JP5140848B2 - 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 produce protocatechuic acid from an inexpensive raw material such as terephthalic acid, phthalic acid, isophthalic acid, or parahydroxybenzoic acid, and a protein having protocatechuic acid 5-position oxidizing activity (hereinafter referred to as protocatechuate 5-position oxidase). ) To provide a method for producing gallic acid at low cost by converting protocatechuic acid to gallic acid.

In order to solve the above-mentioned problems, the present inventors have conducted intensive research and examined the enzyme activity of a wild-type protein having homology with the amino acid sequence of the PAO mutant enzyme. Strong for wild-type proteins having amino acid sequences represented by SEQ ID NOs: 6, 10, 14, 16, 18, 20, 22, 24, 26 and 28 having homology of 1% or more and less than 75.0% It was found that protocatechuic acid has 5-position oxidation activity. Subsequently, the protocatechuic acid 5-position oxidation activity of the protein having the amino acid sequence represented by SEQ ID NO: 16, 26, 30, 32 and 34 having 39.1 to 58.9% homology with each other was measured, and PAO When compared with the activity of the mutant enzyme, it was found that the specific activity of protocatechuic acid 5-position oxidation activity was more than twice as high as that of the PAO mutant enzyme even in the wild type protein.

After constructing a recombinant vector containing a DNA fragment encoding a protein having the amino acid sequence represented by SEQ ID NO: 16, E. coli transformed with the recombinant vector was prepared. As a result, gallic acid was obtained using parahydroxybenzoic acid as a raw material. Has been successfully produced.

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. A transformant having the ability to metabolize terephthalic acid was prepared by transforming E. coli using the recombinant vector and a vector expressing a protein having the amino acid sequence represented by SEQ ID NO: 16 or 30. 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 (15).

(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) Protein having the amino acid sequence represented by SEQ ID NO: 16, 26, 30, 32 or 34 (B) One or more amino acids in the amino acid sequence represented by SEQ ID NO: 16, 26, 30, 32 or 34 A protein having an amino acid sequence containing a substitution, deletion, insertion, or addition of and having an enzyme activity that oxidizes position 5 of protocatechuic acid (SEQ ID NO: 30 does not include a sequence in which phenylalanine at position 385 is substituted or deleted) ).

(2) A culture of microorganisms or a processed 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 manufacturing method of the gallic acid of said (1).

(3) The microorganism has the ability to generate protocatechuic acid from terephthalic acid, and reacts the microorganism with terephthalic acid in a medium containing terephthalic acid to generate and accumulate gallic acid. (1) The method for producing gallic acid according to (1) above.

(4) 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. (3) The manufacturing method of the gallic acid of said.

(5) The microorganism possesses the ability to produce protocatechuic acid from phthalic acid, and reacts the microorganism with phthalic acid in a medium containing phthalic acid to produce and accumulate gallic acid. (1) The method for producing gallic acid according to (1) above.

(6) The microbial culture or the treated product of the culture 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. (5) The manufacturing method of the gallic acid of the said.

(7) The microorganism has the ability to produce protocatechuic acid from isophthalic acid, and reacts the microorganism with isophthalic acid in a medium containing isophthalic acid to produce and accumulate gallic acid, (1) The method for producing gallic acid according to (1) above.

(8) The culture of the microorganism 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. (7) The manufacturing method of the gallic acid of said.

(9) The microorganism is a microorganism having the ability to produce protocatechuic acid from parahydroxybenzoic acid, and reacting the microorganism with parahydroxybenzoic acid in a medium containing parahydroxybenzoic acid, The method for producing gallic acid according to the above (1), wherein the gallic acid is produced, accumulated, and collected.

(10) causing the culture of the microorganism or the treated product of the culture and parahydroxybenzoic acid to exist in an aqueous medium, generating and accumulating gallic acid in the medium, and collecting gallic acid from the medium; (9) The method for producing gallic acid according to (9),

(11) 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 the above (3) or (4), which is obtained by introduction by a method.

(12) The ability to produce protocatechuic acid from phthalic 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 (5) or (6) above, wherein the method is obtained by introduction by a method.

