JP5142268B2 - Improved gallic acid synthase and method for producing gallic acid - Google Patents

Description

Gallic acid, useful as an antioxidant and photographic developer, is industrially produced using a microorganism that expresses a protein having an enzyme activity that oxidizes 5-position of protocatechuic acid (hereinafter referred to as protocatechuic acid 5-position oxidation activity) On how to do.

Gallic acid is an alkaline aqueous solution that has a strong reducing power and is used as a reducing agent and a photographic developer. It is also used as a raw material for tannin synthesis, used in the production of blue inks, and as an ester of propyl gallate, isoamyl gallate, etc., and as an antioxidant for fats and butters.

Regarding the method for producing gallic acid using an enzyme, there is no report on the production of gallic acid using a wild-type enzyme, but position 385 of the wild-type parahydroxybenzoic acid hydroxylase possessed by Pseudomonas aeruginosa PAO strain. It has been reported that gallic acid can be produced via protocatechuic acid starting from parahydroxybenzoic acid in a test tube by using a mutant enzyme (hereinafter abbreviated as PAO mutant enzyme) in which tyrosine is converted to phenylalanine. [Non-Patent Document 1]. However, it has been reported that the specific activity of the PAO mutant enzyme parahydroxybenzoate hydroxylase is reduced to about 1/50 that of the wild-type enzyme, and the protocatechuic acid 5-position oxidation activity for synthesizing gallic acid is also low. ing. In addition, with regard to a method for producing gallic acid using microorganisms, there have been reported examples of producing gallic acid from Escherichia coli using glucose as a raw material and using the PAO mutant enzyme [Patent Document 1 and Non-Patent Document 2]. However, in this example, only a part of the raw material is converted to gallic acid, the fermentation time is long, the precursor 3-dehydroxyshikimic acid accumulates in large quantities, and the production of gallic acid has reached its peak. For this reason, there is a problem in terms of manufacturing cost. When aromatic carboxylic acids such as terephthalic acid, phthalic acid, isophthalic acid and parahydroxybenzoic acid are used as raw materials instead of glucose, gallic acid production can be expected to be close to 100% per mole by enzyme conversion. It has not been reported that gallic acid can be produced by culturing microorganisms using phthalic acid, isophthalic acid and parahydroxybenzoic acid as raw materials.

When gallic acid is produced using an extraction method of plant nullde from a pentaploid, the production cost is high, and therefore a method for industrially producing these compounds is required. Phthalic acids (phthalic acid, terephthalic acid and isophthalic acid) are inexpensive raw materials, and terephthalic acid can be recycled from plastic bottles, so it is an inexpensive raw material. Although biomass is actively used, it is also possible to mass-produce parahydroxybenzoic acid using plants, so parahydroxybenzoic acid is also an inexpensive raw material. From such a background, a method for efficiently producing gallic acid from an inexpensive raw material such as terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid is desired. Since the PAO mutant enzyme has low protocatechuic acid 5-oxidation activity, it is desired to improve protocatechuate 5-oxidation activity in order to efficiently produce gallic acid.
US Patent No. 6,472,190 Entsch, B, Palfey, BA, Ballou, DP and Masey, VJ Biol. Chem. 266, 17341-17349 (1991) Spiros Kambourakis, KM Draths, and JW Frost.J.Am.Chem.Soc. 122, 9042-9043 (2000)

An object of the present invention is to provide a method for producing gallic acid at low cost by converting protocatechuic acid into gallic acid using a protein having protocatechuic acid 5-position oxidation activity (hereinafter referred to as protocatechuic acid 5-position oxidase). is there. Another object of the present invention is to produce protocatechuic acid from an inexpensive raw material such as terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid, and convert protocatechuic acid to gallic acid by protocatechuic acid 5-position oxidase. The object is to provide a method for producing gallic acid at low cost.

Until now, it was known that only the PAO mutant enzyme has protocatechuic acid 5-oxidation activity. However, the present inventors have intensively studied to solve the above-mentioned problems, and Pseudomonas aeruginosa PAO strain. A mutated enzyme obtained by converting leucine at position 199 of wild-type enzyme (hereinafter abbreviated as hydroxylase HFM300, if necessary) to other amino acids such as valine and glycine, as compared to PAO mutant enzyme, position 5 It has been found that the specific activity of the oxidation activity is twice or more higher. In addition, when a double mutant enzyme in which leucine at position 199 of the PAO mutant enzyme is converted to another amino acid such as valine or glycine is produced, the specific activity of protocatechuic acid 5-position oxidation activity is improved, and these double mutant enzymes It was found that even when phenylalanine at position 385 was converted to other amino acids such as valine and alanine, it had strong protocatechuic acid 5-position oxidation activity. The tyrosine at position 385 of the parahydroxybenzoic acid hydroxylase of Corynebacterium glutamicum ATCC13032 represented by SEQ ID NO: 20 (hereinafter abbreviated as hydroxylase HFM145) was substituted with phenylalanine. As for the mutant enzyme (SEQ ID NO: 22), it was found that the specific activity of the 5-position oxidative activity of protocatechuic acid was greatly improved by substitution of leucine at position 200 with valine. The amino acid at position 199 (position 200 in Corynebacterium glutamicum) It has been found that the specific activity of the 5-position oxidation activity of protocatechuic acid by substitution is not limited to the parahydroxybenzoate hydroxylase of Pseudomonas aeruginosa PAO strain.

After constructing a recombinant vector comprising a DNA fragment encoding a protein having the amino acid sequence represented by SEQ ID NO: 24 (sequence in which 385th tyrosine of HFM145 is substituted with phenylalanine and 200th leucine is replaced with valine) Then, when E. coli transformed with the recombinant vector was prepared, it succeeded in efficiently producing gallic acid using parahydroxybenzoic acid as a raw material.

Next, DNA encoding terephthalate dioxygenase, terephthalate dioxygenase reductase, terephthalate 1,2-dihydrodiol dihydrogenase and terephthalate transporter protein was cloned from Rhodococcus RHA1 strain, and the recombinant vector was cloned. It was constructed. Using the recombinant vector and a vector expressing a protein having the amino acid sequence represented by SEQ ID NO: 24, E. coli was transformed to prepare a transformant having acquired the ability to metabolize terephthalic acid. Subsequently, the transformant was successfully used to efficiently produce gallic acid using terephthalic acid as a raw material, and the present invention was completed.

That is, the present invention relates to the following (1) to (19).

(1) A method for producing gallic acid, comprising producing and accumulating gallic acid from protocatechuic acid using a microorganism that expresses the protein according to (A) or (B) below, and collecting the gallic acid.
(A) Enzyme activity having an amino acid sequence in which leucine at position 199 is substituted with another amino acid in SEQ ID NO: 2 or an amino acid sequence having 80% or more identity with SEQ ID NO: 2 and oxidizes position 5 of protocatechuic acid Having a protein.
(B) Enzyme activity having an amino acid sequence in which leucine at position 200 is substituted with another amino acid in SEQ ID NO: 22 or an amino acid sequence having 80% or more identity with SEQ ID NO: 22 and oxidizes position 5 of protocatechuic acid Having a protein.

(2) The method for producing gallic acid according to (1), wherein the amino acid replacing leucine at position 199 or leucine at position 200 is valine or glycine.

(3) In the amino acid sequence having 80% or more identity with SEQ ID NO: 2 or SEQ ID NO: 2, the tyrosine at position 385 is further substituted with another amino acid, (1) or (2 ) Gallic acid production method.

(4) The method for producing gallic acid according to the above (3), wherein the amino acid replacing tyrosine at position 385 is phenylalanine, valine or alanine.

(5) The method for producing gallic acid according to (1), wherein the protein has an amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, 16, 18 or 24 .

(6) The microbial culture or the treated product of the culture and protocatechuic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. The method for producing gallic acid according to any one of (1) to (5) above.

(7) The microorganism is a microorganism having the ability to generate protocatechuic acid from terephthalic acid, and reacting the microorganism with terephthalic acid in a medium containing terephthalic acid to generate and accumulate gallic acid, The method for producing gallic acid according to any one of (1) to (5), wherein the gallic acid is collected.

(8) The culture of the microorganism or the treated product of the culture and terephthalic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. The manufacturing method of the gallic acid as described in said (7).

(9) The microorganism further has a capability of generating protocatechuic acid from phthalic acid, and reacts the microorganism with phthalic acid in a medium containing phthalic acid to generate and accumulate gallic acid, The method for producing gallic acid according to any one of (1) to (5), wherein the gallic acid is collected.

(10) The microbial culture or the treated product of the microorganism and phthalic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. The manufacturing method of the gallic acid as described in said (9).

(11) The microorganism further has a capability of generating protocatechuic acid from isophthalic acid, and reacts the microorganism with isophthalic acid in a medium containing isophthalic acid to generate and accumulate gallic acid, The method for producing gallic acid according to any one of (1) to (5), wherein the gallic acid is collected.

