WO2001005984A2 - Biosynthese de flavanoides - Google Patents

Biosynthese de flavanoides Download PDF

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WO2001005984A2
WO2001005984A2 PCT/GB2000/002701 GB0002701W WO0105984A2 WO 2001005984 A2 WO2001005984 A2 WO 2001005984A2 GB 0002701 W GB0002701 W GB 0002701W WO 0105984 A2 WO0105984 A2 WO 0105984A2
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enzyme
plant
flavonoid
nucleic acid
activity
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Andrea Gaye Prescott
John Leonard Firmin
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Plant Bioscience Ltd
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Plant Bioscience Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis

Definitions

  • the present invention relates to methods for enzymatically manipulating the synthesis of flavonoids. It further relates to materials for use in, and resulting from, such methods.
  • 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.
  • 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.
  • chalcone synthase catalyses the condensation of three molecules of malonyl-CoA with one molecule of 4-coumaroyl-CoA (Ebel and Hahlbrock, 1982) .
  • (25) -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 3 ⁇ -hydroxylase (Britsch and Grisebach, 1986).
  • 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
  • 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. (25)- naringenin is also converted into ( 2R, 3R) -dihydrokaempferol by the action of the enzyme, flavanone 3 ⁇ -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 3 ⁇ -hydroxylase [products are (2R, 3R) -dihydroquercetin and (2R, 3R) - dihydro yricetin respectively] .
  • -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.
  • 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.
  • flavonoid structures 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).
  • flavanone 3 ⁇ -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 3 ⁇ 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 3 ⁇ -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).
  • Flavanone 3 ⁇ -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 vi tro .
  • 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 3 ⁇ - 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) .
  • U72631 (genomic sequence from Landsberg erecta )
  • U84258 (genomic from Col umbia )
  • U84259 (cDNA from Col umbia)
  • U84260 (cDNA from Landsberg erecta ) .
  • the sequence has also been published in Pelletier et al . , (1997).
  • 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) .
  • Enzymes isolated from species lacking 5 ' -hydroxylated flavonoids eg ( 2R r 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.
  • 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) .
  • 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.
  • 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.
  • 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 Escheri chia 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:
  • flavone synthase activity (i) flavone synthase activity, (ii) flavanone 3 -hydroxylase activity (iii) flavanone 3 ⁇ -hydroxylase activity (iv) flavonol synthase activity
  • the enzyme has been shown to utilise substrates which are not found in its plant of origin specifically those based on a resorcinol skeleton [ (25) -liquiritigenin, ( 2R, 3R) - dihydrofisetin, [ 2R, 3R) -dihydrorobinetin] ; 5 ' -hydroxylated dihydroflavonols [ ⁇ 2R, 3R) -dihydromyricetin, ( 2R, 3R) - dihydrorobinetin) ] , 4 ' -hydroxyflavanone and the rare isomer, ( 2R) -naringenin.
  • 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 vi tro, 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.
  • 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 3 -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 .
  • a method of producing a flavonoid, or modifying the production of a flavonoid 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 vi tro and in vi vo production, or manipulation, of one or more flavonoids .
  • the enzyme when used in vi tro 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 .
  • the in vi vo 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 .
  • the enzyme in manipulating flavonoid production, the enzyme will be used in conjunction with other enzymes.
  • the 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.
  • 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' .
  • 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:
  • the encoded enzyme will exhibit 2 or more, preferably 3 or 4 of:
  • the flavonol synthase as any one or more of: (i) a flavone synthase, (ii) a flavanone 3 ⁇ -hydroxylase (iii) a flavanone 3 ⁇ -hydroxylase
  • a method of producing a flavonoid, or modifying the production of a flavonoid 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 comprising use of an enzyme as a flavanone 3 ⁇ -hydroxylase (optionally via use of a nucleic acid molecule encoding the same) .
  • the enzyme is used to manipulate the levels of (-) -epicatechin and (-)- epigallocatechin in plants.
  • 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.
  • the enzyme may use any one or more of the following as substrates:
  • 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:
  • X' can be any amino acid, or may optionally represent the site of insertion or deletion of an amino acid.
  • motifs appear to be present in a number of enzymes designated as flavonol synthases.
  • the enzyme is itself a flavonol synthase, and most preferably it is a flavonol synthase from Arabidopsi s thaliana .
  • 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.
  • the nucleic acid used in the methods of the present invention comprises all or part of Seq ID No 1.
  • nucleic acid and therefore enzyme
  • the nucleic acid 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) :
  • T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% forma ide) - 600/#bp in duplex
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
  • Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
  • Artificial variants may be prepared by those skilled in the art, for instance by site directed or random mutagenesis, or by direct synthesis.
  • 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.
  • 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 . Bi ol . 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.
  • 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.
  • 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.
  • a variant amino acid sequence may include additional amino acids at the C-terminus and/or N-terminus .
  • 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 .
  • 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.
  • variants having non-conservative substitutions are also included.
