Method for producing 4-amino-3-hydroxybenzoic acid or salt thereof

Abstract

To provide a method for producing high-purity 4-amino-3-hydroxybenzoic acids or salts thereof.SOLUTION: A method for producing 4-amino-3-hydroxybenzoic acids or a salt thereof, comprising the step of culturing a microorganism having 4-amino-3-hydroxybenzoic acid-producing ability in a medium containing sucrose as a sugar source.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing 4-amino-3-hydroxybenzoic acids or salts thereof. [Background technology]

[0002] Polybenzoxazole (PBO) is known as an engineering plastic with excellent heat resistance and mechanical strength, and is used for fiber materials, insulating films for semiconductor elements, etc. (Non-Patent Document 1).

[0003] The benzoxazole skeleton is produced by the condensation of an o-aminophenol skeleton with a carboxylic acid. Therefore, 4-amino-3-hydroxybenzoic acids (4,3-AHBA) containing these functional groups in the molecule are expected to be useful as PBO monomers. In fact, the synthesis and property evaluation of polybenzoxazoles using 4,3-AHBA have been investigated (Non-Patent Document 2).

[0004] In recent years, methods for producing compounds by microbial fermentation using renewable resources as raw materials have been attracting attention in order to reduce the burden on the global environment, etc. For example, the microbial production and polymerization of 3-amino-4-hydroxybenzoic acid (3,4-AHBA), which has a structure similar to 4,3-AHBA, has been investigated (Patent Document 1).

[0005] It has been reported that 4,3-AHBA can be produced using 4-aminobenzoic acid (4-ABA) as a precursor using microorganisms capable of expressing polypeptides with 4-aminobenzoic acid hydroxylation activity (Patent Document 2). Considering the use of 4,3-AHBA as a monomer, high purity of 4,3-AHBA during microbial production is desirable. However, it is known that 4,3-AHBA is oxidized and converted to 2-aminophenoxazine-3-one-7-carboxylic acid (Patent Document 3). Accumulation of oxidized products raises concerns about a decrease in the purity of 4,3-AHBA. The presence of oxidized products can be confirmed using the degree of coloration of the culture medium as an indicator. Furthermore, because 4-ABA is detected in the culture supernatant, it is necessary to improve the conversion rate of 4-ABA to 4,3-AHBA to increase the purity of 4,3-AHBA. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5445453 [Patent Document 2] Patent Publication No. 2021-073914 [Patent Document 3] Patent Publication No. 2021-101628 [Non-patent literature]

[0007] [Non-Patent Document 1] Hirotaka Murase, SENI GAKKAISHI (Textiles and Industry), Vol.66, No.6 (2010) [Non-patent document 2] Lon J. Mathias et al., Macromolecules, Vol.18, No.4, pp.616-622 (1985) Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to providing a method for producing highly pure 4-amino-3-hydroxybenzoic acids or salts thereof. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to obtain 4-amino-3-hydroxybenzoic acids using microorganisms capable of producing 4-amino-3-hydroxybenzoic acid. As a result, they have unexpectedly found that by culturing the microorganisms in a medium containing sucrose as a sugar source, the conversion rate of 4-aminobenzoic acids to 4-amino-3-hydroxybenzoic acids can be improved and discoloration of the culture of the microorganisms can be suppressed.

[0010] That is, the present invention relates to the following 1) to 3). 1) A method for producing 4-amino-3-hydroxybenzoic acids or salts thereof, which comprises a step of culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source. 2) A method for improving the conversion rate of 4-aminobenzoic acids or salts thereof to 4-amino-3-hydroxybenzoic acids or salts thereof, which comprises a step of culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source. 3) A method for inhibiting discoloration of a culture of a microorganism capable of producing 4-amino-3-hydroxybenzoic acid, which comprises culturing the microorganism in a medium containing sucrose as a sugar source. [Effects of the Invention]

[0011] According to the present invention, it is possible to produce 4-amino-3-hydroxybenzoic acids or salts thereof with high purity by a fermentation method that places little strain on the environment. [Brief explanation of the drawings]

[0012] [Figure 1]Schematic diagram showing the production pathway of 4-amino-3-hydroxybenzoic acid in a microorganism (coryneform bacterium) capable of producing 4-amino-3-hydroxybenzoic acid. In the figure, aroG and aroF are genes encoding 2-dehydro-3-deoxyarabinoheptonate aldolase, aroB is a gene encoding 3-dehydroquinate synthase, aroD is a gene encoding dehydroquinate dehydratase, aroE3 is a shikimate dehydrogenase, aroA is a gene encoding 5-enolate pyruvylshikimate-3-phosphate synthase, aroC is a gene encoding chorismate synthase, and aroK is a gene encoding shikimate kinase. pabAB is a gene encoding 4-amino-4-deoxychorismate synthase, and pabC is a gene encoding 4-amino-4-deoxychorismate lyase. These are endogenous gene groups of Corynebacterium glutamicum. phbh* is a gene encoding a polypeptide with 4-aminobenzoate 3-hydroxylation activity, a mutant of 4-hydroxybenzoate hydroxylase from Caulobacter vibrioides. This pathway is catabolized by exogenous gene transfer. PEP is phosphoenolpyruvate, DAHP is 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, DHQ is dehydroquinate, DHS is dehydroshikimate, SHK is shikimate, S3P is shikimate-3-phosphate, EPSP is 5-enoylpyruvinylshikimate-3-phosphate, CHA is chorismate, 4ABA is 4-aminobenzoate, and 43AHBA is 4-amino-3-hydroxybenzoate. DETAILED DESCRIPTION OF THE INVENTION

[0013] Herein, 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 analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0014] As used herein, "at least 80% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more.