(12) The microbial culture or the treated product of the culture and isophthalic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. The method for producing gallic acid according to (11) above.

(13) The microorganism further possesses an ability to produce protocatechuic acid from parahydroxybenzoic acid, and reacts the microorganism with parahydroxybenzoic acid in a medium containing parahydroxybenzoic acid, thereby gallic acid. The method for producing gallic acid according to any one of (1) to (5), wherein the gallic acid is collected and collected.

(14) The culture of the microorganism or the treated product of the culture and parahydroxybenzoic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. The method for producing gallic acid according to (13), characterized in that it is characterized in that

(15) The ability to produce protocatechuic acid from terephthalic acid transforms DNA encoding terephthalic acid dioxygenase, terephthalic acid dioxygenase reductase, terephthalic acid 1,2-dihydrodiol dihydrogenase and terephthalic acid transporter proteins The method for producing gallic acid according to (7) or (8) above, wherein the method is obtained by introduction by a method.

(16) The ability to produce protocatechuic acid from phthalate transforms DNA encoding terephthalate dioxygenase, terephthalate dioxygenase reductase, terephthalate 1,2-dihydrodiol dihydrogenase and terephthalate transporter proteins The method for producing gallic acid according to (9) or (10) above, wherein the method is obtained by introduction by a method.

(17) The ability to produce protocatechuic acid from isophthalic acid transforms DNA encoding isophthalic acid dioxygenase, isophthalic acid dioxygenase reductase, isophthalic acid 1,2-dihydrodiol dihydrogenase and isophthalic acid transporter proteins The method for producing gallic acid according to the above (11) or (12), which is obtained by introduction by a method.

(18) The microorganism according to any one of (1) to (17), wherein the microorganism is a microorganism into which a DNA encoding the protein of (A) or (B) is introduced. Method for producing gallic acid.

(19) the microorganism is Escherichia (Escherichia) genus Rhodococcus (Rhodococcus) genus, Acinetobacter (Acinetobacter) spp., Bradyrhizobium (Bradyrhizobium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus, Rhodopseudomonas (Rhodopseudomonas ) genus Sinorhizobium (Sinorhizobium) genus Brevibacterium (Brevibacterium) genus, characterized in that it is a microorganism belonging to Novo sphingomyelin bi um (Novosphingobium) genus or Ralstonia (Ralstonia) genus, wherein (1) (18) The manufacturing method of any one of these.

According to the present invention, there is provided a method for inexpensively producing gallic acid using protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid as a raw material, using an enzyme protein having excellent protocatechuic acid 5-position oxidation activity. can do.

The present invention is described in detail below.

The protocatechuic acid 5-position oxidase protein used in the present invention is the following protein (A) or (B).
(A) Enzyme activity having an amino acid sequence in which leucine at position 199 is substituted with another amino acid in SEQ ID NO: 2 or an amino acid sequence having 80% or more identity with SEQ ID NO: 2 and oxidizes position 5 of protocatechuic acid Having a protein.
(B) Enzyme activity having an amino acid sequence in which leucine at position 200 is substituted with another amino acid in SEQ ID NO: 22 or an amino acid sequence having 80% or more identity with SEQ ID NO: 22 and oxidizes position 5 of protocatechuic acid Having a protein.
Specifically, for example, a protein having an amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 22, or 24 can be mentioned. Here, SEQ ID NO: 6 is the amino acid sequence of the protein in which leucine at position 199 of wild-type parahydroxybenzoate hydroxylase (SEQ ID NO: 2) possessed by Pseudomonas aeruginosa PAO is substituted with valine. Moreover, in SEQ ID NO: 2, the leucine at position 199 may be substituted with an amino acid other than valine, and the amino acid sequence in which leucine at position 199 is substituted with glycine is shown in SEQ ID NO: 8. Furthermore, in SEQ ID NO: 2, in addition to the mutation of leucine at position 199, the tyrosine at position 385 may be replaced with another amino acid, and the tyrosine at position 385 in SEQ ID NO: 6 or SEQ ID NO: 8 is replaced with phenylalanine. The amino acid sequences are shown in SEQ ID NOs: 10 and 12, respectively. Further, amino acid sequences in which tyrosine at position 385 in SEQ ID NO: 6 is substituted with alanine or valine are shown in SEQ ID NOs: 14 and 18, respectively, and amino acid sequences in which tyrosine at position 385 in SEQ ID NO: 8 are substituted with alanine are respectively shown in SEQ ID NO: 16 shows.
The protocatechuic acid 5-position oxidase protein used in the present invention is not limited to Pseudomonas aeruginosa PAO strain, and SEQ ID NOs: 22 and 24 are derived from Corynebacterium glutamicum ATCC13032 having the amino acid sequence represented by SEQ ID NO: 20. It is an amino acid sequence of a protein in which amino acids corresponding to the above mutation points (positions 200 and 385 in Corynebacterium glutamicum) are substituted for wild-type protocatechuic acid 5-position oxidase.
The protein comprises an amino acid sequence having 80% or more identity, preferably 90% or more, particularly preferably 95% or more identity with any one of the amino acid sequences of SEQ ID NO: 2 or 22. A protein having protocatechuic acid 5-position oxidase activity (SEQ ID NO: 22 does not include a sequence in which phenylalanine at position 385 is substituted or deleted), wherein leucine at position 199 or 200 is substituted with another amino acid; it can.

A protein having an amino acid substitution at the above-mentioned site and having protocatechuic acid 5-position oxidase activity is known as Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as Molecular Cloning 2nd Edition). (Abbreviated), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Res., 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Res., 13, 4431 (1985), Proc. Natl. Acad. Sci., USA, 82, Using site-directed mutagenesis described in 488 (1985), etc., for example, by introducing site-directed mutagenesis into DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 20. Can do.

The microorganism used in the present invention is a microorganism that expresses the above-mentioned mutant protocatechuic acid 5-position oxidase protein. For example, to a mutation treatment to microorganisms strains such Pseudomonas (Pseudomonas) genus or Corynebacterium (Corynebacterium) microorganism belonging to the genus, obtainable by isolating those having the mutation. The parent strains of microorganisms are the American Type Culture Collection (hereinafter abbreviated as ATCC as necessary), the National Institute of Technology and Technology, Biological Genetic Resources Department (hereinafter abbreviated as NBRC as necessary), Or it can be obtained from the RIKEN Tsukuba Research Institute BioResource Center.

Specifically, examples of Pseudomonas microorganisms include Pseudomonas aeruginosa PAO strains. Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium glutamicum ATCC13032. However, it is not limited to these strains, and any strain that can achieve its purpose can be used.

The microorganism used in the present invention may be a microorganism transformed with a recombinant DNA containing a DNA encoding the mutant protocatechuate 5-position oxidase protein. A transformant producing a mutant protocatechuic acid 5-position oxidase protein that can be used in the present invention is obtained by, for example, purifying protocatechuate 5-position oxidase protein from the above-mentioned microorganism according to the method described in Molecular Cloning Second Edition. It can be obtained by cloning the encoding DNA, introducing a mutation, ligating it with a vector DNA, producing a recombinant DNA, and transforming a host cell with the recombinant DNA. . Hereinafter, a method for cloning DNA and a method for producing a transformant will be described in detail.

The microorganism belonging to the genus Pseudomonas or Corynebacterium is cultured by a known method. After culturing, the chromosomal DNA of the microorganism is isolated and purified by a known method (for example, the method described in Current Protocols in Molecular Biology). Using this synthetic DNA from the chromosomal DNA, a fragment containing DNA encoding the protocatechuic acid 5-position oxidase protein can be obtained by a hybridization method or a PCR method. The synthetic DNA is a gene encoding a protocatechuic acid 5-position oxidase protein derived from bacteria of the genus Pseudomonas or Corynebacterium (hereinafter referred to as protocatechuate 5-position oxidase gene), preferably Pseudomonas aeruginosa or coryne. It can be designed based on the base sequence of the protocatechuate 5-position oxidase gene derived from Bacterium glutamicum ATCC13032. Specifically, it can be designed based on the nucleotide sequence of the protocatechuate 5-position oxidase gene of Pseudomonas aeruginosa PAO strain or Corynebacterium glutamicum ATCC13032, that is, the nucleotide sequences represented by SEQ ID NOs: 1 and 19. PCR is performed using the synthetic DNA to amplify the gene encoding protocatechuic acid 5-position oxidase protein. By introducing a site-specific mutation into the obtained DNA, a gene encoding a mutant protocatechuic acid 5-position oxidase protein can be obtained.

As a vector for ligating the DNA of the mutant protocatechuic acid 5-position oxidase gene, any plasmid vector or phage vector can be used as long as it is a vector capable of autonomous replication in Escherichia coli K12 strain. PUC19 [Gene, 33,
103 (1985)], pUC18, pBR322, pHelix1 (Roche Diagnostics), ZAP Express (Stratagies, Strategies, 5, 58 (1992)), pBluescript II SK (+) (Stratagene) Nucleic Acids Res., 17, 9494 (1989)], pUC118 (manufactured by Takara Bio Inc.) and the like can be used.

