e. causing or allowing the transcription of a recombinant nucleic acid encoding the enzyme.

Nucleic acid according to the methods of the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e. g. peptide nucleic acid). Where a DNA sequence is specified, e. g. with reference to a figure,

unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.

Nucleic acid molecules according to the present invention may be used isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term"isolated"encompasses all of these possibilities.

The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e. g. using an automated synthesiser.

The sequence of a flavonol synthase cloned by the present inventors is given in Figure 3. The nucleic acid encoding the enzyme is hereinafter termed Seq ID No 1, while the amino acid sequence is termed Seq ID No 2. Preferably the nucleic acid used in the methods of the present invention comprises all or part of Seq ID No 1.

Optionally the nucleic acid (and therefore enzyme) may be a variant of the sequences provided.

A variant nucleic acid molecule shares homology with all or part of Seq ID No 1 discussed above and encodes an enzyme having the required multiple catalytic specificity, or other activity, as appropriate to the various aspects of the invention discussed above. The activity can be tested by analysis of the flavonoid products resulting from the catalyses, for instance by methods analogous to those described hereinafter using HPLC and UV spectra.

Sequence variants may occur naturally, for instance as alleles (which will include polymorphisms or mutations at one or more bases) or pseudoalleles (which may occur at closely linked loci to the flavonol synthase gene). Also included within the scope of the present invention would be use of isogenes, or other homologous genes belonging to the same family as the flavonol synthase gene, and encoding isozymes thereof with the requisite activity.

One method for isolating such variants having appropriate homology may employ a probing approach. A commonly used formula by those skilled on the art for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989): Tm = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C)-0.63 (% formamide) -600/#bp in duplex As an illustration of the above formula, using [Na+] = [0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57°C. The Tm of a DNA duplex decreases by 1-1.5°C with every 1% decrease in homology.

Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.

Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.

It is also well known in the art that the stringency of hybridisation can be increased to reduce the number of positive targets. Suitable conditions include, e. g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na2HPO4, pH 7.2,6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0.1X SSC, 0.1% SDS.

For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na2HPO4, pH 7.2,6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.

Artificial variants (derivatives) may be prepared by those skilled in the art, for instance by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid is generated either directly or indirectly (e. g. via one or more amplification or replication steps) from an original nucleic acid encoding a naturally occuring flavonol synthase, e. g. nucleic acid having Seq ID No 1.

The term variant'nucleic acid as used herein encompasses all of these possibilities. When used in the context of polypeptides or proteins it indicates the encoded expression product of a variant nucleic acid.

Similarity or homology may be as defined and determined by the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standard use in the art, or, and this may

be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711). BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.

Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares homology with Seq ID NO 1 and 2 respectively, most preferably at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.

Homology may be over the full-length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about 20,25,30,33,40,50, 67,133,167,200,233,267,300,333 or more amino acids or codons, compared with the relevant amino acid sequence or nucleotide sequence as the case may be.

Thus a variant amino acid sequence in accordance with the present invention may include within the sequence shown in Seq ID No 2, a single amino acid change or 2,3,4,5,6,7,8, or 9 changes, about 10,15,20,30,40 or 50 changes, or greater than about 50,60,70,80 or 90 changes. In addition to one or more changes within the amino acid sequence shown in Seq ID No 2, a variant amino acid sequence may include additional amino acids at the C-terminus and/or N-terminus. Naturally, changes to the nucleic acid sequence which make no difference to the encoded amino acid sequence (i. e. degeneratively equivalent') are included.

Changes to a sequence, to produce a derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.

Changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; codon usage; other sites which are required for post translation modification; cleavage sites in

the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipoylation etc. Leader or other targetting sequences may be added to the expressed protein to determine its location following expression. All of these may assist in efficiently cloning and expressing an active polypeptide in recombinant form (as described below).

Other desirable mutations may be fashioned by random or site directed mutagenesis in order to alter the activity (e. g. specificity) or stability of the encoded polypeptide.

Changes may be by way of conservative variation, i. e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out.

This is so even when the substitution is in a region which is critical in determining the peptides conformation.

Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure. In regions which may be critical in determining the peptide's conformation or activity (e. g. the conserved motifs discussed above) such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e. g. altered stability or specificity.

In a further aspect of the present invention there is disclosed a method of modifying one or more of the following activities of an enzyme having multiple catalytic specificity, (i) flavone synthase activity, (ii) flavanone 3a-hydroxylase activity (iii) flavanone 3ß-hydroxylase activity,

said method including the step of modifying a nucleic acid encoding the enzyme. Preferably the enzyme is a flavonol synthase, most preferably the nucleic acid which is modified has the sequence shown in Seq ID No 1.

Preferably the nucleic acid is modified such as to alter one of the following motifs within the amino acid sequence of the enzyme encoded by the nucleic acid: (i) IP (X) E (X) IR (X) E (X) EQPA (X) TT (ii) AS (XX) WG (X) FQ (XX) NHGIP (X) E (iii) WPPS (XX) NY (XX) WPK (X) P (XX) YR (XX) NEEY (iv) SNG (X) YK (X) V (X) H (XX) TV (X) K (XX) TR (X) SWP A further aspect of the present invention provides a method of identifying and/or cloning nucleic acids encoding enzymes having multiple catalytic specificity, or other activity described above which method employs a nucleic acid encoding a flavonol synthase, most preferably having all or part of Seq ID No 1 (particularly a part encoding one or more of the motifs discussed above).

When producing enzymes as discussed above for in vitro use, or when the methods are performed in vivo, it may be desirable to use a nucleic acid encoding the enzyme in the form of a recombinant and preferably replicable vector.

"Vector"is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e. g. autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e. g. higher plant, mammalian, yeast or fungal) cells.

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e. g. bacterial, or plant cell. The vector may be a

bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell By"promoter"is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i. e. in the 3'direction on the sense strand of double-stranded DNA).

"Operably linked"means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is"under transcriptional initiation regulation"of the promoter.

Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press.

Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis (see above), sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

The methods of the present invention may therefore include the step of causing or allowing transcription of such vectors as are discussed above.

Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl. Acids Res.

12,8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific

Publishers, pp 121-148).

Promoters used in the vectors may be"inducible". In essence, expression under the control of an inducible promoter is "switched on"or increased in response to an applied stimulus.

The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus.

Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or "switchable") promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero). Upon application of the stimulus, expression is increased (or switched on) to a level which brings about the desired phenotype.

The present invention also provides methods comprising the step of introducing vectors into a plant cell and inducing of expression of an enzyme encoded by the vector by application of a suitable stimulus.

The invention further embraces a method described above comprising the step of transforming a plant cell by introduction of an appropriate vector into the plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the appropriate sequence into the genome.

This may be achieved by standard techniques which are well known to those skilled in the art.

Thus DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP- A-270355, EP-A-0116718, NAR 12 (22) 8711-87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A- 434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Plant Tissue and Cell Culture,

Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e. g.

Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e. g. Kindle, PNAS U. S. A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species, and more recently, in monocots (see e. g. Hiei et al. (1994) The Plant Journal 6, 271-282).

Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium- coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).

If desired, selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.

Generally speaking, following transformation, a plant may be regenerated, e. g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5,158- 162.; Vasil, et al. (1992) Bio/Technology 10,667-674; Vain et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).

Thus the methods of the present invention may be performed in vivo in transgenic plants, or clones, selfed or hybrid progeny, or other descendants (e. g. F1 and F2 descendents) thereof, and any part of any of these, such as cuttings or seeds.

The invention further provides a method of simultaneously influencing a plurality of flavonoid biosynthetic catalytic activities in a plant comprising use of a nucleic acid encoding an enzyme having multiple catalytic specificity. The plurality of catalytic activities may include two or more of the following: (i) flavone synthase activity, (ii) flavanone 3a-hydroxylase activity (iii) flavanone 3ß-hydroxylase activity, and (iv) flavonol synthase activity.

