JP6270110B2 - Sequencer-derived flavonoid-O-methyltransferase (FOMT) and use thereof - Google Patents

Description

  The present invention relates to a seeker-derived flavonoid-O-methyltransferase (hereinafter sometimes abbreviated as FOMT) having an activity of methylating hydroxyl groups of various flavonoids to convert them into methoxy groups. The present invention further relates to a DNA encoding the flavonoid O-methyltransferase, a protein encoded by the DNA, a method for producing the protein, and the like. Furthermore, the present invention relates to a method for producing methylated flavonoids using transformed cells transformed with a vector containing the protein or DNA encoding FOMT.

  Flavonoids are secondary metabolites widely present in plants, and about 8000 species are known. Among them, methylated flavonoids have recently been found to have physiological activities such as anticancer action, hyperlipidemia prevention and anti-inflammatory action, and are expected to be used as functional food materials and pharmaceuticals. Rhamnetin methylated at position 7 of quercetin is more capable of inducing apoptosis in MDA-MB-231 human breast cancer cells than quercetin. Methylated flavonoids are abundant in citrus peel, tea, poplar and the like. However, the content of methylated flavonoids in plants is low and is easily metabolized, which is expensive and hinders utilization.

  Flavonoid methylation is catalyzed primarily by S-adenosylmethionine (SAM) -dependent methyltransferase (FOMT). So far, research on flavonoid methyltransferases has been extensively conducted, and they are roughly divided into two classes depending on their sequences and substrate specificities. That is, it can be divided into a metal ion dependent type (class I) showing methylation activity for caffeic acid CoA ester and a metal ion independent type (class II) showing activity for flavonoids and alkaloids. Poplar-derived 7-O-methyltransferase (POMT7) and tomato-derived 3-O-methyltransferase (3-OMT) (Non-patent Documents 1 and 2) are known. In addition, plant-derived methyltransferases are known (Patent Documents 1 and 2).

  By the way, nobiletin, which is a kind of polymethoxyflavonoid, is abundantly contained in the peels of immature fruits of citrus fruits such as shikwasa, and various studies have been conducted on physiological activity. However, the nobiletin biosynthetic pathway in the seeker has not yet been elucidated.

WO2003 / 062428 (Gene sequence having methyltransferase activity and use therefor) WO2010 / 069004 (Plant with modified flower part)

B. G. Kima, H. Kima, H. G. Hurb, Y. Lima, and J. H. Ahna, (2006) Regioselectivity of 7-O-methyltransferase of poplar to flavones. J. Biotechnol., 126, 241-247. A. Schmids, C. Li, A. D. Jones, and E. Pichersky, (2012) Characterization of a flavonol 3-O-methyltransferase in the trichomes of the wild tomato species Solanum habrochaites.Plant, 236, 839-849. Lavid, N., et al., 2002. O-Methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petala.Plant Physiology, 129: 1899-1907. Tuskan, G.A., et al., 2006. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science, 313: 1596-1604. Pellegrini, L., et al., 1993. Molecular cloning and expression of a new class of orth-diphenol-O-methyltransferases induced in Tobacco leaves by infection or elicitor treatment.Plant Physiology, 103: 509-517. GenBank: ABP94018: Lluch, Y.P., et al., 2007. O-methyltransferase [Citrus sinensis x Citrus reticulata].

  Since plant-derived FOMT has high selectivity for its methylation site, the biosynthesis of highly methylated flavonoids (polymethoxyflavonoids) involves multiple FOMTs with different site selectivity. Guessed. However, the nobiletin biosynthetic pathway in sequencer has not yet been elucidated, nor is FOMT derived from sequencer known.

  Therefore, an object of the present invention is to isolate a novel plant-derived FOMT gene, obtain a transformant expressing the obtained FOMT gene, obtain a plant-derived FOMT from the obtained transformant, It is an object of the present invention to provide a method for producing methylated flavonoids using FOMT or transformants, and to provide a technique associated therewith.

  Creation of various methylated flavonoids (natural and unnatural methylated flavonoids) is expected by using the obtained plant-derived FOMT. Non-naturally occurring methylated flavonoids may have higher bioactivity than natural forms.

  The present inventors have isolated and analyzed methyltransferases of flavonoids from various plants. In the process, two new methyltransferases (named CdFOMT5 and CdFOMT6) were discovered from the Okinawan seeker (Citrus depressa). Furthermore, this methyltransferase was found to catalyze the methyltransferase reaction of various flavonoids, and this fact was the basis for completing the present invention.

Enzymes that methylate phenolic hydroxyl groups such as flavonoids, catechins, and caffeic acids have been known (Non-Patent Documents 3, 4, and 5). However, the novel methyltransferase genes of the present invention were clearly different from each other with low identity. The plant-derived methyltransferases described in Patent Documents 1 and 2 are enzymes that require class I type Mg ²⁺ , and the identity with the enzyme of the present invention was about 6-15%. In addition, one methyltransferase (CdFOMT5) of the present invention has 95% identity with the amino acid sequence (Non-patent Document 6) estimated to be O-methyltransferase in the function estimation from the gene sequence. However, the substrate specificity of the translation product is not known, and even if it is a flavonoid-O-methyltransferase, the regioselectivity of the methyl transfer is not known at all.
The present invention provides the following.

[1] The polynucleotide according to any one of (a) to (f) below;
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(B) a polynucleotide encoding a flavonoid-O-methyltransferase having 96% or more identity with the base sequence described in SEQ ID NO: 1 and having the activity described in (1) below;
(C) A flavonoid-O-methyltransferase that hybridizes to the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 under highly stringent conditions and has the activity described in (1) below. A polynucleotide encoding
(D) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2;
(E) a flavonoid having the activity described in (1) below, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 2 A polynucleotide encoding O-methyltransferase; and (f) a flavonoid -O- having 96% or more identity with the amino acid sequence of SEQ ID NO: 2 and having the activity described in (1) below A polynucleotide encoding a methyltransferase;
(1) The activity of acting on quercetin to produce methoxyquercetin in which at least one hydroxyl group of quercetin is methylated.
[2] The polynucleotide according to [1], wherein the activity according to (1) is an activity to produce methoxyquercetin in which at least one to three hydroxyl groups of quercetin are methylated.
[3] The polynucleotide according to any one of (g) to (l) below;
(G) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3;
(H) a polynucleotide encoding a flavonoid-O-methyltransferase having 90% or more identity with the base sequence set forth in SEQ ID NO: 3 and having the activity described in (2) below;
(I) A flavonoid-O-methyltransferase that hybridizes to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3 under highly stringent conditions and has the activity described in (2) below. A polynucleotide encoding
(J) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 4;
(K) a flavonoid having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 4 and having the activity described in (2) below: A polynucleotide encoding O-methyltransferase; and (l) a flavonoid-O— having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4 and having the activity set forth in (2) below: A polynucleotide encoding a methyltransferase;
(2) The activity of acting on quercetin to produce methoxyquercetin in which the hydroxyl group at the 3 ′ position of quercetin is selectively methylated.
[4] A protein encoded by the polynucleotide according to any one of [1] to [3].
[5] A protein having any one of the following amino acid sequences (m) to (p):
(M) the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing;
(N) an amino acid sequence having 1 to several amino acid deletions, substitutions and / or additions in the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing, and the following (1) or (2) An amino acid sequence encoding a flavonoid-O-methyltransferase having the described activity;
(O) an amino acid sequence encoding a flavonoid-O-methyltransferase having 96% or more identity to the amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing and having the activity described in (1) below Or (p) encodes a flavonoid-O-methyltransferase having 90% or more identity to the amino acid sequence described in SEQ ID NO: 4 in the Sequence Listing and having the activity described in (2) below Amino acid sequence;
(1) The activity of acting on quercetin to produce methoxyquercetin in which at least one hydroxyl group of quercetin is methylated.
(2) The activity of acting on quercetin to produce methoxyquercetin in which the hydroxyl group at the 3 ′ position of quercetin is selectively methylated.
[6] The protein according to [5], wherein the activity according to (1) is an activity to produce methoxyquercetin in which at least one to three hydroxyl groups of quercetin are methylated.
[7] A vector comprising the polynucleotide according to any one of [1] to [3].
[8] A transformed cell into which the polynucleotide according to any one of [1] to [3] or the vector according to [7] has been introduced.
[9] The method includes the steps of culturing the transformed cell according to 8, and recovering a protein encoded by the polynucleotide according to any one of [1] to [3] from the culture. [3] A method for producing a protein encoded by the polynucleotide according to any one of [4] or [4] to [6].
[10] A step of allowing the protein according to any one of [4] to [6] or the transformed cell according to [8] to act on a flavonoid and / or an analog thereof, and a step of recovering the produced compound A process for producing methoxyflavonoids and / or analogs thereof.
[11] Flavonoids and / or analogs thereof are kaempferol, quercetin, myricetin, fisetin, galangin, gosipetin, morin, apigenin, luteolin, naringenin, taxifolin, butyne, eriodictol, pinocembrin, cyanidin, delphinidin, catechin, epi [10] is at least one compound selected from the group consisting of catechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, epiafzerekin, fisetinidol, guibrutinidol, mesquitol, robinetinidol / or analogues thereof. The manufacturing method as described in.
[12] An enzyme agent comprising a protein encoded by the polynucleotide according to any one of [1] to [3] or the protein according to any one of [4] to [6].