Any Escherichia coli used as a host for the recombinant DNA obtained by ligating the above-obtained DNA to the vector can be used as long as it is a microorganism belonging to Escherichia coli. Specifically, Escherichia coli Escherichia coli XL1-Blue MRF '(Stratagies, 5, 81 (1992)), Escherichia coli C600 (Genetics, 39, 440 (1954)), Escherichia coli Y1088 (Science, 222,778 (1983)], Escherichia coli Y1090 (Science, 222, 778 (1983)), Escherichia coli NM522 (J. Mol. Biol., 166, 1 (1983)), Escherichia coli K802 (J. Mol. Biol ., 16, 118 (1966)], Escherichia coli JM105 [Gene, 38, 275 (1985)], Escherichia coli JM109, Escherichia coli BL21 and the like.

Rhodococcus (Rhodococcus) genus, Acinetobacter (Acinetobacter) spp., Bradyrhizobium (Bradyrhizobium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Sinorhizobium (Sinorhizobium) genus, Burebibakuteri um (Brevibacterium) genus, in a microorganism belonging to Novo sphingomyelin bi um (Novosphingobium) genus or Ralstonia (Ralstonia) genus, when introducing a 5-position oxidase gene protocatechuic acid, self replicable vector among these microorganisms Is used. Preferably, the recombinant DNA can be introduced into the host microorganism using a shuttle vector capable of autonomous replication in both microorganisms of both the microorganism and the Escherichia coli K12 strain.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the host cell, for example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110. (1972)], electroporation method [Nucleic Acids Res., 16, 6127 (1988)] and the like.

A recombinant DNA can be extracted from the transformant obtained as described above, and the base sequence of the DNA of the present invention contained in the recombinant DNA can be determined. For determination of the base sequence, a commonly used base sequence analysis method such as dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or 3730xl type DNA analyzer (Applied Biosystems), etc. Can be used.

The target DNA can also be prepared by chemical synthesis using a 8905 type DNA synthesizer manufactured by Perceptive Biosystems based on the DNA base sequence determined above.

A transformant expressing the above mutant protocatechuic acid 5-position oxidase protein can be obtained by expressing the above DNA in a host cell using the following method.

When using the DNA encoding the above-mentioned mutant protocatechuic acid 5-position oxidase protein, a DNA fragment having an appropriate length containing a portion encoding the protein of the present invention can be prepared as necessary. Further, the production rate of the protein can be improved by substituting the base in the base sequence of the protein-encoding part so as to be an optimal codon for host expression. The transformant expressing the DNA of the present invention is prepared by inserting the above DNA fragment downstream of the promoter of an appropriate expression vector to prepare a recombinant DNA, and the recombinant DNA is adapted to the expression vector. Can be obtained by introduction into a host cell.

As a host for expressing the mutant protocatechuic acid 5-position oxidase protein, any bacteria, yeast, animal cells, insect cells, plant cells and the like that can express the target gene can be used.
Preferably, a microorganism having the ability to metabolize terephthalic acid, phthalic acid or isophthalic acid or parahydroxybenzoic acid (ability to produce protocatechuic acid from terephthalic acid, phthalic acid or isophthalic acid or parahydroxybenzoic acid) can be used. More preferably, terephthalic acid, phthalic acid, Escherichia having metabolic capacity of isophthalic acid or para-hydroxybenzoic acid (Escherichia) genus Pseudomonas (Pseudomonas) genus Rhodococcus (Rhodococcus) genus Corynebacterium (Corynebacterium) genus Comamonas ( Comamonas), Bacillus (Bacillu s), Mycobacterium (Mycobacteriu m) the genus Novo sphingolipids bi um (Novosphigobium) genus or Burkholderia (Burkholderia) genus of bacteria. More preferably, mention may be made of a Rhodococcus RHA1 and Comamonas testosteroni (Comamonas testosteroni) E6 with metabolic ability terephthalic acid.
By expressing the mutant protocatechuic acid 5-position oxidase protein in such a microorganism, gallic acid can be produced from terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid via protocatechuic acid. That is, microorganisms having the ability to metabolize terephthalic acid, phthalic acid, isophthalic acid, or parahydroxybenzoic acid and expressing a mutant protocatechuic acid 5-position oxidase protein are represented by terephthalic acid, phthalic acid, isophthalic acid or By reacting with these compounds in a medium containing parahydroxybenzoic acid, gallic acid can be obtained from protocatechuic acid generated from terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid.
The process for producing protocatechuic acid from terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid may be performed using another microorganism. That is, protocatechuic acid produced from terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid by a microorganism capable of metabolizing terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid is converted into a mutant protocatechuic acid 5-position oxidase. The method for producing gallic acid by reacting with a protein-expressing microorganism is also included in the method for producing gallic acid of the present invention.

As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed is used.

When a prokaryotic organism such as a bacterium is used as a host cell, the recombinant DNA containing the DNA of the present invention can be autonomously replicated in the prokaryotic organism, and at the same time, a promoter, a ribosome binding sequence, the DNA of the present invention, It is preferably a recombinant DNA composed of a transcription termination sequence. A gene that controls the promoter may also be included.

A vector for introducing and expressing a mutant protocatechuic acid 5-position oxidase protein or a DNA encoding a fusion protein of the protein with another protein into a microorganism such as Escherichia coli is a so-called multicopy type. Preferably, a plasmid having a replication origin derived from ColE1, such as a pUC-type plasmid, a pBR322-type plasmid, or a derivative thereof can be used. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion, addition and / or inversion. In addition, the modification here includes modification by mutation treatment with a mutation agent, UV irradiation, or natural mutation. More specifically, examples of the vector include pUC19 [Gene, 33, 103 (1985)], pUC18, pBR322, pHelix1 (manufactured by Roche Diagnostics), pKK233-2 (manufactured by Amersham Pharmacia Biotech) ), PSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen), pET-3 (Novagen) pBluescriptII SK (+), pBluescript II KS (+) (Stratagene) PSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.) and the like can be used.

As the promoter, any promoter can be used so long as it can be expressed in a host cell such as Escherichia coli (Escherichia coli). For example, artificially designed and modified such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, etc., promoters derived from E. coli such as T7 promoter, phages, etc., and tac promoter, lacT7 promoter Other promoters can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 5 to 18 bases). In the recombinant DNA of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the host cell, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], electroporation method (Nucleic Acids Res., 16, 6127 (1988)), junction transfer method (JGC Ottow, Ann. Rev. Microbiol., Vol. 29, p. 80 (1975)), Cell fusion method [MH Gabor, J. Bacteriol., Vol.137, p.1346 (1979)] and the like can be mentioned.

When producing gallic acid from terephthalic acid, phthalic acid or isophthalic acid or parahydroxybenzoic acid, a mutant protocatechuic acid 5-position oxidase is produced in a microorganism capable of producing protocatechuic acid from phthalic acid, isophthalic acid or parahydroxybenzoic acid. DNA encoding the protein is introduced.
Microorganisms having the ability to metabolize terephthalic acid, phthalic acid or isophthalic acid or parahydroxybenzoic acid (the ability to produce protocatechuic acid from terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid) used in the present invention are from nature. Can be obtained using an enrichment culture method using terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid as a carbon source. Preferably, a bacterium of the genus Pseudomonas, Rhodococcus, Comamonas, Bacillus, Mycobacterium, Novosphingobium or Burkholderia having the ability to metabolize terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid Can give.
In addition, since a microorganism that expresses protocatechuic acid 5-position oxidase protein, such as Rhodococcus genus RHA1, also has the ability to produce protocatechuate from parahydroxybenzoic acid, protocatechuate 5-position oxidase protein was expressed. By culturing the microorganism in a medium containing parahydroxybenzoic acid, gallic acid can be produced from parahydroxybenzoic acid.

When gallic acid is produced from terephthalic acid, a microorganism having the ability to metabolize terephthalic acid (ability to produce protocatechuic acid from terephthalic acid) into which DNA encoding the mutant protocatechuic acid 5-position oxidase protein is introduced is produced from terephthalic acid. Examples thereof include terephthalate dioxygenase, terephthalate dioxygenase reductase, and terephthalate 1,2-dihydrodiol dihydrogenase and terephthalate transporter-related microorganisms involved in acid metabolism.
When gallic acid is produced from phthalic acid, a microorganism having the ability to metabolize phthalic acid (ability to produce protocatechuic acid from phthalic acid) into which DNA encoding the mutant protocatechuic acid 5-position oxidase protein is introduced is diphthalate Mention may be made of microorganisms capable of producing oxygenase, phthalate dioxygenase reductase, phthalate 4,5-cis-dihydroxydiol dihydrogenase, 4,5-dihydroxyphthalate decarboxylase and phthalate transporter proteins. .
When producing gallic acid from isophthalic acid, a microorganism having the ability to metabolize isophthalic acid (ability to produce protocatechuic acid from isophthalic acid) into which DNA encoding the mutant protocatechuic acid 5-position oxidase protein is introduced is diphthalate. Mention may be made of microorganisms having the ability to produce oxygenase, isophthalate dioxygenase reductase, isophthalate 1,2-dihydrodiol dihydrogenase and isophthalate transporter proteins.
A microorganism having the ability to produce these proteins obtains a transformant that expresses the protocatechuate 5-position oxidase protein after isolating the DNA encoding these proteins using recombinant DNA technology. It can also be obtained using the same method.

