JP2007116975A - Modified aromatic ring dioxygenase and method for producing flavone hydroxide compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a new compound by introducing hydroxy group(s) to a phenyl group (benzene ring) of a flavone compound. <P>SOLUTION: The method for producing a flavone hydroxide compound, comprises the following process: At least one of (a) a modified aromatic ring dioxygenase (modified BphA) and dihydrodiol dehydrogenase (BphB), (b) microorganisms expressing the modified BphA and the BphB and (c) a cultured product of the microorganisms, is added to a sample containing a flavone compound followed by mixing or culture, and from the resultant mixture or cultured product, the objective flavone hydroxide compound is obtained. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a modified aromatic ring dioxygenase large subunit, a nucleic acid encoding the same, a recombinant vector containing the nucleic acid, and a transformant transformed with the recombinant vector. The present invention also relates to a method for producing hydroxylated flavones using the modified aromatic ring dioxygenase large subunit.

Pseudomonas shoe de alkali monocytogenes (Pseudomonas pseudoalcaligenes) KF707 strain, is degrading bacteria isolated biphenyl and polychlorinated biphenyls in Kitakyushu (PCB). From this bacterium, an aromatic ring (biphenyl) degrading enzyme gene group was isolated for the first time in the world (Non-patent Document 1). At present, many researches using aromatic ring-degrading enzyme genes derived from aromatic ring-degrading bacteria have been made due to needs related to environmental purification, and a lot of knowledge about the structure and function of genes and enzymes is accumulated (for example, Non-patent document 2). The functions of the first four reaction aromatic ring decomposing enzymes are shown below (Non-patent Document 3).

When all of the above genes, ie, bphA ( bphA1A2A3A4 ), bphB , bphC , and bphD genes are introduced into E. coli and expressed, the E. coli can metabolize the aromatic ring to produce benzoic acid.

To date, as an aromatic ring dioxygenase gene, Escherichia coli or actinomycetes in which an aromatic ring dioxygenase gene ( bphA ) derived from Pseudomonas pseudoalgenes KF707 strain has been modified and introduced by molecular evolution engineering techniques have been used. It has been reported that dihydrodiols can be prepared from aromatic compounds containing various phenyl groups (see Non-patent Documents 1 and 2 or Patent Document 1 below). That is, the DNA encoding the large subunit (BphA1) in the aromatic ring dioxygenase isolated from Pseudomonas pseudoalgenes KF707 strain, which is an aromatic ring or PCB degrading bacterium, is converted into another aromatic ring degrading bacterium, Burkholderia ( Burkholderia ) DNA shuffling with the DNA encoding the large subunit (BphA1) of the aromatic ring dioxygenase derived from the genus LB400, as a mutant with excellent substrate specificity among mutants By the isolated aromatic ring dioxygenase (group) consisting of the isolated BphA1 (2072) and three components (BphA2A3A4) other than the large subunit of aromatic ring dioxygenase derived from Pseudomonas pseudoalgenes KF707 strain, Contains phenyl group in the molecule It is possible bioconversion of organic low molecular compounds have been found (see Non-Patent Documents 4 and 5). That is, Escherichia coli containing a DNA- shuffled modified aromatic ring dioxygenase gene [ bphA1 (2072) bphA2A3A4 ; bphA (2072)] converts the organic low molecular weight compound, and the position adjacent to the phenyl group (containing a substituent) It is found that an aromatic-cis ( cis ) -dihydrodiol body in which two hydroxyl groups and two hydrogen atoms are introduced in a position-specific manner at positions 2 and 3) is stereoselective. It was. The amino acid sequence of the large subunit [BphA1 (2072)] of this modified aromatic ring dioxygenase is shown in DDBJ / Genbank accession number AB085748. The amino acid sequences of the small subunit (BphA2), ferredoxin (BphA3), and ferredoxin reductase (BphA4) of the KF707 strain are shown in DDBJ / Genbank accession number M83673. Furthermore, an aromatic-cis ( cis ) -dihydrodiol compound synthesized by E. coli having the above bphA (2072) is converted into a diol compound by the aromatic ring dihydrodiol dehydrogenase (BphB) derived from Pseudomonas pseudoalgagenus strain KF707. It is also reported that it is converted (see Patent Document 2). The amino acid sequence of this BphB is also shown in DDBJ / Genbank accession number M83673. The amino acid sequences of aromatic ring diol dioxygenase (BphC) and hydrolase (BphD) that catalyze the next reaction are also shown in DDBJ / Genbank accession number M83673 and DDBJ / Genbank accession number D85851, respectively.

In addition, when E. coli expressing the bphA gene, bphB gene, and bphC gene is mixed and cultured in a synthetic medium with a low molecular weight organic compound having a phenyl group (benzene ring) in the molecule, picolinic acid is not a meta-cleavable compound. It has also been reported that a compound is produced (see Patent Document 3).

Furthermore, it has also been reported that several kinds of flavonoids such as flavone, flavanone and 6-hydroxyflavone can be converted into diol by E. coli having the above bphA (2072) and aromatic ring dihydrodiol dehydrogenase gene ( bphB ) derived from KF707 strain. (Refer nonpatent literature 6).

Japanese Patent Laid-Open No. 2003-269 WO2004 / 0789888 pamphlet WO2005 / 085435 pamphlet Furukawa, K., And Miyazaki, T., Cloning of gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.J. Bacteriol., 166, 392-398, 1986 Furukawa, K., Engineering dioxygenases for efficient degradation of environmental pollutants, Curr. Opinion Biotechnol., 11,244-249.2000 Suenaga, H., Goto, M., And Furukawa, K., Emergence of multifunctional oxygenase activities by random priming recombination.J. Biol. Chem., 276, 22500-22506, 2001 Misawa, N., Shindo, K., Takahashi, H., Suenaga, H., Iguchi, K., Okazaki, H., Harayama, S., and Furukawa, K., Hydroxylation of various molecules including cyclic aromatics using recombinant Escherichia coli cells expressing modified biphenyl dioxygenase genes. “Tetrahedron”, 58, 9605-9612, 2002. Shindo, K., Nakamura, R., Chinda, I., Ohnishi, Y. Horinouchi, S., Takahashi, H., Iguchi, K., Harayama, S., Furukawa, K., and Misawa, N., Hydroxylation of ionized aromatics including carboxylic acid or amine using recombinant Streptomyces lividanscells expressing modified biphenyl dioxygenase genes. “Tetrahedron”, 59, 1895-1900, 2003. Shindo, K., Kagiyama, Y., Nakamura, R., Hara, A., Ikenaga, H., Furukawa, K., and Misawa, N., Enzymatic synthesis of novel antioxidant flavonoids by Escherichia coli cells expressing modified metabolic genes involved in biphenyl catabolism. “J. Mol. Catalysis B: Enzymatic” 23, 9-16, 2003.

  An object of the present invention is to provide a general method for producing a novel compound by introducing a hydroxyl group into two positions (for example, the 2-position and 3-position) of a phenyl group (benzene ring) of a flavone compound.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors obtained a gene encoding an aromatic ring (biphenyl) dioxygenase (modified BphA1A2A3A4: BphA) modified by genetic engineering as a dihydrodiol dehydrogenase (BphB). It has been found that when recombinant Escherichia coli introduced and expressed together with the gene to be encoded and various flavone compounds are mixed and cultured in a synthetic medium, a diol form of the flavone compound is produced, respectively, and the present invention is completed. It was.