(13) 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 (7) or (8) above, wherein the method is obtained by introduction by a method.

(14) The gallic acid according to any one of (1) to (13), wherein the microorganism is a microorganism into which a DNA encoding the protein according to (A) or (B) is introduced. Manufacturing method.

(15) 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) to (14) 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.

Examples of the protocatechuic acid 5-position oxidase protein used in the present invention include a protein in which the specific activity of protocatechuate 5-position oxidase is twice or more superior to the PAO mutant enzyme having the amino acid sequence represented by SEQ ID NO: 4. It is done. For example, the protein which has an amino acid sequence represented by sequence number 16, 26, 30, 32 or 34 is mentioned. Here, SEQ ID NO: 16 is the amino acid sequence of wild-type protocatechuate 5-position oxidase derived from Pseudomonas putida KT2440. SEQ ID NO: 26 is the amino acid sequence of wild-type protocatechuate 5-position oxidase derived from Novosphingobium aromatica bolans DSM 12444. In SEQ ID NO: 16, Tyr at position 386 corresponding to the mutation point of the PAO mutant enzyme may be substituted with Phe, and the amino acid sequence in which the Y386F mutation is introduced in SEQ ID NO: 16 is shown in SEQ ID NO: 32. In SEQ ID NO: 26, Tyr at position 384 corresponding to the mutation point of the PAO mutant enzyme may be substituted with Phe. The amino acid sequence in which the Y384F mutation is introduced in SEQ ID NO: 26 is shown in SEQ ID NO: 34. SEQ ID NO: 30 is the amino acid sequence of wild-type protocatechuate 5-position oxidase derived from Corynebacterium glutamicum ATCC13032, wherein Tyr at position 385 corresponding to the mutation point of the PAO mutant enzyme is substituted with Phe.
The protocatechuic acid 5-position oxidase protein used in the present invention also has an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 16, 26, 30, 32 or 34. And a protein having protocatechuic acid 5-position oxidase activity. However, in the amino acid sequence of SEQ ID NO: 30, phenylalanine at position 385 needs to be conserved, and therefore does not include a sequence in which phenylalanine at position 385 is deleted or substituted.
Preferably, the reason why the protein represented by SEQ ID NOs: 16, 26, 30, 32 and 34 is superior to the PAO mutant enzyme represented by SEQ ID NO: 4 is the amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: Since it is derived from the difference between any of the amino acid sequences of 16, 26, 30, 32 and 34, 1 in the amino acid sequence of SEQ ID NO: 16, 26, 30, 32 or 34 based on the difference of these amino acid sequences. Alternatively, a protein having an amino acid sequence in which several amino acids are deleted, substituted or added and having protocatechuic acid 5-position oxidase activity can be mentioned (in SEQ ID NO: 30, phenylalanine at position 385 was substituted or deleted. Does not contain an array). In addition, the protein includes an amino acid sequence having 75% or more homology, preferably 90% or more, particularly preferably 95% or more homology with any one of SEQ ID NOs: 16, 26, 30, 32 and 34. And a protein having protocatechuic acid 5-position oxidase activity (SEQ ID NO: 30 does not include a sequence in which phenylalanine at position 385 is substituted or deleted). Furthermore, the protein represented by SEQ ID NO: 16, 26, 30, 32 and 34 having homology of 37.8 to 58.9% with each other is more in the protocatechuic acid 5 position than the PAO mutant enzyme represented by SEQ ID NO: 4. Since it has been found in the present invention that the specific activity of the oxidase is two or more times higher, the homology with any one of the proteins represented by SEQ ID NOs: 16, 26, 30, 32 and 34 is 75.0. Even if it is 3% or less, if it is 37.8% or more, an enzyme having a protocatechuic acid 5-position oxidase activity that is at least twice as good as that of the PAO mutant enzyme can be easily found. Therefore, the protocatechuic acid 5-position oxidase protein related to the present invention has 37.8-75.0% homology with the amino acid sequence represented by SEQ ID NOs: 16, 26, 30, 32 and 34. It may be a protein having an amino acid sequence different from that of No. 4 and having an enzyme activity that oxidizes the 5-position of protocatechuic acid.