In a further aspect of the present invention there is disclosed a method of influencing one or more of the following catalytic activities in a plant comprising use of a nucleic acid encoding a flavonol synthase or a variant thereof: (i) flavone synthase activity, (ii) flavanone 3a-hydroxylase activity (iii) flavanone 3ß-hydroxylase activity, In a further aspect of the present invention there is disclosed a method of influencing one or more of the following catalytic activities in a plant comprising use of a nucleic acid encoding an enzyme having the respective activity: (i) flavone synthase activity, (ii) flavanone 3a-hydroxylase activity The above activities will generally be influenced by expression from the nucleic acids (which may be heterologous to the plant, or may have been introduced to increase copy number) to produce enzymes thereby increasing the relevant activities or activity in the plant.

By"heterologous"is meant non-naturally occurring in the plant, for instance something is a heterologous to a plant if its presence in the plant has arisen through human intervention.

Also embraced by the present invention is down-regulation of expression of a gene encoding an enzyme having the relevant activities or activity in the plant.

This may be achieved, for instance by using anti-sense technology or"sense regulation" ("co-suppression").

In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a"reverse orientation"such that transcription yields RNA which is complementary to normal mRNA transcribed from the"sense"strand of the target gene.

See, for example, Rothstein et al, 1987; Smith et al, (1988) Nature 334,724-726; Zhang et al, (1992) The Plant Cell 4,1575- 1588, English et al., (1996) The Plant Cell 8,179-188.

Antisense technology is also reviewed in Bourque, (1995), Plant Science 105,125-149, and Flavell, (1994) PNAS USA 91,3490- 3496.

Thus use of a nucleotide sequence which is complementary to any of those coding sequences discussed above, for modifying the activities discussed above, forms one part of the present invention.

"Complementary to"means that the sequence is capable of base pairing with the coding sequence whereby A is the complement of T (and U); G is the complement of C. A nucleic acid is"the complement"of another nucleic acid to which it is equal in length and complementary.

An alternative is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al., (1990) The Plant Cell 2,291-299; Napoli et al., (1990) The Plant Cell 2,279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, and US-A-5,231,020. See also WO 95/34668 of Biosource; Angell & Baulcombe (1997) The EMBO Journal 16,12: 3675-3684; and Voinnet & Baulcombe (1997) Nature 389: page 553.

The complete sequence corresponding to the coding sequence (in reverse orientation for anti-sense) need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding

sequence to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e. g. a sequence that is characteristic of one or more genes, such as a regulatory sequence.

The sequence employed may be about 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where possible, such as longer than about 600 nucleotides, than about 700 nucleotides, than about 800 nucleotides, than about 1000 nucleotides or more.

It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, though total complementarity or similarity of sequence is not essential.

. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down- regulation of gene expression in accordance with the present invention may be a wild-type sequence (e. g. gene) selected from those available, or a mutant, derivative, or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.

The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the transcribed anti-sense RNA to hybridise with nucleic acid within cells of the plant, though irrespective of whether hybridisation takes place the desired effect is down-regulation of gene expression.

Anti-sense or sense regulation may itself be regulated by employing an inducible promoter in an appropriate construct.

Particular applications The above methods of producing flavonoids, or modifying the production of flavonoids, or using flavonol synthases (or variants thereof), or identifying enzymes having multiple catalytic activities, or modifying (generally enhancing or down-regulating) the various catalytic activities involved in flavonoid biosynthesis, have particular applications which will now be discussed in more detail.

The methods may be used to produce non-naturally occurring flavonoids-these products forming a further aspect of the present invention.

The methods may be used to change various characteristics of those plants in which they are carried out.

As described in the introduction, flavonoid compounds play a role as colouring agents in fruits, flowers and seeds; as signalling molecules in development, pathogenesis, symbiosis and reproduction and as defence compounds against both environmental and biotic stresses (reviewed in Shirley, 1996; Koes et al., 1993).

Thus the present invention includes a method of altering any one or more of these characteristics in a plant, comprising use of a method as described hereinbefore.

Specifically the manipulation of flavanones, flavones and flavonols in transgenic plants may be used to alter flower, fruit, fibre or seed colour. This may be used to increase the commercial value of a horticultural or agricultural species.