The two types of FOMTs of the present invention are common in that they are O-methyltransferases using flavonoids as substrates. However, there are significant differences in the regioselectivity of flavonoids. Although details will be described later, CdFOMT5 has an activity of catalyzing a methyl transfer reaction at a plurality of hydroxyl groups with respect to quercetin (having five hydroxyl groups). On the other hand, CdFOMT6 exhibits a methyl transfer reaction specifically with respect to quercetin only for the hydroxyl group at the 3 ′ position of the B ring.
These enzyme catalysts are extremely useful for the industrial production of methylated flavonoids.

Recombinant CdFOMT5 and CdFOMT6 expression plasmids Analysis of recombinant CdFOMT5 and CdFOMT6 by SDS-PAGE (A: pET-CdFOMT5 and pET-CdFOMT6 expressing cells Cell disruption fluid (M: molecular weight marker, H: host (E. coli BL21 (DE3)), C: control ( pET-21b (+) / E. coli BL21 (DE3)), Cd5: pET-CdFOMT5 / E. coli BL21 (DE3), Cd6: pET-CdFOMT6 / E. coli BL21 (DE3)), B: pMAL-CdFOMT6 Expression cells Cell disruption fluid (M: molecular weight marker, C: control (pMAL-c5x / E. Coli JM109), H: host (E. coli JM109), Cd6: pMALc5-CdFOMT6 / E. Coli JM109)) SDS-PAGE analysis of purified CdFOMT5 and CdFOMT6 (A: pET-CdFOMT5 expressing cells Ni column purification (M: molecular weight marker, C: cell lysate supernatant, U: non-adsorbed fraction, Cd5: purified CdFOMT6), B: pMAL-CdFOMT6 expressing cells Amylose column purification (M: molecular weight marker, C: cell lysate supernatant, U: non-adsorbed fraction, Cd6: purified CdFOMT6 (elution fraction 1-4))) Quercetin methylation with CdFOMT5 and 6 (Q: quercetin, 3MQ: 3-methoxy quercetin, A: azareatin (5-methoxy quercetin), R: rhamnetine (7-methoxy quercetin), I: isorhamnetin (3'-methoxy quercetin) LC-MS analysis result of quercetin methylation product by CdFOMT5 (top: HPLC chromatogram (UV254 nm), A-D: mass spectrum at each peak) Effect of pH on methylation activity of CdFOMT5 and CdFOMT6 (A: CdFOMT5, B: CdFOMT6) Optimal reaction temperature for CdFOMT5 and CdFOMT6 (A: CdFOMT5, B: CdFPMT6) Examination of methylation sites for flavone skeleton compounds of CdFOMT5 (A: 5-hydroxyflavone, B: 6-hydroxyflavone, C: 7-hydroxyflavone, D: 7,8-dihydroxyflavone) Substrate specificity of CdFOMT6 (A: myricetin, B: luteolin, C: epigallocatechin 3-O-gallate, D: epicatechin) LC-MS analysis of quercetin methylation products by CdFOMT5 expressing cells (top: HPLC chromatogram (UV254 nm), A to C: mass spectra of peaks A to C) CdFOMT5 and CdFOMT6 sequences CdFOMT1, CdFOMT3, and CdFOMT4 sequences

[Flavonoid O-methyltransferase (FOMT)]
The present invention relates to CdFOMT5 and CdFOMT6, which are O-methyltransferases (flavonoids: S-adenosyl L-methionine O-methyltransferase) using flavonoids derived from seeker as a substrate, and proteins related thereto. This protein is a protein having any of the following amino acid sequences. In the present specification, CdFOMT5 and CdFOMT6 and proteins related thereto are sometimes referred to as “protein of the present invention”.
(M) the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing;
(N) an amino acid sequence having 1 to several amino acid deletions, substitutions and / or additions in the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing, and the following (1) or (2) An amino acid sequence encoding a flavonoid-O-methyltransferase having the described activity;
(O) an amino acid sequence encoding a flavonoid-O-methyltransferase having 96% or more identity to the amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing and having the activity described in (1) below Or (p) encodes a flavonoid-O-methyltransferase having 90% or more identity to the amino acid sequence described in SEQ ID NO: 4 in the Sequence Listing and having the activity described in (2) below Amino acid sequence;
(1) The activity of acting on quercetin to produce methoxyquercetin in which at least one hydroxyl group of quercetin is methylated.
(2) The activity of acting on quercetin to produce methoxyquercetin in which the hydroxyl group at the 3 ′ position of quercetin is selectively methylated.

  CdFOMT5 and CdFOMT6 are common in that they are O-methyltransferases that use flavonoids as substrates. However, there are significant differences in the regioselectivity of flavonoids. Details will be described later.

  CdFOMT5 is a protein having the amino acid sequence described in SEQ ID NO: 2 in the sequence table of (m) above, and the protein related to CdFOMT5 is 1 in the amino acid sequence described in SEQ ID NO: 2 of the sequence table of (n) above. A protein having an amino acid sequence having a deletion, substitution and / or addition of several amino acids from and having an amino acid encoding a flavonoid-O-methyltransferase having the activity described in (1) above, and A flavonoid-O-methyltransferase having an amino acid sequence having 96% or more identity to the amino acid sequence described in SEQ ID NO: 2 in the sequence listing of (o) and having the activity described in (1) above Is a protein having an amino acid sequence encoding

  The activity described in (1) is an activity that acts on quercetin to produce methoxyquercetin in which at least one hydroxyl group of quercetin is methylated, and this activity is obtained by methylating at least one to three hydroxyl groups of quercetin. It can be an activity to produce methoxy quercetin. Measurement of the activity to act on quercetin to produce methoxyquercetin in which at least one hydroxyl group of quercetin is methylated is carried out as a substrate according to the method described in “1.9 Measurement of FOMT activity” in the experimental method described later. It can be carried out by using quercetin.

The enzymological properties of CdFOMT5 of the present invention are described below. Proteins having the same enzymatic properties as CdFOMT5 are also included in the scope of the present invention.
(1) Action, substrate specificity
CdFOMT5 has an activity of catalyzing a methyl transfer reaction at a plurality of hydroxyl groups with respect to quercetin (having five hydroxyl groups), as specifically described in Examples described later. In the examples, it was confirmed that quercetin was formed by methylating (methoxylating) hydroxyl groups at a maximum of four locations. Furthermore, it has been confirmed in the Examples that CdFOMT5 has an activity of catalyzing a methyl transfer reaction with respect to a hydroxyl group of 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone and 7,8-dihydroxyflavone. The results of the examples suggest that CdFOMT5 methylates at least the 3, 5, 6, 7 OH groups of flavones.

(2) Optimum reaction pH and temperature
The optimum pH of CdFOMT5 when quercetin is used as a substrate is 5.5 to 8.0, and more specifically, pH 6.0 to 7.8. The optimum pH is around 7.0. CdFOMT5 can exhibit about 60% or more of the maximum activity at 25 to 60 ° C. using quercetin as a substrate at an optimum pH. More specifically, it can exhibit about 80% or more of the maximum activity at 35 to 55 ° C. More specifically, it can exhibit about 90% or more of the maximum activity at 42-52 ° C. The optimum temperature is around 45 ° C.

(3) Molecular mass
The molecular mass of CdFOMT5 is calculated to be about 40 kDa from SDS-PAGE.

(4) Other
CdFOMT5 can produce trimethoxyquercetin in which three methyl groups (3,5,7 OH) are methylated when quercetin is used as a substrate. In methylation of hydroxyflavone by CdFOMT5 purified enzyme, methylation to 3, 5, 6, 7-position OH has been confirmed. Furthermore, when CdFOMT5-expressing E. coli is allowed to act on quercetin, 3,3 ′, 5,7-tetramethoxyquercetin can be produced. For continuous quercetin methylation reaction, it is preferable to supply SAM (S-adenosylmethionine).
CdFOMT5 is derived from an organism belonging to the genus Citrus, more specifically a squeaker (Citrus depressa).

  CdFOMT6 is a protein having the amino acid sequence described in SEQ ID NO: 4 in the sequence table of (m) above, and the protein related to CdFOMT6 is 1 in the amino acid sequence described in SEQ ID NO: 4 of the sequence table of (n) above. A protein having an amino acid sequence having a deletion, substitution and / or addition of several amino acids from and having an amino acid encoding a flavonoid-O-methyltransferase having the activity described in (2) above, and A flavonoid-O-methyltransferase having an amino acid sequence having 90% or more identity to the amino acid sequence described in SEQ ID NO: 4 in the sequence listing of (o) and having the activity described in (2) above Is a protein having an amino acid sequence encoding

The activity described in (2) is an activity that acts on quercetin to produce methoxyquercetin in which the hydroxyl group at the 3 ′ position of quercetin is selectively methylated. The activity of producing methoxyquercetin that acts on quercetin to selectively methylate the 3′-position hydroxyl group of quercetin is determined according to the method described in “1.9 Measurement of FOMT activity” in the experimental method described later. It can be carried out by using quercetin as a substrate.