The protein having the amino acid sequence in which one or several amino acids are deleted, substituted or added and having protocatechuic acid 5-position oxidase activity is Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press. (1989) (hereinafter abbreviated as Molecular Cloning 2nd edition), 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, 488 (1985) Obtained by introducing a site-specific mutation into DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NOs: 16, 26, 30, 32 and 34, for example, using the site-directed mutagenesis method described in the above can do. The number of one or several amino acids to be deleted, substituted or added is not particularly limited as long as protocatechuic acid 5-position oxidase activity is maintained, but the amino acid sequence of SEQ ID NO: 4 and SEQ ID NOs: 16, 26, 30, 32 It is desirable that it is within the number of differences between any of the amino acid sequences of and 34, preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5.

The microorganism used in the present invention is a microorganism that expresses the above-described protocatechuic acid 5-position oxidase protein. For example, Acinetobacter (Acinetobacter) spp., Bradyrhizobium (Bradyrhizobium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Sinorhizobium (Sinorhizobium) genus Brevibacterium (Brevibacterium) genus, obtainable by isolation from natural those from microbial strains such as Novo sphingolipids bi um (Novosphingobium) genus or Ralstonia (Ralstonia) microorganism belonging to the genus of the above character. Alternatively, microorganisms having such characteristics may be classified into the American Type Culture Collection (hereinafter abbreviated as ATCC as necessary), the National Institute of Technology and Biogenetics Resources (hereinafter referred to as necessary). (Abbreviated as NBRC), or RIKEN Tsukuba Research Laboratories BioResource Center.

Specifically, examples of the microorganism belonging to the genus Acinetobacter include Acinetobacter calcoaceticus ADP1. Examples of the microorganism belonging to the genus Bradyrhizobium include Bradyrhizobium japonicum USDA110. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium glutamicum ATCC13032. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida KT2440. Examples of microorganisms belonging to the genus Rhodopseudomonas include Rhodopseudomonas palustris CGA009. The Sinorhizobium (Sinorhizobium) microorganism of the genus, there may be mentioned, for example, Sinorhizobium meliloti (Sinorhizobium meliloti) 1021 or the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium linens BL2. Examples of the microorganism belonging to the genus Novosphingobium include Novosphingobium aromaticivorans DSM 12444. Examples of microorganisms belonging to the genus Ralstonia include Ralstonia metallidurans CH34. However, the strain that can be used in the present invention is not limited to this strain, 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 protocatechuic acid 5-position oxidase protein. A transformant that produces a protocatechuic acid 5-position oxidase protein that can be used in the present invention is obtained by, for example, cloning the DNA encoding the protein from the above-described microorganism according to the method described in Molecular Cloning Second Edition, A recombinant DNA can be prepared by ligation with a vector DNA, and can be obtained by transforming a host cell using the recombinant DNA. Hereinafter, a method for cloning DNA and a method for producing a transformant will be described in detail.

