JP7588969B2 - Transformants capable of producing gallic acid - Google Patents

Description

The present invention relates to a transformant capable of producing gallic acid, and a method for producing gallic acid or a salt thereof using the transformant.

Gallic acid has a strong reducing effect, and is therefore used as a raw material for manufacturing photographic developers and blue inks. In addition, esters such as propyl gallate are used as antioxidants for oils and butter. Furthermore, pyrogallol, which is synthesized by decarboxylating gallic acid, is used as an electronic material, an organic synthesis reagent, a photographic developer, and a mordant for woolen fabrics.

Conventional methods for producing gallic acid include extraction from tea, gall nuts, and Chinese gall nut. However, these methods are expensive, and there has been a demand for an industrially inexpensive method for producing gallic acid.
Under these circumstances, a method for producing gallic acid from raw materials such as terephthalic acid, phthalic acid, isophthalic acid, or parahydroxybenzoic acid by fermentation using a microorganism has been reported (Patent Document 1).

Recently, there has been a demand for a method for producing gallic acid using glucose, an inexpensive raw material. For example, a method for producing gallic acid from sugars via protocatechuic acid has been reported (Patent Document 2), in which a transformant containing a gene encoding an enzyme that converts 3-dehydroshikimic acid to protocatechuic acid (3-dehydroshikimate dehydratase) and a gene encoding an enzyme that converts protocatechuic acid to gallic acid (p-hydroxybenzoic acid hydroxylase) has been introduced into a bacterium belonging to the genus Escherichia or Klebsiella.

JP 2009-213392 A U.S. Pat. No. 6,472,190

The present invention relates to a transformant capable of producing gallic acid or a salt thereof using sugars as a raw material, and a method for producing gallic acid or a salt thereof using the transformant.

As a result of extensive research aimed at obtaining a transformant capable of efficiently producing gallic acid or a salt thereof, the present inventors discovered that by introducing a pathway that converts chorismate, the final metabolic product of the shikimate pathway, back to protocatechuate, in addition to the conventional reaction of converting 3-dehydroshikimic acid to protocatechuic acid, a transformant capable of efficiently producing gallic acid or a salt thereof can be obtained.

That is, the present invention relates to the following 1) and 2).
1) A transformant capable of producing gallic acid or a salt thereof, in which expression of (A) a polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity, (B) a polypeptide having 3-hydroxybenzoic acid synthase activity, and (C) a polypeptide having 3-hydroxybenzoic acid hydroxylase activity is enhanced.
2) A method for producing gallic acid or a salt thereof, comprising a step of culturing the transformant of 1) in the presence of a saccharide.

According to the present invention, it is possible to mass-produce gallic acid or its salts at low cost by a fermentation method that places little strain on the environment. In addition, in the transformant of the present invention, the metabolic reactions from chorismic acid are not inhibited and the transformant is non-auxotrophic, so there is no need to add aromatic amino acids or aromatic vitamins when culturing the transformant.

1 is a diagram showing the gallic acid production pathway in the transformant of the present invention when a coryneform bacterium is used as a host, in which the dotted line indicates the metabolic reaction mediated by the foreign gene product.

In the present invention, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the Search homology program of the genetic information processing software GENETYX Ver. 12 with the Unit size to compare (ktup) set to 2.

In the present invention, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

In the present invention, "an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted" refers to an amino acid sequence in which 1 to 10, preferably 1 to 8, more preferably 1 to 5, and even more preferably 1 to 3 amino acids have been deleted, substituted, added, or inserted. In addition, in the present specification, "a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted" refers to a nucleotide sequence in which 1 to 30, preferably 1 to 24, more preferably 1 to 15, and even more preferably 1 to 9 nucleotides have been deleted, substituted, added, or inserted. In the present specification, "addition" of an amino acid or nucleotide includes addition of an amino acid or nucleotide to one end and both ends of a sequence.

In the present invention, "operably linked" between a regulatory region and a gene means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. The procedure for "operably linked" between a gene and a regulatory region is well known to those skilled in the art.

In the present invention, the term "native" used in reference to a function, property, or trait of a cell is used to indicate that the function, property, or trait is present in the wild-type form of the cell. In contrast, the term "exogenous" is used to indicate a function, property, or trait that is not inherently present in the cell, but is introduced from outside. For example, a "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from outside. The exogenous gene or polynucleotide may be from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).

In the present invention, "gallic acid" means 3,4,5-trihydroxybenzoic acid, and examples of salts of gallic acid include salts with alkali metals such as sodium and potassium, and salts with alkaline earth metals such as calcium and magnesium.

In the present invention, the transformant capable of producing gallic acid or a salt thereof is a microbial cell in which expression of (A) a polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity, (B) a polypeptide having 3-hydroxybenzoic acid synthase activity, and (C) a polypeptide having 3-hydroxybenzoic acid hydroxylase activity is enhanced.
That is, in the transformant of the present invention, a pathway is introduced that converts chorismate, the final metabolic product of the shikimate pathway, back to protocatechuate, and gallic acid is produced from the protocatechuate (FIG. 1).
Here, the polypeptide (A) having 3,4-dihydroxybenzoate hydroxylase activity is a polypeptide that catalyzes the reaction of producing gallic acid from protocatechuic acid, and examples thereof include (A1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or (A2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2 and having 3,4-dihydroxybenzoate hydroxylase activity.
Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity, which is a mutant of p-hydroxybenzoate hydroxylase (pobA) derived from Corynebacterium glutamicum (represented as "pobA*" in Figure 1).