The microorganism of the present invention obtained as described above is preferably cultured in a medium containing 1 mM to 1 M protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid, and gallic acid is added to the culture. Gallic acid can be produced by accumulating and collecting from the culture. The method of culturing the microorganism of the present invention in a medium can be performed according to a usual method used for culturing microorganisms.

The culture of the microorganism of the present invention can be performed in a normal nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, various vitamins, etc., and examples of the carbon source include sugars such as glucose, sucrose, and fructose, ethanol, Alcohols such as methanol, organic acids such as citric acid, malic acid and succinic acid, and molasses are used. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea or the like is used alone or in combination. Examples of inorganic salts that can be used include potassium monohydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate. In addition to this, peptone, meat extract, yeast extract, corn stapler,
Nutrients such as various vitamins such as casamino acid and biotin can be added to the medium. Protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid is added as a raw material for producing gallic acid.

The culture is usually carried out under aerobic conditions such as aeration stirring and shaking. The culture temperature is not particularly limited as long as the microorganism of the present invention can grow, and the pH during the culture is not particularly limited as long as the microorganism of the present invention can grow. The pH adjustment during the culture can be performed by adding an acid or an alkali.

After culturing the microorganism of the present invention, adding the culture of the microorganism or a treated product of the culture to an aqueous medium containing any of protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid Thus, gallic acid can be generated and accumulated in the medium, and gallic acid can be collected from the medium.

As a processed product of the culture, a product in which the microorganism of the present invention is immobilized on a carrier may be used. In that case, it is possible to use cells that have been recovered from the culture or washed with an appropriate buffer, such as a phosphate buffer (pH 6 to 10) of about 0.02 to 0.2M. it can. Further, it contains a crushed product obtained by crushing the cells recovered from the culture by means of ultrasonic waves, pressing, etc., and a protocatechuic acid 5-position oxidase protein obtained by extracting the crushed product with water or the like. An extract and a partially purified component of protocatechuic acid 5-position oxidase protein obtained by subjecting the extract to further treatment with ammonium sulfate salting out, column chromatography, etc. are also immobilized on a carrier. Can be used for manufacturing.

Immobilization of these cells, disrupted cells, extracts, or purified enzymes is carried out by immobilizing the cells on an appropriate carrier such as acrylamide monomer, alginic acid, or carrageenan according to a commonly used method known per se. It can be performed by the method of making it.

The aqueous medium used for the reaction can be an aqueous solution containing protocatechuic acid or a suitable buffer, for example, a phosphate buffer (pH 6 to 10) of about 0.02 to 0.2M. When it is necessary to further increase the substance permeability of the cell membrane of the bacterial cells, 0.05 to 2.0% (w / v) of toluene, xylene, nonionic surfactant or the like is added to this aqueous medium. You can also.

The concentration of protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid, which is a reaction raw material in the aqueous medium, is suitably about 0.1 mM to 1 M. The enzyme reaction temperature and pH in the aqueous medium are not particularly limited, but are usually 10 to 60 ° C., preferably 15 to 50 ° C., and the pH in the reaction solution is 5 to 10, preferably about 6 to 9. can do. The pH can be adjusted by adding acid or alkali. The enzyme used in the invention can be obtained by collecting the cell extract as it is or by collecting it by centrifugation, filtration or the like and suspending it in water or a buffer solution. The enzyme thus obtained is reacted with protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid or parahydroxybenzoic acid in the presence of water, but protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid in the reaction solution is reacted. Alternatively, the concentration of parahydroxybenzoic acid is advantageously as high as possible within a range that does not inhibit the activity of the enzyme. The reaction may be performed by any method of standing, stirring and shaking. Alternatively, a method in which an enzyme is immobilized on a suitable support and packed in a column and a solution containing protocatechuic acid, terephthalic acid, phthalic acid, isophthalic acid, or parahydroxybenzoic acid is allowed to flow. The reaction is usually carried out at 10 to 60 ° C., preferably 15 to 50 ° C., pH 5 to 9, preferably p6 to 8.

In addition, when the oxidizing agent is added to the aqueous medium during the reaction, the production yield of gallic acid may be further improved. Examples of the oxidizing agent include nitrates such as sodium nitrite and potassium nitrite, metal salts such as ferric chloride, halogen, peroxo acid, and the like, and preferably sodium nitrite and ferric chloride. The addition concentration varies depending on the type of oxidizing agent, but it is desirable to add it at a concentration that does not inhibit the formation of gallic acid, and is usually 0.001 to 0.05% (W / V), preferably 0.005 to 0.02. %.

Gallic acid from the culture solution or reaction solution after completion of the culture can be isolated and purified by extraction with an organic solvent such as ethyl acetate. In addition, after removing insoluble components such as cells from the culture solution by centrifugation or the like as necessary, for example, a method using activated carbon, a method using an ion exchange resin, a crystallization method, a precipitation method, etc. Gallic acid can be collected in or in combination.

  The method of the present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1. The hydroxylase of Corynebacterium glutamicum ATCC13032 having gallic acid synthesis activity was identified as follows.
(1) Identification of a protein of Corynebacterium glutamicum ATCC13032 that has homology to the amino acid sequence of parahydroxybenzoic acid hydroxylase of Pseudomonas aeruginosa PAO strain Parahydroxybenzoic acid hydroxylase (HFM300) of Pseudomonas aeruginosa PAO strain Is reported to possess the activity to oxidize parahydroxybenzoic acid to produce protocatechuic acid, but not to possess any activity to produce protocatechuic acid 5-position oxidation activity. In addition, the enzyme having the activity to generate gallic acid from protocatechuic acid is a mutant enzyme HFM300Y385F in which the tyrosine at position 385 of wild-type parahydroxybenzoate hydroxylase (HFM300) possessed by Pseudomonas aeruginosa PAO is converted to phenylalanine. Only reported to have activity [Entsch, B, Palfey, BA, Ballou, DP and Masey, VJ Biol. Chem. 266, 17341-17349 (1991)]. On the other hand, from the GenBank (hereinafter abbreviated as GB) database of National Center for Biotechnology Information (hereinafter abbreviated as NCBI), the parahydroxybenzoic acid of Pseudomonas aeruginosa PAO shown in SEQ ID NO: 2 The search was performed using the BLAST homology analysis method using the amino acid sequence of oxyhydroxylase as the query sequence. As a result, the protein HFM145 having 40.1% homology with HFM300 in the genome sequence of Corynebacterium glutamicum ATCC13032 used for industrial production of amino acid fermentation (SEQ ID NO: 20 is the protein sequence number, GB accession) The number was identified as NP_600305.1).

(2) Isolation and purification of chromosomal DNA In order to clone DNA encoding the purified protein HFM145 using the PCR method, the strain Corynebacterium glutamicum ATCC13032 (NBRC number: 12168) was obtained from NBRC and according to the information from NBRC After culturing, chromosomal DNA was isolated and purified from these cultured cells using a DNE tissue kit (Qiagen).

(3) Design and Preparation of PCR Primers The nucleotide sequence encoding the protein HFM145 (GB accession number NC_003450.3; SEQ ID NO: 19) was obtained from the NCBI GenBank database via the Internet. Based on this base sequence, PCR primers for cloning the DNA of each gene using the PCR method were designed and synthesized. The nucleotide sequences of PCR primers are shown in SEQ ID NO: 25 and SEQ ID NO: 26.

(4) Amplification of DNA encoding protein HFM145 by PCR method Expand High Fidelity PCR System purchased from Roche Diagnostics and G.P. purchased from Roche Diagnostics Using the chromosomal DNA obtained above as a template, a DNA encoding the protein HFM145 was amplified using the PCR primer according to the attached instructions, using a GC Rich PCR System. The PCR reaction using the GC rich system was carried out in the presence of demethyl sulfoxide and 7-deasa-dGTP.

(5) Cloning of the gene BD In-Fusion PCR Cloning Kit (BD In-Fusion PCR Cloning Kit), in which the DNA encoding the protein HFM145 obtained above, that is, the DNA of the HFM145 gene was purchased from Clontech The restriction enzyme site Nco I and the restriction enzyme site Sma of plasmid pROX1 (obtained from Roche Applied Science; the base sequence is shown in SEQ ID NO: 27), which is an expression vector for E. coli using the E. coli T7 promoter. The plasmid pROX_HFM145 was cloned between I and expressing the HFM145 gene. These expression plasmids are enzyme proteins in which a 10 amino acid peptide of Gly-Gly-Gly-Ser-His-His-His-His-His-His (SEQ ID NO: 28) is added to the C-terminus of each wild-type enzyme. Is expressed.