[0015] In the present invention, "an amino acid sequence in which one or several amino acids have been deleted, substituted, added, or inserted" refers to an amino acid sequence in which 1 to 78, preferably 1 to 59, more preferably 1 to 39, even more preferably 1 to 20, still more preferably 1 to 16, even more preferably 1 to 12, even more preferably 1 to 8, even more preferably 1 to 4, and even more preferably 1 to 2 amino acids have been deleted, substituted, added, or inserted. Furthermore, in the present invention, a "nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted" refers to a nucleotide sequence in which 1 to 235, preferably 1 to 176, more preferably 1 to 118, even more preferably 1 to 59, even more preferably 1 to 47, even more preferably 1 to 35, even more preferably 1 to 24, even more preferably 1 to 12, and even more preferably 1 to 6 nucleotides have been 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 or both ends of a sequence. In the present invention, "deletion" of an amino acid or nucleotide includes deletion of an amino acid or nucleotide from one or both ends of a sequence, but is preferably deletion of an amino acid or nucleotide within the sequence. Furthermore, in the present invention, the deletion, substitution, addition, or insertion of an amino acid or nucleotide is preferably a deletion, substitution, or insertion, and more preferably a deletion, substitution, or insertion of an amino acid or nucleotide within the sequence.

[0016] As used herein, a "corresponding position" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 31) to maximize homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the Clustal website operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI) [www.ebi.ac.uk / index.html], and the DNA Data Bank of Japan (DDBJ) website operated by the National Institute of Genetics [www.ddbj.nig.ac.jp / searches-j.html]. The position of the target sequence aligned to any position in the reference sequence by the above-mentioned alignment is considered to be the "position corresponding to" that position.

[0017] Those skilled in the art can further fine-tune the alignment of amino acid sequences obtained above to optimize it. Such optimal alignment is preferably determined taking into account the similarity of the amino acid sequences, the frequency of inserted gaps, and other factors. Here, amino acid sequence similarity refers to the percentage (%) of the number of positions at which identical or similar amino acid residues exist in both aligned amino acid sequences relative to the total number of amino acid residues in the two sequences. Similar amino acid residues refer to amino acid residues among the 20 amino acids that constitute proteins that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Groups of such similar amino acid residues are well known to those skilled in the art, and include, but are not limited to, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; and leucine and isoleucine.

[0018] As used herein, the term "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. Procedures for "operably linking" a gene and a regulatory region are well known to those skilled in the art.

[0019] As used herein, "upstream" and "downstream" in relation to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the 5' region of the gene on the DNA sense strand.

[0020] As used herein, the term "native" when used with respect to a cellular function, property, or trait 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. An exogenous gene or polynucleotide 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 or polynucleotide).

[0021] The method of the present invention for producing 4-amino-3-hydroxybenzoic acids or salts thereof comprises the step of culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source.

[0022] In the present invention, a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is used to produce 4-amino-3-hydroxybenzoic acids or salts thereof. Examples of microbial species include Bacillus subtilis, actinomycetes, Pantoea bacteria, Escherichia bacteria, Pseudomonas bacteria, Streptococcus bacteria, Lactobacillus bacteria, fungi (such as Neurospora, Aspergillus, and Trichoderma), and yeasts (such as Saccharomyces, Kluyveromyces, Schizosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Pichia, and Candida), with actinomycetes, Pantoea bacteria (such as Pantoea ananatis), or Escherichia bacteria (such as Escherichia coli).

[0023] Preferred actinomycetes are a group of microorganisms defined as coryneform bacteria (Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974)), specifically including bacteria of the genus Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, Micrococcus, etc. Of these, bacteria of the genus Corynebacterium are preferred. Examples of bacteria of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, etc. Of these, Corynebacterium glutamicum is preferred.

[0024] In addition to wild-type strains, coryneform bacteria may also be mutant strains or artificially engineered strains, such as strains in which genes encoding lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, malate dehydrogenase, etc. are disrupted.

[0025] Microorganisms capable of producing 4-amino-3-hydroxybenzoic acid refer to microorganisms that inherently have the ability to produce 4-amino-3-hydroxybenzoic acid and microorganisms to which the ability to produce 4-amino-3-hydroxybenzoic acid has been imparted. Preferred examples include (A) microorganisms in which the expression of a polypeptide having activity of hydroxylating 3-position of 4-aminobenzoic acid has been enhanced, and further, in addition to (A), (B) microorganisms in which the expression of a polypeptide necessary for the biosynthesis of 4-aminobenzoic acid from chorismate has been enhanced, and further, in addition to (A) and (B), microorganisms in which the expression of (C) a polypeptide necessary for the biosynthetic pathway of chorismate from phosphoenolpyruvate has been enhanced (see Figure 1).

[0026] The polypeptide (A) having 4-aminobenzoic acid 3-hydroxylating activity is a polypeptide that catalyzes the reaction of producing 4-amino-3-hydroxybenzoic acid from 4-aminobenzoic acid, and examples thereof include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 31, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 31 and having 4-aminobenzoic acid 3-hydroxylating activity. Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 31 is a polypeptide having 4-aminobenzoic acid 3-hydroxylation activity, which is a mutant of 4-hydroxybenzoic acid hydroxylase derived from Caulobacter vibrioides (M106A, T294S double mutant: JP 2024-14569 A) (HFM122_M106A_T294S in the Examples below, and represented as "phbh*" in Figure 1).