The sequence may be used as a genetic marker to aid breeding to improve these traits.

Flavonoids also have important roles in human and animal health and diet (reviewed by Middleton, 1996).

In general, flavonoids act as anti-oxidants (eg. Rice-Evans et al., 1995; Vanacker et al., 1996) and provide an important component to both taste and colour in food and beverages (eg.

Lancaster, 1992; Noble, 1994; Mayen et al., 1995). In addition, certain flavonoids (oligomers of proanthocyanidins) can affect palatability and nutritive value of food and forage (Ortiz et al., 1994; Jackson et al., 1996). Proanthocyanidins

can also affect food and drink manufacturing processes (Horsley et al., 1991; Naczk et al., 1996) due to their ability to cross-link proteins (commonly called tanning).

The importance of flavonoids to human health is demonstrated by a wealth of scientific studies demonstrating that specific flavonoids act as phyto-oestrogens and anti-cancer, antithrombic and anti-hypertensive agents (reviewed by Formica and Regelson, 1995; Cook and Samman, 1996). In addition, high levels of flavonoids are a common denominator in many proposed healthy diets, such as those of the Mediterranean region (low levels of cardiovascular disease) and of Japan (low levels of breast cancer). Increasing the level of flavonoids ingested by humans for health reasons has been advocated by nutritionalists and dieticians (Goldbohm et al., 1995.

Thus use of the methods described above to manipulate flavanones, flavonols, flavones and proanthocyanidins in transgenic plants to alter the flavour, palatability, astringency and nutritive value, or ease of manufacturing, of feedstuffs and beverages for human and animal consumption is specifically included. Such modified plant products form a further aspect of the present invention.

Specific examples of applications in which alteration of flavour, colour, astringency or nutritional (or nutraceutical') value of a plant or plant product include the alteration of flavonols, catechins, gallocatechins and other flavonoids in black and green teas (see Harbowy and Balentine, 1997) or in grapes and grape products e. g. wine (Noble, 1994).

Increasing proanthocyanadin levels in some forage legumes may be used to decrease the occurrence of bloat in grazing livestock (see Douglas et al., 1995). Conversely, decreasing proanthocyanadin levels may be advantageous in less palatable forage or grain species (see Reed, 1995; Robbins et al, 1997).

Manipulation of flavonoid and tannin levels may also be used to reduce plant disease and pest predation (see Skadhauge et al., 1997; Hedin and Waage, 1986).

Food manufacturing processes may be improved by removing flavanoids and proanthocyanidins from the raw plant material to prevent flavonoid-protein complex formation. For example, beer haze formation during the brewing process can be avoided by utilising barley varieties deficient in proanthocyanidin

synthesis (von Wettstein et al., 1977). Flavonoids may also be used as food colourants (see Harborne and Grayer, 1988) and food additives e. g. anti-oxidants and stabilizers (Neito et al, 1993).

It may be particularly desirable to synthesise flavones, flavonols, dihydroflavonols proanthocyanidins and other polyphenolic compounds (including flavonoids and proanthocyanidins) which are novel, or rare or difficult to isolate from natural sources for use as pharmaceuticals, chemical feedstocks or additives to food and drink.

Similarly the methods of the invention may be used in the manipulation of proanthocyanidin levels to affect timber processing and the manufacture of timber products eg. wood- based adhesives.

The methods and materials of the present invention will now be described in more detail with reference to the following Figures and Examples. Other embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these.

TABLE AND FIGURES Table 1 This details the identification of products formed from racemic naringenin Figure 1 The basic flavonoid skeleton showing the ring structure (A, B and C) and the numbering of the carbon atoms. The two commonest hydroxylation patterns, the phloroglucinol and resorcinol types, are also shown.

Figure 2 A scheme showing the biosynthetic pathway to the common phloroglucinol-based flavones, flavonols and anthocyanidins.

The grid system is the result of the action of the flavonoid 3'-hydroxylase (F3'OH) and flavonoid 3', 5'-hydroxylase (F3', 5'OH). The other enzyme activities shown are the flavone synthase (FS), flavanone 3ß-hydroxylase (F3H), flavonol

synthase (FLS), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS).