The above Acinetobacter (Acinetobacter) spp., Bradyrhizobium (Bradyrhizobium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Sinorhizobium (Sinorhizobium) genus Brevibacterium (Brevibacterium) genus, culturing Novo sphingolipids bi um (Novosphingobium) genus or Ralstonia (Ralstonia) microorganism belonging to the genus by known methods. 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. Incidentally, the synthetic DNA is Acinetobacter (Acinetobacter) genus Bradyrhizobium (Bradyrhizobium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus Pseudomonas (Pseudomonas) genus Rhodopseudomonas (Rhodopseudomonas) genus Sinorhizobium (Sinorhizobium) genus Brevibacterium (Brevibacterium) the genus, Novo sphingolipids bi um (Novosphingobium) genus or Ralstonia (Ralstonia) gene encoding protocatechuic acid 5-position oxidase protein derived from bacteria of the genus (hereinafter 5-position protocatechuic acid oxidation enzyme gene called a), preferably Acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Bradyrhizobium japonicum (Bradyrhizobium japonicum), Corynebacterium glutamicum (Corynebacterium glutamicum), Shrewsbury Pseudomonas putida (Pseudomonas putida), Rod Pseudomonas pulse tris (Rhodopseudomonas palustris), Sinorhizobium meliloti (Sinorhizobium meliloti), Novo sphingolipid bi Umm aroma Atlantica borane scan (Novosphingobium aromaticivorans), Ralstonia methallyl radiodurans (Ralstonia metallidurans), or It can be designed based on the base sequence of protocatechuate 5-position oxidase gene derived from Brevibacterium linens . Specifically, Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) ADP1, Bradyrhizobium japonicum (Bradyrhizobium japonicum) USDA110, Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032, Pseudomonas putida (Pseudomonas putida) KT2440, Rod Pseudomonas pulse tris (Rhodopseudomonas palustris) CGA009, Sinorhizobium meliloti (Sinorhizobium meliloti) 1021, Brevibacterium Rinenzu (Brevibacterium linens) BL2, Novo sphingolipid bi Umm aroma Atlantica borane scan (Novosphingobium aromaticivorans) DSM 12444 or Ralstonia methallyl Durance (Ralstonia metallidurans) CH34 nucleotide sequence of protocatechuic acid 5-position oxidase gene, i.e. SEQ ID NO: 5,9,13,15,17,19,21,23,25 and 27 It can be designed based on that nucleotide sequence.

As a vector for ligating the protocatechuic acid 5-position oxidase gene DNA, 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 (Stratagene, Strategies, 5, 58 (1992)), pBluescript II SK (+) [Manufactured by 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 protocatechuic acid 5-position oxidase protein can be obtained by expressing the above DNA in a host cell using the following method.

When using DNA encoding the 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 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), 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, Rhodococcus bacterium RHA1 and Comamonas testosteroni E6 having the ability to metabolize terephthalic acid can be mentioned. By expressing 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 that have the ability to metabolize terephthalic acid, phthalic acid, isophthalic acid, or parahydroxybenzoic acid and that express protocatechuic acid 5-position oxidase protein, are treated with terephthalic acid, phthalic acid, isophthalic acid, or parahydroxyl, respectively. By reacting with these compounds in a medium containing benzoic 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, and protocatechuic acid 5-position oxidase protein A method of obtaining gallic acid by reacting with an expressed 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.

As a vector for introducing and expressing a protocatechuic acid 5-position oxidase protein or a DNA encoding a fusion protein of the protein and another protein into a microorganism such as Escherichia coli, a so-called multicopy type is preferable, Examples include plasmids having a replication origin derived from ColE1, such as pUC series plasmids, pBR322 series plasmids, and derivatives thereof. 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.

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. More preferably, Rhodococcus genus RHA1 expressing protocatechuic acid 5-position oxidase protein can be mentioned.
As shown in the examples described later, since the microorganism expressing the protocatechuic acid 5-position oxidase protein also has the ability to produce protocatechuic acid from parahydroxybenzoic acid, the protocatechuate 5-position oxidase protein is used. By culturing the expressed microorganism in a medium containing parahydroxybenzoic acid, gallic acid can be produced from parahydroxybenzoic acid.

Microorganisms having the ability to metabolize terephthalic acid (the ability to produce protocatechuic acid from terephthalic acid) used in the present invention include terephthalic acid dioxygenase, terephthalic acid dioxygenase reductase, and terephthalic acid involved in the metabolism of terephthalic acid to protocatechuic acid. Mention may be made of microorganisms capable of producing proteins of 1,2-dihydrodiol dihydrogenase and terephthalic acid transporter.
Microorganisms having the ability to metabolize phthalic acid (the ability to produce protocatechuic acid from phthalic acid) used in the present invention are phthalate dioxygenase, phthalate dioxygenase reductase, phthalate 4,5-cis-dihydroxydiol dihydrogenase And microorganisms having the ability to produce 4,5-dihydroxyphthalate decarboxylase and phthalate transporter proteins.
Microorganisms having the ability to metabolize isophthalic acid used in the present invention (capability of producing protocatechuic acid from isophthalic acid) include isophthalic acid dioxygenase, isophthalic acid dioxygenase reductase, isophthalic acid 1,2-dihydrodiol dihydrogenase and Mention may be made of microorganisms having the ability to produce isophthalic acid transporter proteins. Microorganisms having the ability to produce these proteins obtain the transformant expressing the above 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 as