[0027] Amino acid sequences having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 31 include, for example, amino acid sequences in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 31.

[0028] Examples of polypeptides that consist of an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 31 and have the activity of hydroxylating 3-position of 4-aminobenzoic acid include mutants of 4-hydroxybenzoate hydroxylase derived from bacteria of the genus Caulobacter, specifically mutants of 4-hydroxybenzoate hydroxylase derived from Caulobacter rhizosphaerae and 4-hydroxybenzoate hydroxylase derived from Caulobacter sp. Specific examples of mutants include one or more mutants selected from the mutants described in JP 2021-073914 A, the mutants described in JP 2021-101626 A, the mutants described in JP 2021-101627 A, and the mutants described in JP 2022-047939 A, preferably one or more mutants selected from the following in the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 31: a mutant in which position 47 or a corresponding position in the amino acid sequence shown in SEQ ID NO: 31 is leucine, a mutant in which position 106 or a corresponding position is alanine, a mutant in which position 201 or a corresponding position is phenylalanine, a mutant in which position 222 or a corresponding position is phenylalanine, and a mutant in which position 294 or a corresponding position is serine.

[0029] (B) Polypeptides necessary for the biosynthesis of 4-aminobenzoic acid from chorismate include (B1) 4-amino-4-deoxychorismate synthase and (B2) 4-amino-4-deoxychorismate lyase. Therefore, enhancing expression of polypeptides necessary for the biosynthesis of 4-aminobenzoic acid from chorismate can be achieved by enhancing expression of one or more of these polypeptides. 4-Aminobenzoic acid is produced from chorismate via 4-amino-4-deoxychorismate. The conversion of chorismate to 4-amino-4-deoxychorismate involves para-aminobenzoate synthetase component II (PabA) and para-aminobenzoate synthetase component I (PabB), and 4-amino-4-deoxychorismate is converted to 4-aminobenzoic acid by 4-amino-4-deoxychorismate lyase (PabC). The (B1) 4-amino-4-deoxychorismate synthase is a polypeptide that catalyzes the reaction of producing 4-amino-4-deoxychorismate from chorismate, and examples thereof include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 32 and having 4-amino-4-deoxychorismate synthase activity. The (B2) 4-amino-4-deoxychorismate lyase is a polypeptide that catalyzes the reaction of producing 4-aminobenzoic acid from 4-amino-4-deoxychorismate, and examples thereof include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 33, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 33 and having 4-amino-4-deoxychorismate lyase activity. Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32 is 4-amino-4-deoxychorismate synthase derived from Corynebacterium glutamicum and is known as "pabAB", and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 33 is 4-amino-4-deoxychorismate lyase derived from Corynebacterium glutamicum and is known as "pabC".

[0030] Amino acid sequences having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 32 or 33 include, for example, amino acid sequences in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 32 or 33.

[0031] (C) Polypeptides necessary for the biosynthetic pathway from phosphoenolpyruvate to chorismate include (C1) a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, (C2) a polypeptide having 3-dehydroquinate synthase activity, (C3) a polypeptide having dehydroquinate dehydratase activity, (C4) a polypeptide having shikimate dehydrogenase activity, (C5) a polypeptide having shikimate kinase activity, (C6) a polypeptide having 5-enolate pyruvylshikimate-3-phosphate synthase activity, and (C7) a polypeptide having chorismate synthase activity. Therefore, enhancing expression of polypeptides necessary for the biosynthetic pathway from phosphoenolpyruvate to chorismate includes enhancing expression of any one or more of these polypeptides. In one example, expression of (C1) a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity is enhanced. Examples of the polypeptide (C1) include a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 34, a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 34 and having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 35, and a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 35 and having 2-dehydro-3-deoxyarabinoheptonate aldolase activity. Here, the polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 34 is a mutant of 2-dehydro-3-deoxyarabinoheptonate aldolase derived from Escherichia coli and has 2-dehydro-3-deoxyarabinoheptonate aldolase activity (aroGec_D146N in the Examples below).The polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 35 is a mutant of 2-dehydro-3-deoxyarabinoheptonate aldolase derived from Corynebacterium glutamicum and has 2-dehydro-3-deoxyarabinoheptonate aldolase activity (aroF_P155L in the Examples below).

[0032] Amino acid sequences having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 34 or 35 include, for example, amino acid sequences in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 34 or 35.

[0033] Methods for introducing mutations such as deletion, substitution, addition, or insertion of amino acids into the amino acid sequence of the polypeptide include, for example, methods for introducing mutations such as deletion, substitution, addition, or insertion of nucleotides into the nucleotide sequence encoding the amino acid sequence. Techniques for introducing mutations into nucleotide sequences include, for example, 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; the method described by Dieffenbach et al. (Cold Spring Harbar Laboratory Press, New York, pp. 581-621, 1995); and genome editing using artificial DNA cleaving enzymes (artificial DNA nucleases or programmable nucleases). Examples of site-specific mutagenesis methods include 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, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, the Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio Inc.), Transformer TM Commercially available site-directed mutagenesis kits such as Site-Directed Mutagenesis Kit (Clontech) and KOD-Plus-Mutagenesis Kit (Toyobo) can also be used.