That is, the present invention includes the following inventions.
(1) The following polypeptide (a) or (b).
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, or (b) one or several amino acids other than the 324th or 325th amino acid in the amino acid sequence shown in SEQ ID NO: 2 or 4 A polypeptide comprising an amino acid sequence substituted, deleted, inserted or added, and having the same aromatic ring dioxygenase large subunit (BphA1) activity as (a) (2) The following (a) or (b) Nucleic acids.
(A) a nucleic acid encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, or (b) one or a number other than the 324th or 325th amino acid in the amino acid sequence shown in SEQ ID NO: 2 or 4 Nucleic acid (3) or less encoding a polypeptide comprising an amino acid sequence in which one amino acid is substituted, deleted, inserted or added, and having the same aromatic ring dioxygenase large subunit (BphA1) activity as (a) The nucleic acid of (a) or (b).
(A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1, or (b) a nucleic acid comprising a sequence complementary to the base sequence represented by SEQ ID NO: 1 under a stringent condition, and an aromatic ring Nucleic acid encoding a polypeptide having dioxygenase large subunit (BphA1) activity

(4) A recombinant vector comprising the nucleic acid of (2) or (3) above.
(5) A transformant transformed with the recombinant vector of (4) above.
(6) A method for producing a polypeptide having aromatic ring dioxygenase large subunit activity (BphA1), which comprises culturing the transformant of (5) and collecting the polypeptide from the resulting culture.

(7) The set according to (4), further comprising a nucleic acid encoding a polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG) and a nucleic acid encoding a polypeptide having dihydrodiol dehydrogenase activity (BphB) Replacement vector.

(8) Expressing the polypeptide (BphA1) of (1) above, and expressing a polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG) and a polypeptide having dihydrodiol dehydrogenase activity (BphB) Microorganism characterized by
(9) The microorganism according to (8), which is transformed with the recombinant vector according to (4) or (7).
(10) The microorganism according to (8) or (9) above, which is capable of converting a flavone compound into a hydroxylated flavone compound.
(11) The microorganism according to any one of (8) to (10) above, wherein the microorganism is Escherichia coli.

(12) The activity of converting a flavone compound into a hydroxylated flavone compound, characterized by culturing the microorganism of any one of (8) to (11) above and collecting a polypeptide from the resulting culture. The manufacturing method of the enzyme group which has.

(13) The following (a) to (c):
(A) Polypeptide (BphA1) of the above (1), polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG), and polypeptide having dihydrodiol dehydrogenase activity (BphB)
(B) The microorganism of any one of (8) to (11) above (c) At least one of the cultures of the microorganism of any of (8) to (11) above is added to a sample containing a flavone compound. Mixing or culturing, and obtaining a hydroxylated flavone compound from the resulting mixture or culture, a method for producing a hydroxylated flavone compound.
(14) The flavone compound is selected from flavone, 6-hydroxyflavone, 7-hydroxyflavone, 5,7-dihydroxyflavone, 6-hydroxyflavanone, 7-hydroxyflavanone, chalcone, 1-phenylnaphthalene, and 2-phenylnaphthalene. The method according to (13), wherein the method is selected from the group consisting of:
(15) The method according to (13) or (14) above, wherein the hydroxylated flavone compound is a diol having two hydroxyl groups introduced at the 2-position and 3-position of the phenyl group of the flavone compound.

(16) A compound represented by the following formula II.

(17) The following (a) to (c):
(A) Polypeptide (BphA1) of the above (1), polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG), and polypeptide having dihydrodiol dehydrogenase activity (BphB)
(B) The microorganism of any one of (8) to (11) (c) At least one culture of the microorganism of any of (8) to (11) above is added to a sample containing chalcone and mixed Alternatively, a method for producing a compound represented by the formula II, wherein the compound represented by the formula II is obtained from a mixture or culture obtained by culturing.
(18) An antioxidant comprising at least one selected from the group consisting of compounds represented by the following formulas II, III, and IV.

  According to the present invention, a novel method for producing a hydroxylated flavone compound (particularly a diol having a hydroxyl group at two positions) from an existing flavone compound is provided. Such conversion products can be provided as raw materials for organic chemical synthesis of industrial products such as various pharmaceuticals and agricultural chemicals. In addition, the method of the present invention can introduce a hydroxyl group specifically at two positions (position 2 and position 3) of the phenyl group (benzene ring) of the flavone compound, and is a useful reaction in organic chemical synthesis.

Hereinafter, the present invention will be described in detail.
The method for producing a hydroxylated flavone compound according to the present invention comprises a flavone compound having a phenyl group (benzene ring), a polypeptide having an aromatic ring dioxygenase large subunit activity according to the present invention and a known aromatic ring decomposition system. By reacting with an enzyme, the phenyl group (benzene ring) is hydroxylated and converted into a hydroxylated flavone compound.

  Specifically, an enzyme comprising a polypeptide having an aromatic ring dioxygenase large subunit activity according to the present invention (hereinafter also referred to as “modified BphA1”) and another subunit of aromatic ring dioxygenase (BphA2A3A4 or BphEFG). [Modified BphA] and aromatic ring dihydrodiol dehydrogenase (BphB) are allowed to act sequentially to produce flavones in which the phenyl group (benzene ring) is hydroxylated.

1. Aromatic ring decomposing enzyme and gene encoding the same In the present invention, the first enzyme of the aromatic ring (biphenyl) decomposing system, namely, aromatic ring (biphenyl) dioxygenase (BphA); Are also used for the activity of the modified aromatic ring dioxygenase (modified BphA) modified by genetic engineering and the second enzyme dihydrodiol dehydrogenase (BphB). BphA is composed of four subunits (BphA1, BphA2, BphA3 and BphA4; or BphA1, BphE, BphF and BphG). In the present invention, “modified aromatic ring dioxygenase” refers to a large subunit ( BphA1) is modified by genetic engineering.

(1) Modified BphA (aromatic ring dioxygenase)
In the present invention, the modified aromatic ring dioxygenase (modified BphA) is not limited, but large subunits thereof are BphA1 (1-22) (SEQ ID NO: 2) and BphA1 (2-2) ( The thing of sequence number 4) can be used. These modified BphA1 are, but not limited to, encoded by the bphA1 (1-22) gene (SEQ ID NO: 1) and the bphA1 (2-2) gene (SEQ ID NO: 3), respectively.

bphA1 (1-22) gene and bphA1 (2-2) gene are Pseudomonas putida KF715 (Hayase, N., Taira, K. and Furukawa, K., Pseudomonasputida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: Cloning analysis, and expression in soil bacteria. J. Bacteriol., 172, the bphA1 sequences isolated from 1990) has in the central part, is a hybrid bphA1 gene with at both ends bphA1 sequence derived from Pseudomonas shoe de alkali monocytogenes KF707 bphA1 (715 -707) Based on the gene, some amino acid residues are substituted. The bphA1 (715-707) gene is constructed by isolating the bphA1 sequence from Pseudomonas putida KF715 strain using the cassette PCR method (Japanese Patent Application Laid-Open No. 2000-125871) (Patent Document 3).

The present inventors consider that the 324th and 325th sites (amino acid residues tyrosine and isoleucine, respectively) of the amino acid sequence encoded by the bphA1 (715-707) gene are important for determining the substrate specificity of BphA, Random mutations were introduced into this site, and as a mutant strain excellent in conversion of flavone compounds, amino acids 324 and 325 in the amino acid sequence of bphA1 (715-707) were changed to alanine and leucine residues. Obtained [ bphA1 (1-22)] and [ bphA1 (2-2)] in which amino acids 324 and 325 were changed to leucine and isoleucine residues, and the fragrance encoded by these modified bphA1 The large ring dioxygenase subunit is replaced with another subunit (BphA2A3 4) and was expressed with BphB, it was confirmed that it is possible to introduce a hydroxyl group into the phenyl group of flavones.