The polypeptide (B) having 3-hydroxybenzoate synthase activity is a polypeptide that catalyzes the reaction of producing 3-hydroxybenzoic acid from chorismate, and examples thereof include (B1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or (B2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 6 and having 3-hydroxybenzoate synthase activity.
Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:6 is 3-hydroxybenzoate synthase derived from Streptomyces hygroscopicus and is known as "hyg5."

The polypeptide (C) having 3-hydroxybenzoate hydroxylase activity is a polypeptide that catalyzes the reaction of producing protocatechuic acid from 3-hydroxybenzoic acid, and examples thereof include (C1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:8, or (C2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:8 and having 3-hydroxybenzoate hydroxylase activity.
Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:8 is 3-hydroxybenzoate hydroxylase derived from Comamonas testosteroni and is known as "mobA."

Furthermore, in order to increase the production amount of gallic acid or a salt thereof, the transformant of the present invention can further enhance the expression of (D) a polypeptide having 3-dehydroshikimate dehydratase activity.
The polypeptide (D) having 3-dehydroshikimic acid dehydratase activity is a polypeptide that catalyzes a reaction to produce protocatechuic acid from 3-dehydroshikimic acid, and examples thereof include (D1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10, or (D2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 10 and having 3-dehydroshikimic acid dehydratase activity.
Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:10 is 3-dehydroshikimate dehydratase derived from Corynebacterium glutamicum and is known as "qsuB."

The transformant of the present invention can further enhance the expression of at least one polypeptide selected from the group consisting of (E) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase activity, (F) a polypeptide having 3-dehydroquinate dehydratase activity, and (G) a polypeptide having transketolase activity.
The polypeptide (E) having DAHP synthase activity is a polypeptide that produces DAHP, the initial metabolic product in the aromatic compound biosynthetic pathway, from erythrose-4-phosphate and phosphoenolpyruvate. Examples of the polypeptide include (E1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14, and (E2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14 and having DAHP synthase activity.
The polypeptide consisting of the amino acid sequence shown in SEQ ID NO:12 is a DAHP synthase derived from Corynebacterium glutamicum and is known as "aroF."
The polypeptide consisting of the amino acid sequence shown in SEQ ID NO:14 is a DAHP synthase derived from Corynebacterium glutamicum and is known as "aroG."

The polypeptide (F) having 3-dehydroquinate dehydratase activity is a polypeptide that catalyzes the conversion of 3-dehydroquinic acid to 3-dehydroshikimic acid, and examples thereof include (F1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16, or (F2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 16 and having 3-dehydroquinate dehydratase activity.
The polypeptide consisting of the amino acid sequence shown in SEQ ID NO:16 is 3-dehydroquinate dehydratase derived from Corynebacterium glutamicum and is known as "aroD."

The polypeptide having transketolase activity (G) has activity to catalyze two types of reactions. The first reaction is a reaction that catalyzes the conversion of D-xylulose-5-phosphate to glyceraldehyde-3-phosphate and the conversion of D-ribose-5-phosphate (R5P) to sedoheptulose-7-phosphate (S7P) in the nonoxidative pentose phosphate pathway. These reactions are reversible and coupled. The second reaction is a reaction that catalyzes the conversion of D-fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P) and the conversion of glyceraldehyde-3-phosphate to D-xylulose-5-phosphate. These reactions are reversible and coupled. Examples of polypeptides having transketolase activity include (G1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 18, or (G2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 18 and having transketolase activity.
The polypeptide consisting of the amino acid sequence shown in SEQ ID NO:18 is a transketolase derived from Corynebacterium glutamicum and is known as "tkt."

Examples of amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, and 18 include amino acid sequences in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequences shown in SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, and 18.

(A2) Examples of polypeptides having 3,4-dihydroxybenzoate hydroxylase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:2 include polypeptides having 3,4-dihydroxybenzoate hydroxylase activity derived from bacteria of the genus Corynebacterium, specifically 4-hydroxybenzoate-3-monooxygenase derived from Corynebacterium glutamicum, 4-hydroxybenzoate-3-monooxygenase derived from Corynebacterium suranareeae, and 4-hydroxybenzoate-3-monooxygenase derived from Corynebacterium crudilactis.

(B2) Examples of polypeptides having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO:6 and having 3-hydroxybenzoate synthase activity include 3-hydroxybenzoate synthase derived from bacteria of the genus Streptomyces, specifically 3-hydroxybenzoate synthase derived from Streptomyces hygroscoipicus, imine deaminase derived from Streptomyces malaysiensis, etc.

(C2) Examples of polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:8 and that have 3-hydroxybenzoate hydroxylase activity include 3-hydroxybenzoate hydroxylases derived from bacteria of the genus Comamonas, specifically 3-hydroxybenzoate hydroxylase derived from Comamonas testosteroni and phenol-2-monooxygenase derived from Comamonas thiooxydans.

(D2) Examples of polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:10 and that have 3-dehydroshikimate dehydratase activity include 3-dehydroshikimate dehydratase derived from bacteria of the genus Corynebacterium, specifically 3-dehydroshikimate dehydratase derived from Corynebacterium glutamicum and 3-dehydroshikimate dehydratase derived from Corynebacterium sulanareae.

