Method for producing 4-hydroxybenzoic acid

Abstract

To provide a method for producing a 4-hydroxybenzoic acid using biomass resources.SOLUTION: Provided is a method for producing a 4-hydroxybenzoic acid represented by general formula (2), the method comprising a step of bringing a 4-hydroxyisophthalic acid represented by general formula (1) into contact with: a polypeptide having an amino acid sequence selected from a specific amino acid sequence group, or a polypeptide having an amino acid sequence with at least 90% identity to the sequence, wherein the polypeptide has 4-hydroxyisophthalic acid decarboxylase activity, or with a microorganism that produces such a polypeptide. (In the formulas, R1 and R2 may be the same or different, and represent a hydrogen atom, a hydroxy group, a halogen atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, an amino group, or a carboxy group).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing 4-hydroxybenzoic acids using biomass resources. [Background technology]

[0002] 4-Hydroxybenzoic acid is widely used industrially as a constituent monomer of liquid crystal materials, a raw material for preservatives, etc. Currently, it is known that 4-hydroxybenzoic acid can be produced by the Kolbe-Schmitt reaction using phenol salts and carbon dioxide as raw materials.

[0003] Meanwhile, chemical recycling technologies that convert discarded chemical materials into new chemical raw materials have been actively developed in recent years. Phthalic acids in particular are used as constituent monomers for polyethylene terephthalate and thermoplastic resins, so if 4-hydroxybenzoic acid can be obtained from these phthalic acids as a starting material, it will increase the recyclability of chemical materials and provide a new method for supplying 4-hydroxybenzoic acid.

[0004] One possible chemical method for obtaining 4-hydroxybenzoic acid from phthalic acids as a starting material would be to selectively decarboxylate the carboxy group at the 3-position of 4-hydroxyisophthalic acid, but such a reaction has not been reported to date.

[0005] Metallo-decarboxylases are decarboxylases that have a divalent metal ion, such as zinc, manganese, or magnesium, in their active center, and are known to catalyze the decarboxylation reaction by forming a complex with benzoic acids that have a hydroxyl group at the ortho position (Non-patent Document 1). However, it has not been known that such metallo-decarboxylases can catalyze the selective decarboxylation of 4-hydroxyisophthalic acid as a substrate. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Stefan E. Payer et al., Adv. Synth. Catal. 2019, 361, 2402-2420 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a method for producing 4-hydroxybenzoic acids using biomass resources. [Means for solving the problem]

[0008] The present inventors have found that a specific polypeptide having 5-carboxyvanillic acid decarboxylase activity exhibits selective decarboxylation activity at the 3-position of 4-hydroxyisophthalic acid, and that 4-hydroxybenzoic acids can be efficiently produced by using the polypeptide or a microorganism that produces the polypeptide.

[0009] The present invention relates to the following. The following general formula (1):

[0010] [ka]

[0011] [In the formula, R 1 and R 2 may be the same or different and represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an amino group, or a carboxy group. The 4-hydroxyisophthalic acid represented by the following (A) or (B): (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or 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 4-hydroxyisophthalic acid decarboxylase activity; (B) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4, and having 4-hydroxyisophthalic acid decarboxylase activity; or a microorganism producing the same,

[0012] [ka]

[0013] [In the formula, R 1 and R 2 indicates the same as above. A method for producing 4-hydroxybenzoic acids represented by the following formula: Effect of the Invention

[0014] According to the method of the present invention, 4-hydroxybenzoic acids can be efficiently produced using biomass resources. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] 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.

[0016] In the present invention, "at least 90% identity" with respect to amino acid sequences and nucleotide sequences means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, and even more preferably 99% or more.

[0017] In the present invention, "an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted" refers to an amino acid sequence in which 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are deleted, substituted, added, or inserted. In addition, "a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted" refers to a nucleotide sequence in which 1 to 60, preferably 1 to 45, more preferably 1 to 30, more preferably 1 to 24, more preferably 1 to 15, and even more preferably 1 to 9 nucleotides are deleted, substituted, added, or inserted. In the present invention, "addition" of an amino acid or nucleotide includes addition of an amino acid or nucleotide to one end and both ends of a sequence. Introduction of such mutations, such as deletion, substitution, insertion, and addition, can be carried out by introducing a mutation into the target nucleotide sequence, for example, by site-specific mutagenesis.

[0018] In the present invention, "upstream" and "downstream" of a gene refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter in the DNA sense strand, and "upstream" of a gene means the 5' region of the gene in the DNA sense strand.