  • 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.
  • 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.
  • changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.
  • the method including the step of modifying a nucleic acid encoding the enzyme.
  • the enzyme is a flavonol synthase, most preferably the nucleic acid which is modified has the sequence shown in Seq ID No 1.
  • 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:
  • 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) .
  • 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 Agrobacteri um 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) .
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • the methods of the present invention may therefore include the step of causing or allowing transcription of such vectors as are discussed above.
  • 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.
  • 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.
  • 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) .
  • 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 .
  • DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by
  • Agrobacteri um 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) icroinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al .
  • Agrobacteri um 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 Agrobacteri um is inefficient or ineffective.
  • a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacteri um- coated microparticles (EP-A-486234 ) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacteri um (EP-A-486233) .
  • selectable genetic markers 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.
  • 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 Cul ture and Soma ti c Cell Geneti cs of Plants, Vol I, II and III, Labora tory Procedures and Their Applica ti ons, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Mol ecular Bi ol ogy, 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 Opini on in Bi otechnol ogy 5, 158- 162.; Vasil, et al . (1992) Bi o/Technol ogy 10, 667-674; Vain et al., 1995, Bi otechnol ogy Advances 13 (4): 653-671; Vasil, 1996, Na ture Bi otechnology 14 page 702) .
  • the methods of the present invention may be performed in vi vo in transgenic plants, or clones, selfed or hybrid progeny, or other descendants (e.g. FI 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • Antisense technology is also reviewed in Bourque, (1995), Plant Sci ence 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490- 3496.
  • “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.
  • the complete sequence corresponding to the coding sequence 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
  • 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.
  • 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%,
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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) .
  • 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).
  • 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) .
  • flavonoids act as phyto-oestrogens and anti-cancer, antithrombic and anti-hypertensive agents (reviewed by Formica and Regelson, 1995; Cook and Samman, 1996) .
  • 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 .
  • 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.
  • 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) .
  • 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.
  • 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 basic flavonoid skeleton showing the ring structure (A, B and C) and the numbering of the carbon atoms.
  • 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 3 ⁇ -hydroxylase (F3H) , flavonol synthase (FLS) , dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) .
  • Nucleic acid sequence of the full-length cDNA insert contained in agp4e 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.
  • 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 Al and A2 or Cl and C2 respectively.
  • [Peak B is the substrate (25) -naringenin] . Peaks Al, A2, Cl and C2 were subjected to further characterisation.
  • Example 1- Isolation of the putative flavonol synthase from Arabidopsis thaliana
  • Isolation of a partial cDNA clone encoding a putative flavonol synthase from Arabidopsi s 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.
  • EST Arabidopsis expressed sequence tag
  • 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).
  • oligonucleotide primers used for the 5 '-RACE PCR were OLIGO 1 (general primer for G-tailed cDNAs) 5 ' -TTCTAGAATTCGGATCCCCCCCCCC-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 Hindi (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 .
  • oligonucleotide primer was designed which would place an Ndel 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 pSPORTl (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 Arabi dopsis 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 Ndel and BamHI (GibcoBRL life Technologies) and cloned into pET3a (Novagen) using the host strain DH5 (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 Escheri chia coli host strain BL21 (DE3 ) pLysE (Novagen; Studier and Moffat, 1986) . 100ml cultures of Luria-Bertani media (see Maniatis et al . , 1982) containing O.l ⁇ g/ ⁇ l 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.
  • 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, ImM dithiothreitol (DTT) ] with lO ⁇ g/ml leupeptin. (All buffer components were from Sigma) .
  • Q-Sepharose buffer 25mM Tricine pH 7.3, 10% (v/v) glycerol, 2mM EDTA, ImM dithiothreitol (DTT)
  • All buffer components were from Sigma.
  • the bacterial suspension was passed three times through a French pressure cell (SLM-Aminco, SLM Instruments) at lOOOpsig and the resulting suspension diluted 4 fold with ice cold buffer (as above) .
  • 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 lOO ⁇ l aliquots to a reaction containing lOOmM Tricine pH 7.3, 10% (v/v) glycerol, 0. ImM ferrous sulphate, lOmM 2-oxoglutaric acid, lOmM ascorbic acid, 0. Img/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 ImM 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) .
  • 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, ImM DTT. 1ml 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.
  • 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 lOOmM Tricine pH 7.3, 10% (v/v) glycerol, 0. ImM ferrous sulphate, lOmM 2-oxoglutaric acid, lOmM ascorbic acid, 0. Img/ml bovine serum albumin, 0.5mg/ml catalase, lOmM of flavonoid substrate (substrates from Sigma, Carl Roth and Apin Chemicals) and 1- lO ⁇ g 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 5 ⁇ m 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 1ml per minute.
  • 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