Figure 3 Nucleic acid sequence of the full-length cDNA insert contained in agp4e. The translated product of the cDNA is shown using the single letter amino acid code. Seq ID No 1 is the coding nucleic acid sequence; Seq ID No 2 is the encoded amino acid sequence.

Figure 4 The products formed from an incubation of (2S)-naringenin with the enzyme. (2R, 3S)-dihydrokaempferol and derivatives have not been described previously as either natural or synthetic products.

Figure 5 The products formed from an incubation of (2S)-liquiritigenin with the enzyme. The (2R, 3R)-dihydroflavonol and the flavonol products have not been described previously as either natural or synthetic products. The dotted arrows show the products which are inferred to be synthesized by the enzyme by analogy with the reaction with (2S)-naringenin which has been studied in detail.

Figure 6 The products formed from an incubation of 4'-hydroxyflavanone with the enzyme. The (2R, 3R)-dihydroflavonol and the flavonol products have not been described previously as either natural or synthetic products. The dotted arrows show the products which are inferred to be synthesized by the enzyme by analogy with the reaction with (2S)-naringenin which has been studied in detail.

Figure 7 The products formed from an incubation of (2R)-naringenin with the enzyme. The (2S, 3S)-isomer of dihydrokaempferol has not been described previously as either a natural or synthetic product.

Figure 8 The synthesis of the flavonols kaempferol, quercetin, and myricetin from their respective (2R, 3R)-dihydroflavonol precursors.

Figure 9 The synthesis of the flavonols fisetin and robinetin from their respective (2R, 3R)-dihydroflavonol precursors.

Figure 10 HPLC traces showing the purification of flavonoid products from an incubation of racemic naringenin with the recombinant enzyme. Crude mixtures of flavonoids were separated using a Spherisorb column (traces a and b). Traces a) and b) are from the same injection of the HPLC but show the results at different wavelengths (trace a-290nm, trace b-265nm).

Peaks A and B were isolated and separated further using a Chiraspher NT column to yield peaks A1 and A2 or Cl and C2 respectively. [Peak B is the substrate (2S)-naringenin]. Peaks Al, A2, Cl and C2 were subjected to further characterisation.

EXAMPLES Example 1-Isolation of the putative flavonol svnthase from Arajbidojpsis thaliana Isolation of a partial cDNA clone encoding a putative flavonol synthase from Arabidopsis thaliana leaf tissue was accomplished by analysis of the Arabidopsis expressed sequence tag (EST) database for nucleic acid sequences showing homology to the Petunia flavonol synthase. A single cDNA clone (accession number T22434) was identified with 43% nucleic acid identity to the Petunia flavonol synthase (Holton et al., 1993). The partial clone was obtained from the Arabidopsis Stock Center, Ohio USA.

The partial cDNA clone T22434 was sequenced using standard methods (Sanger et al., 1977) and Sequenase v2.1 (Amersham). The sequence was used to design an oligonucleotide primer which would enable the 5'end of the cDNA clone to be isolated by 5'-

RACE polymerase chain reaction (PCR, Frohman et al., 1988, Hirzmann et al., 1993). The sequence of the oligonucleotide primers used for the 5'-RACE PCR were OLIGO 1 (general primer for G-tailed cDNAs) 5'-TTCTAGAATTCGGATCCCCCCCCCCCC-3'and OLIGO 2 (specific primer for the putative flavonol synthase) 5'- TCGTATCAGCTCCGTCG-3'.

Oligonucleotide primers were prepared using an Expedite nucleic acid synthesis system (Perseptive Biosystems) according to the manufacturer's instructions. PCR was carried out according to Hirzmann et al., (1993) using cDNA prepared from mRNA isolated from Arabidopsis plants by the method of Prescott and Martin, (1987). The amplified cDNA fragments were digested with the restriction enzymes BamHI and HincII (GibcoBRL Life Technologies) and cloned into an ml3-based vector using standard techniques (Maniatis et al., 1982). The resulting clones were sequenced using standard techniques (Sanger et al., 1977) and the sequences examined to determine the DNA sequence surrounding the ATG codon at the start of the open reading frame thought to encode a flavonol synthase. An oligonucleotide primer was designed which would place an NdeI site at the ATG codon by site-directed mutagenesis, (OLIGO 3, 5'-CAAAAAACATATGGAGGTC-3').