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, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, 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. Wild-type hydroxylase having gallic acid synthesizing activity was identified as follows.
(1) Database search of proteins having homology with the amino acid sequence of Pseudomonas arginosa PAO strain parahydroxybenzoate hydroxylase Pseudomonas arginosa PAO strain parahydroxybenzoate hydroxylase oxidizes parahydroxybenzoic acid. It has been known that it possesses the activity to produce protocatechuic acid but not the activity to produce protocatechuic acid 5-position oxidation activity [Entsch, B, Palfey, BA, Ballou, DP and Masey, VJ Biol. Chem. 266, 17341-17349 (1991)]. Among proteins having homology with the hydroxylase but not having a large homology value, Pseudomonas arginosa PAO strain has an activity different from that of parahydroxybenzoate hydroxylase, that is, protocatechuic acid 5-position oxidation activity. I decided to find out if I had the protein I had. Specifically, the protein having 75% or less homology with the hydroxylase is GenBank (hereinafter abbreviated as GB) of National Center for Biotechnology Information (hereinafter abbreviated as NCBI). The database was searched using the BLAST homology analysis method using the amino acid sequence of parahydroxybenzoic acid hydroxylase of Pseudomonas arginosa PAO strain shown in SEQ ID NO: 2 as a query sequence. As a result, 12 types of proteins shown in Table 1 were obtained. Abbreviations of each protein and Pseudomonas arginosa PAO
Table 1 shows the percent homology of the strains with parahydroxybenzoate hydroxylase.

(2) Isolation and purification of chromosomal DNA In order to clone DNAs encoding the 12 proteins listed in Table 1 using the PCR method, chromosomal DNA of each strain was prepared or obtained as follows.

The strain Acinetobacter calcoaceticus ADP1 (ATCC number: 33305) was obtained from ATCC and the strain Corynebacterium glutamicum ATCC13032 (NBRC number: 12168) was obtained from NBRC. These strains were cultured according to information obtained from each institution. Strain Bradyrizobium japonicam USDA110 and strain Sinorizobium meriroti 1021 were distributed by Dr. Daisuke Shibata of Kazusa DNA Research Institute and cultured according to the method taught by Dr. Kazusa. Chromosomal DNA was isolated and purified from these cultured cells using DNeasy tissue kit (Qiagen). Chromosomal DNA of strain Caulobacter crescents CB15 (ATCC number: 19089D), Chromosomal DNA of the strain Pseudomonas putida KT2440 (ATCC number: 47054D), Chromosomal DNA of the strain Rhodopomonas pultris CGA009 (ATCC number: BAA-98D), Chromosomal DNA of the strain Brevibacterium linens BL2 (ATCC number: 9175D) The chromosomal DNA of the strain Novosphingobium aromaticaborans DSM 12444 (ATCC number: 700278D) and the chromosomal DNA of the strain Ralstonia metalidurance CH34 (ATCC number: 43123D) were obtained from ATCC.

(3) PCR primer design and preparation For 12 types of genes shown in Table 2, nucleotide sequence data (SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, NCBI GenBank database) 25 and 27) were obtained via the Internet. The GB accession number of each base sequence data is shown in Table 2. Based on these base sequences, PCR primers for cloning the DNA of each gene using the PCR method were designed and synthesized. The base sequence of each PCR primer is shown in Table 2.