The nucleotide sequence of the DNA incorporated into these plasmids was requested by Dr. Daisuke Shibata of the Kazusa DNA Research Institute, the dideoxychain termination method (Sanger, F. et al., Proc. Natl. Acad). Sci. USA, Vol. 74, p.5463, (1977)], and confirmed that each plasmid contained the gene of interest.

(6) Construction and culture of transformant The transformant BL21 / pROX__HFM145 is constructed by introducing the plasmid pROX_HFM145 described above into Escherichia coli K-12 BL21 (DE3) using a transformation method using calcium ions. did. The BL21 (DE3) strain was obtained as strain number ME9026 from the National Institute of Genetics, National Institute of Genetics, National Institute of Genetics, Information and Systems Research Organization.

(7) Production of gallic acid using the above transformed strain The transformed strain BL21 / pROX_HFM145 was cultured overnight in 5 ml of LB medium. Ampicillin was added to a final concentration of 100 ppm to maintain the plasmid. Inoculate 1/100 volume of fresh LB medium, incubate at 25 ° C, and add IPTG (isopropyl-1-thio-β-D-galactopyranoside) to a final concentration of 1 mM in the logarithmic growth phase for 3 hours of protein induction went. After protein induction, E. coli was collected by centrifugation. The supernatant was discarded and suspended in 500 μl of HEPES buffer (50 mM HEPES-NaOH (pH 7.5), 10% glycerol). The E. coli suspension was disrupted with an ultrasonic disrupter. The E. coli suspension was centrifuged (4 ° C., 10 minutes, 20000 × g) to separate into a supernatant and a precipitate, and the supernatant was used as a crude enzyme solution. The protein concentration of the crude enzyme solution is measured using the Bio-Rad protein assay (Bio-Rad Laboratories, CA, USA) based on the Bradford method, and is adjusted to 1 mg / ml with HEPES buffer. Diluted.

The gallic acid synthesis reaction was performed in 100 μl in HEPES buffer containing 0.5 mM substrate (protocatechuic acid or parahydroxybenzoic acid), 0.01 mM FAD, 0.01 mM FMN (flavin mononucleotide), 2.5 mM NADH, 25 mM NADPH. The reaction system was carried out. When protocatechuic acid was used as a substrate, 68 μg of crude enzyme was added to the reaction solution to start the reaction. The enzyme reaction solution was reacted at 30 ° C., and the reaction was stopped with 1 ml of ethyl acetate after 0 and 1 hour. When parahydroxybenzoic acid was used as a substrate, 10 μg of crude enzyme was added to the reaction solution to start the reaction. The enzyme reaction solution was reacted at 30 ° C., and after 0 and 30 minutes, the reaction was stopped with 1 ml of ethyl acetate. Further, 150 μl of HEPES buffer and 1.5 μl of 2N HCl were added and mixed vigorously for 5 minutes, followed by centrifugation (room temperature, 5 minutes, 20000 × g). 800 μl of the upper layer (ethyl acetate layer) of the reaction solution / ethyl acetate mixture separated into two layers was collected and transferred to a new 1.5 ml tube.

After drying with a vacuum centrifugal dryer, 10 μl of acetonitrile was added, mixed vigorously for 5 minutes, further diluted with 190 μl of water, filtered through a 0.2 μm pore size filter (Millex-LG), and injected into a vial. The vial was set in an LC-TOF type mass spectrometer (Waters, LCT Premier XE) and analyzed by the LC-TOF type mass spectrometer. Gallic acid was detected using an LC-TOF mass spectrometer. The gallic acid produced by combining the retention time of high performance liquid chromatography and the mass value from the mass spectrometer was compared with the standard gallic acid.

Hydroxylase HFM145 synthesized gallic acid when protocatechuic acid was used as a substrate. The gallic acid synthesis per hour per 1 mg of crude enzyme protein was 249 μM / hr / mg crude enzyme protein.

Example 2 The activity of hydroxylase HFM145 and the activity of PAO mutant enzyme were compared as follows.
(1) Construction of polycistron-type expression plasmid In order to efficiently express the above HFM145 gene in E. coli JM109 strain, E. coli JM109 (DE3) strain or E. coli BL21 (DE3) strain without depending on the transcription and translation efficiency of the gene. Transcription of the gene depends on the pseudogene, and an expression system was constructed in which the target gene was also translated while maintaining the translation efficiency of the pseudogene. More specifically, in order to insert the T7 promoter sequence, the pseudo gene, and the target gene into the pUC19 plasmid DNA through the HindIII site and the SphI site, four synthetic DNAs represented by SEQ ID NOs: 29 to 32 were synthesized. . These synthetic DNAs were inserted between the HindIII site and SphI site of pUC19 (manufactured by Takara Bio Inc.) to construct the expression vector pUTCH19.

(2) Construction of PAO Mutant Enzyme and Integration into a Vector Parahydroxybenzoate hydroxylase gene nucleotide sequence data (NC — 002516) of Pseudomonas aeruginosa PAO strain was obtained from NCBI GenBank database via the Internet. Based on this base sequence, a forward PCR primer represented by SEQ ID NO: 33 and a reverse PCR primer represented by SEQ ID NO: 34 for designing the DNA of each gene using the PCR method were designed and synthesized. Pseudomonas aeruginosa PAO strain chromosomal DNA (ATCC No .: 47085D-5) was obtained from ATCC, using this as a template, PCR using two kinds of PCR primers and PrimeSTAR enzyme (manufactured by Takara Bio Inc.). After amplifying the DNA of the oxyhydroxylase gene, a treatment for adding an A residue to the 3 ′ end with Taq polymerase (Takara Bio Inc.) was performed. After purifying the target DNA after gel electrophoresis, the plasmid pT7Blue_HFM300 carrying DNA encoding the parahydroxybenzoic acid hydroxylase protein HFM300 was constructed by incorporating it into the pT7Blue-T vector. Plasmid pUTCH_HFM300 was constructed by excising DNA encoding each enzyme protein from pT7Blue_HFM300 using restriction enzyme HindIII and restriction enzyme XbaI and incorporating it into expression vector pUTCH19.

Similarly, a mutant enzyme protein HFM300Y385F (PAO mutation in which the 385th tyrosine of the enzyme protein HFM300 is replaced with phenylalanine by PCR using a forward PCR primer represented by SEQ ID NO: 35 and a reverse PCR primer represented by SEQ ID NO: 36. Plasmid pT7Blue_HFM300Y385F carrying DNA encoding protein HFM300Y385F was constructed by amplifying DNA encoding enzyme) and incorporating it into the pT7Blue-T vector. Plasmid pUTCH_HFM300Y385F was constructed by cutting out DNA encoding each enzyme protein from pT7Blue_HFM300Y385F using restriction enzyme HindIII and restriction enzyme XbaI and incorporating it into expression vector pUTCH19.

(3) Construction of plasmid expressing enzyme protein HFM145 From the results of Example 1, good gallic acid production was observed when a culture extract of a transformant expressing enzyme protein HFM145 was used. Therefore, a plasmid that efficiently expresses the enzyme protein HFM145 was constructed. The enzyme protein HFM145 produced from the plasmid pROX_HFM145 has a 10 amino acid peptide (Gly-Gly-Gly-Ser-His-His-His-His-His-His (SEQ ID NO: 28)) added to the C-terminus. ing. In order to construct a plasmid expressing an enzyme protein from which these peptides were removed, two types of PCR primers shown in SEQ ID NOs: 37 and 38 were designed and synthesized.

The target DNA was amplified by PCR using the pROX_HFM145 plasmid DNA as a template and PCR primers shown in the above table and PrimeSTAR enzyme (Takara Bio), and then A was added to the 3 ′ end by Taq polymerase (Takara Bio). Processing to give residues was performed. After purifying the target DNA after gel electrophoresis, the plasmid pT7Blue_HFM145 was constructed by incorporating it into the pT7Blue-T vector. A plasmid pUTCH_HFM145 was constructed by cutting out DNA encoding each enzyme protein from these plasmids using restriction enzymes HindIII and restriction enzymes XbaI or KpnI and incorporating them into the expression vector pUTCH19.

(4) Construction of a plasmid that expresses the mutant enzyme protein HFM145Y385F The enzyme that has been proven to have the activity of generating gallic acid from protocatechuic acid is a mutant enzyme in which the 385th tyrosine of the enzyme protein HFM300 is substituted with phenylalanine. Only protein HFM300Y385F. Thus, a plasmid expressing the mutant enzyme protein HFM145Y385F in which the 385th tyrosine of the enzyme protein HFM145 was substituted with phenylalanin was constructed by the same method as described above.