The enzymological properties of CdFOMT6 of the present invention are described below. Proteins having the same enzymatic properties as CdFOMT6 are also included in the scope of the present invention.
(1) Action, substrate specificity
CdFOMT6 has an activity of selectively catalyzing the methyl transfer reaction of the hydroxyl group at the 3 ′ position with respect to quercetin (having five hydroxyl groups), as specifically described in Examples described later. In the examples, the production of quercetin (isoramnetin (3′-methoxyquercetin)) in which the hydroxyl group at the 3 ′ position of quercetin is methylated (methoxylated) has been confirmed. Furthermore, it was also confirmed in the Examples that CdFOMT6 has an activity of catalyzing a methyl transfer reaction to the hydroxyl group of catechin, myricetin, luteolin and flavanol as epicatechin and epigallocatechin 3-O-gallate (EGCg) as flavones. ing. However, methylation activity was not shown for kaempferol (3,5,7,4′-tetrahydroxyflavone) as flavones and caffeic acid, resveratrol and gallic acid methyl ester as other substrates. The lack of methylation activity against kaempferol (3,5,7,4'-tetrahydroxyflavone) suggests that CdFOMT6 specifically methylates the 3'OH of flavones and flavans. It was. In addition, since the activity for luteolin and epicatechin having two hydroxyl groups in the B-ring of flavonoids is higher than that for myricetin and EGCg having three hydroxyl groups, the OH group of the B-ring is a high substrate for two substrates. It is suggested to show specificity.

(2) Optimum reaction pH and temperature
The optimum pH of CdFOMT6 when quercetin is used as a substrate is 7.0 to 9.6, and more specifically, pH 7.5 to 8.5. The optimum pH is around 8.0. CdFOMT6 can exhibit about 60% or more of the maximum activity at 37 to 53 ° C. using quercetin as a substrate at an optimum pH. More specifically, it can exhibit an activity of about 80% or more at the maximum at 38 to 51 ° C. More specifically, it can exhibit about 90% or more of the maximum activity at 40-49 ° C. The optimum temperature is around 45 ° C.

(3) Molecular mass as fusion protein The molecular mass of CdFOMT6 expressed as a fusion protein with maltose binding protein (MBP: molecular mass 42.5 kDa) is calculated to be about 80 kDa from SDS-PAGE.

(4) Others For continuous quercetin methylation reaction, it is preferable to supply SAM (S-adenosylmethionine).
CdFOMT5 is derived from an organism belonging to the genus Citrus, more specifically a squeaker (Citrus depressa).

  The range of “1 to several” in “amino acid sequence having deletion, substitution and / or addition of 1 to several amino acids” in the above (l) is not particularly limited, but for example, 1 to 20, preferably Means from 1 to 10, more preferably from 1 to 7, even more preferably from 1 to 5, particularly preferably 1, 2, 3 or 4.

When substituting amino acid residues in proteins, it is particularly preferable to perform so-called conservative amino acid substitutions with amino acids having similar side chain chemical properties. Amino acids are classified according to their side chain chemistry, for example:
(1) Neutral hydrophobic side chain (alanine, tryptophan, valine, phenylalanine, proline, methionine, leucine);
(2) Neutral polar side chain (asparagine, glycine, glutamine, cysteine, serine, tyrosine, threonine);
(3) basic side chains (arginine, histidine, lysine);
(4) acidic side chains (aspartic acid, glutamic acid);
(5) aliphatic side chain (alanine, isoleucine, glycine, valine, leucine);
(6) Aliphatic hydroxyl side chain (serine, threonine);
(7) amine-containing side chains (asparagine, arginine, glutamine, histidine, lysine);
(8) aromatic side chains (tyrosine, tryptophan, phenylalanine); and
(9) Sulfur-containing side chains (cysteine, methionine).

  In the present invention, when referring to `` identity '' with respect to amino acid sequences, except when specifically described, when the two sequences are aligned in an optimal manner, the percentage of the number of matched amino acids shared between the two sequences is calculated. means. That is, identity = (number of matched positions / total number of positions) × 100, which can be calculated using a commercially available algorithm. Such an algorithm is also described in Altschul et al. , J .; Mol. Biol. 215 (1990) 403-410, incorporated into the NBLAST and XBLAST programs. More specifically, search / analysis regarding amino acid sequence identity can be performed by algorithms or programs (for example, BLASTN, BLASTP, BLASTX, ClustalW) well known to those skilled in the art. Those skilled in the art can appropriately set parameters when using a program, and the default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

  The identity in the “o amino acid sequence having 96% or more identity to the amino acid sequence described in SEQ ID NO: 2 in the sequence listing” in the above (o) is not particularly limited as long as it is 96% or more, but more preferably 97% or more, more preferably 98% or more, and further preferably 99%.

  The identity in the “amino acid sequence having 90% or more identity to the amino acid sequence described in SEQ ID NO: 4 in the sequence listing” in the above (p) is not particularly limited as long as it is 90% or more, but more preferably It is 95% or more, more preferably 96% or more, further preferably 97%, more preferably 98%, and particularly preferably 99% or more.

  The method for obtaining the protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique. When producing a recombinant protein, first, a polynucleotide encoding the protein of the present invention described later is obtained. The protein of the present invention can be produced by using a transformed cell obtained by introducing this polynucleotide into an appropriate expression system. The expression of the protein in the expression system is also described later in this specification.

  Proteins produced by genetic engineering techniques are recovered from biological materials containing the proteins. For example, when the protein is secreted outside the host cell, the protein is recovered from the medium in which the cell is cultured. When the host is a transgenic organism, the target protein can be recovered from the body fluid. Alternatively, when the protein is produced intracellularly, the protein is recovered from the lysate obtained by lysing the cell.

The recovered protein can be purified by the same means as when the protein is purified from cells that naturally produce the protein. In other words, known purification methods such as salting out, distillation, various chromatography, gel electrophoresis, gel filtration, ultrafiltration, recrystallization, acid extraction, dialysis, immunoprecipitation, solvent precipitation, solvent extraction, ammonium sulfate or ethanol precipitation, etc. In combination, the target protein can be purified. A person skilled in the art can use, for example, the following various types of chromatography in combination. For these chromatography, liquid phase chromatography systems such as HPLC and FPLC can be used.
Affinity chromatography,
Ion exchange chromatography such as anion or cation exchange,
Reverse phase chromatography,
Adsorption chromatography,
Gel filtration,
Hydrophobic chromatography,
Hydroxyapatite chromatography

  Moreover, if the protein of the present invention (FOMT) is expressed as a fusion protein with a tag, it can be recovered using a column that binds to the tag. For example, a fusion protein having a GST tag can be easily separated using a glutathione column. Alternatively, the fusion protein with a histidine tag can be purified using a nickel column. A protease recognition sequence can be inserted between the tag and the protein (FOMT). For example, thrombin or factor Xa can be used as the protease. After binding the fusion protein to the column, the column is washed if necessary. Subsequently, when these proteases are allowed to act, the target protein is cleaved from the tag. Thereafter, the target protein can be easily purified by recovering the cleaved protein.

  The protein of the present invention may be made into an enzyme agent (a composition containing an enzyme and other components) by adding components other than the enzyme such as a stabilizer, a buffer, a solubilizing agent, an antioxidant, and a diluent. it can.

[Flavonoid O-methyltransferase (FOMT) gene]
The present invention relates to polynucleotides encoding the amino acid sequences of CdFOMT5 and CdFOMT6 and their associated proteins.

The polynucleotide encoding the amino acid sequence of CdFOMT5 and related proteins is the polynucleotide described in any of (a) to (f) below.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(B) a polynucleotide encoding a flavonoid-O-methyltransferase having 96% or more identity with the base sequence described in SEQ ID NO: 1 and having the activity described in (1) below;
(C) A flavonoid-O-methyltransferase that hybridizes to the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 under highly stringent conditions and has the activity described in (1) below. A polynucleotide encoding
(D) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2;
(E) a flavonoid having the activity described in (1) below, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 2 A polynucleotide encoding O-methyltransferase; and (f) a flavonoid -O- having 96% or more identity with the amino acid sequence of SEQ ID NO: 2 and having the activity described in (1) below A polynucleotide encoding a methyltransferase;
(1) The activity of acting on quercetin to produce methoxyquercetin in which at least one hydroxyl group of quercetin is methylated.

  A flavonoid comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 2 of (e) and having the activity described in (1) below -O-methyltransferase, and (e) a flavonoid-O-methyltransferase having 96% or more identity with the amino acid sequence described in SEQ ID NO: 2 and having the activity described in (1) below: Furthermore, the activity described in (1) is as described in the description of CdFOMT5 as a protein.