[0034] Enhancing the expression of the polypeptide includes, for example, enhancing the expression of a polynucleotide encoding the polypeptide. Methods for enhancing the expression of a polynucleotide encoding the polypeptide include introducing a polynucleotide encoding the polypeptide into a host (the microorganism) so that the polynucleotide is expressible, or modifying the regulatory region of the polynucleotide encoding the polypeptide on the host genome to increase the transcription level of the polynucleotide. The polypeptide may be foreign or may be inherent to the host. Introducing the polynucleotide so that the polynucleotide is expressible includes introducing the polynucleotide so that the level of expression is enhanced. Specific examples include introducing a vector or DNA fragment containing the polynucleotide and a regulatory region operably linked to it, or replacing the regulatory region of the polynucleotide with a strong regulatory region. (A) Preferred polynucleotides encoding polypeptides having 4-aminobenzoic acid 3-hydroxylating activity include a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 36, and a polynucleotide consisting of a nucleotide sequence at least 80% identical to the nucleotide sequence set forth in SEQ ID NO: 36, which encodes a polypeptide having 4-aminobenzoic acid 3-hydroxylating activity. The polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 36 is an example of a polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 31. (B1) Preferred polynucleotides encoding polypeptides having 4-amino-4-deoxychorismate synthase activity include a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 37, and a polynucleotide consisting of a nucleotide sequence at least 80% identical to the nucleotide sequence set forth in SEQ ID NO: 37, which encodes a polypeptide having 4-amino-4-deoxychorismate synthase activity. The polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 37 is an example of a polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 32. (B2) Preferred polynucleotides encoding a polypeptide having 4-amino-4-deoxychorismate lyase activity include a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 38, and a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 38 and encoding a polypeptide having 4-amino-4-deoxychorismate lyase activity. The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 38 is an example of a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 33.(C1) Preferred polynucleotides encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity include a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 39, a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 39 and encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, and a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 40, and a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 40 and encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity. The polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 39 is an example of a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 34, and the polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 40 is an example of a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 35. Nucleotide sequences having at least 80% identity with the nucleotide sequence shown in any one of SEQ ID NOs: 36 to 40 include, for example, nucleotide sequences in which one or several nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence shown in any one of SEQ ID NOs: 36 to 40.

[0035] The polynucleotide may be in single-stranded or double-stranded form, and may be DNA or RNA. The DNA may be artificial DNA such as cDNA or chemically synthesized DNA.

[0036] The polynucleotide may be incorporated into a vector. Preferably, the vector containing the polynucleotide is an expression vector. Also preferably, the vector is an expression vector that can introduce the polynucleotide into a host microorganism and express the polynucleotide in the host microorganism. Preferably, the vector contains the polynucleotide and a control region operably linked thereto. The vector may be a vector that is capable of autonomous replication and replication outside a chromosome, such as a plasmid, or may be a vector that is integrated into a chromosome.

[0037] Specific examples of vectors include pBluescript II SK(-) (Stratagene), pUC vectors such as pUC18 / 19 and pUC118 / 119 (Takara Bio), pET vectors (Takara Bio), pHSG 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), and pDJB2 (DJBallance et 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 (CDSkory et al., Mol Genet Genomics, 268, 397-406, 2002), pFG1 (Gruber, F. et al., Curr Genet, 18, 447-451, 1990), and the like.

[0038] The polynucleotide may also be constructed as a DNA fragment containing the polynucleotide. Examples of such DNA fragments include PCR-amplified DNA fragments and restriction enzyme-cleaved DNA fragments. Preferably, the DNA fragment may be an expression cassette containing the polynucleotide and a control region operably linked thereto.

[0039] The control region contained in the vector or DNA fragment is a sequence for expressing the polynucleotide in a host cell into which the vector or DNA fragment has been introduced, and examples thereof 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 contain a selection marker such as an antibiotic resistance gene or an amino acid synthesis-related gene.

[0040] To introduce a vector or DNA fragment into a host cell, a common transformation method such as electroporation, transformation, transfection, conjugation, protoplast method, particle gun method, Agrobacterium method, etc. can be used.

[0041] Modified microorganisms 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 supplemented with the antibiotic. Furthermore, if the selection marker is an amino acid synthesis-related gene, the gene can be introduced into a host cell requiring the amino acid, and then cells into which a vector or DNA fragment of interest has been introduced can be selected using the presence or absence of the amino acid requirement as an indicator. Alternatively, introduction of a vector or DNA fragment of interest can be confirmed by examining the DNA sequence of transformed cells using PCR or other methods.

[0042] Furthermore, examples of strong regulatory regions include known high expression promoters such as the T7 promoter, lac promoter, tac promoter, trp promoter, tuf promoter, gap promoter, SPL13 promoter, and the promoter of the cg2875 gene, but are not particularly limited to these. Furthermore, the strong regulatory region can be an inducible promoter derived from a prokaryote, 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 (hereinafter abbreviated as Pben), which is induced by the addition of benzoic acid; 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 in the genome of a host cell with a strong regulatory region include introducing a DNA fragment containing the strong regulatory region and a polynucleotide sequence of a selection marker into the host cell and selecting cells transformed by homologous recombination, non-homologous recombination, or the like.

[0043] The level of 4-amino-3-hydroxybenzoic acid production ability of the modified microorganism can be confirmed by measuring the amount of 4-amino-3-hydroxybenzoic acid produced in a culture of the microorganism and comparing it with the amount of 4-amino-3-hydroxybenzoic acid produced by the microorganism before modification. The amount of 4-amino-3-hydroxybenzoic acid produced can be determined by culturing the microorganism and measuring the amount of 4-amino-3-hydroxybenzoic acid produced by HPLC or the like. Based on the measurement results, a microbial strain having the desired ability to produce 4-amino-3-hydroxybenzoic acid can be obtained.