(E2) Examples of polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14 and that have DAHP synthase activity include DAHP synthases derived from bacteria of the genus Corynebacterium, specifically DAHP synthases derived from Corynebacterium glutamicum, Corynebacterium thalanaeae, and Corynebacterium efficiens.

(F2) Examples of polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:16 and that have 3-dehydroquinate dehydratase activity include 3-dehydroquinate dehydratases derived from bacteria of the genus Corynebacterium, specifically 3-dehydroquinate dehydratases derived from Corynebacterium glutamicum, Corynebacterium curdilactis, and Corynebacterium desertii.

Examples of polypeptides having transketolase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:18 of (G2) include transketolase derived from bacteria of the genus Corynebacterium, specifically transketolase derived from Corynebacterium glutamicum, transketolase derived from Corynebacterium curdilactis, transketolase derived from Corynebacterium thalanareae, etc.

Examples of methods for introducing mutations such as deletion, substitution, addition, or insertion of amino acids into the amino acid sequence of the above-mentioned polypeptide include methods for introducing mutations such as deletion, substitution, addition, or insertion of nucleotides into the nucleotide sequence encoding the amino acid sequence. Examples of methods for introducing mutations into nucleotide sequences include mutagenesis using chemical mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, and nitrous acid, or physical mutagens such as ultraviolet light, X-rays, gamma rays, and ion beams, site-directed mutagenesis, and the method described in Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995), and the like. Examples of the site-specific mutagenesis technique include a method using splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), the ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and the Kunkel method (Kunkel, T. A., Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, commercially available site-directed mutagenesis kits such as Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), Transformer ^™ Site-Directed Mutagenesis Kit (Clonetech), and KOD-Plus-Mutagenesis Kit (Toyobo) can also be used.

In the present invention, the transformed cell in which the expression of at least one polypeptide selected from the group consisting of (A) a polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity, (B) a polypeptide having 3-hydroxybenzoic acid synthase activity, and (C) a polypeptide having 3-hydroxybenzoic acid hydroxylase activity, optionally (D) a polypeptide having 3-dehydroshikimate dehydratase activity, optionally (E) a polypeptide having DAHP synthase activity, (F) a polypeptide having 3-dehydroquinate dehydratase activity, and (G) a polypeptide having transketolase activity is enhanced is specifically a cell into which a polynucleotide necessary for the expression of the polypeptide has been introduced so as to be expressible, and the polypeptide may be foreign or may be inherent to the cell. For example, a cell into which the polynucleotide has been introduced so as to be expressible, and a cell in which the degree of expression of the polynucleotide has been enhanced are included. Specifically, examples of such cells include cells into which a vector or DNA fragment containing the polynucleotide and a control region operably linked thereto has been introduced, and cells in which the control region of the polynucleotide has been replaced with a strong control region.

Here, the polynucleotides include (a) the polynucleotide (a1) or (a2) below as a polynucleotide encoding a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity, (b) the polynucleotide (b1) or (b2) below as a polynucleotide encoding a polypeptide having 3-hydroxybenzoate synthase activity, (c) the polynucleotide (c1) or (c2) below as a polynucleotide encoding a polypeptide having 3-hydroxybenzoate hydroxylase activity, and (d) the polynucleotide encoding a polypeptide having 3-dehydroshikimate dehydratase activity. Examples of the polynucleotide that encodes a polypeptide having DAHP synthase activity include the polynucleotide (d1) or (d2) below; (e) a polynucleotide that encodes a polypeptide having DAHP synthase activity includes the polynucleotide (e1) or (e2) below; (f) a polynucleotide that encodes a polypeptide having 3-dehydroquinate dehydratase activity includes the polynucleotide (f1) or (f2) below; and (g) a polynucleotide that encodes a polypeptide having transketolase activity includes the polynucleotide (g1) or (g2) below (these polynucleotides are also referred to as "polynucleotides of the present invention").

(a1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or (a2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 and encoding a polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity. Note that, here, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:3 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:4, which is identical to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:2.
(b1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:5, or (b2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:5, and encoding a polypeptide having 3-hydroxybenzoate synthase activity.
(c1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7, or (c2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 7, and encoding a polypeptide having 3-hydroxybenzoate hydroxylase activity.
(d1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:9, or (d2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:9 and encoding a polypeptide having 3-dehydroshikimate dehydratase activity.
(e1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:11 or SEQ ID NO:13, or (e2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:11 or SEQ ID NO:13, and encoding a polypeptide having DAHP synthase activity.
(f1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15, or (f2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 15, and encoding a polypeptide having 3-DEHYDROQUINATE DEHYDRATASE activity.
(g1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17, or (g2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 17, and encoding a polypeptide having transketolase activity.

Examples of nucleotide sequences having at least 90% identity to the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 include nucleotide sequences in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17. Methods for introducing mutations such as deletion, substitution, addition, or insertion of nucleotides into a nucleotide sequence are as described above. The polynucleotide may be in the form of a single strand or a double strand, or may be DNA or RNA. The DNA may be artificial DNA such as cDNA or chemically synthesized DNA.