Two types of PCR primers shown in SEQ ID NOs: 39 and 40 were designed and synthesized. The target DNA was amplified by the PCR method using the plasmid DNA of pROX_HFM145 as a template and the PCR primers in Table 4 and PrimeSTAR enzyme (Takara Bio), and then the 3 ′ end by Taq polymerase (Takara Bio) The process which provides A residue to was performed. After purifying the target DNA after gel electrophoresis, the plasmid pT7Blue_HFM145Y385F was constructed by incorporating it into the pT7Blue-T vector. A plasmid pUTCH_HFM145Y385F was constructed by cutting out DNA encoding each enzyme protein from these plasmids using restriction enzymes HindIII and restriction enzymes XabI or KpnI and incorporating them into the expression vector pUTCH19.

(5) Production of proteins HFM300, HFM145 and HFM145Y385F by E. coli Plasmids pUTCH_HFM300, pUTCH_HFM145 and pUTCH_HFM145Y385F were introduced into E. coli JM109 by transformation to produce recombinant E. coli JM109 / pUTCH_HFM300, JM109 / pUTCH_HFM145 and JHF109M145Y These transformants were cultured at 37 ° C. for 14 hours using Overnight Express Instant TB (manufactured by Novagen; hereinafter, abbreviated as OEI-TB medium), which is a lac promoter-inducible expression medium. did. After collection, the cells were suspended in 0.5 ml of HEPES-glycine buffer. The suspension was subjected to ultrasonic crushing treatment and centrifuged, and the supernatant was recovered to obtain a crude enzyme solution. Subsequently, the protein concentration of the crude enzyme solution was measured by the Bradford method and adjusted to 1 mg / ml.

(6) Measurement of gallic acid synthase activity The gallic acid synthase activity of the crude enzyme solution prepared in 5 above was measured as follows. The enzyme reaction was performed by protocatechuic acid (substrate) 500 μM, FAD 10 μM, FMN 10 μM, NADH
30 in 100 μl reaction solution containing 2.5 mM, NADP 2.5 mM, and crude enzyme solution 68 μg
1 hour at 0 ° C. After the reaction, the amount of gallic acid produced was measured using an LC-TOF mass spectrometer (Waters, LCT Premier XE). As a result, 133 μM, 172 μM, and 865 μM gallic acid were detected per 1 mg protein in recombinant E. coli JM109 / pUTCH_HFM300, JM109 / pUTCH_HFM145, and JM109 / pUTCH_HFM145Y385F, respectively. From the activity measurement results, the activity ratio of each gallic acid synthase was as shown in the table below.

In the expression vector pUTCH19, the gene incorporated in the vector is designed to produce a protein with the same expression efficiency. In order to confirm this, the gallic acid synthase protein in the crude enzyme solution was used. The amount of was measured as follows. Β-mercaptoethanol (0.25 μl of 99.5%, 1.25 μl of 10% sodium dodecyl sulfate solution and 1.25 μl of staining solution) was added to 10 μL (10 μg) of the crude enzyme protein solution prepared to 1 mg / ml as described above. After boiling for 5 minutes, the cooled sample for electrophoresis was subjected to polyacrylamide gel (ATTO).
e-PAGEL E-T1020L) was applied to the well and electrophoresed at 20 mA for 80 minutes. The gel was subjected to protein staining using Coomassie brilliant blue solution (ATTO EzStainAqua). It was fully decolorized with distilled water and the images were captured electronically using a scanner. Compared with the vector control, a signal with a molecular weight (about 44 kD) predicted from the amino acid sequence of the HFM enzyme protein was seen in the newly expressed protein. When the electronically captured images were analyzed with the image processing software NIH ImageJ (http://www.bioarts.co.jp/~ijjp/ij/), the expression level of each HFM enzyme protein was observed to differ between samples. Was not.

Example 3 An amino acid substitution mutant at position 199 of hydroxylase HFM300 was constructed as follows, and the enzyme activity was measured.
(1) Types of amino acid substitutions in a mutant having an amino acid substitution at position 199 of hydroxylase HFM300 As described above, tyrosine at position 385 of hydroxylase HFM300 of Pseudomonas aeruginosa PAO strain, or Corynebacterium glutamicum ATCC13032 It was shown that gallic acid synthesis activity was improved by introducing a mutation into tyrosine at position 385 of the hydroxylase HFM145. An attempt was made as follows to improve gallic acid synthesis activity by introducing mutations into other amino acid residues of these enzymes. Specifically, eight types of mutants (shown in Table 2) were constructed by substituting the leucine residue at position 199 of HFM300 and the leucine residue at position 200 of HFM145 with other amino acid residues.

(2) Construction of a plasmid expressing the mutant enzyme protein HFM300_L199V A plasmid expressing the mutant enzyme protein HFM300_L199V in which the leucine at position 199 of the enzyme protein HFM300 was replaced with valine was constructed as follows. In order to prepare a gene encoding the mutant enzyme protein HFM300_L199V based on the base sequence data (SEQ ID NO: 1) encoding the enzyme protein HFM300, the forward PCR primer represented by SEQ ID NO: 41 and SEQ ID NO: 42 are used. Reverse PCR primers were designed and synthesized. Mutation in which the 199th leucine of the enzyme protein HFM300 is replaced with valine by PCR using the plasmid pUTCH_HFM300 carrying the DNA encoding the hydroxylase protein HFM300 as a template and two PCR primers and Pfu enzyme (Stratagene) The DNA of the enzyme protein HFM300_L199V was amplified and cloned into a protein expression vector to construct pUTCH_HFM300_L199V.

(3) Site-directed mutagenesis PCR method using a forward PCR primer represented by SEQ ID NO: 43 and a reverse PCR primer represented by SEQ ID NO: 44 as in the construction of a plasmid expressing the mutant enzyme protein HFM300_L199G (2) Was used to amplify DNA encoding the mutant enzyme protein HFM300_L199G in which the 199th leucine of the enzyme protein HFM300 was replaced with glycine, and cloned into a protein expression vector to construct pUTCH_HFM300__L199G.

(4) Production of the proteins HFM300, HFM300_L199G, and HFM300_L199V by E. coli Plasmids pUTCH_HFM300_L199G and pUTCH_HFM300_L199V were introduced into E. coli JM109 using a transformation method using calcium ions to produce recombinant E. coli JM109 / pUTCH_HFM300_L199G and JM109 / pUTL_199. As a control for comparison, JM109 / pUTCH_HFM300 was also used for the experiment. These transformants were used at 37 ° C. for 14 hours using Overnight Express Instant TB (manufactured by Novagen; hereinafter abbreviated as OEI-TB medium), which is a medium for expression of lac promoter induction. Cultured. Ampicillin was added to a final concentration of 100 ppm to maintain the plasmid. After collection, the cells were suspended in 0.5 ml of HEPES-glycine buffer. The suspension was subjected to ultrasonic crushing treatment and centrifuged, and the supernatant was recovered to obtain a crude enzyme solution. Subsequently, the protein concentration of the crude enzyme solution was measured using a Bio-Rad protein assay (Bio-Rad Laboratories, CA, USA) based on the Bradford method, and HEPES was adjusted to 1 mg / ml. Dilute with buffer.

また酵素蛋白質HFM300との各没食子酸合成酵素の活性比は表５のとおりであった。

(5) Measurement of gallic acid synthase activity The gallic acid synthase activity of the crude enzyme solution prepared above was measured as follows. The enzymatic reaction was carried out at 30 ° C. for 1 hour in 100 μl of a reaction solution containing protocatechuic acid (substrate) 500 μM, FAD 10 μM, FMN 10 μM, NADH 2.5 mM, NADP 2.5 mM, and crude enzyme solution 68 μg. After the reaction, the amount of gallic acid produced was measured using an LC-TOF mass spectrometer (Waters, LCT Premier XE).
The gallic acid synthesis reaction was carried out in a 100 μl reaction system in a HEPES buffer containing 0.5 mM substrate (protocatechuic acid or parahydroxybenzoic acid), 0.01 mM FAD, 0.01 mM FMN, 2.5 mM NADH, 25 mM NADPH. It was. When protocatechuic acid was used as a substrate, 68 μg of crude enzyme was added to the reaction solution to start the reaction. The enzyme reaction solution was reacted at 30 ° C., and the reaction was stopped with 1 ml of ethyl acetate after 0 and 1 hour. Further, 150 μl of HEPES buffer and 1.5 μl of 2N HCl were added and mixed vigorously for 5 minutes, followed by centrifugation (room temperature, 5 minutes, 20000 × g). 800 μl of the upper layer (ethyl acetate layer) of the reaction solution / ethyl acetate mixture separated into two layers was collected and transferred to a new 1.5 ml tube. After drying with a vacuum centrifugal dryer, 10 μl of acetonitrile was added, mixed vigorously for 5 minutes, further diluted with 190 μl of water, filtered through a 0.2 μm pore size filter (Millex-LG), and injected into a vial. The vial was set in an LC-TOF type mass spectrometer (Waters, LCT Premier XE) and analyzed by the LC-TOF type mass spectrometer. Gallic acid was detected using an LC-TOF mass spectrometer. The gallic acid produced by combining the retention time of high performance liquid chromatography and the mass value from the mass spectrometer was compared with the standard gallic acid.
As a result, the activity per 1 mg protein / hour was as shown in Table 3 and Table 4 for recombinant E. coli JM109 / pUTCH_HFM300_L199G, JM109 / pUTCH_HFM300_L199V, and JM109 / pUTCH_HFM300, respectively.