  “Highly stringent conditions” in “hybridizes under high stringency conditions to the polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1” described in (c) is, for example, ECL Using direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech), the conditions described in the manual (for example, wash: 42 ° C, primary wash buffer containing 0.5 × SSC. Is hybridized in 150 mM sodium chloride and 15 mM sodium citrate). More specific “highly stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS conditions, preferably 50 ° C., 2 × SSC, 0.1% SDS conditions. More preferably, the conditions are 65 ° C., 0.1 × SSC and 0.1% SDS. Alternatively, hybridization can be performed at 45 ° C. in the presence of 2 × SSC and 50% formamide followed by washing the filter at 60 ° C. with 0.1 × SSC, or 2 × SSC and 50% formamide. After hybridization at 50 ° C. in the presence, a condition of washing the filter at 65 ° C. using a 0.1 × SSC solution may be used. In addition to the salt concentration and temperature, multiple factors including probe concentration, probe length, and reaction time affect the stringency of hybridization. Those skilled in the art can adjust stringency by appropriately selecting these elements.

  For example, the base sequence of the probe used for hybridization can be selected from the base sequences described in SEQ ID NO: 1 or 3. For example, a polynucleotide having at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive base sequences can be used as a probe. Alternatively, a polynucleotide having the full length of the base sequence described in SEQ ID NO: 1 can be used as a probe.

  A polynucleotide that can hybridize under highly stringent conditions is likely to encode an amino acid sequence having high identity (homology) with the amino acid sequence set forth in SEQ ID NO: 2. Specifically, a polynucleotide encoding an amino acid sequence having 96% or more identity with the amino acid sequence shown in SEQ ID NO: 2 is included in the polynucleotide of the present invention. Such proteins with high identity are likely to have the same or similar activities.

The polynucleotide encoding the amino acid sequence of CdFOMT6 and related proteins is the polynucleotide described in any of (g) to (l) below.
(G) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3;
(H) a polynucleotide encoding a flavonoid-O-methyltransferase having 90% or more identity with the base sequence set forth in SEQ ID NO: 3 and having the activity described in (2) below;
(I) A flavonoid-O-methyltransferase that hybridizes to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3 under highly stringent conditions and has the activity described in (2) below. A polynucleotide encoding
(J) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 4;
(K) a flavonoid having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 4 and having the activity described in (2) below: A polynucleotide encoding O-methyltransferase; and (l) a flavonoid-O— having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4 and having the activity set forth in (2) below: A polynucleotide encoding a methyltransferase;
(2) The activity of acting on quercetin to produce methoxyquercetin in which the hydroxyl group at the 3 ′ position of quercetin is selectively methylated.

  The amino acid sequence of SEQ ID NO: 4 described in (k) includes an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and has the activity described in (2) Flavonoid-O-methyltransferase and (j) a flavonoid-O-methyltransferase having 90% or more identity with the amino acid sequence of SEQ ID NO: 4 and having the activity of (2) Further, the activity described in (2) is as described in the description of CdFOMT6 as the above-mentioned protein.

  “Highly stringent conditions” in “hybridize under high stringency conditions to the polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 4” described in (i) is CdFOMT5 ( Same as “Highly stringent conditions” for c).

  A polynucleotide capable of hybridizing under highly stringent conditions is likely to encode an amino acid sequence having high identity (homology) with the amino acid sequence set forth in SEQ ID NO: 4. Specifically, a polynucleotide encoding an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4 is included in the polynucleotide of the present invention. Such proteins with high identity are likely to have the same or similar activities.

  In the present invention, when referring to “identity” with respect to a base sequence (sometimes referred to as a nucleotide sequence), it is shared between two sequences when the two sequences are aligned in an optimal manner, unless otherwise specified. Means the percentage of the number of matched nucleotides. That is, identity = (number of matched positions / total number of positions) × 100, which can be calculated using a commercially available algorithm. Such an algorithm is also described in Altschul et al. , J .; Mol. Biol. 215 (1990) 403-410, incorporated into the NBLAST and XBLAST programs. More specifically, search / analysis regarding nucleotide sequence identity can be performed by algorithms or programs (for example, BLASTN, BLASTP, BLASTX, ClustalW) well known to those skilled in the art. Those skilled in the art can appropriately set parameters when using a program, and the default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

  The identity in the above (b) is not particularly limited as long as it is 96% or more, but is more preferably 97% or more, further preferably 98% or more, and more preferably 99%.

  The identity in the above (h) is not particularly limited as long as it is 90% or more, more preferably 95% or more, more preferably 96% or more, further preferably 97%, more preferably 98%, particularly preferably 99. % Or more.

  The method for obtaining the polynucleotide of the present invention is not particularly limited. By preparing appropriate probes and primers based on the amino acid sequence and nucleotide sequence information described in SEQ ID NOs: 1 to 4 in the sequence listing in the present specification, and using them to screen a cDNA library of seeker, The polynucleotides of the invention can be isolated. A cDNA library can be prepared from cells such as a seeker leaf by a conventional method.

  The polynucleotide of the present invention can also be obtained by PCR. PCR is carried out using a chromosomal DNA library or cDNA library of a sequencer as a template and a pair of primers designed to amplify the base sequence described in SEQ ID NO: 1 or 2. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C for 30 seconds (denaturation), 55 ° C for 30 seconds to 1 minute (annealing), and 72 ° C for 2 minutes (extension) As an example, there may be mentioned conditions for carrying out the reaction at 72 ° C. for 7 minutes after 30 cycles. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

  Operations such as preparation of the probe or primer, construction of the cDNA library, screening of the cDNA library, and cloning of the target gene are known to those skilled in the art and can be performed according to conventional methods.

  In addition, it encodes a protein having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence set forth in SEQ ID NO: 3 or 4 in the sequence listing and having a predetermined methyltransferase activity And a nucleotide sequence having a deletion, substitution and / or addition of one to several bases in the nucleotide sequence set forth in SEQ ID NO: 1 or 2 in the sequence listing, and encodes a predetermined methyltransferase For genes having base sequences (hereinafter, these genes are referred to as mutant genes), chemical synthesis, genetic engineering techniques or mutagenesis based on the amino acid sequence and base sequence information described in SEQ ID NOs: 1 to 4 Or any other method known to those skilled in the art.

  For example, it can be carried out using a method of contacting a DNA having the base sequence set forth in SEQ ID NO: 1 or 2 in the sequence listing with a drug that becomes a mutagen, a method of irradiating with ultraviolet rays, a genetic engineering method, . Site-directed mutagenesis, which is one of genetic engineering techniques, is useful because it can introduce a specific mutation at a specific position.

[Recombinant vector]
The polynucleotide of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes. Preferably, the vector used in the present invention is an expression vector. Examples of suitable vectors include various vectors such as plasmids, cosmids, viruses, and bacteriophages. In the expression vector, the gene of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.

  The recombinant vector of the present invention can be constructed according to techniques commonly used in the fields of molecular biology, biotechnology and genetic engineering. In order to express the polynucleotide of the present invention using a microorganism or the like as a host, it is first necessary to introduce the DNA into a plasmid vector or a phage vector stably present in the microorganism, and to transcribe and translate the genetic information. There is. For that purpose, a promoter is usually arranged 5 'upstream of the polynucleotide of the present invention. A promoter is a unit that controls transcription and translation.

  And it is preferable to arrange | position a terminator 3 'downstream of the polynucleotide of this invention. As the promoter and terminator, those that function in the microorganism used as the host can be selected. In addition, control sequences necessary for transcription and / or translation such as enhancer, operator sequence, initiation signal, polyadenylation signal, and ribosome binding site can be incorporated as necessary. The vector of the present invention preferably contains all the components of the regulatory sequences required for the expression of the inserted polynucleotide of the present invention. Furthermore, the vector of the present invention can contain a selection marker for selecting a host cell into which the vector has been introduced.

  In the present invention, a sequence encoding a signal peptide can also be added to the polynucleotide of the present invention. By adding a signal peptide, proteins expressed in the host cell can be transferred to the endoplasmic reticulum lumen. Alternatively, when a gram-negative bacterium is used as a host, a protein expressed in the host cell can be transferred into the periplasm or out of the cell by a signal peptide. Any signal peptide that can function in the host cell utilized can be utilized. Therefore, a signal peptide derived from a different species for the host can be used. Furthermore, a linker, an initiation codon (ATG), a termination codon (TAA, TAG or TGA), etc. can be added when introducing the polynucleotide of the present invention into a vector, if necessary.