  Therefore, in the modified BphA1 of the present invention, in the known amino acid sequence of BphA1, the amino acids corresponding to the 324th and 325th amino acids of the amino acid sequence of BphA1 (715-707) are respectively modified to alanine and isoleucine. Or by modifying to leucine and isoleucine, respectively.

Known BphA1s include, but are not limited to, BphA1 derived from Pseudomonas pseudoalgenes KF707, BphA1 derived from Burkholderia LB400, bphA1 from Pseudomonas pseudoalgenes KF707 and bphA1 from Burkholderia LB400 BphA1 encoded by BphA1 encoded by bphA1 gene [ bphA1 (2072)] obtained by performing DNA shuffling between them (DDBJ / Genbank accession number AB085748), bphA1 gene derived from Pseudomonas putida KF715 strain in the middle A hybrid bphA1 gene [ bphA1 (715) having a bphA1 gene at both ends of Pseudomonas pseudoalgenes KF707 strain. -707)] encoded by BphA1 (Patent Document 3) and the like.

Moreover, as long as it has the same aromatic ring dioxygenase large subunit activity as the polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 or 4 exemplified in the present specification, the amino acid sequence other than the 324th and 325th amino acids. A plurality of, preferably 1 or 2 amino acids may have mutations such as deletion, substitution and addition. Equivalent aromatic ring dioxygenase large subunit activity is responsible for substrate recognition and its oxygenation reaction as a major constituent protein in the aromatic ring dioxygenase enzyme possessed by the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. It means having a function. This function is to be co-expressed in a microorganism together with a gene encoding another subunit of aromatic ring dioxygenase ( bphA2A3A4 or bphEFG ) and a gene encoding dihydrodiol dehydrogenase ( bphB ) to conduct a biotransformation experiment of the substrate. Can be confirmed.

  For example, 1 to 2 amino acids of the amino acid sequence shown in SEQ ID NO: 2 or 4 may be deleted, or 1 to 2 amino acids may be added to the amino acid sequence shown in SEQ ID NO: 2 or 4. Alternatively, a protein in which one or two amino acids of the amino acid sequence shown in SEQ ID NO: 2 or 4 are substituted with other amino acids is also included in the protein having the aromatic ring dioxygenase large subunit activity. However, the 324th and 325th amino acids in SEQ ID NO: 2 or 4 do not contain the mutation.

  The element other than the modified BphA1 constituting the modified aromatic ring dioxygenase (modified BphA) is BphA2A3A4 or BphEFG depending on the microorganism, and any element known in the art can be used. The subunit of aromatic ring dioxygenase (BphA2A3A4 or BphEFG) can be obtained from a microorganism that naturally produces this enzyme or a microorganism into which a gene encoding the enzyme has been introduced. Or it can obtain from the microorganisms which introduce | transduced the gene which codes the variant of this enzyme, or the modification by the molecular evolution engineering technique (patent document 3 etc.). For example, the amino acid sequences of the small subunit (BphA2), ferredoxin (BphA3), and ferredoxin reductase (BphA4) of Pseudomonas pseudoalkagenes KF707 strain are shown in DDBJ / Genbank accession number M83673. The amino acid sequences of the small subunit (BphE), ferredoxin (BphF), and ferredoxin reductase (BphG) of Burkholderia xenovorans LB400 strain are shown in DDBJ / Genbank accession number M86348.

(2) BphB (dihydrodiol dehydrogenase)
Dihydrodiol dehydrogenase (BphB) can be obtained from a microorganism that naturally produces the enzyme or a microorganism into which a gene encoding the enzyme has been introduced. Or it can obtain from the microorganisms which introduce | transduced the gene which codes the variant of this enzyme, or the modification by the molecular evolution engineering technique. For example, but not limited to, the amino acid sequence of BphB of Pseudomonas pseudoalkanegenes KF707 strain is shown in DDBJ / Genbank accession number M83673. In addition, the amino acid sequence of BphB of Pseudomonas pseudoalgenes KF707 strain of Burkholderia xenovorans LB400 strain is shown in DDBJ / Genbank accession number M86348.

(3) Gene encoding modified BphA or BphB The above-described gene encoding modified aromatic ring dioxygenase (modified bphA ) is modified bphA1 , bphA2 , bphA3 , and bphA4 , or modified bphA1 , bphE , bphF. , And bphG , and the gene encoding dihydrodiol dehydrogenase consists of bphB .

The modified bphA gene is not limited, but the gene encoding the large subunit (BphA1) is bphA1 (1-22) gene (SEQ ID NO: 1) and bphA1 (2-2) gene (SEQ ID NO: 3). ) Is preferable. That is, the modified bphA1 gene is modified so that the amino acids corresponding to the 324th and 325th amino acids in the known amino acid sequence of BphA1 are alanine and isoleucine, or leucine and isoleucine, respectively. It is a thing.

Other genes (ie, bphA2 , bphA3 , bphA4 and bphB , or bphE , bphF , and bphG ) are not only derived from microorganisms naturally having these genes, but also occur, for example, naturally or artificially. And a gene encoding a variant obtained by performing a molecular evolution engineering technique such as DNA shuffling between these genes.

Usable genes, like for example from Pseudomonas pseudotuberculosis alkali monocytogenes KF707 strain bphA2, bphA3, bphA4, and bphB genes or Burkholderia Zenoboransu (Burkholderia xenovorans) from LB400 strain bphE,, bphF, bphG and bphB genes It is done. The nucleotide sequences of the bphA2 , bphA3 , bphA4 and bphB genes of the KF707 strain are shown in DDBJ / Genbank accession number M83673. The base sequences of bphE , bphF , bphG and bphB genes derived from Burkholderia xenovorans LB400 strain are shown in DDBJ / Genbank accession number M83648.

  Further, the gene encoding the above-mentioned aromatic ring dioxygenase or dihydrodiol dehydrogenase may be a mutant into which a mutation has been introduced by site-directed mutagenesis and the like and has the above function or activity. In order to introduce a mutation into a gene, a known method such as Kunkel method or Gapped duplex method or a method based thereon can be employed. For example, mutations are introduced using a mutation introduction kit (for example, Mutan-K (manufactured by TAKARA) or Mutan-G (manufactured by TAKARA)) using site-directed mutagenesis. Moreover, gene mutation introduction and chimera genes can be constructed by techniques such as error introduction PCR and DNA shuffling. Error introduction PCR and DNA shuffling are techniques known in the art. For example, for error introduction PCR, Chen K, and Arnold FH. 1993, Proc. Natl. Acad. Sci. USA, 90: 5618-5622 In addition, molecular evolution engineering methods such as DNA shuffling and cassette PCR are described in, for example, Kurtzman, AL, Govindarajan, S., Vahle, K., Jones, JT, Heinrichs, V., Patten PA, Advances in directed protein evolution. by recursive genetic recombination: applications to therapeutic proteins. Curr. Opinion Biotechnol., 12, 361-370, 2001, and Okuta, A., Ohnishi, A. and Harayama, S., PCR isolation of catechol 2,3-dioxygenase See Gene fragments from environmental samples and their assembly into functional genes. Gene, 212, 221-228, 1998.

Whether or not a recombinant protein produced by a nucleic acid having a mutation has a target function (ie, aromatic ring degradation enzyme activity) is determined by transforming the mutant nucleic acid by a method known in the art as described above. It can be confirmed by conducting a substrate bioconversion experiment using the recombinant protein obtained by In addition, in order to retain the activity of hydroxylating flavones, the modified bphA1 gene encoding modified BphA1 has the amino acids 324 and 325 in the amino acid sequence encoded thereby to be alanine and isoleucine, respectively. Alternatively, the mutations are introduced so as to encode amino acid sequences that become leucine and isoleucine, respectively.