The polynucleotide may be incorporated into a vector. Preferably, the vector containing the polynucleotide of the present invention is an expression vector. Also preferably, the vector is an expression vector capable of introducing the polynucleotide of the present invention into a host microorganism and expressing the polynucleotide in the host microorganism. Preferably, the vector contains the polynucleotide of the present invention and a control region operably linked thereto. The vector may be a vector capable of autonomously replicating and replicating outside a chromosome, such as a plasmid, or may be a vector that is incorporated into a chromosome.

Specific examples of vectors include pBluescript II SK(-) (Stratagene), pUC18/19, pUC118/119 and other pUC vectors (Takara Bio), pET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), and pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2):93-103), pBR322 (Takara Bio), pRS403 (Stratagene), pMW218/219 (Nippon Gene), pRI-based vectors such as pRI909/910 (Takara Bio), pBI-based vectors (Clontech), IN3-based vectors (Implanta Innovations), pPTR1/2 (Takara Bio), pDJB2 (D.J.Ballance et al., Gene,36,321-331,1985), pAB4-1 (van Hartingsveldt W et al., Mol Gen Genet,206,71-75,1987), pLeu4 (M.I.G.Roncero et al., al., Gene, 84, 335-343, 1989), pPyr225 (C.D.Skory et al., Mol Genet Genomics, 268, 397-406, 2002), pFG1 (Gruber, F. et al., Curr Genet, 18, 447-451, 1990), etc.

The polynucleotide may be constructed as a DNA fragment containing the polynucleotide. Examples of the DNA fragment include a PCR-amplified DNA fragment and a restriction enzyme-cleaved DNA fragment. Preferably, the DNA fragment may be an expression cassette containing the polynucleotide of the present invention and a control region operably linked thereto.

The control region contained in the vector or DNA fragment is a sequence for expressing the polynucleotide of the present invention in a host cell into which the vector or DNA fragment has been introduced, and examples of such control regions include expression control regions such as promoters and terminators, and replication origins. The type of control region can be appropriately selected depending on the type of host microorganism into which the vector or DNA fragment is introduced. If necessary, the vector or DNA fragment may further have a selection marker such as an antibiotic resistance gene or an amino acid synthesis-related gene.

To introduce a vector or DNA fragment into a host cell, a general transformation method such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, or Agrobacterium can be used.

Transformed cells into which a vector or DNA fragment of interest has been introduced can be selected using a selection marker. For example, if the selection marker is an antibiotic resistance gene, cells into which a vector or DNA fragment of interest has been introduced can be selected by culturing the cells in a medium containing the antibiotic. For example, if the selection marker is an amino acid synthesis-related gene, the gene can be introduced into a host cell that requires the amino acid, and then the presence or absence of the amino acid requirement can be used as an indicator to select cells into which a vector or DNA fragment of interest has been introduced. Alternatively, the introduction of a vector or DNA fragment of interest can be confirmed by examining the DNA sequence of transformed cells using PCR or the like.

Furthermore, examples of strong regulatory regions include known high expression promoters such as T7 promoter, lac promoter, tac promoter, trp promoter, tu promoter, and gap promoter, but are not particularly limited to these.
Furthermore, as the strong regulatory region, an inducible promoter derived from a prokaryote can be used, and examples thereof include, but are not limited to, the vanA promoter which is induced by the addition of ferulic acid, vanillic acid, or vanillin, the rhcH promoter which is induced by the addition of resorcinol or 2,4-dihydroxybenzoic acid, the pcaI promoter which is induced by the addition of 4-hydroxybenzoic acid, the promoter of the nagI (cg3351) gene which is induced by the addition of 3-hydroxybenzoic acid, the promoter of the benA (cg2637) gene which is induced by the addition of benzoic acid (hereinafter, abbreviated as Pben), or the promoter of the cg2118 gene or the promoter of the ptsS (cg2925) gene which are induced by the addition of either fructose or sucrose.
Methods for replacing the regulatory region of the polynucleotide present on the genome of a host cell with a strong regulatory region include a method in which a DNA fragment containing the strong regulatory region and a polynucleotide sequence of a selection marker is introduced into a host cell, and cells transformed by homologous recombination or non-homologous recombination are selected.

In the present invention, the host cell may be any suitable cell for producing gallic acid or a salt thereof, such as Escherichia coli, Bacillus subtilis, actinomycetes, bacteria of the genus Pseudomonas, bacteria of the genus Streptococcus, bacteria of the genus Lactobacillus, fungi (such as those of the genus Neurospora, Aspergillus, and Trichoderma), yeasts (such as those of the genus Saccharomyces, Kluyveromyces, Schizosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Pichia, and Candida), but actinomycetes are preferred.

As actinomycetes, a group of microorganisms defined as coryneform bacteria (Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974)) is preferred, and specific examples thereof include bacteria of the genus Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, Micrococcus, etc.
Examples of bacteria of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum.
Examples of the Brevibacterium genus include Brevibacterium ammoniagenes.
Examples of the Arthrobacter genus include Arthrobacter globiformis.
Examples of bacteria belonging to the genus Mycobacterium include Mycobacterium bovis, and examples of bacteria belonging to the genus Micrococcus include Micrococcus freudenreichii, Micrococcus leuteus, Micrococcus ureae, Micrococcus roseus, and the like.
Among the coryneform bacteria, the genus Corynebacterium is preferred, and Corynebacterium glutamicum is more preferred.