[0019] In the present invention, the "operable linkage" between a control region such as a promoter and a gene means that the gene and the control region are linked so that the gene can be expressed under the control of the control region. The procedure for "operably linking" a gene and a control region is well known to those skilled in the art.

[0020] In the present invention, a "foreign gene" refers to an exogenous gene introduced into a cell from outside. A foreign gene may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene).

[0021] The method for producing a 4-hydroxybenzoic acid compound of the present invention comprises reacting a compound represented by the following general formula (1):

[0022] [ka]

[0023] [In the formula, R 1 and R 2 may be the same or different and represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an amino group, or a carboxy group.

[0024] The 4-hydroxyisophthalic acid represented by the following (A) or (B): (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or 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 4-hydroxyisophthalic acid decarboxylase activity; (B) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4, and having 4-hydroxyisophthalic acid decarboxylase activity; or a microorganism producing the same,

[0025] [ka]

[0026] [In the formula, R 1 and R 2 indicates the same as above. This is a method for producing 4-hydroxybenzoic acids represented by the following formula:

[0027] In formula (1) or (2), R 1 , R 2Examples of the halogen atom represented by the formula (I) include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom, a chlorine atom and a bromine atom, and more preferably a fluorine atom. Preferred examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, and a tert-butyl group, and more preferred are a methyl group and an ethyl group. Preferred examples of the alkoxy group having 1 to 4 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, and a butoxy group, and a methoxy group is more preferred.

[0028] R 1 , R 2 are each more preferably a hydrogen atom, a hydroxyl group, a methoxy group, a fluorine atom or a methyl group, and R 1 and R 2 More preferably, both are hydrogen atoms.

[0029] The 4-hydroxyisophthalic acids used as the raw material may be commercially available products, or may be produced, for example, from hydroxybenzoic acids by utilizing the Kolbe-Schmitt reaction (JP Patent Publication 06-100494).

[0030] The polypeptide represented by (A) or (B) above (referred to as the "polypeptide of the present invention") has 4-hydroxyisophthalic acid decarboxylase activity, which selectively decarboxylates the carboxy group at the 3-position of 4-hydroxyisophthalic acid. (A) The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is a peptide known as 5-carboxyvanillic acid decarboxylase derived from Sphingomonas paucimobilis, and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is a peptide known as 5-carboxyvanillic acid decarboxylase derived from Novosphingobium aromaticivorans. 5-carboxyvanillic acid decarboxylase is an enzyme that catalyzes the reaction of decarboxylating the carboxyl group at the 5-position of 5-carboxyvanillic acid to produce vanillic acid (Applied and Environmental Microbiology Volume 68, Issue 9, 4407-4415).

[0031] In the polypeptide of the present invention, the identity with the amino acid sequence shown in SEQ ID NO: 2 or 4 is preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and still more preferably 99% or more. Examples of amino acid sequences having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 or 4 include amino acid sequences in which one or several amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 2 or 4.

[0032] 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 Harbar Laboratory Press, New York, 581-621, 1995). Examples of site-specific mutagenesis techniques 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), the Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488), etc. Alternatively, commercially available site-specific mutagenesis kits such as the Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio Inc.), Transformer (trademark) Site-Directed Mutagenesis Kit (Clonetech Inc.), and KOD-Plus-Mutagenesis Kit (Toyobo Co., Ltd.) can also be used.

[0033] As shown in the Examples below, 4-hydroxyisophthalic acid decarboxylase activity can be determined, for example, by contacting 4-hydroxyisophthalic acid with the polypeptide of the present invention or a microorganism producing the polypeptide and measuring the amount of 4-hydroxybenzoic acid produced by HPLC or the like.

[0034] The polypeptide of the present invention can be produced by culturing a recombinant microorganism into which a gene encoding the polypeptide shown in (A) or (B) has been introduced. The gene encoding the polypeptide shown in (A) or (B) preferably includes the polynucleotide shown in (a) or (b) below.

[0035] (a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1; a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a polypeptide having 4-hydroxyisophthalic acid decarboxylase activity (b) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3; a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a polypeptide having 4-hydroxyisophthalic acid decarboxylase activity. Here, (a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 is known as a gene encoding 5-carboxyvanillic acid decarboxylase derived from Sphingomonas paucimobilis, and (b) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 is known as a gene encoding 5-carboxyvanillic acid decarboxylase derived from Novosphingobium aromaticivorans.