Table 5 shows the activity ratio of each gallic acid synthase to the enzyme protein HFM300.

Example 4 A double mutant enzyme protein in which mutations were introduced into the amino acid residues at positions 199 and 385 of the enzyme protein HFM300 was constructed as follows.
(1) Construction of HFM300_L199G_Y385F By site-directed mutagenesis PCR using a forward PCR primer represented by SEQ ID NO: 41 and a reverse PCR primer represented by SEQ ID NO: 42, using plasmid pUTCH_HFM300_Y385F as a template, the 199th enzyme protein HFM300_Y385F A DNA encoding a mutant enzyme protein HFM300_L199V_Y385F in which leucine was replaced with valine was amplified and cloned into a protein expression vector to construct pUTCH_HFM300_L199V_Y385F.

(2) The 199th of the enzyme protein HFM300_Y385F was obtained by site-directed mutagenesis PCR using the forward PCR primer represented by SEQ ID NO: 43 and the reverse PCR primer represented by SEQ ID NO: 44 using the plasmid pUTCH_HFM300_Y385F as a template for construction of HFM300_L199V_Y385F. DNA encoding the mutant enzyme protein HFM300_L199G_Y385F in which leucine was replaced with glycine was cloned into a protein expression vector to construct pUTCH_HFM300_L199G_Y385F.

(3) Similarly to the construction of HFM300_L199V_Y385A, by the site-directed mutagenesis PCR method using the forward PCR primer represented by SEQ ID NO: 45 and the reverse PCR primer represented by SEQ ID NO: 46, using the plasmid pUTCH_HFM300_L199V as a template, the enzyme protein HFM300_Y385F The DNA encoding the mutant enzyme protein HFM300_L199V_Y385A in which the 385th tyrosine was replaced with alanine was amplified and cloned into a protein expression vector to construct pUTCH_HFM300_L199V_Y385A.

(4) Similarly to the construction of HFM300_L199G_Y385A, by the site-directed mutagenesis PCR method using the forward PCR primer represented by SEQ ID NO: 45 and the reverse PCR primer represented by SEQ ID NO: 46, using the plasmid pUTCH_HFM300_L199G as a template, the enzyme protein HFM300_L199G The DNA encoding the mutant enzyme protein HFM300_L199G_Y385A in which the 385th tyrosine was replaced with alanine was amplified and cloned into a protein expression vector to construct pUTCH_HFM300_L199G_Y385A.

(5) Similarly to the construction of HFM300_L199V_Y385V, by the site-directed mutagenesis PCR method using the forward PCR primer represented by SEQ ID NO: 47 and the reverse PCR primer represented by SEQ ID NO: 48, the plasmid pUTCH_HFM300_L199V was used as a template and the enzyme protein HFM300_L199V A DNA encoding a mutant enzyme protein HFM300_L199V_Y385V in which the 385th tyrosine was replaced with valine was amplified and cloned into a protein expression vector to construct pUTCH_HFM300_L199V_Y385V.

(6) the double mutant enzyme protein HFM300_L199V_Y385F, mutant enzyme protein HFM300_L199G_Y385F, mutant enzyme protein HFM300_L199V_Y385A, mutant enzyme protein HFM300_L199G_Y385A and mutant enzyme protein HFM300_L199V_Y385V of E. coli by the production plasmid pUTCH_HFM300_L199V_Y385F, pUTCH_HFM300_L199G_Y385F, pUTCH_HFM300_L199V_Y385A, transforming pUTCH_HFM300_L199G_Y385A and pUTCH_HFM300_L199V_Y385 each E. coli JM109 Recombinant E. coli JM109 / pUTCH_HFM300_L199V_Y385F, recombinant E. coli JM109 / pUTCH_HFM300_L199G_Y385F, recombinant E. coli JM109 / pUTCH_HFM300_L199V_Y385A, recombinant E. coli JM109 / pUTCH_HFM300_L199G_Y385A and recombinant E. coli JM109_P As a control for comparison, JM109 / pUTCH_HFM300 was also used for the experiment. These transformants were cultured at 37 ° C. for 14 hours using Overnight Express Instant TB (overnight express instant TB; manufactured by Novagen; hereinafter abbreviated as OEI-TB medium), which is a lac promoter-inducible expression medium. did. After collection, the cells were suspended in 0.5 ml of HEPES-glycine buffer. The suspension was subjected to ultrasonic crushing treatment and centrifuged, and the supernatant was recovered to obtain a crude enzyme solution. Subsequently, the protein concentration of the crude enzyme solution was measured by the Bradford method and adjusted to 1 mg / ml.

また酵素蛋白質HFM300との各没食子酸合成酵素の活性比は表８のとおりであった。

(7) Measurement of gallic acid synthase activity The gallic acid synthase activity of the crude enzyme solution prepared in (6) above was measured as follows. The enzymatic reaction was carried out at 30 ° C. for 1 hour in 100 μl of a reaction solution containing protocatechuic acid (substrate) 500 μM, FAD 10 μM, FMN 10 μM, NADH 2.5 mM, NADP 2.5 mM, and crude enzyme solution 68 μg. After the reaction, the amount of gallic acid produced was measured using an LC-TOF mass spectrometer (Waters, LCT Premier XE). As a result, recombinant E. coli JM109 / pUTCH_HFM300_L199V_Y385F, recombinant E. coli JM109 / pUTCH_HFM300_L199G_Y385F, recombinant E. coli JM109 / pUTCH_HFM300_L199V_Y385A, recombinant E. coli JM109 / pUTCH_HFM300_L199G_Y385A, recombinant E. coli JM109 / pUTCH_HFM300_L199G_Y385A, It was as Table 6 and Table 7.

Table 8 shows the activity ratio of each gallic acid synthase to the enzyme protein HFM300.

Example 5 FIG. A 200-position amino acid substitution mutant of hydroxylase HFM145 was constructed as follows, and the enzyme activity was measured.
(1) Construction of a plasmid that expresses the double mutant enzyme protein HFM145_L200V_Y385F The double mutant introduction of the enzyme protein HFM300 was found to significantly increase the activity of producing gallic acid from protocatechuic acid. The following is the preparation of a double mutation-introducing enzyme in which the 385-position tyrosine of HFM145 corresponding to the 385-position tyrosine is converted to phenylalanine, and the 200-position leucine in the HFM145 corresponding to the 199-position leucine of HFM300 is converted to valine. I went as follows.
First, two types of PCR primers shown in SEQ ID NO: 49 and SEQ ID NO: 50 were designed and synthesized. Site-directed mutagenesis PCR using these PCR primers and PrimeSTAR enzyme (manufactured by Takara Bio Inc.) Double mutagenesis using the plasmid DNA of pUTCH_HFM145_Y385F as a template and leucine at position 200 of mutated enzyme protein HFM145_Y385F replaced with valine DNA encoding the enzyme protein HFM145_L200V_Y385F was amplified and cloned into a protein expression vector to construct pUTCH_HFM145_L200V_Y385F.

(2) Production of the double mutant enzyme protein HFM145_L200V_Y385F by E. coli Plasmid pUTCH_HFM145_L200V_Y385F was introduced into E. coli JM109 by transformation to construct recombinant E. coli JM109 / pUTCH_HFM145_L200V_Y385F. For comparison, JM109 / pUTCH_HFM300 and JM109 / pUTCH_HFM145_Y385F were also used for the experiment at the same time. These transformants were cultured at 37 ° C. for 14 hours using OEI-TB medium. After collection, the cells were suspended in 0.5 ml of HEPES-glycine buffer. The suspension was subjected to ultrasonic crushing treatment and centrifuged, and the supernatant was recovered to obtain a crude enzyme solution. Subsequently, the protein concentration of the crude enzyme solution was measured by the Bradford method and adjusted to 1 mg / ml.

また酵素蛋白質HFM300との各没食子酸合成酵素の活性比は表１０のとおりであった。

(3) Measurement of gallic acid synthase activity The gallic acid synthase activity of the crude enzyme solution prepared above was measured as follows. The enzymatic reaction was carried out at 30 ° C. for 1 hour in 100 μl of a reaction solution containing protocatechuic acid (substrate) 500 μM, FAD 10 μM, FMN 10 μM, NADH 2.5 mM, NADP 2.5 mM, and crude enzyme solution 68 μg. After the reaction, the amount of gallic acid produced was measured using an LC-TOF mass spectrometer (Waters, LCT Premier XE). As a result, Table 9 shows the activity per 1 mg protein in recombinant E. coli JM109 / HFM145_Y385F, recombinant E. coli JM109 / HFM145_L200V_Y385F, and JM109 / pUTCH_HFM300.

Table 10 shows the activity ratio of each gallic acid synthase to the enzyme protein HFM300.