In addition to wild-type strains, coryneform bacteria may be mutants or artificially genetically modified strains. For example, they may be strains in which genes such as lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, and malate dehydrogenase are disrupted.

The enhanced activity of each of the enzymes (A) to (G) in the prepared transformant can be confirmed by measuring the activity of each enzyme in a cell extract of the transformant. The prepared transformed cells are then cultured, the productivity of gallic acid or a salt thereof is evaluated, and an appropriate transformed cell is selected to obtain a useful strain producing gallic acid or a salt thereof. The product can be measured according to the method described in the Reference Example below.

The method for producing gallic acid or a salt thereof of the present invention is carried out by culturing the above-mentioned transformed cells in the presence of sugars.

As the saccharide, glucose is preferred, but in addition to monosaccharides such as fructose, mannose, arabinose, xylose, galactose, etc., saccharides that can generate glucose through metabolism can also be used. Such saccharides include oligosaccharides or polysaccharides having glucose units, such as disaccharides such as cellobiose, sucrose (cane sugar), lactose, maltose, trehalose, cellobiose, xylobiose, etc.; polysaccharides such as dextrin or soluble starch, etc.
Molasses can also be used as a raw material containing these raw material compounds. In addition, inedible agricultural waste such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw, etc.), bagasse, corn stover, etc., energy crops such as switchgrass, napier grass, and miscanthus, wood chips, waste paper, etc. can be saccharified with a saccharification enzyme or the like to produce a saccharified liquid containing multiple sugars such as glucose.

The medium for culturing the transformed cells may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. and is a medium in which the transformed cells of the present invention can be efficiently cultured.
As the carbon source, the above-mentioned sugars or molasses or saccharified solution containing them can be used, but in addition to the above-mentioned sugars, sugar alcohols such as mannitol, sorbitol, xylitol, and glycerin, organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid, alcohols such as ethanol and propanol, hydrocarbons such as normal paraffin, etc. The carbon source can be used alone or in combination of two or more kinds.
The concentration of the saccharide as the raw material compound in the culture medium is preferably 1 to 20 w/v %, more preferably 2 to 10 w/v %, and even more preferably 2 to 5 w/v %.

Examples of nitrogen sources that can be used include peptone, meat extract, yeast extract, casein hydrolysate, alkaline extract of soybean meal, alkylamines such as methylamine, nitrogen-containing organic compounds such as amino acids, ammonia or its salts (inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate), urea, aqueous ammonia, sodium nitrate, and potassium nitrate.

Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate.
Furthermore, if necessary, vitamins, antifoaming agents, etc. can be added. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.

Examples of media for coryneform bacteria include A medium [J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)], BT medium [J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)], and CGXII medium [Patent No. 6322576]. These media can be used with the sugar concentration in the above range.

Prior to the reaction or culture containing sugars, it is preferable to grow the transformant in a similar medium under aerobic conditions at a temperature of about 25 to 38°C for about 12 to 48 hours.

The culture temperature or reaction temperature is preferably 15 to 45°C, more preferably 25 to 37°C.
The culture or reaction time is 24 to 168 hours, preferably 24 to 96 hours, more preferably 24 to 72 hours, and can be performed with stirring or shaking as necessary. During culture, antibiotics such as ampicillin and kanamycin may be added to the medium as necessary.
The culture may be any of a batch system, a fed-batch system, and a continuous system, of which the batch system is preferred.
The culture or reaction may be carried out under aerobic conditions or reducing conditions, but is preferably carried out under aerobic conditions.
When the reaction or culture is carried out under aerobic conditions, it is preferable to carry out the reaction or culture under conditions that suppress excessive growth of the transformant, in terms of the efficiency of production of gallic acid or a salt thereof.
In order to prevent oxidation of gallic acid, it is preferable to carry out the culture under conditions of low dissolved oxygen concentration, specifically, a dissolved oxygen concentration of 0.1 to 3 ppm is preferable, and 0.1 to 1 ppm is more preferable.

There are no particular limitations on the method for recovering and purifying gallic acid or its salts from the culture. In other words, it can be carried out by combining well-known ion exchange resin methods, precipitation methods, crystallization methods, recrystallization methods, concentration methods, and other methods. For example, gallic acid or its salts can be obtained by removing the bacterial cells by centrifugation or the like, removing ionic substances with cation and anion exchange resins, and concentrating the mixture. Gallic acid or its salts accumulated in the culture may be used as is without isolation.