OLIGO 3 together with OLIGO 4, the ml3-40 forward primer, (5'- GTTTTCCCAGTCACGAC-3') were used to amplify a full-length cDNA clone (using the method of Frohman et al., 1988) from an Arabidopsis cDNA library cloned into pSPORT1 (GibcoBRL Life Technologies). The Arabidopsis cDNA library was made using a Superscript cDNA library kit (GibcoBRL Life Technologies) from random Arabidopsis cDNAs made from mRNA isolated from tissues derived from Arabidopsis thaliana ecotype Landsberg erecta plants at various stages in growth. The mRNA was prepared initially by the method of Prescott and Martin, (1987) and subsequently the poly A+ fraction was isolated using a PolyAtract kit (Promega) The amplified full-length cDNAs were digested with NdeI and BamHI (GibcoBRL life Technologies) and cloned into pET3a (Novagen) using the host strain DH5a (Hanahan, 1983). The sequence of the insert contained by a single clone was determined (Figure 3) and the clone named agp4e.

Recombinant protein was expressed by transforming agp4e into the Escherichia coli host strain BL21 (DE3) pLysE (Novagen; Studier and Moffat, 1986). 100ml cultures of Luria-Bertani

media (see Maniatis et al., 1982) containing O. lug/ul ampicillin (Sigma) were inoculated with a single colony of bacteria containing agp4e and grown overnight at 37°C in an orbital shaker (New Brunswick) at 180rpm. 8mls of overnight culture were used to inoculated flasks containing 500mls of fresh media with ampicillin and the cultures grown at 37°C under identical conditions until the OD595 was approximately 0.6. Cultures were induced with 0.4mM IPTG (isopropylthiogalactoside, Sigma) and grown for a further 5 hours at 30°C in an orbital shaker. Bacteria from these cultures were harvested by centrifugation at 10,000g (Beckman centrifuge, model J2-21). The pellets were washed in 100mM Tris-HCL pH 8.0, lOmM EDTA and frozen at-80°C.

Recombinant enzyme was prepared by resuspending the frozen bacterial pellet in Q-Sepharose buffer [25mM Tricine pH 7.3, 10% (v/v) glycerol, 2mM EDTA, lmM dithiothreitol (DTT)] with lOug/ml leupeptin. (All buffer components were from Sigma).

The bacterial suspension was passed three times through a French pressure cell (SLM-Aminco, SLM Instruments) at 1000psig and the resulting suspension diluted 4 fold with ice cold buffer (as above). After centrifugation for 20 minutes at 37,000g at 4°C, the soluble protein fraction was recovered and polethylenimine (Sigma) added to a final concentration of 1% (v/v). The suspension was stirred at 4°C for 10 minutes and recentrifuged. The soluble protein was recovered and loaded onto a Q-Sepharose column (column dimensions 2.6cm x 20cm, Pharmacia) previously equilibrated in Q-Sepharose buffer using an FPLC machine (Pharmacia). After washing the column with 3 volumes of Q-Sepharose buffer, 3ml fractions were collected as a gradient of 0-350mM sodium chloride in Q-Sepharose buffer A was applied over a volume of 120mls.

Fractions were assayed for activity by adding 100ul aliquots to a reaction containing 100mM Tricine pH 7.3,10% (v/v) glycerol, O. lmM ferrous sulphate, lOmM 2-oxoglutaric acid, lOmM ascorbic acid, O. lmg/ml bovine serum albumin, 0.5mg/ml catalase and lOmM (+/-)-dihydroquercetin (dissolved in methanol) in a total volume of 0.5ml. All components for the reaction mix were purchased from Sigma. The reactions were carried out at 30°C for 30 minutes in open tubes in a Microtherm incubator (Camlab) and examined visually for a change in colour to green. Those fractions which gave a positive result were pooled and dialysed in 500ml 25mM MES pH 6.15,10% (v/v) glycerol, 2mM EDTA and lmM DTT (Mono Q buffer) overnight at 4°C.