Example 6 Production of gallic acid from terephthalic acid by E. coli was performed as follows. 1. DNA cloning encoding terephthalate dioxygenase, terephthalate dioxygenase reductase, terephthalate 1,2-dihydrodiol dihydrogenase and terephthalate transporter protein Rhodococcus sp. RHA1 strain terephthalate dioxygenase, terephthalate Acid dioxygenase reductase, 1,2-dihydrodiol terephthalate base sequence data (NC_008268, NC_008269, NC_008270, NC_008271) of the gene gene were obtained from the NCBI GenBank database via the Internet. Based on this base sequence, a forward PCR primer represented by SEQ ID NO: 51 and a reverse PCR primer represented by SEQ ID NO: 52 were designed and synthesized for cloning the DNA of each gene using the PCR method. Using chromosomal DNA from Rhodococcus sp. RHA1 as a template, PCR using two PCR primers and PrimeSTAR enzyme (manufactured by Takara Bio Inc.), terephthalate dioxygenase, terephthalate dioxygenase reductase, terephthalate 1,2- After amplifying the DNA of the dihydrodiol dihydrogenase enzyme gene, a treatment for adding an A residue to the 3 ′ end with Taq polymerase (manufactured by Takara Bio Inc.) was performed. Plasmid that carries DNA encoding terephthalate dioxygenase, terephthalate dioxygenase reductase, terephthalate 1,2-dihydrodiol dihydrogenase by purifying the target DNA after gel electrophoresis and then incorporating it into the pT7Blue-T vector pT7Blue_TPACB1 was created. Plasmid pUTCH_TPACB1 was constructed by excising DNA encoding each enzyme protein from pT7Blue_TPACB1 using restriction enzymes HindIII and XbaI and incorporating it into the expression vector pUTCH19.
The Rhodococcus sp. RHA1 strain was distributed by Dr. Masao Fukuda of Nagaoka University of Science and Technology and cultured according to the method taught by him. Chromosomal DNA was isolated and purified from the cultured cells using a DNeasy tissue kit (Qiagen).

2. Synthesis of gallic acid from terephthalic acid
PUTCH_TPACB1 and pRTCH_HFM145_Y385F, pUTCH_TPACB1 and PRTCH_HFM145_L200V_Y385F, E. coli JM109 (DE3) was transformed with PUTCH_TPACB1 and pRTCH_HFM300 or PUTCH_TPACB1 and pRTCH_HFM300_Y385F,, recombinant E. coli JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_Y385F), JM109 (DE3) / (pUTCH_TPACB1 , pRTCH_HFM145_L200V_Y385F), JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM300), and JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM300_Y385F).
Recombinant E. coli JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_Y385F), JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_L200V_Y385F), JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM300), _ Was cultured at 37 ° C. for 16 hours using 5 ml of OEI-TB medium. After collection, the cells were suspended in 3 ml of LB medium (kanamycin 50 ppm, ampicillin 100 ppm, IPTG 1 mM, pH 5.95) and cultured at 25 ° C. for 4 hours. 500 μl of the bacterial suspension was taken and transferred to a 1.5 ml tube. Terephthalic acid was added to a final concentration of 1 mM. At 0, 2, and 20 hours, 100 μl was taken from the bacterial suspension, 1 ml of ethyl acetate, 150 μl of HEPES buffer 5 μl of 2N HCl was added, vigorously suspended for 5 minutes, and centrifuged. 800 μl of the upper layer (ethyl acetate layer) divided into two layers was taken, transferred to a new 1.5 mM tube, and centrifuged. The dried sample was vigorously suspended in 10 μL of acetonitrile and diluted with 190 μL of water. It filtered with the filter of the hole diameter 0.2micrometer, and analyzed with LC-TOF type | mold mass spectrometer (Waters, LCT Premier XE).

As a result, the recombinant Escherichia coli JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_Y385F), JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_L200V_Y385F), JM109 (DE3) / (pUTCH_TPACB1, _RM_F3) _JR109 / JR109 / HF3F Production of 1.2 μM, 3.7 μM, 1.0 μM and 0.3 μM gallic acid was observed for (pUTCH_TPACB1, pRTCH_HFM300), respectively.

Claims (19)

A method for producing gallic acid, comprising producing and accumulating gallic acid from protocatechuic acid using a microorganism that expresses the protein described in (A) or (B) below, and collecting the gallic acid.
(A) Enzyme activity which has an amino acid sequence in which leucine at position 199 is substituted with another amino acid in SEQ ID NO: 2 or an amino acid sequence having 90 % or more identity with SEQ ID NO: 2 and oxidizes position 5 of protocatechuic acid Having a protein.
(B) Enzyme activity having an amino acid sequence in which leucine at position 200 is substituted with another amino acid in SEQ ID NO: 22 or an amino acid sequence having 90 % or more identity with SEQ ID NO: 22 and oxidizes position 5 of protocatechuic acid Having a protein. The method for producing gallic acid according to claim 1, wherein the amino acid replacing leucine at position 199 or leucine at position 200 is valine or glycine. 3. The gallic acid according to claim 1, wherein tyrosine at position 385 is further substituted with another amino acid in SEQ ID NO: 2 or an amino acid sequence having 90 % or more identity with SEQ ID NO: 2. Manufacturing method. The method for producing gallic acid according to claim 3, wherein the amino acid replacing tyrosine at position 385 is phenylalanine, valine or alanine. The method for producing gallic acid according to claim 1, wherein the protein has an amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, or 24. The microbial culture or the treated product of the culture and protocatechuic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. The method for producing gallic acid according to any one of 1 to 5. The microorganism further possesses the ability to generate protocatechuic acid from terephthalic acid. By reacting the microorganism with terephthalic acid in a medium containing terephthalic acid, gallic acid is generated and accumulated, and this is collected. The method for producing gallic acid according to any one of claims 1 to 5, wherein: The culture of the microorganism or the treated product of the culture and terephthalic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. 8. The method for producing gallic acid according to 7. The microorganism further possesses the ability to generate protocatechuic acid from phthalic acid. By reacting the microorganism with phthalic acid in a medium containing phthalic acid, gallic acid is generated, accumulated, and collected. The method for producing gallic acid according to any one of claims 1 to 5, wherein: The microbial culture or the treated product of the culture and phthalic acid are present in an aqueous medium, gallic acid is produced and accumulated in the medium, and gallic acid is collected from the medium. 9. The method for producing gallic acid according to 9. The microorganism further possesses the ability to produce protocatechuic acid from isophthalic acid. By reacting the microorganism with isophthalic acid in a medium containing isophthalic acid, gallic acid is produced, accumulated, and collected. The method for producing gallic acid according to any one of claims 1 to 5, wherein: The microbial culture or the treated product of the culture and isophthalic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. 11. A method for producing gallic acid according to 11. The microorganism further possesses the ability to produce protocatechuic acid from parahydroxybenzoic acid, and reacts the microorganism with parahydroxybenzoic acid in a medium containing parahydroxybenzoic acid to produce gallic acid, The method for producing gallic acid according to any one of claims 1 to 5, wherein the gallic acid is accumulated and collected. The culture of microorganisms or the treated product of the culture and parahydroxybenzoic acid are present in an aqueous medium, gallic acid is generated and accumulated in the medium, and gallic acid is collected from the medium. The method for producing gallic acid according to claim 13. The ability to produce protocatechuic acid from terephthalic acid is introduced by transformation of DNA encoding terephthalate dioxygenase, terephthalate dioxygenase reductase, terephthalate 1,2-dihydrodiol dihydrogenase and terephthalate transporter proteins The method for producing gallic acid according to claim 7 or 8, wherein the gallic acid is obtained. The ability to produce protocatechuic acid from phthalic acid is introduced by transformation methods into DNA encoding terephthalate dioxygenase, terephthalate dioxygenase reductase, terephthalate 1,2-dihydrodiol dihydrogenase and terephthalate transporter proteins The method for producing gallic acid according to claim 9 or 10, wherein the gallic acid is obtained. The ability to produce protocatechuic acid from isophthalic acid is introduced by transformation methods into DNA encoding isophthalate dioxygenase, isophthalate dioxygenase reductase, isophthalate 1,2-dihydrodiol dihydrogenase and isophthalate transporter proteins The method for producing gallic acid according to claim 11, wherein the gallic acid is obtained. The method for producing gallic acid according to any one of claims 1 to 17, wherein the microorganism is a microorganism into which a DNA encoding the protein (A) or (B) is introduced. Microbe Escherichia (Escherichia) genus Rhodococcus (Rhodococcus) genus, Acinetobacter (Acinetobacter) spp., Bradyrhizobium (Bradyrhizobium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Sinorhizobium (Sinorhizobium) genus Brevibacterium (Brevibacterium) genus, characterized in that it is a microorganism belonging to Novo sphingomyelin bi um (Novosphingobium) genus or Ralstonia (Ralstonia) genus, to any one of claims 1 18 The manufacturing method described.