[0036] Examples of nucleotide sequences having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 include nucleotide sequences in which one or several nucleotides are deleted, substituted, added, or inserted relative to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. 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 contain a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF). The polynucleotide may be codon-optimized according to the species of the transformant used to produce the polypeptide of the present invention. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0037] Means for introducing such a polynucleotide (a) or (b) into a microorganism include, for example, introducing a vector or a DNA fragment containing each polynucleotide into a host microorganism (parent strain). Here, the vector containing each polynucleotide is an expression vector, preferably an expression vector capable of introducing the polynucleotide into a host microorganism and expressing the polynucleotide in the host. The vector preferably contains the polynucleotide and a control region operably linked thereto. The vector may be an extrachromosomal vector capable of autonomously replicating and replicating, such as a plasmid, or may be a vector that is integrated into a chromosome. Here, "operably linked" means that a gene and a 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 linking" a gene and a regulatory region is well known to those skilled in the art.

[0038] Specific examples of vectors include pBluescript II SK(-) (Stratagene), pUC vectors such as pUC18 / 19 and pUC118 / 119 (Takara Bio), pET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2):93-103), pBR322 (Takara Bio), pRS403 (Stratagene), pMW218 / 219 (Nippon Gene), pRI vectors such as pRI909 / 910 (Takara Bio), pBI vectors (Clontech), IN3 vectors (Implanta Innovations), pPTR1 / 2 (Takara Bio), pDJB2 (DJBallance et al., 2011). al., Gene, 36, 321-331, 1985), pAB4-1 (van Hartingsveldt W et al., Mol Gen Genet, 206, 71-75, 1987), pLeu4 (MIGRoncero et al., Gene, 84, 335-343, 1989), pPyr225 (CD Skory et al., Mol Genet Genomics, 268, 397-406, 2002), pFG1 (Gruber, F. et al., Curr Genet, 18, 447-451, 1990), and the like.

[0039] The polynucleotide of the present invention 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 and a control region operably linked thereto.

[0040] The control region contained in the vector or DNA fragment is a sequence for expressing a polynucleotide encoding the polypeptide 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 regulatory 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 (e.g., resistance genes for drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol).

[0041] The polynucleotide of the present invention can be linked to the above-mentioned control region or marker gene sequence by the above-mentioned SOE-PCR method or the like. The procedure for introducing a gene sequence into a vector is well known in the art. The types of control regions such as promoter regions, terminators, and secretion signal regions are not particularly limited, and promoters and secretion signal sequences that are commonly used can be appropriately selected and used depending on the host to be introduced.

[0042] Suitable examples of the regulatory region include strong regulatory regions that can enhance expression compared to the wild type, such as known high expression promoters such as the T7 promoter, lac promoter, tac promoter, and trp promoter, but are not particularly limited to these.

[0043] The target polynucleotide and the control region contained in the vector or DNA fragment may be introduced into the nucleus of the host, or may be introduced into the host genome. Alternatively, the target polynucleotide contained in the vector or DNA fragment may be directly introduced into the host genome and operably linked to a high expression promoter on the genome. A method for introducing a polynucleotide into a genome includes homologous recombination.

[0044] To introduce a vector or DNA fragment into the above-mentioned host cells, a common transformation method such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, or Agrobacterium can be used.

[0045] A recombinant microorganism into which a vector or DNA fragment of interest has been introduced can be selected using a selection marker. For example, when the selection marker is an antibiotic resistance gene, transformed 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, when the selection marker is an amino acid synthesis-related gene, the gene is introduced into a microbial strain that requires the amino acid, and then the microbial strain into which the vector or DNA fragment of interest has been introduced can be selected based on the presence or absence of the amino acid requirement. Alternatively, the introduction of the vector or DNA fragment of interest can be confirmed by examining the DNA sequence of the recombinant strain by PCR or the like.

[0046] By the above procedure, a recombinant microorganism in which the polynucleotide of the present invention is introduced into a host microbial strain can be prepared. The recombinant microorganism has the ability to produce 4-hydroxyisophthalic acid decarboxylase of the present invention.

[0047] The microorganism used as the host (parent strain) may be any of fungi, yeast, actinomycetes, Escherichia coli, Bacillus subtilis, etc., but is preferably Escherichia coli, yeast, or actinomycetes. As the 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, and Micrococcus. Among these, preferred are bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium callunae, etc.), and more preferred is Corynebacterium glutamicum.

[0048] The polypeptide of the present invention can be obtained by culturing the above-mentioned recombinant microorganism in any medium containing a carbon source, a nitrogen source, inorganic salts, etc., producing and accumulating the polypeptide of the present invention in the microorganism, and recovering the polypeptide from the microorganism. The polypeptide of the present invention can also be obtained by culturing a recombinant microorganism constructed so as to secrete the polypeptide of the present invention extracellularly, producing and accumulating the polypeptide of the present invention in the culture supernatant, and recovering the polypeptide from the culture supernatant.