[Host and transformed cells]
The host for expressing the polynucleotide of the present invention is not particularly limited as long as it is an organism that can be transformed with a recombinant vector containing the polynucleotide and can express a protein having a methyltransferase. The present invention provides a transformant (transformed cell) transformed with the polynucleotide of the present invention or the vector of the present invention. Examples of the microorganism that is a target of the transformant of the present invention include the following microorganisms.
(1) Bacteria for which host vector systems have been developed: Escherichia genus, Bacillus genus, Pseudomonas genus, Serratia genus, Brevibacterium genus, Corynebacterium genus ( Corynebacterium genus, Streptococcus genus, Lactobacillus genus, etc.
(2) Actinomycetes, Rhodococcus genus, Streptomyces genus, etc. for which host vector systems have been developed
(3) Yeast, Saccharomyces genus, Kluyveromyces genus, Schizosaccharomyces genus, Zygosaccharomyces genus, Yarrowia genus, Trichosporon (Trichosporon) genus, Rhodosporidium genus, Pichia genus, Candida genus, etc.
(4) Host vectors have been developed, such as molds, Neurospora, Aspergillus, Cephalosporium, Trichoderma

  For example, in the genus Escherichia, especially Escherichia coli, plasmid vectors such as pET, pBR, and pUC can be used, and lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp fusion) ), Promoters derived from λ phage PL, PR, etc. can be used. Moreover, as the terminator, a terminator derived from trpA, phage, or rrnB ribosomal RNA can be used. In particular, a vector pSE420D obtained by partially modifying the multicloning site of commercially available pSE420 (manufactured by Invitrogen) is a suitable vector when an Escherichia bacterium is used as a host.

  In the genus Bacillus, pUB110 series plasmids, pC194 series plasmids and the like can be used as vectors. When these vectors are used, the polynucleotide of the present invention can be integrated into the host chromosome. As the promoter and terminator, apr (alkaline protease), npr (neutral protease), amy (α-amylase) and the like can be used.

  In the genus Pseudomonas, host vector systems have been developed for Pseudomonas putida, Pseudomonas cepacia, and the like. Wide host range vector (including genes necessary for autonomous replication derived from RSF1010 etc.) based on plasmid TOL plasmid involved in degradation of toluene compounds pKT240 etc. can be used, derived from lipase gene as promoter / terminator Can be used.

  In the genus Brevibacterium, particularly Brevibacterium lactofermentum, plasmid vectors such as pAJ43 (Gene (1985) 39: 281) can be used. As the promoter / terminator, the promoter and terminator used in Escherichia coli can be used as they are.

  In the genus Corynebacterium, particularly Corynebacterium glutamicum, plasmid vectors such as pCS11 (Japanese Patent Laid-Open No. 57-183799) and pCB101 (Mol Gen Genet (1984) 196: 175) can be used.

  In the genus Streptococcus, pHV1301 (FEMS Microbiol Lett (1985) 26: 239), pGK1 (Appl Environ Microbiol (1985) 50:94) and the like can be used as plasmid vectors.

  In the genus Lactobacillus, pAMβ1 (J Bacteriol (1979) 137: 614) developed for Streptococcus can be used as a vector, and a promoter used in Escherichia coli can be used.

  In the genus Rhodococcus, plasmid vectors isolated from Rhodococcus rhodochrous can be used in addition to Rhodococcus opacus and Rhodococcus erythropolis (J Gen Microbiol (1992) 138: 1003).

  In the genus Streptomyces, a plasmid can be constructed according to the method described in Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual Cold Spring Harbor Laboratories (1985). In particular, in Streptomyces lividans, pIJ486 (Mol Gen Genet (1986) 203: 468-78), pKC1064 (Gene (1991) 103: 97-9), pUWL-KS (Gene (1995) 165: 149-50) can be used. A similar plasmid can also be used in Streptomyces virginiae (Actinomycetol (1997) 11: 46-53).

  In the genus Saccharomyces, particularly Saccharomyces cerevisiae, YRp, YEp, YCp, and YIp plasmids can be used. In addition, an integration vector (such as EP537456) using homologous recombination with ribosomal DNA present in multiple copies in a chromosome is extremely useful because it can introduce a gene in multiple copies and can stably hold the gene. ADH (alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK (phosphoglycerate kinase), ENO (enolase) Promoter terminators such as are available.

  In the genus Klyberomyces, particularly Kluyveromyces lactis, a 2 μm-derived plasmid derived from Saccharomyces cerevisiae, a pKD1-based plasmid (J Bacteriol (1981) 145: 382-90), pGKl1 involved in killer activity A plasmid derived from the gene, an autonomously proliferating gene KARS plasmid in the genus Kleiberymyces, a vector plasmid (EP537456, etc.) that can be integrated into the chromosome by homologous recombination with ribosomal DNA and the like can be used. In addition, promoter / terminators derived from ADH, PGK and the like can be used.

  In the genus Schizosaccharomyces, plasmid vectors containing ARS (genes involved in autonomous replication) derived from Schizosaccharomyces pombe and selection markers that complement the auxotrophy derived from Saccharomyces cerevisiae are available. (Mol Cell Biol (1986) 6:80). Further, an ADH promoter derived from Schizosaccharomyces pombe can be used (EMBO J (1987) 6: 729). In particular, pAUR224 is commercially available from Takara Shuzo and can be easily used.

  In the genus Tigosaccharomyces, plasmid vectors derived from pSB3 (Nucleic Acids Res. 13: 4267 (1985)) derived from Zygosaccharomyces rouxii can be used, and the PHO5 promoter derived from Saccharomyces cerevisiae, and Tigo A promoter of Saccharomyces rouxi-derived GAP-Zr (glyceraldehyde-3-phosphate dehydrogenase) (Agri Biol Chem (1990) 54: 2521) and the like can be used.

  In the genus Pichia, a host vector system using Pichia angusta (former name: Hansenula polymorpha) has been developed. As vectors, genes involved in autonomous replication derived from Pichia Angusta (HARS1, HARS2) can be used, but they are relatively unstable, so multicopy integration into the chromosome is effective (Yeast (1991) 7 : 431-43). In addition, AOX (alcohol oxidase), FDH (formate dehydrogenase) promoter, etc. induced by methanol or the like can be used. In addition, for Pichia pastoris, etc., a host vector system utilizing genes (PARS1, PRS2), etc. involved in Pichia-derived autonomous replication has been developed (Mol Cell Biol (1985) 5: 3376). A strong promoter such as AOX that can be induced by concentration culture and methanol can be used (Nucleic Acids Res (1987) 15: 3859).

  In the genus Candida, host vector systems have been developed for Candida maltosa, Candida albicans, Candida tropicalis, Candida utilis, etc. ing. In Candida maltosa, ARS derived from Candida maltosa has been cloned (Agri Biol Chem (1987) 51: 1587), and a vector using this has been developed. Further, in Candida utilis, a strong promoter has been developed for a chromosome integration type vector (Japanese Patent Laid-Open No. 08-173170).

  In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae and the like have been most studied. Plasmids using these as hosts can be used, and integration of desired genes into chromosomes can be performed. Promoters derived from extracellular proteases and amylases are also available (Trends in Biotechnology (1989) 7: 283-7).

  In the genus Trichoderma, a host vector system using Trichoderma reesei has been developed, and an extracellular cellulase gene-derived promoter or the like can be used (Biotechnology (1989) 7: 596-603).

  In addition to microorganisms, various host vector systems using plants and animals as hosts have been developed. For example, insect vector systems using moths (Nature (1985) 315: 592-4) and plant vector systems such as rapeseed, corn and potato have been developed as systems for expressing large amounts of heterologous proteins. Available to:

  Introduction of the polynucleotide of the present invention into a vector can be performed by a ligase reaction utilizing a restriction enzyme site. In addition, in consideration of the codon usage frequency of the host to be used, the polynucleotide sequence of the present invention may be modified as necessary to design a vector having high expression efficiency.

  As described above, various cells have been established as host cell lines. Methods for introducing an expression vector suitable for each cell line are also known, and those skilled in the art can select a method suitable for each selected host cell. For example, for prokaryotic cells, calcium treatment, transformation by electroporation and the like are known. For plant cells, methods using Agrobacterium are known, and for mammalian cells, calcium phosphate precipitation can be exemplified. The present invention is not particularly limited to these methods, and other known nuclear microinjection, protoplast fusion, DEAE-dextran method, cell fusion, electric pulse perforation method, lipofectamine method (GIBCO BRL) depending on the selected host. The expression vector can be introduced by various known methods including a method using FuGENE6 reagent (Boehringer-Mannheim).

  By culturing the transformant transformed with the recombinant vector containing the polynucleotide of the present invention as described above, the protein having the predetermined methyl transfer activity of the present invention can be produced. Therefore, as a preferred embodiment of the present invention, there is provided a method for producing a methyltransferase, comprising the steps of culturing the transformant of the present invention and recovering the protein encoded by the polynucleotide from the culture. . The method for culturing the transformant is not particularly limited, and it is desirable to select conditions such as a medium, temperature, and time that are suitable for the growth of each selected host cell and most suitable for the production of the enzyme of the present invention. For example, the conditions described in the examples can be mentioned, but the present invention is not limited thereto.

[Protein production method]
The present invention
The protein encoded by the polynucleotide of the present invention or the protein of the present invention (FOMT), comprising the step of culturing the transformed cell of the present invention and recovering the protein encoded by the polynucleotide of the present invention from the culture. It relates to the manufacturing method.

[Method for producing methoxyflavonoid and / or its analog]
The present invention provides a process for producing a methoxyflavonoid and / or an analog thereof, comprising the steps of causing the protein of the present invention or the transformed cell of the present invention to act on a flavonoid and / or an analog thereof, and recovering the produced compound. Regarding the method.