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Abstract

Cette invention se rapporte à des procédés servant à influencer l'activité catalytique de biosynthèse des flavanoïdes dans une cellule et consistant à modifier dans cette cellule l'activité d'une enzyme ayant: (i) une activité de flavone-synthase et/ou (ii) une activité de flavanone-3α-hydroxylase, plus éventuellement (iii) une activité de flavanone-3β-hydroxylase et (iv) une activité de flavonol-synthase. Comme exemples d'enzymes, cette invention propose une flavonol-synthase (numéro d'identification de séquence-2) pouvant être obtenue à partir de l'Arabidopsis thaliana ou à partir de variants de cet organisme, déployant de nouvelles activités et combinaisons d'activités et pouvant utiliser une grande variété de substrats à l'état naturel et non naturel. Les procédés faisant l'objet de cette invention concernent l'utilisation d'acides nucléiques pour exprimer ou réguler à la baisse cette enzyme dans des cellules végétales et dans des plantes. Ce procédé peut servir à modifier une ou plusieurs caractéristiques dans une plante, telles que la couleur, la valeur nutritive et la résistance aux contraintes. Cette invention concerne également de nouvelles utilisations de cette enzyme, de nouvelles flavanoïdes pouvant être utilisées à l'aide de cette enzyme et des oligonucléotides.
PCT/GB2000/002701 1999-07-16 2000-07-13 Biosynthese de flavanoides Ceased WO2001005984A2 (fr)

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Cited By (7)

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WO2002086110A3 (fr) * 2001-04-20 2002-12-12 Univ Muenchen Tech Enzymes de flavone synthase i et leur utilisation
EP1442122A4 (fr) * 2001-10-05 2007-02-14 Agriculture Victoria Serv Pty Manipulation de la biosynthese des flavonoides chez les plantes
CN103756982A (zh) * 2013-12-16 2014-04-30 上海交通大学 郁金香黄酮醇合成酶TfFLS蛋白及其编码基因
CN107164459A (zh) * 2016-12-07 2017-09-15 中国农业科学院茶叶研究所 一种鉴定和筛选高儿茶素指数茶树的功能标记及其应用
CN109512814A (zh) * 2018-10-27 2019-03-26 李定文 一种抗雾霾药品及其抗霾口罩和二氢杨梅素在防治雾霾及治疗季节性流感中的用途
CN115247156A (zh) * 2020-10-27 2022-10-28 聊城大学 一种重组苹果黄酮醇合酶及其制备方法和用途
CN117737015A (zh) * 2023-12-13 2024-03-22 杭州佳嘉乐生物技术有限公司 一种提高黄烷酮-3-羟化酶催化活性的方法及其应用

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Cited By (13)

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WO2002086110A3 (fr) * 2001-04-20 2002-12-12 Univ Muenchen Tech Enzymes de flavone synthase i et leur utilisation
US9523089B2 (en) 2001-10-05 2016-12-20 Agriculture Victoria Services Pty Ltd Manipulation of flavonoid biosynthesis in plants
AU2002333038B2 (en) * 2001-10-05 2008-02-07 Agresearch Limited Manipulation of flavonoid biosynthesis in plants
US7767416B2 (en) 2001-10-05 2010-08-03 Agriculture Victoria Services Pty Ltd Manipulation of flavonoid biosynthesis in plants
US8569064B2 (en) 2001-10-05 2013-10-29 Agriculture Victoria Services Pty Ltd. Manipulation of flavonoid biosynthesis in plants
EP1442122A4 (fr) * 2001-10-05 2007-02-14 Agriculture Victoria Serv Pty Manipulation de la biosynthese des flavonoides chez les plantes
CN103756982A (zh) * 2013-12-16 2014-04-30 上海交通大学 郁金香黄酮醇合成酶TfFLS蛋白及其编码基因
CN107164459A (zh) * 2016-12-07 2017-09-15 中国农业科学院茶叶研究所 一种鉴定和筛选高儿茶素指数茶树的功能标记及其应用
CN107164459B (zh) * 2016-12-07 2020-07-07 中国农业科学院茶叶研究所 一种鉴定和筛选高儿茶素指数茶树的功能标记及其应用
CN109512814A (zh) * 2018-10-27 2019-03-26 李定文 一种抗雾霾药品及其抗霾口罩和二氢杨梅素在防治雾霾及治疗季节性流感中的用途
CN115247156A (zh) * 2020-10-27 2022-10-28 聊城大学 一种重组苹果黄酮醇合酶及其制备方法和用途
CN117737015A (zh) * 2023-12-13 2024-03-22 杭州佳嘉乐生物技术有限公司 一种提高黄烷酮-3-羟化酶催化活性的方法及其应用
CN117737015B (zh) * 2023-12-13 2024-11-12 杭州佳嘉乐生物技术有限公司 一种提高黄烷酮-3-羟化酶催化活性的方法及其应用

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