In relation to the above-described embodiment, the present invention further discloses the following aspects.
<1> A transformant capable of producing gallic acid or a salt thereof, in which expression of (A) a polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity, (B) a polypeptide having 3-hydroxybenzoic acid synthase activity, and (C) a polypeptide having 3-hydroxybenzoic acid hydroxylase activity is enhanced.
<2> The transformant according to <1>, comprising, in an expressible state, a polynucleotide encoding (A) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity, (B) a polypeptide having 3-hydroxybenzoate synthase activity, and (C) a polypeptide having 3-hydroxybenzoate hydroxylase activity.
<3> The transformant according to <2>, wherein the polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is a polypeptide represented by the following (A1) or (A2), the polypeptide having 3-hydroxybenzoate synthase activity is a polypeptide represented by the following (B1) or (B2), and the polypeptide having 3-hydroxybenzoate hydroxylase activity is a polypeptide represented by the following (C1) or (C2).
(A1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:2. (A2) A polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:2, and having 3,4-dihydroxybenzoic acid hydroxylase activity. (B1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:6. (B2) A polypeptide consisting of a peptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:6, and having 3-hydroxybenzoic acid synthase activity. (C1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:8. (C2) A polypeptide consisting of a peptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8 , and having 3-hydroxybenzoic acid hydroxylase activity. <4> The transformant according to <2>, wherein the polynucleotide encoding the polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is a polynucleotide represented by the following (a1) or (a2), the polynucleotide encoding the polypeptide having 3-hydroxybenzoate synthase activity is a polynucleotide represented by the following (b1) or (b2), and the polynucleotide encoding the polypeptide having 3-hydroxybenzoate hydroxylase activity is a polynucleotide represented by the following (c1) or (c2).
(a1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3; (a2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and encoding a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity; (b1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:5; (b2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:5, and encoding a polypeptide having 3-hydroxybenzoate synthase activity; (c1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:7; (c2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:7, and encoding a polypeptide having 3-hydroxybenzoate hydroxylase activity. <5> The transformant according to any one of <1> to <4>, further comprising (D) enhanced expression of a polypeptide having 3-dehydroshikimate dehydratase activity.
<6> (D) The transformant according to <5>, which contains, in an expressible state, a polynucleotide encoding a polypeptide having 3-dehydroshikimate dehydratase activity.
<7> The transformant according to <6>, wherein the polypeptide having 3-dehydroshikimate dehydratase activity (D) is a polypeptide represented by the following (D1) or (D2):
(D1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10. (D2) A polypeptide consisting of a peptide sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 10 and having 3-dehydroshikimate dehydratase activity. <8> (d) The transformant according to <6>, wherein the polynucleotide encoding the polypeptide having 3-dehydroshikimate dehydratase activity is a polynucleotide shown in the following (d1) or (d2).
(d1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:9; (d2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:9, and encoding a polypeptide having 3-dehydroshikimate dehydratase activity. <9> The transformant according to any of <1> to <8>, further comprising enhanced expression of at least one polypeptide selected from the group consisting of (E) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity, (F) a polypeptide having 3-dehydroquinate dehydratase activity, and (G) a polypeptide having transketolase activity.
<10> The transformant according to <9>, comprising, in an expressible state, a polynucleotide encoding at least one polypeptide selected from the group consisting of (E) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity, (F) a polypeptide having 3-dehydroquinate dehydratase activity, and (G) a polypeptide having transketolase activity.
<11> The transformant according to <10>, wherein the polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity is a polypeptide represented by the following (E1) or (E2), the polypeptide having 3-dehydroquinate dehydratase activity is a polypeptide represented by the following (F1) or (F2), and the polypeptide having transketolase activity is a polypeptide represented by the following (G1) or (G2).
(E1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14. (E2) A polypeptide consisting of a peptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14, and having DAHP synthase activity. (F1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:16. (F2) A polypeptide consisting of a peptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:16, and having 3-dehydroquinate dehydratase activity. (G1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:18. (G2) A polypeptide consisting of a polypeptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:18, and having transketolase activity. <12> The transformant according to <10>, wherein the polynucleotide encoding the polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity is a polynucleotide represented by the following (e1) or (e2), the polynucleotide encoding the polypeptide having 3-dehydroquinate dehydratase activity is a polynucleotide represented by the following (f1) or (f2), and the polynucleotide encoding the polypeptide having transketolase activity is a polynucleotide represented by the following (g1) or (g2).
(e1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:11 or SEQ ID NO:13. (e2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:11 or SEQ ID NO:13, and encoding a polypeptide having DAHP synthase activity. (f1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:15. (f2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:15, and encoding a polypeptide having 3-DEHYDROQUINATE DEHYDRATASE activity. (g1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:17. (g2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:17, and encoding a polypeptide having transketolase activity. <13> The transformant according to any one of <1> to <12>, which is a coryneform bacterium.
<14> The transformant according to <13>, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.
<15> The transformant according to <14>, wherein the bacterium of the genus Corynebacterium is Corynebacterium glutamicum.
<16> A method for producing gallic acid or a salt thereof, comprising a step of culturing the transformant according to any one of <1> to <15> in the presence of a saccharide.

Example 1 Production of gallic acid (1) Preparation of transformant into which a polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity has been introduced 1) Construction of a plasmid for replacing the cg0620 gene region with a polypeptide gene having 3,4-dihydroxybenzoic acid hydroxylase activity The 5' upstream region of the cg0620 gene region (SEQ ID NO: 19) was amplified with two types of DNA primers (SEQ ID NO: 20 and 21), and the 3' downstream region of the cg0620 gene region (base number 22) was amplified with two types of DNA primers (SEQ ID NO: 23 and 24) to obtain a DNA fragment. In addition, a DNA fragment (SEQ ID NO: 25) containing the promoter of the tuf gene (cg0587) of Corynebacterium glutamicum ATCC13032 strain (hereinafter referred to as tu promoter) was prepared by artificial gene synthesis, and amplified with two types of DNA primers (SEQ ID NO: 26 and 27) to obtain a DNA fragment. In addition, two types of DNA fragments (SEQ ID NOs: 28 and 29) containing a polypeptide gene having 3,4-dihydroxybenzoic acid hydroxylase activity (hereinafter abbreviated as hfm145VF) were prepared by artificial gene synthesis. Each DNA fragment was used as a template and amplified with two types of DNA primers (SEQ ID NOs: 30 and 31, and 32 and 33), respectively, to obtain two types of DNA fragments. In addition, pHKPsacB1 (referenced in Japanese Patent Application No. 2014-523757) was used as a template and amplified with two types of DNA primers (SEQ ID NOs: 34 and 35), and the obtained PCR products were treated with DpnI (Takara Bio). The six types of PCR products obtained were each purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated using In-Fusion HD Cloning Kit (Takara Bio) to produce the plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt.