(4) Amplification of DNA encoding each enzyme by PCR method Expand High Fidelity PCR System purchased from Roche Diagnostics and G. purchased from Roche Diagnostics Using the GC Rich PCR System, the chromosomal DNA obtained in 2 above as a template, and using the DNA primers listed in Table 2 and the DNA of the genes shown in Table 2 according to the attached instructions Was amplified. 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 The DNA of the HFM5 gene, the DNA of the HFM77 gene, the DNA of the HFM86 gene, the DNA of the HFM122 gene, the DNA of the HFM145 gene, the DNA of the HFM305 gene, the DNA of the HFM339 gene, the DNA of the HFM388 gene BD In-Fusion PCR Cloning Kit (BD In-Fusion PCR Cloning Kit) purchased from Clontech, DNA, HFM544 DNA, HFM545 DNA, HFM689 DNA, and HFM737 DNA The restriction enzyme site Nco I and the restriction enzyme site Sma I of plasmid pROX1 (obtained from Roche Applied Science; the base sequence is shown in SEQ ID NO: 59), which is an expression vector for E. coli using the E. coli T7 promoter. Plasmids pROX_HFM5, pROX_HFM77, pRO that express the respective genes X_HFM86, pROX_HFM122, pROX_HFM145, pROX_HFM305, pROX_HFM339, pROX_HFM388, pROX_HFM544, pROX_HFM545, pROX_HFM689 and pROX_HFM737 were obtained. 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: 82) 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 transformants The plasmids pROX_HFM5, pROX_HFM77, pROX_HFM86, pROX_HFM122, pROX_HFM145, pROX_HFM305, pROX_HFM339, pROX_HFM388, pROX_HFM544, pROX_HFM544, pROX_HFM544, By introducing the transformation method using calcium ions into the transformed strains BL21 / pROX_HFM5, BL21 / pROX_HFM77, BL21 / pROX_HFM86, BL21 / pROX_HFM122, BL21 / pROX_HFM145, BL21 / pROX_HFM305, BL21 / pROX_388MBL, BL21 / pROX_HFM544, BL21 / pROX_HFM545, BL21 / pROX_HFM689, and BL21 / pROX_HFM737 were constructed. 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 transformed strain The above 12 types of transformants were 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 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. 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.

10 types of 12 types of HFM enzymes (HFM5, HFM86, HFM145, HFM305, HFM339, HFM388
, HFM544, HFM545, HFM689 and HFM737) synthesized gallic acid when protocatechuic acid was used as a substrate. The gallic acid synthesis per hour per mg of each crude enzyme protein is HFM5 (151 μM / hour / mg crude enzyme protein), HFM86 (30 μM / hour / mg crude enzyme protein), HFM145 (249 μM / hour / mg crude enzyme protein). ), HFM305 (359 μM / hour / mg crude enzyme protein), HFM339 (20 μM / hour / mg crude enzyme protein), HFM388 (11 μM / hour / mg crude enzyme protein), HFM544 (35 μM / hour / mg crude enzyme protein), HFM545 (8 μM / hour / mg crude enzyme protein), HFM689 (350 μM / hour / mg crude enzyme protein) and HFM737 (41 μM / hour / mg crude enzyme protein). HFM 145, HFM 305 and HFM689 showed particularly strong gallic acid synthesis activity.

Example 2 The activity of the isolated hydroxylase was compared with the activity of the PAO mutant enzyme as follows.
1. Construction of polycistron-type expression plasmid The above HFM145 enzyme gene, HFM305 enzyme gene and HFM689 enzyme gene can be efficiently used in E. coli JM109 strain, E. coli JM109 (DE3) strain and E. coli BL21 (DE3) strain independent of the transcription and translation efficiency of each gene. In order to perform expression, transcription of the target gene was dependent 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, four synthetic DNAs represented by SEQ ID NOs: 60 to 63 were synthesized in order to insert a T7 promoter sequence, a pseudo gene, and a target gene into pUC19 plasmid DNA through a HindIII site and a SphI site. . 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 arginosa 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: 64 and a reverse PCR primer represented by SEQ ID NO: 65 for cloning DNA of each gene using the PCR method were designed and synthesized. Pseudomonas arginosa 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, mutant enzyme protein HFM300Y385F (PAO) in which 385th tyrosine of enzyme protein HFM300 is replaced with phenylalanine by PCR using a forward PCR primer represented by SEQ ID NO: 66 and a reverse PCR primer represented by SEQ ID NO: 67. A plasmid pT7Blue_HFM300Y385F carrying DNA encoding the protein HFM300Y385F was constructed by amplifying DNA encoding the mutant 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. Enzyme protein HFM145, enzyme protein HFM305 and. Construction of Plasmid Expressing Enzyme Protein HFM689 From the results of Example 1, when cultivated extracts of enzyme protein HFM145, enzyme protein HFM305, and transformant expressing enzyme protein HFM689 were used, good gallic acid was produced. Observed. Therefore, plasmids were constructed that efficiently expressed the enzyme protein HFM145, the enzyme protein HFM305, and the enzyme protein HFM689. The enzyme protein HFM145, enzyme protein HFM305, and enzyme protein HFM689 produced from the plasmids pROX_HFM145, pROX_HFM305 and pROX_HFM689 are all peptides of 10 amino acids (Gly-Gly-Gly-Gly-Ser-His-His-His-His- His-His (SEQ ID NO: 82)) is added. In order to construct a plasmid that expresses an enzyme protein from which these peptides were removed, PCR primers shown in Table 3 were first designed and synthesized.