[0044] In the present invention, the production of 4-amino-3-hydroxybenzoic acids or salts thereof comprises a step of culturing the above-mentioned microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source, and is carried out by recovering 4-amino-3-hydroxybenzoic acids or salts thereof from the medium. In culturing the above-mentioned microbial species exemplified as species capable of producing 4-amino-3-hydroxybenzoic acid, glucose is typically used as a sugar source. On the other hand, as shown in the Examples below, when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured in a medium containing sucrose as a sugar source, the conversion rate of 4-aminobenzoic acids to 4-amino-3-hydroxybenzoic acids is improved compared to when the microorganism is cultured in a medium not containing sucrose as a sugar source. More specifically, when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured in a medium containing sucrose as a sugar source, the conversion rate of 4-aminobenzoic acids to 4-amino-3-hydroxybenzoic acids is improved compared to when the microorganism is cultured in a medium containing only glucose as a sugar source. Here, the conversion rate of 4-aminobenzoic acids to 4-amino-3-hydroxybenzoic acids refers to the percentage of the amount of 4-amino-3-hydroxybenzoic acids relative to the sum of the amounts of 4-amino-3-hydroxybenzoic acids and 4-aminobenzoic acids. Furthermore, when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured in a medium containing sucrose as a sugar source, discoloration of the cultured product of the microorganism is suppressed compared to when the microorganism is cultured in a medium not containing sucrose as a sugar source. More specifically, when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured in a medium containing sucrose as a sugar source, discoloration of the cultured product of the microorganism is suppressed compared to when the microorganism is cultured in a medium containing only glucose as a sugar source. Here, since discoloration of the cultured product is thought to be due to oxidation of 4-amino-3-hydroxybenzoic acids, the suppression of discoloration of the cultured product means that oxidation of 4-amino-3-hydroxybenzoic acids is suppressed. Therefore, according to the method of the present invention, the target 4-amino-3-hydroxybenzoic acids or salts thereof can be produced with high purity.

[0045] In the present invention, the 4-amino-3-hydroxybenzoic acids include those represented by the following general formula (1):

[0046] [ka]

[0047] [In the formula, R 1 represents a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH3), an amino group (-NH2), a fluorine atom (-F), a chlorine atom (-Cl), a bromine atom (-Br), an iodine atom (-I), a carboxy group (-COOH), a methyl group (-CH3) or an ethyl group (-CH2CH3), and R 2 represents a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH3), an amino group (-NH2), a fluorine atom (-F), a chlorine atom (-Cl), a bromine atom (-Br), an iodine atom (-I), a carboxy group (-COOH), a methyl group (-CH3) or an ethyl group (-CH2CH3), and X 1 and X 2 represents a hydrogen atom or a hydroxy group, and at least one of them represents a hydroxy group. Examples of the 4-amino-3-hydroxybenzoic acid include 4-amino-3-hydroxybenzoic acid represented by the following formula:

[0048] R 1 The functional group represented by is preferably a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH3), a fluorine atom (-F) or a methyl group (-CH3). R 2 The functional group represented by is preferably a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH3), a fluorine atom (-F) or a methyl group (-CH3). R 1 and R 2 and are more preferably both hydrogen atoms. Also, X 1 and X 2 may both be hydroxy groups, but at least X 1 or X 2 One of the groups is a hydroxy group.

[0049] Salts of 4-amino-3-hydroxybenzoic acids include base addition salts, acid addition salts, etc. Examples of base addition salts include salts with alkali metals such as sodium and potassium, and salts with alkaline earth metals such as calcium and magnesium, while examples of acid addition salts include mineral acid salts such as hydrochlorides, sulfates, nitrates, and phosphates.

[0050] In the present invention, the 4-aminobenzoic acids specifically include those represented by the following general formula (2):

[0051] [ka]

[0052] [In the formula, R 1 and R 2 indicates the same as above.] and 4-aminobenzoic acid or a derivative thereof represented by the following formula:

[0053] Salts of 4-aminobenzoic acids include base addition salts, acid addition salts, etc. Examples of base addition salts include salts with alkali metals such as sodium and potassium, and salts with alkaline earth metals such as calcium and magnesium, while examples of acid addition salts include mineral acid salts such as hydrochlorides, sulfates, nitrates, and phosphates.

[0054] The medium used for culture contains sucrose (cane sugar) as a sugar source. "Containing sucrose as a sugar source in the medium" means that part or all of the sugar source in the medium is sucrose at least at one point during culture. For example, the medium may contain sucrose as a sugar source at the start of culture, or sucrose may be added as a sugar source during culture, but it is preferable for the medium to contain sucrose as a sugar source at the start of culture. The sucrose content in the medium may be more than 0% by weight (hereinafter also referred to as wt.) of the total amount of sugar sources in the medium, and is preferably 1 wt.% or more, more preferably 2 wt.% or more, even more preferably 5 wt.% or more, even more preferably 20 wt.% or more, and even more preferably 30 wt.% or more and 100 wt.% or less. The sucrose content in the medium may be more than 0 wt% and not more than 100 wt% of the total amount of sugar sources in the medium, and is preferably 1 to 100 wt%, more preferably 2 to 100 wt%, even more preferably 5 to 100 wt%, even more preferably 20 to 100 wt%, and even more preferably 30 to 100 wt%. When sucrose is added at the start of culture or during culture, the medium at the time of addition should have the above-mentioned sucrose content. In this specification, weight is calculated based on the standard gravitational acceleration (9.80665 m / s 2 ) means the weight at

[0055] The medium used for the culture preferably contains only sucrose as a sugar source, but may also contain sugars other than sucrose as sugar sources. Examples of sugars that may be contained in the medium used for the culture include monosaccharides such as glucose, fructose, mannose, arabinose, xylose, and galactose, as well as sugars (excluding sucrose) that can be metabolized to produce glucose. These sugars include oligosaccharides or polysaccharides having glucose units, such as disaccharides such as cellobiose, lactose, maltose, trehalose, cellobiose, and xylobiose; and polysaccharides such as dextrin or soluble starch. Molasses can also be used as a raw material containing these raw material compounds. In addition, saccharified solutions containing multiple sugars such as glucose, which are obtained by saccharifying inedible agricultural waste such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw, etc.), bagasse, and corn stover, energy crops such as switchgrass, napier grass, and miscanthus, wood chips, and waste paper using saccharifying enzymes, etc., can also be used. Among these, glucose is preferred as a sugar source other than sucrose.