  Flavonoids and / or analogs thereof include kaempferol, quercetin, myricetin, fisetin, galangin, gosipetin, morin, apigenin, luteolin, naringenin, taxifolin, butyne, eriodictyol, pinocembrin, cyanidin, delphinidin, catechin, epicatechin, epicatechin It is at least one compound selected from the group consisting of gallocatechin, epicatechin gallate, epigallocatechin gallate, epiafzerequin, fisetinidol, guiburtinidol, mesquitol, robinetinidol / analogues thereof.

The step of allowing the protein of the present invention or the transformed cell of the present invention to act on a flavonoid and / or an analog thereof can be performed, for example, under the following conditions.
Solvent: M9 minimal medium (containing 1% glucose and 10 mM L-methionine)
pH: 7.0
Temperature: 30 ° C
Time: 3 hours Use amount of protein or transformed cell of the present invention: 1% (w / v)
Substrate: 100 μM to 10 mM

The step of recovering the compound produced in the above step can be specifically performed as follows.
An equal amount of ethyl acetate is added to the reaction solution after completion of the reaction and mixed, and then the ethyl acetate phase is separated and recovered. This is repeated several times, and the recovered ethyl acetate phases are combined, dehydrated with sodium sulfate, and then the solvent is removed under reduced pressure to obtain a dried product. The obtained dried product is again dissolved in ethyl acetate, and then separated and purified using silica gel chromatography or the like to obtain the desired methoxyflavonoid.
In this way, methoxyflavonoids and / or analogs thereof can be produced.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

1 Experimental method
1.1 Host used, vector
As a FOMT gene source, a seeker from Okinawa Prefecture was used. Escherichia coli JM109 was used as a cloning host, and E. coli JM109 and E. coli BL21 (DE3) were used as expression hosts. PGEM-T Easy (Promega) was used as a cloning vector, and pET-21b (+) (Novagen) and pMAL-c5x (New England BioLabs) were used as expression vectors, respectively.

1.2 Preparation of genomic DNA Genomic DNA was prepared from a sequencer according to the technique of Dellaporta et al. 10 grams of Sikuwasa leaves were ground with a mortar pestle under liquid nitrogen, and 40 ml of CTAB extract solution was added and mixed. After incubating at 65 ° C. for 60 minutes, an equal volume of chloroform-isoamyl alcohol (24: 1) was added and mixed. The mixture was separated by centrifugation (7,500 rpm, 5 minutes), and the upper aqueous phase was collected in a new tube. 1/10 volume of CTAB / NaCl solution was added to the recovered aqueous phase, and the mixture was kept warm and stirred at 65 ° C for 30 minutes. An equal amount of chloroform-isoamyl alcohol was added and mixed, separated by centrifugation (7,500 rpm, 5 minutes), and the upper layer was recovered. An equal amount of CTAB precipitation solution was added to the upper layer, mixed, and kept at 65 ° C. for 30 minutes. Nucleic acid was precipitated by centrifugation (500 × g, 4 ° C., 5 minutes), and the supernatant was removed. 1 ml of high salt TE buffer was added to the nucleic acid pellet and dissolved. After dissolving the pellet, 0.6 volumes of isopropyl alcohol was added and mixed, and the nucleic acid was precipitated by centrifugation (7,500 rpm, 4 ° C., 5 minutes). The nucleic acid pellet was washed with an 80% ethanol solution, dried, dissolved in 1 ml of TE buffer, and used as a genomic DNA sample.

1.3 Preparation of Total RNA Total RNA was prepared from Sequencer using TRI Reagent ^™ from Cosmo Bio. The extraction work was performed according to the recommended protocol of Cosmo Bio.

1.4 Isolation of FOMT gene by PCR Alignment was made based on the amino acid sequence of FOMT derived from various plants obtained from the database, and degenerate primers corresponding to the conserved region were made. The generated degenerate primers are listed in Table 1. Using these primers in any combination, PCR was performed using the genomic DNA of the seeker as a template, and a gene fragment containing a partial sequence of the FOMT gene was amplified. The amplified DNA fragments were separated by agarose gel electrophoresis and then extracted and collected from the gel. The collected PCR fragment was cloned into pGEM-T Easy vector (Promega) and transformed into E. coli JM109. A plasmid was extracted from the obtained transformant, and the sequence of the PCR amplified fragment inserted by sequencing was confirmed. After confirming that the fragment having a sequence similar to known FOMT is be cloned, based on the obtained DNA sequence was created primers used in TAIL-PCR (T hermal A symmetric I nter l aced PCR) ( Table 1). After determining the full-length sequence of the genes corresponding to 5 types of FOMT (FOMT1,3,4,5,6) by TAIL-PCR, a primer was designed based on the putative ORF both end sequences and RT-PCR was performed. Each FOMT cDNA fragment was amplified and cloned by RT-PCR, and the sequence and intron were determined.

1.5 Construction of Recombinant CdFOMT5 and CdFOMT6 Expression Plasmids CdFOMT5 and CdFOMT6 PCR fragments amplified using the cDNA amplification primers shown in Table 1 were restricted with restriction enzymes BamH I / Sal I (CdFOMT5) and Sma I / BamHI ( CdFOMT6) and ligated with pET-21b (+) and pMAL-c5x which were similarly treated with restriction enzymes. The ligated DNA sample was introduced into E. coli JM109 by electroporation, spread on LB agar medium containing antibiotics and cultured at 37 ° C. The obtained transformant was isolated, extracted with a plasmid, and treated with a restriction enzyme to confirm that the target insert was inserted into an arbitrary vector. The constructed expression plasmids were designated as pET-CdFOMT5 and pMAL-CdFOMT6, respectively, and pET-CdFOMT5 was transformed into E. coli BL21 (DE3) as an expression host. pMAL-CdFOMT6 used E. coli JM109 as an expression host as it was. The obtained transformant was isolated and subjected to subsequent expression experiments.

1.6 Recombinant enzyme expression
Culture of transformed Escherichia coli containing pET-CdFOMT5 and pMAL-CdFOMT6 and induction of expression of the recombinant enzyme were performed according to the following procedures.

1.6.1 Induction of recombinant CdFOMT5 expression
E. coli BL21 (DE3) / pET-CdFOMT5 recombinant cells were platinized in 4 ml of LB medium (50 μg / ml ampicillin) and pre-cultured overnight at 37 ° C. 500 μl of the preculture solution was inoculated into 500 ml LB medium (50 μg / ml ampicillin) and cultured at 37 ° C. and 200 rpm. When OD ₆₆₀ reached 0.5, IPTG (isopropyl-β-thio-D-galactoside) as an inducer was added to a final concentration of 0.1 mM, and induction was performed at 18 ° C. and 200 rpm for 24 hours. After the induction, the cells were collected by centrifugation (4 ° C., 12,000 rpm, 10 minutes) and washed with 50 mM potassium phosphate buffer (KPB, pH 7.2). After washing, the cells were collected again by centrifugation and used for the subsequent experiments.

1.6.2 Induction of recombinant CdFOMT6 expression
E. coli JM109 / pMAL-CdFOMT6 Recombinant cells were platinized in 4 ml of LB medium (50 μg / ml ampicillin, 2% (w / v) glucose) and pre-cultured at 30 ° C. overnight. 500 μl of the preculture solution was inoculated into 500 ml LB medium (50 μg / ml ampicillin, 2% (w / v) glucose) and cultured at 30 ° C. and 200 rpm. When OD ₆₆₀ reached 2.0, IPTG as an inducer was added to a final concentration of 0.3 mM, and induction was performed at 18 ° C. and 200 rpm for 24 hours. After the induction, the cells were collected by centrifugation (4 ° C., 12,000 rpm, 10 minutes) and washed with 50 mM KPB (pH 7.5). After washing, the cells were collected again by centrifugation and used for the subsequent experiments.

1.7 Purification of recombinant enzyme Recombinant CdFOMT5 and CdFOMT6 were purified as follows.

1.7.1 Purification of recombinant CdFOMT5
1. Recombinant cells whose expression was induced in 1.6.1 were suspended in Ni-Sepharose binding buffer (50 mM KPB, 200 mM NaCl, 10% glycerol, 10 mM imidazole, pH 7.2) and sonicated. The cells were crushed. The cell lysate was centrifuged (4 ° C., 17,000 rpm, 20 minutes), and the supernatant was transferred to a new tube. Centrifugation was performed again under the same conditions, and the supernatant was collected. The cell lysate supernatant was applied to a Ni-Sepharose column (10 ml bed) equilibrated with binding buffer to adsorb the target protein. After washing the column with 100 ml of binding buffer, the target protein was eluted with an dilution buffer (50 mM KPB, 200 mM NaCl, 10% glycerol, 250 mM imidazole, pH 7.2). Fractionation was 10 ml x 10. After quantifying the protein concentration by the Brad-ford method, the target protein elution fraction was subjected to subsequent SDS-PAGE and activity measurement experiments.