  Furthermore, it hybridizes under stringent conditions with a sequence complementary to all or part of the nucleic acid comprising the above base sequence, and the activity of the aromatic ring dioxygenase large subunit or aromatic ring dioxygenase subunit, Alternatively, a nucleic acid encoding a protein having dihydrodiol dehydrogenase activity can also be used in the present invention. Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, it refers to conditions under which DNA having high homology (homology is 60% or more, preferably 80% or more) hybridizes. Such hybridization conditions are known in the art and are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.

In addition, the gene group ( bphA1A2A3A4 gene or bphA1EFG gene, and bphB gene) encoding the above aromatic ring-degrading enzyme is usually present in the genomic DNA or plasmid DNA in the immediate vicinity, so either If a clone containing the sequence of one gene, such as a cosmid clone, is isolated by colony hybridization or the like, other genes can often be easily isolated together.

  However, since many genes encoding proteins having similar enzyme activities have been reported and are well known to those skilled in the art, the present invention is limited to those utilizing enzymes produced from recombinant microorganisms having these gene sequences. Is not to be done.

2. Expression of Aromatic Ring Degrading Enzyme in Microorganism In the present invention, a microorganism capable of expressing a modified aromatic ring dioxygenase gene (modified bphA ) and a dihydrodiol dehydrogenase gene ( bphB ) and hydroxylating a flavone compound is prepared. In addition, the microorganism or an enzyme produced by the microorganism can be used for hydroxylating a flavone compound.

Specifically, for example, the above-described modified bphA1 gene is introduced into a microorganism that expresses all or part of BphA2, BphA3, and BphA4 (or BphE, BphF, and BphG), or a microorganism that expresses BphB, and These polypeptides can be expressed in microorganisms. Alternatively, the modified bphA1 gene described above is introduced into a microorganism together with the bphA2 gene, bphA3 gene, bphA4 gene, and bphB gene, or together with the bphE gene, bphF gene, bphG gene, and bphB gene, and these polypeptides are used in the microorganism. Can be expressed. Therefore, as long as modified BphA and BphB are expressed, polypeptides naturally present in microorganisms may be used, or polypeptides may be expressed by introducing these genes into microorganisms.

Methods for introducing and expressing a gene of interest in a microorganism are known in the art. In one embodiment of the present invention, a microorganism is transformed with a recombinant vector containing a modified bphA1 gene. In another embodiment, transformation is performed using a recombinant vector containing a modified bphA1 gene and another gene ( bphA2A3A4 or bphEFG and further bphB gene). In yet another embodiment, a microorganism is co-transformed with a recombinant vector containing a modified bphA1 gene and a recombinant vector containing another gene. As long as a microorganism that expresses the target polypeptide is prepared, the type of gene contained in the recombinant vector, the order of transformation, and the like are not particularly limited.

The recombinant vector used in the present invention can be obtained by ligating (inserting) the above gene, for example, modified bphA1 into an appropriate vector. The vector used in the present invention is not particularly limited as long as it can replicate in the host microorganism. Examples include plasmids, cosmids, bacteriophages, phagemids, etc., which may be linear or circular. A shuttle vector can also be used.

  As plasmid DNA, plasmids derived from actinomycetes (for example, pK4, pRK401, pRF31, etc.), plasmids derived from E. coli (for example, pET, pBR322, pBR325, pUC118, pUC119, pUC18, etc.), plasmids derived from Bacillus subtilis (for example, pUB110, pTP5). Etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.) and the like, and phage DNAs include λ phage (λgt10, λgt11, λZAP, etc.).

  In order to insert a gene into a vector, first, a method in which the purified gene is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector, and linked to the vector, etc. are employed.

  The gene needs to be integrated into the vector so that the polypeptide of interest is expressed. Thus, in addition to a promoter and a target gene, a cis element such as an enhancer, a selectable marker, and the like can be linked to the vector used in the present invention as desired. Any promoter may be used as long as it can be expressed in a host microorganism such as Escherichia coli.

For example, the plasmid pJHF18 (Hirose, J., Auyama, A., Hayashida, S., Furukawa, K., Gene, 128, 27-33, expressing the bphA1A2A3A4-bphB-bphC gene group of Pseudomonas pseudoalkagenes KF707 strain, 1994), or bphA1 and (715-707) bphA2A3A4, and replace the bphA1 gene in plasmid PBPA715-707BC (Patent Document 3) expressing bphB and bphC in modified bphA1 of the present invention, also removes bphC gene by any Can be produced. In addition, for example, pBluescript II SK can be used as an E. coli vector, and the lac promoter can be used for expression of bphA (1-22) bphB gene and bphA (2-2) bphB gene.

Transformation of a microorganism can be performed by introducing the above recombinant vector into a host microorganism so that the target polypeptide can be expressed. Here, the host microorganism is not particularly limited. For example, it is known that microorganisms belonging to the genus Escherichia, preferably E. coli , and naturally express an aromatic ring degrading enzyme. Pseudomonas (Pseudomonas) genus Comamonas (Comamonas) the genus Burkholderia (Burkholderia) genus Sphingomonas (Sphingomonas) genus Rhodococcus (Rhodococcus) genus, and Ralstonia a (Ralstonia) microorganism belonging to the genus or the like. Specific examples of the microorganism, Escherichia coli JM109 strain, Pseudomonas pseudoephedrine alkali monocytogenes (Piesuiudomonaspseudoalcaligenes), for example Pseudomonas pseudotype alkali monocytogenes KF707 strain (Non-Patent Document 1), Comamonas testosteroni (Comamonas testosteroni), Burg formate terrier (Burkholderia) genus LB400 strain (Erickson, BD, Mondello, FJ, Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxgenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J. Bacteriol., 174, 2903-2912, 1992), Sphingomonas aroma tea grain lance (Sphingomonasaromaticivorans), Rhodococcus Guroberurasu (Rodococcus globerulus), mention may be made of Ralstonia Okisaratika (Ralstoniaoxalatica) and the like. In addition, the above gene can be introduced and expressed in Streptomyces genus bacteria, which are actinomycetes, and such transformed recombinant microorganisms can also be used (Chun, HK, Ohnishi). , Y., Shindo, K., Misawa, N., Furukawa, K., Horinouchi, S., Biotransformation of flavone and flavanone by Streptomycelividans cells carrying shuffled biphenyl dioxygenase genes.J. Mol. Catalysis B: Enzymatic, 21, 113 -121, 2003). These microorganisms can be obtained from culture collections such as ATCC and DSMZ. However, the microorganisms that can be used in the present invention are not limited to those described above, and any microorganism that can express the target polypeptide can be used.

  Methods for introducing and expressing foreign genes into microorganisms can be performed by conventional methods (eg, Sambrook, J., Russell, DW, “Molecular cloning -A laboratory manual”, Third edition, Cold Spring Harbor Laboratory Press, 2001). . For example, as a method for introducing a recombinant vector into a microorganism, for example, a method using calcium ions (Cohen, SNet al .: Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972)), electro The poration method is exemplified. Selection of transformants can also be performed by a method known in the art, for example, selection using a drug resistance gene.

  Since the microorganism obtained as described above produces an aromatic ring-degrading enzyme that hydroxylates flavone compounds, it must be used in the production of such enzymes or in the production of hydroxylated flavone compounds. Can do.