2) Preparation of a strain carrying a polypeptide gene having 3,4-dihydroxybenzoic acid hydroxylase activity Using a transformation method by electroporation, the above-mentioned plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt was introduced into the CY44 strain (the CY44 strain is a tkt strain described in Reference Example 14 of Japanese Patent Application No. 2014-523757, in which the expression of the aroF, aroG, aroB, aroD and tkt genes is enhanced by controlling the transcription of the aroF, aroG, aroB, aroD and tkt genes with the tu promoter. In addition, the expression of the qsuB gene is enhanced by controlling the transcription of the Pben promoter.) was introduced and selected for kanamycin resistance to obtain the KC148sr strain. When the KC148sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of SEQ ID NOs: 20 and 36, the expected results were obtained, confirming that the KC148sr strain is a single-crossover homologous recombination transformant in which the plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt has been introduced into the cg0620 gene region.
The KC148sr strain was cultured in 1 mL of LB liquid medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) for 24 hours, and a portion of the culture was smeared and cultured on LB agar medium containing 20% sucrose to obtain the KC148 strain. By PCR (Sapphire Amp (Takara Bio)) using primers of SEQ ID NOs: 36 and 37, it was confirmed that the KC148 strain was a double-crossover homologous recombinant in which the Ptu-hfm145VF-hfm145VFop gene was introduced into the cg0620 gene region, as expected.

(2) Preparation of 3-hydroxybenzoate-4-hydroxylase and 3-hydroxybenzoate synthase and transformant 1) Construction of a plasmid for replacing the cg1625 gene region with the 3-hydroxybenzoate-4-hydroxylase gene and the 3-hydroxybenzoate synthase gene The 5' upstream region of the cg1625 gene region (SEQ ID NO: 38) was amplified with two types of DNA primers (SEQ ID NO: 39 and 40), and the 3' downstream region of the cg1625 gene region (base number 41) was amplified with two types of DNA primers (SEQ ID NO: 42 and 43) to obtain a DNA fragment. In addition, a DNA fragment (SEQ ID NO: 44) of a promoter (hereinafter referred to as tytu promoter) described in a prior literature was prepared by artificial gene synthesis (Eurofins Genomics), and amplified with two types of DNA primers (SEQ ID NO: 27 and 45) to obtain a DNA fragment. In addition, a 3-hydroxybenzoate-4-hydroxylase gene (SEQ ID NO: 46) was prepared by artificial gene synthesis (Eurofins Genomics), and amplified with two types of DNA primers (SEQ ID NOs: 47 and 48) to obtain a DNA fragment. In addition, a DNA fragment (SEQ ID NO: 49) containing a 3-hydroxybenzoate synthase gene was prepared by artificial gene synthesis (Eurofins Genomics), and amplified with two types of DNA primers (SEQ ID NOs: 50 and 51) to obtain a DNA fragment. In addition, pHKPsacB1 (Patent Application No. 2014-523757) was used as a template, and amplified with two types of DNA primers (SEQ ID NOs: 34 and 35), and the obtained PCR product was treated with DpnI (Takara Bio). The six types of PCR products obtained were each purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated using In-Fusion HD Cloning Kit (Takara Bio) to produce the plasmid pHKPsacB_cg1625-Ptytu-mobA-hyg5.

2) Preparation of strains carrying 3-hydroxybenzoate-4-hydroxylase gene and 3-hydroxybenzoate synthase gene The above-mentioned plasmid pHKPsacB_cg1625-Ptytu-mobA-hyg5 was introduced into the KC148 strain by electroporation transformation, and the strain was selected for kanamycin resistance to obtain the KC152sr strain. The KC152sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of SEQ ID NO: 39 and 52, and the expected results were obtained. It was therefore confirmed that the KC152sr strain is a single-crossover homologous recombinant in which the plasmid pHKPsacB_cg1625-Ptytu-mobA-hyg5 was introduced into the cg1625 gene region.
The KC152sr strain was cultured in 1 mL of LB liquid medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) for 24 hours, and a portion of the culture was smeared on a 20% sucrose-containing LB agar medium to obtain the KC152 strain. By PCR (Sapphire Amp (Takara Bio)) using primers of SEQ ID NOs: 52 and 53, it was confirmed that the KC152 strain was a double-crossover homologous recombinant in which the Ptytu-mobA-hyg5 gene was introduced into the cg1625 gene region, as expected.