Using the plasmid DNA of pROX_HFM145, pROX_HFM305, and pROX_HFM689 as a template, the target DNA was amplified by PCR using the PCR primers in the above table and PrimeSTAR enzyme (manufactured by Takara Bio Inc.), and then Taq polymerase (manufactured by Takara Bio Inc.) A treatment for adding an A residue to the 3 ′ end was performed. After purifying the target DNA after gel electrophoresis, the plasmid pT7Blue_HFM145, plasmid pT7Blue_HFM305, and plasmid pT7Blue_HFM689 were constructed by incorporating the DNA into the pT7Blue-T vector. Plasmids pUTCH_HFM145, pUTCH_HFM305, and pUTCH_HFM689 were 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 plasmids expressing mutant enzyme protein HFM145Y385F, mutant enzyme protein HFM305Y386F and mutant enzyme protein HFM6895Y384 The enzyme that has been proven to have the activity of generating gallic acid from protocatechuic acid is the 385th tyrosine of enzyme protein HFM300. The mutant enzyme protein HFM300Y385F was substituted only with phenylalanine. Therefore, the mutant protein HFM145Y385F in which the 385th tyrosine of the enzyme protein HFM145 is substituted with phenylalanin, the mutant enzyme protein HFM305Y386F in which the 386th tyrosine of the enzyme protein HFM305 is replaced with phenylalanthin, and the 384th tyrosine of the enzyme protein HFM689 A plasmid expressing the mutant enzyme protein HFM689Y384F in which is substituted with phenylalanin was constructed by the same method as described in 3 above.