3. Isolation and purification of aromatic ring decomposing enzyme In the present invention, for the conversion reaction described later, the enzyme group is isolated and purified from a microorganism that produces aromatic ring decomposing enzyme (modified BphA and BphB), You can use it.

  The aromatic ring-degrading enzyme can be obtained by culturing the microorganism having the gene encoding it and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The method of culturing the microorganism of the present invention in a medium is performed according to a usual method used for culturing microorganisms.

  As a medium for culturing microorganisms such as bacteria, a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism and can cultivate microorganisms efficiently. Any of these may be used.

  As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, and aromatic hydrocarbons such as dibenzofuran, gentisic acid and salicylic acid are used. As the nitrogen source, ammonia, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, peptone, meat extract, corn steep liquor, or other nitrogen-containing compounds are used. As the inorganic substance, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

  The culture is usually performed at 20 to 40 ° C. under aerobic conditions such as shaking culture or aeration and agitation culture. During the incubation period, the pH is maintained at about 5-9. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  After the culture, when proteins (modified BphA and BphB) of the target aromatic ring-degrading enzyme group are produced in the cells or cells, the proteins are extracted by disrupting the cells or cells. When the target protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, The target protein can be isolated and purified from

  Whether or not the target protein has been obtained can be confirmed by SDS-polyacrylamide gel electrophoresis or the like. Furthermore, various tests can be performed to examine the physicochemical properties or functions of the target protein. Test items include X-ray crystal analysis, CD spectrum analysis, NMR analysis, and the like.

4). Conversion of flavone compounds In the present invention, the flavone compounds can be hydroxylated using the above-mentioned modified aromatic ring dihydrogenase (modified BphA) and dihydrodiol dehydrogenase (BphB).

  Furthermore, it is also easy to produce necessary enzymes from the above-mentioned recombinant microorganisms and to convert flavone compounds using them. That is, the present invention not only produces a hydroxylated flavone compound by mixed culture of a recombinant microorganism and a flavone substrate to be converted, but also an enzyme produced by the recombinant microorganism or a non-recombinant microorganism having an equivalent gene. It is possible to produce a hydroxylated flavone compound by extraction and using the extracted enzyme.

  Therefore, in the present invention, at least one of a modified aromatic ring dioxygenase (modified BphA) and dihydrodiol dehydrogenase (BphB), a microorganism that produces these enzymes, or a culture of the microorganism is used as a flavone compound. It is added to a sample to be mixed and mixed or cultured, and a hydroxylated flavone compound is obtained from the resulting mixture or culture.

  Here, the “flavone compounds” used as a substrate for the conversion reaction are compounds having a flavone skeleton and compounds derived therefrom, specifically, flavones, isoflavones, 6-hydroxyflavones, 7-hydroxyflavones, 5,7-dihydroxyflavone (chrysin); flavanone, 6-hydroxyflavanone, 7-hydroxyflavanone; a compound in which a cyclized hydrocarbon group and a phenyl ring are linked by biphenyl (1-phenylnaphthalene, 2-phenylnaphthalene); Examples include compounds having a substituted or unsubstituted alkenylene group (preferably a substituted alkenylene group having 3 carbon atoms) between phenyl groups ((trans-) chalcone, chalcone, dihydrochalcone). The flavone compound that can be used as a substrate in the present invention is a monocyclic or bicyclic ring containing one or more hetero atoms selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom as a ring atom. It may have a group and may be substituted with a substituent. Moreover, as an alkenylene group, vinylene group, 1-propenylene group, 1-butenylene group, 2-butenylene group etc. are mentioned, for example.

  The flavone compounds (substrates) particularly preferably used in the present invention are not limited, but include flavone, chalcone, 7-hydroxyflavone, chrysin, flavanone, 6-hydroxyflavone, 6-hydroxyflavanone, 7- Examples include hydroxyflavanone, 1-phenylnaphthalene, and 2-phenylnaphthalene.

  In the present invention, the above flavone compound is reacted with a modified aromatic ring dioxygenase (modified BphA) and dihydrodiol dehydrogenase (BphB) as a substrate, or cultured with a microorganism producing these enzymes, or By reacting with the culture, it can be converted into a compound in which a hydroxyl group is introduced into the phenyl group (benzene ring) of the flavone compound. In particular, according to the present invention, the flavone compound can be converted into a diol having a hydroxyl group introduced at two positions of the phenyl group (benzene ring), preferably at the 2-position and 3-position.

  Specifically, as shown in FIG. 1, compounds 1 to 4 represented by Formulas I to VI can be obtained from chalcone, 7-hydroxyflavone and chrysin, which are flavone compounds, by the method of the present invention. it can. Of these, Compound 2 is a novel substance. In addition, compounds 2 to 4 represented by formulas II to VI have antioxidant activity as shown in Example 3. Therefore, the composition containing these compounds 2 to 4 can be used as an antioxidant.

  Reaction of a flavone compound with a modified aromatic ring dioxygenase (modified BphA) and dihydrodiol dehydrogenase (BphB) (or a disrupted microorganism, microorganism culture solution, crude enzyme, purified enzyme, etc. containing these enzymes), or The method of culturing together with the microorganisms that produce the above three enzymes can be carried out by a conventional method in the same manner as in the usual enzyme reaction or culture method.

For example, the reaction of the above flavone compound with the modified aromatic ring dioxygenase (modified BphA) and dihydrodiol dehydrogenase (BphB) is performed by mixing the modified BphA and BphB in a solution containing the flavone compound. The flavone compounds can be converted by incubating at a suitable temperature for a certain period of time.
In addition, for example, a method for culturing a microorganism that produces a modified aromatic ring dioxygenase (modified BphA) and dihydrodiol dehydrogenase (BphB) together with the flavones can be performed as follows.

  The medium for cultivating the microorganism is usually a medium that can grow the microorganism, and specific examples include LB medium, M9 medium, KB medium, YM medium, KY medium, F101 medium, and the like.

  Any carbon source can be used as long as it is a carbon compound that can be assimilated and grown by cells. As the nitrogen source, for example, inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, and ammonium nitrate, and organic nitrogen sources such as yeast extract, peptone, and meat extract can be used. In addition to these, inorganic salts, metal salts, vitamins, and the like can be added as necessary.

  Culture is usually at a temperature of 20 to 40 ° C, more preferably 25 to 35 ° C, and a pH of 5 to 9 is preferred. Moreover, it is good also as a shaker culture and rotation culture suitably. After completion of the culture, the culture solution is centrifuged, the supernatant is collected, and the supernatant solution is extracted using an organic solvent such as ethyl acetate.

  To purify hydroxylated flavone compounds from the above mixture or culture, general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. Can be used alone or in appropriate combination.

  According to the method of the present invention, a flavone compound, particularly a flavone compound such as 7-hydroxyflavone, 5,7-dihydroxyflavone (chrysin), 1-phenylnaphthalene, which has been difficult to convert, can be efficiently converted into a flavone hydroxide. It can be converted into a similar compound. In the method of the present invention, a hydroxyl group can be specifically introduced into the 2-position and 3-position of the phenyl group (benzene ring) of the flavone compound. The hydroxylated flavone compound (diol form) produced in this manner can be used as a raw material for various industrial products such as pharmaceuticals and agricultural chemicals.

  Furthermore, the hydroxylated flavone compound (diol body) converted as described above is converted into a picolinic acid compound by BphC, an aromatic ring decomposing enzyme, and a carboxylic acid compound by BphC and BphD, an aromatic ring decomposing enzyme. Can be converted to

  The following examples are intended to explain the present invention more specifically and are not intended to limit the present invention.