[0056] When the medium contains sugars other than sucrose in addition to sucrose as a sugar source, the content of sugars other than sucrose in the medium can be more than 0 wt%, for example, 1, 5, 10, 30, 50, 60, 70, 90, 95, 98, 99 wt% or more, and less than 100 wt%, relative to the total amount of sugar sources in the medium. Furthermore, the content of sugars other than sucrose in the medium may be greater than 0 wt% and less than 100 wt%, 1 wt% or more and less than 100 wt%, 5 wt% or more and less than 100 wt%, 10 wt% or more and less than 100 wt%, 30 wt% or more and less than 100 wt%, 50 wt% or more and less than 100 wt%, 60 wt% or more and less than 100 wt%, 70 wt% or more and less than 100 wt%, 90 wt% or more and less than 100 wt%, 95 wt% or more and less than 100 wt%, 98 wt% or more and less than 100 wt%, or 99 wt% or more and less than 100 wt%, relative to the total amount of sugar sources in the medium. The remainder is sucrose. When sucrose is added at the start of culture or during culture, the medium at the time of addition should satisfy the above-mentioned content of sugars other than sucrose.

[0057] The concentration of the sugar source in the medium is preferably 1 weight / volume % (hereinafter also referred to as w / v%) or more, more preferably 5 w / v% or more, and preferably 20 w / v% or less, more preferably 15 w / v% or less, and preferably 1 to 20 w / v%, more preferably 5 to 15 w / v%. In this specification, the volume refers to the volume at 25°C and 1 atmosphere.

[0058] The medium used for the culture may be either a natural medium or a synthetic medium, as long as it contains, in addition to the above sugar source, a nitrogen source, inorganic salts, etc., and is a medium that can efficiently culture a microorganism capable of producing 4-amino-3-hydroxybenzoic acid. Furthermore, although the medium contains the above sugar source as a carbon source, it may also contain a carbon source other than the above sugar source.

[0059] 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.

[0060] 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. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.

[0061] Carbon sources other than the above sugar sources include 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. Carbon sources can be used singly or in combination of two or more.

[0062] Examples of media include A medium [J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)], BT medium [J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)], and CGXII medium [Japanese Patent No. 6322576]. These media may be used with the sugar source content within the above range.

[0063] The culture temperature or reaction temperature is preferably 15° C. or higher, more preferably 25° C. or higher, and is preferably 45° C. or lower, more preferably 37° C. or lower, and is preferably 15 to 45° C., more preferably 25 to 37° C. The culture or reaction time is preferably 24 hours or more and 168 hours or less, more preferably 96 hours or less, and even more preferably 72 hours or less. It is 24 to 168 hours, preferably 24 to 96 hours, and more preferably 24 to 72 hours. The culture or reaction can be carried out with stirring or shaking as needed. Antibiotics such as ampicillin or kanamycin may be added to the medium during culture as needed. The culture may be performed by any of batch, fed-batch, and continuous methods, with the batch method being preferred. The culture or reaction may be carried out under aerobic conditions or under 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 microorganisms, for example, under conditions that suppress aeration and agitation to suppress the amount of oxygen supply, in terms of the production efficiency of 4-amino-3-hydroxybenzoic acids or salts thereof.

[0064] The method for recovering and purifying 4-amino-3-hydroxybenzoic acids or salts thereof from the culture is not particularly limited. That is, the recovery and purification can be carried out by combining well-known methods such as ion exchange resin methods, precipitation methods, crystallization methods, recrystallization methods, concentration methods, and others. For example, 4-amino-3-hydroxybenzoic acids or salts thereof can be obtained by removing the bacterial cells by centrifugation or the like, followed by crystallization and separation. The 4-amino-3-hydroxybenzoic acids or salts thereof accumulated in the culture may be used as is without isolation.

[0065] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A method for producing 4-amino-3-hydroxybenzoic acids or salts thereof, comprising the step of culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source. <2> The conversion rate of 4-aminobenzoic acids or salts thereof to 4-amino-3-hydroxybenzoic acids or salts thereof is improved compared to when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured using a medium that does not contain sucrose as a sugar source. <1> The method described. <3> The coloring of the culture is suppressed compared to when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured in a medium that does not contain sucrose as a sugar source. <1> The method described. <4> The method further comprises recovering 4-amino-3-hydroxybenzoic acids or salts thereof from the culture. <1> ~ <3> The method according to any one of the preceding claims. <5> A method for improving the conversion rate of 4-aminobenzoic acids or salts thereof to 4-amino-3-hydroxybenzoic acids or salts thereof, comprising a step of culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source. <6> The conversion rate of 4-aminobenzoic acids or salts thereof to 4-amino-3-hydroxybenzoic acids or salts thereof is improved compared to when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured using a medium that does not contain sucrose as a sugar source. <5> The method described. <7> A method for inhibiting discoloration of a culture of a microorganism capable of producing 4-amino-3-hydroxybenzoic acid, comprising the step of culturing the microorganism in a medium containing sucrose as a sugar source. <8> The coloring of the culture is suppressed compared to when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured in a medium that does not contain sucrose as a sugar source. <7> The method described. <9> A method for improving the purity of 4-amino-3-hydroxybenzoic acids or salts thereof in a culture of a microorganism capable of producing 4-amino-3-hydroxybenzoic acid, comprising a step of culturing the microorganism in a medium containing sucrose as a sugar source. <10> The purity of 4-amino-3-hydroxybenzoic acids or salts thereof in the culture is improved compared to when a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is cultured using a medium that does not contain sucrose as a sugar source. <9> The method described.