[0049] In the present invention, 4-hydroxybenzoic acids are produced by contacting 4-hydroxyisophthalic acids with the polypeptide of the present invention or the above-mentioned microorganism producing the polypeptide. Here, when the polypeptide of the present invention is used, the polypeptide may be an aqueous liquid containing the polypeptide, such as a cell suspension or cell disruption solution containing medium components in which a microorganism producing the polypeptide is cultured, or one obtained by drying the same, or one that does not contain these and is essentially composed of the polypeptide itself. For example, the polypeptide may be an immobilized product from an aqueous liquid containing the polypeptide produced by culturing the above-mentioned recombinant microorganism, disrupting the cells as necessary, and removing the cells, or a powder of the aqueous liquid containing the polypeptide. Examples of immobilized polypeptides include those in which the polypeptide is immobilized on a carrier such as silica, celite, diatomaceous earth, perlite, polyvinyl alcohol, anion exchange resin, phenol adsorption resin, hydrophobic carrier, cation exchange resin, chelating resin, etc. Examples of powdered polypeptides include those obtained by drying and powdering a polypeptide-containing aqueous liquid by a method such as spray drying, freeze drying, or drying after solvent precipitation.

[0050] The contact conditions between the polypeptide of the present invention and 4-hydroxyisophthalic acids are not particularly limited, but the contact can usually be performed at 20°C to 50°C for 5 minutes to 72 hours, preferably 1 hour to 60 hours, and more preferably 1 hour to 24 hours, with stirring or shaking as necessary.

[0051] When a microorganism is used, contact with 4-hydroxyisophthalic acids can be achieved by culturing the above-mentioned recombinant microorganism in a medium containing 4-hydroxyisophthalic acids. The medium for culturing the recombinant microorganism 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 can efficiently culture the recombinant microorganism. Examples of the carbon source that can be used include sugars such as glucose, polyols such as glycerin, alcohols such as ethanol, and organic acids such as pyruvic acid, succinic acid, and citric acid. Examples of the nitrogen source that can be used include peptone, meat extract, yeast extract, casein hydrolysate, alkaline extract of soybean meal, alkylamines such as methylamine, and ammonia or a salt thereof. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and antifoaming agents may also be used as necessary.

[0052] The culture can usually be performed at 10° C. to 40° C. for 6 to 72 hours, preferably 9 to 60 hours, and more preferably 12 to 48 hours, with stirring or shaking as necessary. During the culture, antibiotics such as ampicillin and kanamycin may be added to the medium as necessary.

[0053] The method for recovering and purifying 4-hydroxybenzoic acids from the reaction system is not particularly limited, and can be carried out by combining well-known methods such as ion exchange resin method, precipitation method, crystallization method, recrystallization method, concentration method, and others. For example, in the case of production using recombinant microorganisms, 2,4-pyridinedicarboxylic acids 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. 4-Hydroxybenzoic acids accumulated in the culture may be used as they are without isolation.

[0054] The present invention also includes the following substances, manufacturing methods, uses, methods, etc. as exemplary embodiments, but the present invention is not limited to these embodiments. <1> The following general formula (1):

[0055] [ka]

[0056] [In the formula, R 1 and R 2 may be the same or different and represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an amino group, or a carboxy group. The 4-hydroxyisophthalic acid represented by the following (A) or (B): (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or 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 4-hydroxyisophthalic acid decarboxylase activity; (B) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4, and having 4-hydroxyisophthalic acid decarboxylase activity; or a microorganism producing the same,

[0057] [ka]

[0058] [In the formula, R 1 and R 2 indicates the same as above. A method for producing 4-hydroxybenzoic acids represented by the following formula: <2> The microorganism producing the polypeptide represented by (A) or (B) is a recombinant microorganism into which a gene encoding the polypeptide has been introduced. <1> How to. <3> The gene encoding the polypeptide represented by (A) or (B) is a polynucleotide represented by the following (a) or (b): <2> How to. (a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1; a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a polypeptide having 4-hydroxyisophthalic acid decarboxylase activity (b) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3; a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a polypeptide having 4-hydroxyisophthalic acid decarboxylase activity. <4> The microorganism is Escherichia coli, yeast or coryneform bacteria; <1> ~ <3> Either way. <5> Recovering 4-hydroxybenzoic acids. <1> ~ <4> Either way. <6> In general formulas (1) and (2), R 1 and R 2 each represents a hydrogen atom, a hydroxyl group, a methoxy group, a fluorine atom, or a methyl group; <1> ~ <5> Either way. <7> In general formulas (1) and (2), R 1 and R 2 are both hydrogen atoms, <1> ~ <5> How to. EXAMPLES