First, PCR primers shown in Table 4 were designed and synthesized. Using the plasmid DNA of pROX_HFM145, pROX_HFM305, and pROX_HFM689 as a template, the target DNA was amplified by PCR using the PCR primers in Table 4 and PrimeSTAR enzyme (manufactured by Takara Bio Inc.), and then Taq polymerase (manufactured by Takara Bio Inc.). The A residue was added to the 3 ′ end by. After purifying the target DNA after gel electrophoresis, the plasmid pT7Blue_HFM145Y385F, the plasmid pT7Blue_HFM305Y386F, and the plasmid pT7Blue_HFM689Y384F were constructed by incorporating into the pT7Blue-T vector. Plasmids pUTCH_HFM145Y385F, pUTCH_HFM305Y386F, and pUTCH_HFM689Y384F were constructed by excising 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, HFM300Y385F, HFM145, HFM145Y385F, HFM305, HFM305Y386F, HFM689, and HFM689Y384F in Escherichia coli E. coli JM109 / pUTCH_HFM300, JM109 / pUTCH_HFM300Y385F, JM109 / pUTCH_HFM145, JM109 / pUTCH_HFM145Y385F, JM109 / pUTCH_HFM305, JM109 / pUTCH_HFM305Y386F, JM109 / pUTCH_HFM6F 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
The reaction was performed at 30 ° C. for 1 hour in a 100 μl reaction solution containing 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, JM109 / pUTCH_HFM300Y385F, JM109 / pUTCH_HFM145, JM109 / pUTCH_HFM145Y385F, JM109 / pUTCH_HFM305, JM109 / pUTCH_HFM305Y386F, JM109 / pUTCH_HFM3 865 μM, 1466 μM, 408 μM, 500 μM, and 428 μM gallic acid were detected. From the activity measurement results, the activity ratio of each gallic acid synthase is 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 The production of gallic acid by Escherichia coli using parahydroxybenzoic acid as a raw material was performed as follows.
Recombinant Escherichia coli JM109 / pUTCH_HFM300, JM109 / pUTCH_HFM300Y385F and JM109 / pUTCH_HFM305 were cultured overnight at 25 ° C. using OEI-TB medium, and then collected and the medium was discarded. 3 ml of a fresh LB medium (pH 5.95) containing 0.5 mM parahydroxybenzoic acid and 1 mM IPTG was added and cultured at 25 ° C. for 20 hours. 0.1 ml of the culture solution was taken and 1 ml of ethyl acetate was added, followed by stirring for 5 minutes. After adding 0.15 ml of HEPES-glycine buffer and 5 μl of 2N HCl, centrifugation was performed for 5 minutes. 800 μl of the upper layer was collected and dried. The dried product was dissolved in 10 μl of acetonitrile. After adding 190 μl of water, the amount of gallic acid produced was measured using an LC-TOF mass spectrometer (Waters, LCT Premier XE). As a result, the amounts of gallic acid produced by recombinant Escherichia coli JM109 / pUTCH_HFM300, JM109 / pUTCH_HFM300Y385F and JM109 / pUTCH_HFM305 were 0.4 μM, 2.5 μM and 42 μM, respectively. Therefore, the gallic acid productivity of JM109 / pUTCH_HFM305 was 9.5 times that of JM109 / pUTCH_HFM300 and 17.1 times that of JM109 / pUTCH_HFM300Y385F.

Example 4 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: 80 and a reverse PCR primer represented by SEQ ID NO: 81 were designed and synthesized for cloning 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
E. coli JM109 (DE3) was transformed with pUTCH_TPACB1 and pRTCH_HFM145_Y385F or pUTCH_TPACB1 and pRTCH_HFM305 to obtain recombinant E. coli JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_Y385F), and JM109 (DE3TCH_TP3TP_BCH).
The obtained recombinant Escherichia coli JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_Y385F) and JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM305) were cultured at 37 ° C. for 14 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 0.5 mM. At 0.5 and 24 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, when the culture time in the OEI-TB medium was 4 hours, recombinant E. coli JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_Y385F) and JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM305) were 2 μM and 39 μM gallic acid, respectively. Production was observed. When the incubation time in OEI-TB medium is 20 hours, recombinant E. coli JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM145_Y385F) and JM109 (DE3) / (pUTCH_TPACB1, pRTCH_HFM305) are 66 μM and 9 μM gallic acid, respectively. Production was observed.

Claims (15)

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) Protein having the amino acid sequence represented by SEQ ID NO: 16, 26, 30, 32 or 34 (B) In the amino acid sequence represented by SEQ ID NO: 16, 26, 30, 32 or 34, one or several A protein having an amino acid sequence including amino acid substitution, deletion, insertion or addition and having an enzyme activity that oxidizes the 5-position of protocatechuic acid (for SEQ ID NO: 30, includes a sequence in which phenylalanine at position 385 is substituted or deleted) Absent). 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 1. 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 claim 1, 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. 4. The method for producing gallic acid according to 3. 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 claim 1, 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. 5. The method for producing gallic acid according to 5. 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 claim 1, 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. 8. The method for producing gallic acid according to 7. 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, It accumulates and this is extract | collected, The manufacturing method of the gallic acid of Claim 1 characterized by the above-mentioned. 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 manufacturing method of the gallic acid of Claim 9. 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 3 or 4, 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 5, 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 7 or 8, wherein the gallic acid is obtained. The method for producing gallic acid according to any one of claims 1 to 13, wherein the microorganism is a microorganism into which 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, the first term any of claims 1 to 14 The manufacturing method described.