1.7.2 Purification of recombinant CdFOMT6
1. Recombinant bacterial cells whose expression has been induced in 1.6.2 are suspended in amylose resin binding buffer (20 mM Tris-HCl, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, pH 7.4). In the same manner as in 1.7.1, the cells were disrupted by sonication and the supernatant was collected. The lysate supernatant was applied to an amylose regin column (2 ml bed) equilibrated with Amylose resin binding buffer, and the non-adsorbed fraction was collected. After washing the column with 12 ml binding buffer, the adsorbed protein was eluted using an elution buffer containing 20 mM maltose. Fractionation was 2 ml x 10. After quantifying the protein concentration by the Brad-ford method, the fraction containing the target protein was subjected to subsequent SDS-PAGE and activity measurement experiments.

1.8 Confirmation of recombinant protein by SDS-PAGE
The recombinant CdFOMT protein purified in 1.7 was confirmed by SDS-PAGE. SDS-PAGE was performed according to a conventional method, and the acrylamide gel concentrations were 12.5% (CdFOMT5) and 10% (CdFOMT6), respectively. The cell lysate corresponding to 20 μg protein, the non-adsorbed column fraction, and the purified fraction corresponding to 2 μg protein were each subjected to SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Brilliant Blue to confirm the expression and purification of the target protein.

1.9 Measurement of FOMT activity
The activity of FOMT was measured based on the following reaction conditions, and the conditions were changed appropriately for each experimental item.

Reaction solution composition
50 mM KPB (pH 7.5)
100 μM substrate (add 5 μl of 10 mM DMF solution)
500μM SAM (S-adenosylmethionine)
100 μl enzyme sample
Total 500μl

After reacting at 30 ° C for 1 hour, add 500 µl of methanol and vortex to stop the reaction.
After centrifugation at 4 ° C., 15,000 rpm for 20 minutes, the supernatant was collected and analyzed by HPLC.

HPLC conditions
The Shimadzu LC-10 system was used for HPLC analysis.
Intakt Cadenza CD-C18 (4.6 mm × 75 mm, particle size 3 μm) was used as the analytical column.
The analysis was performed based on the following conditions.

Mobile phase A: 0.01% formic acid
B: 100% acetonitrile flow rate 0.5 ml / min
Column temperature 40 ° C
Sample injection volume 1 μl
Program 0-10 min 15%
(% B) 10-25 min 15-55%
25-40 min 55-75%
40-45 min 75-15%
45-55 min 15%
Detector UV 254 nm to 375 nm

1.10 Analysis of methylated products by LC-MS
LC-MS analysis was performed using MicrOTOF (Burker daltonics) and Agilent HPLC 1200. HPLC was performed under the same conditions as in 1.9. ESI was used as the ion source, and data was collected in positive mode.

1.11 Production of polymethoxyquercetin using cells expressing CdFOMT5
Quercetin methylation reaction was performed using CdFOMT5-expressing cells cultured and induced according to 1.6.1. The collected cells were washed with M9 medium, centrifuged and collected, and suspended again in M9 medium. At this time, the bacterial cell concentration was adjusted to 1% (wet weight / vol). The conversion reaction was performed with the following composition.

M9 medium
10 mM L-methionine
100 μM quercetin
1% (w / v) glucose
1% (w / v) CdFOMT5 recombinant cell
(Organic solvent / surfactant)
total 50 mL

The reaction was carried out at 30 ° C. and 200 rpm for 3 hours.
500 μl of the sample was collected, an equal amount of methanol was added, the reaction was stopped by vortexing, and the supernatant was collected by centrifugation at 4 ° C., 15,000 rpm for 10 minutes. The supernatant was centrifuged again and analyzed as an HPLC sample.

1.12 NMR analysis NMR analysis of methylated products was performed using a Bruker 400 MHz NMR spectrometer. Heavy methanol (containing 0.01% TMS) was used as the solvent.
2 Experimental results
2.1 Isolation of FOMT gene from sequencer PCR was carried out using the degenerate primers listed in Table 1 in any combination and using genomic DNA prepared from the true leaf of sequencer as a template (1st PCR). As a result, amplification of a plurality of bands was confirmed. Therefore, primers based on the internal sequence were further combined, nested PCR was performed using the 1st PCR product as a template, and the amplification band was narrowed down (2nd PCR). The band narrowed down by the two PCR operations was purified and then cloned into the pGEM-T Easy vector to confirm the base sequence. As a result, it was clarified that some PCR fragments showed homology with known plant-derived FOMT amino acid sequences. Therefore, primers were designed based on these nucleotide sequences, and the surrounding region was cloned by TAIL-PCR. As a result, the full-length sequences of four types of FOMT genes could be obtained (CdFOMT1,3,4,5). From the deduced amino acid sequence, a start codon and a stop codon were estimated, and a primer for obtaining a full-length cDNA sequence was designed. Reverse transcription reaction was performed using total RNA prepared from Sequoccer true leaves, and PCR was performed using the synthesized 1st strand cDNA as a template. As a result of RT-PCR, CdFOMT6, which is considered to be a homologue of CdFOMT3, was newly cloned, and thereby the full-length cDNA sequence and amino acid sequence of five types of FOMT were determined (FIGS. 11 and 12). Moreover, the insertion position of the intron was determined by comparing the previously determined genomic sequence with the cDNA sequence.

  CdFOMT1 consisted of an ORF with a total length of 1254 base pairs containing one intron inside and encoded 362 amino acid residues (presumed 40505.9 Da). CdFOMT3 consisted of an ORF with a total length of 2514 base pairs including three introns inside, and encoded 352 amino acid residues (presumed 38849.9 Da). CdFOMT4 consisted of an ORF with a total length of 1664 base pairs including one intron inside and encoded 335 amino acid residues (presumed 37459.8 Da). CdFOMT5 consisted of a 1586 base pair ORF with 3 introns inside and encoded 353 amino acid residues (presumed 38977.6 Da). CdFOMT6 consisted of an ORF of 1056 base pairs and encoded 352 amino acid residues (presumed 39024.4 Da). Each amino acid sequence was analyzed by BLAST search (NCBI BLAST.P, Matrix .: BLOSUM62, Gap lots: Existence 11, Extension 1). As a result, it was about 60-95% identical to known plant-derived FOMT and catechol methyltransferase. (Table 2). The amino acid sequences of these genes showed about 60-95% identity with known plant-derived O-methyltransferases. In addition, CdFOMT had about 30 to 80% identity with each other. All of these FOMTs belonged to the class II O-methyltransferase group independent of metal ions.

2.2 Different host expression of recombinant CdFOMT The constructed expression plasmid (FIG. 1) was expressed in E. coli BL21 (DE3) and E. coli JM109 as hosts. Initially, both genes attempted to be expressed by pET-21b (+), but the target band of approximately 40 kDa was confirmed only with CdFOMT5, and CdFOMT6 could obtain a significant recombinant enzyme in the soluble protein fraction. Could not (Fig. 2a). Therefore, when using pMAL-c5x as an expression vector that can be expressed as a fusion protein with maltose binding protein (MBP), which has a higher solubilizing ability of the recombinant protein, a significant soluble protein band around 80 kDa. Could be obtained (Figure 2b). When these recombinant proteins were purified using an affinity column, purified proteins of about 40 kDa and 80 kDa could be obtained, respectively (FIG. 3ab). Subsequent activity measurements were performed using these purified enzymes.

2.3 Flavonoid methylation activity of recombinant CdFOMT Using purified recombinant enzymes, methylation activity was confirmed using quercetin as a substrate. As a result of the analysis of the enzyme reaction sample by HPLC, both enzymes obtained specific peaks presumed to be due to quercetin methylation (Fig. 4). Comparison with the standard compound revealed that CdFOMT6 methylated the OH group at the 3 ′ position of quercetin and converted it into isorhamnetin (3′-O-methoxyquercetin). On the other hand, CdFOMT5 was confirmed to have multiple peaks, suggesting that OH groups were methylated at multiple sites rather than at one site. These peaks were compared with the standard compounds, suggesting that they were 3-O-methoxy quercetin, azareatine (5-O-methoxy quercetin) or rhamnetin (7-O-methoxy quercetin) (Azaleatine and rhamnetin are HPLC It has not been identified due to difficulty in peak separation by In addition, multiple peaks specific to the reaction product with CdFOMT5 were found, suggesting the production of polymethoxyquercetin with methylated OH groups at multiple sites.

2.4 LC-MS analysis of quercetin methylation products with CdFOMT5
In order to confirm the methylation activity of OH groups at several locations in quercetin by CdFOMT5, LC-MS analysis of the reaction sample was performed (Fig. 5). Due to the positive mode measurement, the molecular weight of quercetin is 302.24 + 1. Therefore, it is estimated that the methylated product has a molecular weight of 303.24 + 14n (n = methylation site). As a result of analyzing the molecular weight by LC-MS, peaks indicating molecular weights of 317, 331, and 345 were obtained. This suggests that the specific peak in the CdFOMT5 reaction product is obtained by methylation of 1 to 3 OH groups.

2.5 Effect of pH on methylation activity of CdFOMT5 and CdFOMT6 The optimum pH of each enzyme when quercetin was used as a substrate was examined (Fig. 6). When quercetin was used as a substrate, CdFOMT5 and CdFOMT6 had pH 7 and pH 8, respectively.