  The normal gene recombination experiments used here are standard methods (Sambrook, J., Fritsch, EF, Maniatis, T., “Molecular cloning -A laboratory manual.” Cold unless otherwise stated. Spring Harbor Laboratory Press, 1989).

  In this example, the modified aromatic ring dioxygenase [bphA (1-22)] / aromatic dihydrodiol dehydrogenase plasmid pBPA (1-22) B and the modified aromatic ring dioxygenase [bphA (2- 2)] / Plasma pBPA (2-2) B for simultaneous expression of aromatic ring dihydrodiol dehydrogenase was prepared.

The present inventors previously used BphA1 (715-707), which is a hybrid BphA1 of an aromatic ring dioxygenase large subunit (BphA1) derived from Pseudomonas pseudoalgenes KF707 strain and BphA1 derived from Pseudomonas putida KF715, cassette PCR, It was produced using a so-called technique (Japanese Patent Laid-Open No. 2000-125871). The present inventors produced plasmid pBPA715-707BC expressing this BphA1 (715-707) and bphA2A3A4-bphB-bphC gene group [ bphA1 (715-707) bphA2A3A4 gene] derived from KF707 strain, and this plasmid was introduced. Various bioconversion experiments were conducted using Escherichia coli JM109 strain, and as a result, the production of picolinic acid was confirmed as disclosed in Patent Document 3.

  BphA1 (715-707) has a broad substrate specificity compared to BphA1 derived from KF707 strain. BphA1 (715-707) is different from BphA1 derived from KF707 strain in the two amino acid residues 324-325, and the one derived from KF707 strain is serine residue-valine residue, threonine residue-isoleucine residue It is the basis.

  Therefore, in this example, based on BphA1 (715-707), a mutant strain in which the two amino acid residues at positions 324 to 325 were randomly substituted was prepared with the aim of obtaining wider substrate specificity. Random mutations were introduced by using a gene encoding BphA1 (715-707) as a template, and a gene library in which the codon part encoding amino acid residues 324 to 325 was randomly substituted was prepared by PCR. First, PCR was performed using 715TINNK-F (SEQ ID NO: 7) and BphA1BglIIR (SEQ ID NO: 6) as primers, and PCR using BphA1SacIF (SEQ ID NO: 5) and 715TINNK-R (SEQ ID NO: 8) as primers, respectively. The amplification products (about 10 kb and about 5 kb for each PCR) were separated and purified by agarose gel electrophoresis.

The primer sequences used are as follows:
BphA1SacIF: 5'-CC GAATTC AAGGAGACGTTGAATCAT GAGCTC AGC -3 '(SEQ ID NO: 5)
BphA1BglIIR: 5'-TT GAATTC TTCCGGTTGAC AGATCT -3 '(SEQ ID NO: 6)
715TINNK-F: 5'-GCATGTTCGGCCAGCACATG NNKNNK TTCCCGACCTGTTC-3 '(SEQ ID NO: 7)
715TINNK-R: 5'-CATGTGCTGGCCGAACATGC-3 '(SEQ ID NO: 8)

BphA1SacIF (SEQ ID NO: 5) and BphA1BglIIR (SEQ ID NO: 6) encode sequences containing the vicinity of the N-terminal, the vicinity of the C-terminal, and the outside of BphA1 (715-707), respectively. BphA1SacIF (SEQ ID NO: 5) Sac I site in the, in BphA1BglIIR (SEQ ID NO: 6) has a Bgl II site, further Eco RI site on either side are applied (both are shown underlined) .

  The underlined part in the sequence of 715TINNK-F (SEQ ID NO: 7) corresponds to the position of amino acid residues 324 to 325 of BphA1 (715-707), and a mixture of random codons (NNK; N represents A, T, G, C Equal mixing, K is equivalent to mixing G and T).

The PCR conditions were the same for both amplification of about 10 kb and about 5 kb, and 30 cycles were performed at 94 ° C. for 1 minute, 61 ° C. for 1 minute, 72 ° C. for 1 minute 30 seconds, and KOD Dash polymerase (TOYOBO) was used. Amplified products were separated by agarose gel electrophoresis, and bands of about 10 kb and about 5 kb were cut out and purified. Next, these two gene fragments were mixed, and further PCR was performed by adding primers BphA1SacIF (SEQ ID NO: 5) and BphA1BglIIR (SEQ ID NO: 6) to obtain an amplification product corresponding to a total length of about 1.5 kb of bphA1 . The PCR reaction conditions are the same as those for amplification of about 10 kb and about 5 kb. Amplified products were separated by agarose gel electrophoresis, and a band of about 1.5 kb was cut out and purified to obtain a bphA1 (715-707) mutant library.

P. Expression plasmid pJHF18 (Hirose, J., Auyama, A., Hayashida, S., Furukawa, K.) containing the bphA1A2A3A4-bphB-bphC gene group (registered in GenBank accession number M83673) of pseudoalkalinenes KF707 strain , Gene, 128, 27-33, 1994), the reaction proceeds until meta-cleavage. Therefore, when biphenyl is used as a substrate, 2-hydroxy-6-oxo-6-phenylhexa-2 is obtained as a meta-cleavage product. , 4-dienoic acid (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid) is considered to be produced. In general, meta-cleavage products have a yellow color and can be monitored at a wavelength around 434 mm.

Since there is one Mlu I site in bphA1 in plasmid pJHF18, plasmid pJHF18ΔM1uI in which only bphA1 was destroyed was prepared by digestion with Mlu I, blunt end treatment, and ligation again (T. Kumamaru, H Suenaga, M. Mitsuoka, T. Watanabe, K. Furukawa, Nature Biotechnology, 16, 663-666, 1998).

Then, by a double digest PJHF18ΔM1uI with Sac I / Bgl II, Δ bphA1 gene except 1.39kb fragment containing only, instead, bphA1 prepared in the above (715-707) mutant library (Sac I / After double digestion with Bgl II), a modified aromatic ring dioxygenase gene library ( bphA1 mutation library :: bphA2A3A4 gene) and a plasmid containing the bphBbphC gene were obtained.

E. coli JM109 having this plasmid was filled with biphenyl vapor, and colonies capable of exhibiting yellow color were selected by meta-cleavage. This means that the bphA1 (715-707) mutant gene can function normally in colonies that can exhibit a yellow color by meta-cleavage.

Secondary screening was carried out using 200 E. coli transformants that were able to exhibit a yellow color by biphenyl vapor. The secondary screening was performed by growing mutant colonies on an agar medium mixed with either 1-phenylnaphthalene or 2-phenylnaphthalene as a substrate and confirming the coloration of the meta-cleavage product. As a result of selection based on the high decomposition efficiency of meta-cleavage for 1-phenylnaphthalene and 2-phenylnaphthalene, 5 mutants were found. Two of them (the plasmids contained in this Escherichia coli are named pSHF1-22ABC and pSHF2-2ABC, respectively) are 7-hydroxyflavones that cannot be decomposed by the Kph707- derived bphA1 gene or bphA1 (715-707) . 7-Dihydroxyflavone could also be decomposed by meta-cleavage.

The bphA1 (715-707) mutant genes derived from the plasmids pSHF1-22ABC and pSHF2-2ABC are hereinafter referred to as bphA1 (1-22) gene and bphA1 (2-2), respectively.

When the sequence of the bphA1 (1-22) gene was analyzed, the two amino acid residues from 324 to 325 , which were mutated based on bphA1 (715-707) , were the residues of bphA1 (715-707). The group-isoleucine residue was an alanine residue-leucine residue. In addition, when the sequence of the bphA1 (2-2) gene was analyzed, the two amino acid residues from 324 to 325 , which were mutated based on bphA1 (715-707), were found to be of bphA1 (715-707) . A threonine residue-isoleucine residue was a leucine residue-isoleucine residue. The sequences of the bphA1 (1-22) gene and the bphA1 (2-2) gene are shown in SEQ ID NOs: 1 and 3, respectively.