[0059] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0060] Test Example 1: Production of 4-hydroxybenzoic acid The gene encoding 5-carboxyvanillic acid decarboxylase derived from Sphingomonas paucimobilis (SEQ ID NO: 1) and the gene encoding 5-carboxyvanillic acid decarboxylase derived from Novosphingobium aromaticivorans (SEQ ID NO: 3) were inserted between the NdeI site and the BamHI site of the commercially available E. coli expression vector pET21a(+) to obtain plasmids pET21a-SpCVDC and pET21a-NaCVDC expressing 5-carboxyvanillic acid decarboxylase. These genes (SEQ ID NO: 1, 3) were sequences that were codon-optimized for E. coli, and artificially synthesized ones were used. Each plasmid was used to transform ECOS competent cells BL21(DE3) (Nippon Gene), which were then spread on LBAmp agar medium and left to stand overnight at 37°C. The colonies obtained were used as test strains. Similarly, colonies obtained by transforming the pET21a vector were used as control strains. Each strain was inoculated into 750μL of OXTBAmp medium (per 1L: Overnight Express TB medium (Merck) 60g, glycerin 10mL, ampicillin sodium 50mg) and cultured at 37℃ for 24 hours. 400μL of the culture was centrifuged to obtain bacterial cells, and 400μL of 100mM potassium phosphate buffer (pH7.4) was added, and 100μL of 1M Tris-HCl buffer (pH8.0) solution (10g / L) of 4-hydroxyisophthalic acid (Tokyo Chemical Industry) was added, and the mixture was shaken at 30℃ for 24 hours. 30μL of the supernatant after shaking was mixed with 270μL of 0.1(v / v)% phosphoric acid aqueous solution, and insoluble matter was removed using an AcroPrep 96 filter plate (0.2μm, WWPTFE membrane, Nippon Pole) and subjected to high performance liquid chromatography (HPLC). The HPLC equipment used was Chromaster (Hitachi High-Tech Science). The analytical column used was an L-column ODS (4.6 mm ID x 150 mm, Chemicals Evaluation and Research Institute, Japan). Gradient elution was performed at a flow rate of 1.0 mL / min and a column temperature of 40°C, with eluent A being 0.1 (v / v)% phosphoric acid solution of 0.1 M potassium dihydrogen phosphate and eluent B being 70 (v / v)% methanol.A UV detector (detection wavelength 280 nm) was used to detect 4-hydroxyisophthalic acid and 4-hydroxybenzoic acid, and a concentration calibration curve was prepared using the standard sample "4-hydroxybenzoic acid (seller code H0207, Tokyo Chemical Industry)", and quantification was performed based on the concentration calibration curve. As shown in Table 1, the strains expressing 5-carboxyvanillic acid decarboxylase derived from Sphingomonas paucimobilis and 5-carboxyvanillic acid decarboxylase derived from Novosphingobium aromaticivorans produced 4-hydroxybenzoic acid when contacted with 4-hydroxyisophthalic acid.

[0061] [Table 1]

Claims

1. The following general formula (1): 【Chemistry 1】 [In the formula, R １ and R ２ may be the same or different and represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an amino group, or a carboxy group. The 4-hydroxyisophthalic acid represented by the following (A) or (B): (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or 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 4-hydroxyisophthalic acid decarboxylase activity; (B) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4, and having 4-hydroxyisophthalic acid decarboxylase activity; or a microorganism producing the same, 【Chemistry 2】 [In the formula, R １ and R ２ indicates the same as above. The present invention relates to a method for producing a 4-hydroxybenzoic acid represented by the following formula:

2. 2. The method according to claim 1, wherein the microorganism producing the polypeptide represented by (A) or (B) is a recombinant microorganism into which a gene encoding said polypeptide has been introduced.

3. 3. The method according to claim 1 or 2, wherein the microorganism is Escherichia coli, yeast or coryneform bacteria.

4. The method according to any one of claims 1 to 3, further comprising a step of recovering the 4-hydroxybenzoic acids.

5. In the general formulas (1) and (2), R １ and R ２ The method according to any one of claims 1 to 4, wherein each of them is a hydrogen atom, a hydroxy group, a methoxy group, a fluorine atom or a methyl group.

6. In the general formulas (1) and (2), R １ and R ２ The method according to any one of claims 1 to 4, wherein both are hydrogen atoms.

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