The dialysed material was recovered and loaded onto a Mono Q HR 5/5 column (Pharmacia) which had previously been equilibrated with Mono Q buffer using an FPLC machine (Pharmacia). After washing the column with 3 volumes of Mono Q buffer, 0.5ml fractions were collected as a gradient of 0-120mM sodium chloride in Mono Q buffer was applied over a volume of 15mls.

Fractions were assayed as before (incubation time increased to 1 hour) and those giving a positive result were pooled.

The recombinant protein was passed down a Superdex 75 column (dimensions 1.6cm x 70cm, Pharmacia at a flow rate of 0.5ml/min) equilibrated in 25mM Tricine pH 7.3,150mM sodium chloride, 10% (v/v) glycerol, 2mM EDTA, 1mM DTT. lml fractions containing eluted protein were collected and concentrated using Centricon-30 columns (Amicon) according to the manufacturer's instructions. Fractions containing pure recombinant protein, as determined by analysis on SDS/PAGE mini-gels stained with Coomassie Blue using standard techniques (Laemmli, 1970), were kept for use in experimental assays.

Example 2-Catalvtic specificity of flavonol synthase Flavonoid assays were carried out in a total volume of 0.5ml in open 1.5ml Eppendorf tubes, incubated at 30°C in a shaking incubator (Camlab) at 50rpm. The assay mixture contained 100mM Tricine pH 7.3,10% (v/v) glycerol, O. lmM ferrous sulphate, lOmM 2-oxoglutaric acid, lOmM ascorbic acid, O. lmg/ml bovine serum albumin, 0.5mg/ml catalase, lOmM of flavonoid substrate (substrates from Sigma, Carl Roth and Apin Chemicals) and 1- l0ug of recombinant protein. The reactions were stopped by the addition of an equal volume of methanol before analysis of the products.

Flavonoid products were separated by HPLC using a Spherisorb 5um S50DS2 (Anachem) column with a gradient of 45-100% methanol/55-0% formic acid solution (4.5% v/v formic acid in distilled water) formed over 22 minutes. The flow rate of the solvent through the column was 2mls per minute. Further separation of complex mixtures of flavonoids was achieved by collecting fractions from the Spherisorb column and re-running them on a Chiraspher NT chiral column (Merck) with isocratic concentrations of 60-80% (v/v) methanol in distilled water.

The flow rate of solvent through the column was lml per minute.

The reactions catalysed by the recombinant protein are shown in detail in Figures 4-9. The enzyme utilises (2S)-naringenin (Figure 4), (2S)-liquiritigenin (Figure 5), 4'-hydroxyflavanone (Figure 6) and (2R)-naringenin (Figure 7) as flavanone substrates. The common feature which determines which flavanones are substrates is the presence of a single hydroxyl group at the 4'position of the B ring of the flavanone.

Flavanones with more than 1 hydroxyl group on the B ring are not substrates for this enzyme.

The enzyme also utilises dihydroflavonols as substrates including the (2R, 3R)-isomers of dihydrokaempferol, dihydroquercetin, dihydromyricetin (Figure 8), dihydrofisetin and dihydrorobinetin (Figure 9). All dihydroflavonols which are commercially available have been shown to be substrates for this enzyme.

Flavonoid products were identified by standard techniques including retention time on HPLC, ultra violet spectral characteristics, molecular mass, circular dichroism spectra and nuclear magnetic resonance spectra. A detailed example showing the purification and identification of the products formed using racemic naringenin as a substrate is shown in Figure 10 and Table 1.