Moreover, the only exception bphC from this pSHF1-22ABC and pSHF2-2ABC, bphA1 (1-22) plasmid PBPA (1-22) expressing bphA2A3A4bphB gene B and bphA1 (2-2) plasmid expressing the bphA2A3A4bphB gene PBPA (2-2) B was produced.

  In this example, a conversion experiment was performed using the modified gene prepared in Example 1, and the obtained conversion product was identified and purified.

(1) Conversion experiment 700 ml to 1400 ml of a mixed culture of E. coli JM109 having the plasmid pBPA (1-22) B or pBPA (2-2) B prepared in Example 1 and the substrate whose conversion was confirmed in Example 1 An equal amount of methanol was added to the mixture and stirred at room temperature for 2 hours. This was centrifuged at 7,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was concentrated under reduced pressure to 300 ml to 500 ml and extracted twice with an equal amount of ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to obtain a product-containing extract. The extract was subjected to thin layer chromatography (TLC) using silica gel [0.25 nm Silica Gel 60 (Merck)], and after confirming the conversion product, a silica gel column [20 × 250 mm, Silica Gel 60 (Merck)] was applied. The product was subjected to the column chromatography used to obtain a pure product.

In addition, the developing solvent of TLC in each substrate is as follows:
Chalcone, hexane -EtOAc (4: 1); 7- hydroxy _{flavone, CH 2 Cl 2 -MeOH (10} : 1); _{chrysin, CH 2 Cl 2 -MeOH (20} : 1)
The developing solvent for column chromatography on each substrate is as follows:
Chalcone, hexane -EtOAc (3: 1); 7- hydroxy _{flavone, CH 2 Cl 2 -MeOH (15} : 1); _{chrysin, CH 2 Cl 2 -MeOH (20} : 1)

(2) Identification of conversion product of chalcone Crude extracts obtained by conducting [(trans-) chalcone] conversion experiments using Escherichia coli JM109 having plasmid pBPA (1-22B) or pBPA (2-2) B, respectively. (105.8 mg) was subjected to TLC, and it was found that a product having an Rf value of 0.2 and an Rf value of 0.1 was produced for both. Both products were purified by silica gel chromatography to obtain pure products of Compound 1 (27.6 mg) and Compound 2 (5.2 mg).

Compound 1 was obtained by direct comparison with a standard product by TLC and HPLC using 3- (2,3) dihydroxyphenyl) -1-phenylpropan-1-one (3- (2,3-dihydroxyphenyl) -1-phenylpropan-1 -one) (FIG. 1).

The molecular formula of Compound 2 was determined to be C ₁₅ H ₁₄ O ₄ from API-MS (m / z 258 (M +)), ¹ H, ¹³ C NMR. From the resulting molecular formula and ¹ of Compound 2 ^H, 13 ^C NMR, compound 2 two on the A ring and B ring of chalcone, it was suggested that compounds which four phenolic hydroxyl groups have been introduced . In the DQF COSY spectrum of the compound, two sets of three consecutive sp ² vicinal spin bonds (δ 7.02 (d, J = 7.9 Hz) −δ 6.74 (δ, J = 7.9, 8.0 Hz)) − δ 7 .36 (d, J = 8.0 Hz) and δ 6.56 (d, J = 7.3 Hz) −δ 6.52 (δ, J = 7.0, 7.3 Hz) −δ 6.63 (d, J = 7.0 Hz)) was observed, and it was determined that Compound 2 was a substance in which four phenolic hydroxyl groups were introduced at the 5th and 6th positions of the A ring and the 1 ′ and 2 ″ positions of the B ring. It was.

From the above results, Compound 2 was found to be 1,3-bis- (2,3-dihydroxy-phenyl) -propan-1-one (1,3-bis- (2,3-dihydroxy-phenyl) -propan-1- one) (FIG. 1). Compound 2 was a novel compound. The NMR data of Compound 2 are shown below.

(3) Identification of the conversion product of 7-hydroxyflavone (7-hydroxyflavone) Conversion of 7-hydroxyflavone with E. coli JM109 having plasmid pBPA (1-22) B or pBPA (2-2) B When the extract (93.2 mg) was subjected to TLC, it was found that products having an Rf value of 0.5 were produced for both. This product was purified by silica gel chromatography to obtain a pure compound 3 (4.5 mg).

The molecular formula of Compound 3 was determined to be C ₁₅ H ₁₀ O ₅ from API-MS (m / z 270 (M ⁺ )), ¹ H, ¹³ C NMR. From the resulting molecular formula and ¹ of Compound 3 ^H, 13 ^C NMR, Compound 3 was suggested to be a compound in which two phenolic hydroxyl groups introduced into the 7-hydroxy flavone. In the DQF COSY spectrum of the compound, three consecutive sp ² vicinal spin bonds (δ 7.29 (d, J = 8.5 Hz) −δ 8.80 (δ, J = 7.4, 8.5 Hz) −δ 6.95 ( d, J = 7.4 Hz) was observed, which revealed that two phenolic hydroxyl groups were introduced at the 1 ′ and 2 ″ positions of the B ring.

From the above results, Compound 3 was found to be 2- (2,3-dihydroxy-phenyl) -7-hydroxy-chromen-4-one (2- (2,3-dihydroxy-phenyl) -7-hydroxy-chromen-4- one) (FIG. 1).

Compound 3 is a known substance whose structure is registered in CAS, but there is no report that it can be produced by an aromatic ring dioxygenase + desaturase reaction. Therefore, the method for producing Compound 3 according to the present invention is new and effective. The NMR data of Compound 3 are shown below.

(4) Identification of chrysin conversion product A crude extract (126.3 mg) obtained by conducting a chrysin conversion experiment using E. coli JM109 having plasmid pBPA (1-22B) or pBPA (2-2) B was subjected to TLC. It was found that products having an Rf value of 0.3 were produced for both. Both products were purified by silica gel chromatography to obtain a pure product of Compound 4 (4.2 mg).

The molecular formula of Compound 4 was determined to be C ₁₅ H ₁₀ O ₆ from API-MS (m / z 286 (M ⁺ )), ¹ H, ¹³ C NMR. From the resulting ¹ H molecular formula and compounds ^4, 13 C NMR, compound 4 is two phenolic hydroxyl groups it was suggested that the compound introduced into chrysin. In the DQF COSY spectrum of the compound, three consecutive sp ² vicinal spin bonds (δ 7.30 (d, J = 8.0 Hz) −δ 6.81 (δ, J = 7.6, 8.0 Hz) −δ 6.98 ( (d, J = 7.6 Hz) was observed, it was clarified that two phenolic hydroxyl groups were introduced at the 1 ′ and 2 ″ positions of the B ring.

From the above results, compound 4 is 2- (2,3-dihydroxy-phenyl) -5,7-dihydroxy-chromen-4-one (2- (2,3-dihydroxy-phenyl) -5,7-dihydroxy- chromen-4-one) (FIG. 1).

Although compound 4 is a known substance having a structure registration in CAS, this is the first report that it can be produced by an aromatic ring dioxygenase + desaturase reaction. Therefore, the method for producing compound 4 according to the present invention is novel and effective. The NMR data of Compound 4 are shown below.