[0066] <11> The sucrose content in the medium is more than 0 wt% and less than 100 wt% based on the total amount of sugar sources in the medium; <1> ~ <10> The method according to any one of the preceding claims. <12> The sucrose content in the medium is preferably 1 to 100 wt%, more preferably 2 to 100 wt%, even more preferably 5 to 100 wt%, even more preferably 20 to 100 wt%, and even more preferably 30 to 100 wt%, based on the total amount of sugar sources in the medium. <1> ~ <11> The method according to any one of the preceding claims. <13> The sucrose content in the medium is preferably 1 wt% or more, more preferably 2 wt% or more, even more preferably 5 wt% or more, even more preferably 20 wt% or more, even more preferably 30 wt% or more and 100 wt% or less, based on the total amount of sugar sources in the medium; <1> ~ <11> The method according to any one of the preceding claims. <14> The medium contains sugars other than sucrose as a sugar source. <1> ~ <13> The method according to any one of the preceding claims. <15> The content of sugars other than sucrose in the medium is greater than 0 wt% and less than 100 wt%, 1 wt% or more and less than 100 wt%, 5 wt% or more and less than 100 wt%, 10 wt% or more and less than 100 wt%, 30 wt% or more and less than 100 wt%, 50 wt% or more and less than 100 wt%, 60 wt% or more and less than 100 wt%, 70 wt% or more and less than 100 wt%, 90 wt% or more and less than 100 wt%, 95 wt% or more and less than 100 wt%, 98 wt% or more and less than 100 wt%, or 99 wt% or more and less than 100 wt%, <14> The method described. <16> The sugars other than sucrose are monosaccharides selected from glucose, fructose, mannose, arabinose, xylose and galactose, or sugars (excluding sucrose) that can produce glucose through metabolism. <14> or <15> The method described. <17> the saccharide capable of producing glucose through metabolism is an oligosaccharide or polysaccharide having a glucose unit, and is a disaccharide selected from cellobiose, lactose, maltose, trehalose, cellobiose, and xylobiose, or a polysaccharide selected from dextrin and soluble starch; <16> The method described. <18> The sugar other than sucrose is glucose, <16> The method described. <19> The medium not containing sucrose as a sugar source is a medium containing only glucose as a sugar source; <2> , <3> , <6> , <8> or <10> The method described. <20> The concentration of the sugar source in the medium is preferably 1 w / v% or more, more preferably 5 w / v% or more. <1> ~ <19> The method according to any one of the preceding claims. <21> The concentration of the sugar source in the medium is preferably 20 w / v% or less, more preferably 15 w / v% or less. <1> ~ <20> The method according to any one of the preceding claims. <22> The concentration of the sugar source in the medium is preferably 1 to 20 w / v%, more preferably 5 to 15 w / v%. <1> ~ <19> The method according to any one of the preceding claims.