2.6 Effect of temperature on methylation activity of CdFOMT5 and CdFOMT6 When quercetin was used as a substrate, each enzyme had a reaction temperature of 45 ° C (Fig. 7).

2.7 Methylation site specificity of CdFOMT5
For CdFOMT5, methylation sites were examined using various substrates. As substrates, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone and 7,8-dihydroxyflavone were used. Specific peaks were found when reacted with CdFOMT5 on each substrate. From comparison with standard compounds, it was revealed that each corresponding OH group was a monomethoxyflavone methylated. These results suggest that CdFOMT5 methylates at least the 3, 5, 6, and 7 OH groups of flavones.

2.8 Substrate specificity of CdFOMT6
Using CdFOMT6, methylation activity against various flavonoids was examined. Kaempferol, quercetin, myricetin and luteolin were used as flavones, and epicatechin and epigallocatechin 3-O-gallate (EGCg) were used as flavanols. As other substrates, caffeic acid, resveratrol, and gallic acid methyl ester were used. Among these, peaks that appeared to be methylated with respect to quercetin, myricetin, luteolin, epicatechin, and EGCg were found in the reaction product (FIG. 9). The lack of methylation activity against kaempferol (3,5,7,4'-tetrahydroxyflavone) suggests that CdFOMT6 specifically methylates the 3'OH of flavones and flavans. It was. In addition, the activity against luteolin and epicatechin is higher than the activity against myricetin and EGCg, suggesting that the OH group of the B ring of the flavonoid shows high activity against two substrates.

2.9 Production of polymethoxyquercetin using CdFOMT5-expressing cells
A quercetin methylation reaction was carried out using CdFOMT5-expressing cells, and polymethoxyquercetin production was attempted. Since multiple peaks presumed to have undergone methylation were obtained, analysis by LC-MS was performed. As a result, production of polymethoxyflavonoids in which 1 to 4 OH groups were methylated was confirmed (FIG. 10). In methylation of hydroxyflavone by CdFOMT5 purified enzyme, methylation to 3, 5, 6, 7-position OH has been confirmed. From this, when quercetin is used as a substrate, trimethoxyquercetin in which three methyl groups (3,5,7 OH) are methylated should be obtained. However, in the reaction using E. coli BL21 (DE3) as a control, the peak of isorhamnetin methylated at the 3 'OH was found (Fig. 4), indicating that E. coli itself has a catecholmethyltransferase. These reactions suggest that 3'OH is methylated. Therefore, in order to confirm the structure of this product, the reaction product presumed to be methylated at four OH groups was purified by HPLC and analyzed by NMR. The analysis results are shown below.

¹ H NMR (acetone-d ₆ , 400 MHz), δ (ppm): 7.79 (dd, J = 2.1, 8.6 Hz, 1H), 7.74 (d, J = 2.1 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H), 6.66 (d, J = 2.2 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H) , 3.83 (s, 3H).

  As a result of NMR spectrum analysis, four peaks derived from a methoxy group were obtained at 3.83 to 3.93 ppm. From this, it was revealed that the separated compound was tetramethoxyquercetin in which four OH groups of quercetin were methylated. Since the site selectivity exhibited by CdFOMT5 and E. coli itself methylates 3′OH of quercetin, it is speculated that the isolated product is 3,3 ′, 5,7-tetramethoxyquercetin.

  Quercetin is presumed to be one of the factors that has low water solubility and is difficult to cause methylation reaction. Therefore, the effects of various additives (organic solvents, surfactants) as auxiliary solvents for increasing water solubility were examined. When comparing the production of 3-methoxyquercetin in the presence of each additive, the highest production was obtained in the sample to which 0.1% MEGA-9 was added. On the other hand, the production amount of the reaction product presumed to be 3,3 ′, 5,7-tetramethoxyquercetin decreased. When other polar organic solvents were added, there was no significant increase in both products compared to the control sample, or a decrease in productivity was confirmed (Table 3).

  The present invention is useful in the technical field relating to flavonoid-O-methyltransferase (FOMT).

SEQ ID NO: 1 CdFOMT5 cDNA sequence
SEQ ID NO: 2 CdFOMT5 amino acid sequence
SEQ ID NO: 3 CdFOMT6 cDNA sequence
SEQ ID NO: 4 CdFOMT5 amino acid sequence
SEQ ID NO: 71 CdFOMT1 cDNA sequence
SEQ ID NO: 72 CdFOMT1 amino acid sequence
SEQ ID NO: 73 CdFOMT3 cDNA sequence
SEQ ID NO: 74 CdFOMT3 amino acid sequence
SEQ ID NO: 75 CdFOMT4 cDNA sequence
SEQ ID NO: 76 CdFOMT4 amino acid sequence

Claims (11)

At least 1 to 3 flavonoids comprising a protein encoded by the polynucleotide according to any one of (a) to (f) below or a protein having any one of the following amino acid sequences from (g) to (i): Enzyme agent that methylates the hydroxyl group.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(B) a polynucleotide encoding a flavonoid-O-methyltransferase having 96% or more identity with the base sequence described in SEQ ID NO: 1 and having the activity described in (1) below;
(C) A flavonoid-O-methyltransferase that hybridizes to the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 under highly stringent conditions and has the activity described in (1) below. A polynucleotide encoding
(D) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2;
(E) A flavonoid having the activity described in (1) below, including an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 2 A polynucleotide encoding -O-methyltransferase; and (f) a flavonoid-O having 96% or more identity with the amino acid sequence of SEQ ID NO: 2 and having the activity described in (1) below A polynucleotide encoding a methyltransferase;
(G) the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing;
(H) a flavonoid having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing and having the activity described in (1) below An amino acid sequence encoding -O-methyltransferase; and
(I) an amino acid sequence encoding a flavonoid-O-methyltransferase having 96% or more identity to the amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing and having the activity described in (1) below ;
(1) The activity of acting on quercetin to produce methoxyquercetin in which at least one hydroxyl group of quercetin is methylated. 2. The enzyme agent according to claim 1, wherein the activity described in (1) is an activity for producing methoxy quercetin in which at least one to three hydroxyl groups of quercetin are methylated. A method for producing a methoxyflavonoid and / or an analog thereof, comprising the steps of causing the enzyme agent according to claim 1 or 2 to act on a flavonoid and / or an analog thereof, and recovering a produced compound. The polynucleotide according to any one of ( j ) to ( o ) below:
( J ) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3;
( K ) a polynucleotide encoding a flavonoid-O-methyltransferase having 90% or more identity with the base sequence described in SEQ ID NO: 3 and having the activity described in (2) below;
( L ) A flavonoid-O-methyltransferase that hybridizes to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3 under highly stringent conditions and has the activity described in (2) below. A polynucleotide encoding
( M ) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 4;
( N ) A flavonoid comprising the amino acid sequence of SEQ ID NO: 4, wherein one or several amino acids are substituted, deleted, inserted, and / or added, and have the activity described in (2) below A polynucleotide encoding -O-methyltransferase; and ( o ) a flavonoid -O having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4 and having the activity set forth in (2) below A polynucleotide encoding a methyltransferase;
(2) The activity of acting on quercetin to produce methoxyquercetin in which the hydroxyl group at the 3 ′ position of quercetin is selectively methylated. A protein having any one of the following amino acid sequences ( p ) to ( r ):
( P ) the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing;
( Q ) A flavonoid having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing and having the activity described in (2) below An amino acid sequence encoding -O-methyltransferase;
( R ) an amino acid sequence encoding a flavonoid-O-methyltransferase having 90% or more identity to the amino acid sequence described in SEQ ID NO: 4 of the Sequence Listing and having the activity described in (2) below ;
(2) The activity of acting on quercetin to produce methoxyquercetin in which the hydroxyl group at the 3 ′ position of quercetin is selectively methylated. A vector comprising the polynucleotide of claim 4 . A transformed cell into which the polynucleotide according to claim 4 or the vector according to claim 6 has been introduced. Culturing the transformed cell according to claim 7, comprising the step of recovering the protein encoded by the polynucleotide of claim 4 from the culture, the protein encoded by the polynucleotide of claim 4 Or the manufacturing method of the protein of Claim 5 . A step of allowing the protein of claim 5 or the transformed cell of claim 7 to act on a flavonoid and / or an analog thereof, and a step of recovering the produced compound, and a methoxyflavonoid and / or an analog thereof Body manufacturing method. Flavonoids and / or analogs thereof are kaempferol, quercetin, myricetin, fisetin, galangin, gosipetin, morin, apigenin, luteolin, naringenin, taxifolin, butyne, eriodictyol, pinocembrin, cyanidin, delphinidin, catechin, epicatechin, epicatechin 10. At least one compound selected from the group consisting of gallocatechin, epicatechin gallate, epigallocatechin gallate, epiafzerequin, fisetinidol, guibrutinidol, mesquitol, robinetinidol / or an analogue thereof, according to claim 3 or 9 . The manufacturing method as described. , Enzyme preparation comprising the protein according to 請 Motomeko 5.