(5) Other conversion products (same compounds as 2072AB conversion)
(5-1) Flavanone
_{TLC CH 2 Cl 2 -MeOH (20} : 1) Rf0.6
Ethyl acetate extract weight 212.1mg
Silica column CH ₂ Cl ₂ -MeOH (20: 1) 68.6 mg

(5-2) 6-OH Flavone
_{TLC CH 2 Cl 2 -MeOH (10} : 1) Rf0.3
Ethyl acetate extract weight 77.0mg
Silica column _{CH 2 Cl 2 -MeOH (10:} 1) 20.3mg

(5-3) 6-OH Flavanone
_{TLC CH 2 Cl 2 -MeOH (20} : 1) Rf0.3
Ethyl acetate extract weight 155.9mg
Silica column _{CH 2 Cl 2 -MeOH (20:} 1) 60.3mg

(5-4) 7-OH Flavanone
_{TLC CH 2 Cl 2 -MeOH (20} : 1) Rf0.5
Ethyl acetate extract weight 149.3mg
Silica column CH ₂ Cl ₂ -MeOH (20: 1) 31.5 mg

(5-5) 1-phenylnaphthalene (conversion with bphA, the same conversion product as 2072A)
_{TLC CH 2 Cl 2 -MeOH (50} : 1) Rf0.3
Ethyl acetate extract weight 114.9mg
Silica column _{CH 2 Cl 2 -MeOH (50:} 1) 60.9mg

(5-6) 2-phenylnaphthalene (conversion with bphA, the same conversion product as 2072A)
_{TLC CH 2 Cl 2 -MeOH (50} : 1) Rf0.2
Ethyl acetate extract weight 114.9mg
Silica column CH ₂ Cl ₂ -MeOH (50: 1) 65.5 mg

(5-7) Naringenin
Conversion reaction does not proceed.

The biological component most damaged by the antioxidant active active oxygen is lipid (particularly polar lipid). Rat brain tissue is rich in polar lipids and also contains an oxidoreductase system involved in the reaction of active oxygen, so it is an excellent material for investigating the damage of active oxygen to biological lipids (cause of arteriosclerosis).

  Thus, the brain lipid peroxidation inhibitory action of compounds 2 to 4 as conversion compounds was examined by the TBA (thiobarbituric acid) method. This method is described in K. Shindo, Y. Kagiyama, R. Nakamura, A. Hara, H. Ikenaga, K. Furukawa, N. Misawa, J. Mol. Catal. B: Enzym., 23, 9 (2003) Are listed. Antioxidant activity of compounds 2 to 4 was measured (rat brain lipid peroxidation inhibition IC50). Briefly, the lipid peroxidation inhibitory action when each compound is added is measured using a rat brain homogenate.

The results are shown below:
Compound 2 0.7 μM
Compound 3 2.9 μM
Compound 4 4.8 μM
From the above results, it was shown that compounds 2 to 4 have antioxidant activity, particularly antioxidant activity on lipids.

  According to the present invention, a novel method for producing a hydroxylated flavone compound (particularly a diol having a hydroxyl group at two positions) from an existing flavone compound is provided. Such conversion products can be provided as raw materials for organic chemical synthesis of industrial products such as various pharmaceuticals and agricultural chemicals. In addition, the method of the present invention can introduce a hydroxyl group specifically at two positions (position 2 and position 3) of the phenyl group (benzene ring) of the flavone compound, and is a useful reaction in organic chemical synthesis.

1 shows structures (formulas I to IV) of compounds 1 to 4 obtained by a conversion experiment using a modified aromatic ring dioxygenase.

SEQ ID NOs: 1 and 3: synthetic polynucleotides SEQ ID NOs: 2 and 4: synthetic polypeptides SEQ ID NOs: 5-8: synthetic oligonucleotides
N in SEQ ID NO: 7 represents any nucleotide.

Claims (18)

The following polypeptide (a) or (b).
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, or (b) one or several amino acids other than the 324th or 325th amino acid in the amino acid sequence shown in SEQ ID NO: 2 or 4 A polypeptide comprising an amino acid sequence substituted, deleted, inserted or added, and having the same aromatic ring dioxygenase large subunit (BphA1) activity as (a) The following nucleic acid (a) or (b):
(A) a nucleic acid encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, or (b) one or a number other than the 324th or 325th amino acid in the amino acid sequence shown in SEQ ID NO: 2 or 4 Nucleic acid encoding a polypeptide comprising an amino acid sequence in which one amino acid is substituted, deleted, inserted or added, and having the same aromatic ring dioxygenase large subunit (BphA1) activity as (a) The following nucleic acid (a) or (b):
(A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1, or (b) a nucleic acid comprising a sequence complementary to the base sequence represented by SEQ ID NO: 1 under a stringent condition, and an aromatic ring Nucleic acid encoding a polypeptide having dioxygenase large subunit (BphA1) activity   A recombinant vector comprising the nucleic acid according to claim 2 or 3.   A transformant transformed with the recombinant vector according to claim 4.   A method for producing a polypeptide having an aromatic ring dioxygenase large subunit activity (BphA1), wherein the transformant according to claim 5 is cultured, and the polypeptide is collected from the resulting culture.   The recombinant vector according to claim 4, further comprising a nucleic acid encoding a polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG) and a nucleic acid encoding a polypeptide having dihydrodiol dehydrogenase activity (BphB).   It expresses the polypeptide (BphA1) according to claim 1, and expresses a polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG) and a polypeptide having dihydrodiol dehydrogenase activity (BphB). Microorganisms.   The microorganism according to claim 8, which is transformed with the recombinant vector according to claim 4 or 7.   The microorganism according to claim 8 or 9, which is capable of converting a flavone compound into a hydroxylated flavone compound.   The microorganism according to any one of claims 8 to 10, wherein the microorganism is Escherichia coli.   An enzyme having an activity of converting a flavone compound into a hydroxylated flavone compound, which comprises culturing the microorganism according to any one of claims 8 to 11 and collecting a polypeptide from the obtained culture. Group manufacturing method. The following (a) to (c):
(A) The polypeptide according to claim 1 (BphA1), the polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG), and the polypeptide having dihydrodiol dehydrogenase activity (BphB)
(B) The microorganism according to any one of claims 8 to 11 (c) At least one of the microorganism cultures according to any one of claims 8 to 11 is used as a sample containing a flavone compound. A method for producing a hydroxylated flavone compound comprising adding and mixing or culturing, and obtaining a hydroxylated flavone compound from the resulting mixture or culture.   The flavone compound is selected from the group consisting of flavone, 6-hydroxyflavone, 7-hydroxyflavone, 5,7-dihydroxyflavone, 6-hydroxyflavanone, 7-hydroxyflavanone, chalcone, 1-phenylnaphthalene, and 2-phenylnaphthalene. 14. The method of claim 13, wherein the method is selected.   The method according to claim 13 or 14, wherein the hydroxylated flavone compound is a diol having two hydroxyl groups introduced at the 2-position and the 3-position of the phenyl group of the flavone compound. Formula II below:

A compound represented by The following (a) to (c):
(A) The polypeptide according to claim 1 (BphA1), the polypeptide having aromatic ring dioxygenase subunit activity (BphA2A3A4 or BphEFG), and the polypeptide having dihydrodiol dehydrogenase activity (BphB)
(B) The microorganism according to any one of claims 8 to 11 (c) At least one of the microorganism cultures according to any one of claims 8 to 11 is added to a sample containing chalcone. Mixed or cultured, and the resulting mixture or culture is represented by the following formula II:

A process for producing a compound represented by the formula II, characterized in that a compound represented by formula II is obtained. Formulas II, III, and IV below:

An antioxidant comprising at least one selected from the group consisting of compounds represented by:

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