The methodology used to identify the flavonoid products varied according to whether a standard could be obtained for comparison and on any identification data already present in the literature (Mabry et al., 1970). The following description of the procedures used is based on the data presented in Figure 10 and Table 1 as an example. For peaks Cl and C2, the products were compared with authentic pure samples of apigenin and kaempferol acquired from Apin Chemicals. The flavonoid products contained in these peaks had identical retention times on HPLC to the appropriate standard flavonoids and the ultra violet spectra of standard and product in methanol were identical (Mabry et al., 1970). The molecular mass and fragmentation patterns of the flavonoids contained in peaks Cl and C2 were assessed by electron impact mass spectrometry and found to be consistent with their identification as apigenin and kaempferol. A proton nuclear magnetic resonance (NMR) spectrum using deuterated dimethylsulphoxide as a solvent (DMSO, Aldrich Chemicals) was also determined for the C2 peak and found to be consistent with those published previously (Mabry et al., 1970; Markham and Geiger, 1993). The NMR was

carried at 21°C using a JEOL GX400 spectrometer with a 45° pulse angle and a total recycle time of 5 seconds.

Characterisation of the flavonoids found in peaks A1 and A2 was done using a sample of (2R, 3R)-dihydrokaempferol as a standard.

Although peak A2 was subsequently found to be a different isomer of dihydrokaempferol, the W spectrum, retention time on HPLC and molecular mass and fragmentation pattern of product A2 was identical to that of (2R, 3R)-dihydrokaempferol. The identification of the peak as the (2S, 3S)- isomer of dihydrokaempferol was based on an analysis of the proton NMR spectrum in comparison with the NMR spectra of (2R, 3R)- dihydrokaempferol and of isomers of related dihydroflavonols (Foo, 1986; Foo, 1987; Nonaka et al., 1987; Lundgren and Theander, 1988) and on its circular dichroism spectra (Gaffield, 1970; Nonaka et al., 1987; Lundgren and Theander, 1988).

Characterisation of the novel flavonoid found in peak A1 was carried out by careful comparison of the UV spectrum, retention time on HPLC, molecular mass circular dichroism spectra and proton NMR spectrum with all possible flavonoid derivatives of (2S)-naringenin which have been described previously. As no match was found, the structure was determined from first principles using the guidelines published previously (Mabry et al., 1970; Harborne et al., 1975; Markham and Geiger, 1993).

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Table 1 Identification of flavonoid products formed from a mixture of (2S)- and (2: Identification Flavonoid Peak (as shown in Figure 10) Technique A1 A2 C1 C2 Standard Only (2R, 3R) - Only (2R, 3R) - Standard from Standard available for isomer available isomer available Apin Chemicals from Apin comparison (Apin Chemicals) (Apin Chemicals) Chemicals Retention time 11.8 13.1 6.6 8.0 on HPLC (chiral colum) /min UV spectrum in 293 290 267, 336 266, >360 methanol (# max, nm) Molecular mass 288.4 288.4 270.4 286.5 Circular Identical Opposite spectrum Not applicable Not dichroism spectrum to to (2R, 3R)- applicable (2R, 3R)- dihydrokaempferol dihydrokaempferol standard standard Identification Flavonoid Peak (as shown in Figure 10) Technique NMR spectrum in # 7.28 (d, J = # 7.29 (d, J = not determined # 6.17 (d, J DMSO-d6 8.6Hz, H-2',H-6') ; 8.6Hz, H-2', H-6') ; = 1.6Hz, H- 6.74 (d, J = 6.76 (d, J = 6); 6.42 8.6Hz, H-3', H- 8.3Hz, H-3', H-5') ; (indistinct 5') ; 6.56 (s, H- 5.83 (indistinct doublet, H- 3) ; 5.74-5.75 doublet, H-8) ; 8) ; 6.91 (broad peak, H-8, 5.78 (indistinct (d, J = H-6) doublet, H-6) ; 8.9Hz, H- 5.00 (d, J = 3',H-5') ; 11.4 Hz, H-2) ; 4.53 8.03 (d, J (dd, J = 11.4Hz = 8.9Hz, H- and 11.4Hz, H-3) 2', H-6') Identity (2R, 3S) - (2S, 3S) - Apigenin Kaempferol Dihydrokaempferol Dihydrokaempferol