(3) Cultivation of the transformant: Sodium benzoate was added to 1.5 mL of the CGXII medium shown in the table to a final concentration of 1 mM, and the transformant obtained above was inoculated and cultivated at 32° C. The culture solution was collected after 24 hours and 32 hours, appropriately diluted with dilute sulfuric acid, and centrifuged to remove the bacterial cells, and the supernatant was collected. The glucose and gallic acid concentrations in the supernatant were quantified.

(4) Results Comparing the KC148 strain and the KC152 strain, it was confirmed that the KC152 strain had an improved sugar consumption yield, indicating that enhancing mobA and hyg5 has an advantageous effect on gallic acid production.

(5) Method for calculating the consumed sugar yield The amount of glucose consumed from 24 to 32 hours of culture was calculated from the amount of glucose in the culture supernatant 24 and 32 hours after culture. The amount of gallic acid produced from 24 to 32 hours of culture was calculated from the concentration of gallic acid in the culture supernatant 24 and 32 hours after culture. The consumed sugar yield (%) was calculated using the following formula.
Consumed sugar yield (%: g/g) = Gallic acid production / glucose consumption x 100

(6) Analytical Conditions Insoluble matter was removed from the collected supernatant using an AcroPrep 96 filter plate (0.2 μm GHP membrane, Nippon Pall), and the reaction solution was subjected to HPLC.
The HPLC device used was a Chromaster (Hitachi High-Tech Science). For the analysis of gallic acid, an L-column ODS (4.6 mm ID x 150 mm, Chemicals Evaluation and Research Institute, Japan) was used, and gradient elution was performed under the conditions of a flow rate of 1.0 mL/min and a column temperature of 40°C, with eluent A being a 0.1% phosphoric acid solution of 0.1 M potassium dihydrogen phosphate and eluent B being 70% methanol. For the detection of gallic acid, a UV detector (detection wavelength 280 nm) was used.
Glucose was analyzed using ICsep ION-300 (7.8 mm x 300 mm, Tokyo Chemical Industry Co., Ltd.), and detection was performed using a 37 mM sulfuric acid solution as the eluent at a flow rate of 0.5 mL/min and a column temperature of 50° C. RI was used for glucose detection.

Claims (10)

A transformant capable of producing gallic acid or a salt thereof, in which expression of (A) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity, (B) a polypeptide having 3-hydroxybenzoate synthase activity, and (C) a polypeptide having 3-hydroxybenzoate hydroxylase activity is enhanced,
the polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is a polypeptide represented by the following (A1) or (A2), the polypeptide having 3-hydroxybenzoate synthase activity is a polypeptide represented by the following (B1) or (B2), the polypeptide having 3-hydroxybenzoate hydroxylase activity is a polypeptide represented by the following (C1) or (C2), and the transformant is a bacterium of the genus Corynebacterium.
(A1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:2.
(A2) A polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity, which comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:2.
(B1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:6.
(B2) A polypeptide having 3-hydroxybenzoate synthase activity, comprising a peptide sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:6.
(C1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:8.
(C2) A polypeptide consisting of a peptide sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:8 and having 3-hydroxybenzoic acid hydroxylase activity . The transformant according to claim 1, comprising, in an expressible state, a polynucleotide encoding (A) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity, (B) a polypeptide having 3-hydroxybenzoate synthase activity, and (C) a polypeptide having 3-hydroxybenzoate hydroxylase activity. The transformant according to claim 1 or 2 , further comprising enhanced expression of a polypeptide (D) having 3-dehydroshikimate dehydratase activity. The transformant according to claim 3 , which comprises (D) a polynucleotide encoding a polypeptide having 3-dehydroshikimate dehydratase activity in an expressible state. The transformant according to claim 4 , wherein the polypeptide (D) having 3-dehydroshikimate dehydratase activity is a polypeptide represented by the following (D1) or (D2):
(D1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10. (D2) A polypeptide consisting of a peptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 10 and having 3-dehydroshikimate dehydratase activity. The transformant according to any one of claims 1 to 5, further comprising enhanced expression of at least one polypeptide selected from the group consisting of (E) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity, (F) a polypeptide having 3- dehydroquinate dehydratase activity, and (G) a polypeptide having transketolase activity. The transformant according to claim 6, comprising, in an expressible state, a polynucleotide encoding at least one polypeptide selected from the group consisting of (E) a polypeptide having 3-deoxy-D-arabino-heptulosonate- 7 -phosphate synthase activity, (F) a polypeptide having 3-dehydroquinate dehydratase activity, and (G) a polypeptide having transketolase activity. The transformant according to claim 7, wherein the polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity is a polypeptide represented by the following (E1) or (E2), the polypeptide having 3 -dehydroquinate dehydratase activity is a polypeptide represented by the following (F1) or (F2), and the polypeptide having transketolase activity is a polypeptide represented by the following (G1) or (G2).
(E1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14. (E2) A polypeptide consisting of a peptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:14, and having DAHP synthase activity. (F1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:16. (F2) A polypeptide consisting of a peptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:16, and having 3-dehydroquinate dehydratase activity. (G1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:18. (G2) A polypeptide consisting of a polypeptide sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:18, and having transketolase activity. The transformant according to any one of claims 1 to 8 , wherein the bacterium of the genus Corynebacterium is Corynebacterium glutamicum. A method for producing gallic acid or a salt thereof, comprising a step of culturing the transformant according to any one of claims 1 to 9 in the presence of a saccharide.