[0067] <23> The microorganism capable of producing 4-amino-3-hydroxybenzoic acid is (A) a microorganism in which expression of a polypeptide having activity of hydroxylating 3-position of 4-aminobenzoic acid is enhanced, or (B) a microorganism in which expression of a polypeptide necessary for biosynthesis of 4-aminobenzoic acid from chorismate is enhanced in addition to (A). <1> ~ <22> The method according to any one of the preceding claims. <24> (A) The polypeptide having 4-aminobenzoic acid 3-hydroxylating activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 31, and having 4-aminobenzoic acid 3-hydroxylating activity. <23> The method described. <25> (A) The polypeptide having 4-aminobenzoic acid 3-hydroxylating activity consists of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 31, and is a polypeptide having 4-aminobenzoic acid 3-hydroxylating activity, and is one or more mutant polypeptides selected from a mutant in which position 47 or a position corresponding thereto of the amino acid sequence shown in SEQ ID NO: 31 is leucine, a mutant in which position 106 or a position corresponding thereto is alanine, a mutant in which position 201 or a position corresponding thereto is phenylalanine, a mutant in which position 222 or a position corresponding thereto is phenylalanine, and a mutant in which position 294 or a position corresponding thereto is serine. <23> or <24> The method described. <26> (A) A microorganism in which the expression of a polypeptide having an activity of hydroxylating 3-position of 4-aminobenzoic acid is enhanced is a microorganism in which the expression of a polynucleotide encoding the polypeptide is enhanced. <23> ~ <25> The method according to any one of the preceding claims. <27> (A) the polynucleotide encoding the polypeptide having the activity of hydroxylating 3-position of 4-aminobenzoic acid is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 36, or a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 36 and encoding the polypeptide having the activity of hydroxylating 3-position of 4-aminobenzoic acid; <26> The method described. <28> (B) The polypeptide necessary for biosynthesis of 4-aminobenzoic acid from chorismate is (B1) a polypeptide having 4-amino-4-deoxychorismate synthase activity or (B2) a polypeptide having 4-amino-4-deoxychorismate lyase activity. <23> ~ <27> The method according to any one of the preceding claims. <29> (B1) The polypeptide having 4-amino-4-deoxychorismate synthase activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 32, and is a polypeptide having 4-amino-4-deoxychorismate synthase activity. <28> The method described. <30> (B1) A microorganism in which expression of a polypeptide having 4-amino-4-deoxychorismate synthase activity is enhanced is a microorganism in which expression of a polynucleotide encoding the polypeptide is enhanced. <28> or <29> The method described. <31> (B1) The polynucleotide encoding a polypeptide having 4-amino-4-deoxychorismate synthase activity is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 37, or a polynucleotide having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 37, and encoding a polypeptide having 4-amino-4-deoxychorismate synthase activity. <30> The method described. <32> (B2) The polypeptide having 4-amino-4-deoxychorismate lyase activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 33, and is a polypeptide having 4-amino-4-deoxychorismate lyase activity. <28> ~ <31> The method described. <33> (B2) A microorganism in which expression of a polypeptide having 4-amino-4-deoxychorismate lyase activity is enhanced is a microorganism in which expression of a polynucleotide encoding the polypeptide is enhanced. <28> or <32> The method described. <34> (B2) The polynucleotide encoding a polypeptide having 4-amino-4-deoxychorismate lyase activity is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 38, or a polynucleotide having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% to the nucleotide sequence shown in SEQ ID NO: 38, and encoding a polypeptide having 4-amino-4-deoxychorismate lyase activity. <33> The method described. <35> The microorganism capable of producing 4-amino-3-hydroxybenzoic acid is (C) a microorganism in which expression of a polypeptide required for the biosynthetic pathway from phosphoenolpyruvate to chorismate is enhanced. <23> ~ <34> The method according to any one of the preceding claims. <36> (C) The polypeptide required for the biosynthetic pathway from phosphoenolpyruvate to chorismate is selected from (C1) a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, (C2) a polypeptide having 3-dehydroquinate synthase activity, (C3) a polypeptide having dehydroquinate dehydratase activity, (C4) a polypeptide having shikimate dehydrogenase activity, (C5) a polypeptide having shikimate kinase activity, (C6) a polypeptide having 5-enolate pyruvylshikimate-3-phosphate synthase activity, and (C7) a polypeptide having chorismate synthase activity. <35> The method described. <37> (C1) The polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence set forth in SEQ ID NO: 34, and a polypeptide having acid aldolase activity, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 35, or a polypeptide consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 35, and having 2-dehydro-3-deoxyarabinoheptonate aldolase activity; <36> The method described. <38> (C1) A microorganism in which expression of a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity is enhanced is a microorganism in which expression of a polynucleotide encoding the polypeptide is enhanced. <36> or <37> The method described. <39> (C1) A polynucleotide encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, still more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and still more preferably 99.5% identity to the nucleotide sequence set forth in SEQ ID NO: 39, and or a polynucleotide encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, or a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 40, or a polynucleotide consisting of a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the nucleotide sequence set forth in SEQ ID NO: 40, and encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity. <38> The method described. <40> The microorganism is a microorganism belonging to the genus Corynebacterium, Pantoea, or Escherichia. <1> ~ <39> The method according to any one of the preceding claims. <41> The microorganism is Corynebacterium glutamicum, Pantoea ananatis, or Escherichia coli; <1> ~ <40> The method according to any one of the preceding claims.

[0068] <42> The 4-amino-3-hydroxybenzoic acid is represented by the following general formula (1):

[0069] [ka]

[0070] [In the formula, R 1represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group; R 2 represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group; X 1 and X 2 represents a hydrogen atom or a hydroxy group, and at least one of them represents a hydroxy group. 4-amino-3-hydroxybenzoic acid or a derivative thereof represented by the formula: <1> ~ <41> The method according to any one of the preceding claims. <43> The 4-aminobenzoic acid compound is represented by the following general formula (2):

[0071] [ka]

[0072] [In the formula, R 1 and R 2 indicates the same as above.] 4-aminobenzoic acid or a derivative thereof represented by <2> , <5> or <6> The method described.

[0073] <44> Use of sucrose for producing 4-amino-3-hydroxybenzoic acids or salts thereof in a microorganism capable of producing 4-amino-3-hydroxybenzoic acid. <45> Use of sucrose to improve the conversion rate from 4-aminobenzoic acids or salts thereof to 4-amino-3-hydroxybenzoic acids or salts thereof in a microorganism capable of producing 4-amino-3-hydroxybenzoic acid. <46> Use of sucrose for inhibiting discoloration in a culture of a microorganism capable of producing 4-amino-3-hydroxybenzoic acid. <47> Use of sucrose to improve the purity of 4-amino-3-hydroxybenzoic acids or salts thereof in a culture of a microorganism capable of producing 4-amino-3-hydroxybenzoic acid.

[0074] <48> Sucrose is used in an amount of more than 0 wt% and not more than 100 wt% based on the total amount of sugar sources in a medium for culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid; <44> ~ <47> The use according to any one of the preceding claims. <49> Sucrose is used in an amount of preferably 1 to 100 wt%, more preferably 2 to 100 wt%, even more preferably 5 to 100 wt%, even more preferably 20 to 100 wt%, and even more preferably 30 to 100 wt%, based on the total amount of sugar sources in a medium for culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid. <44> ~ <48> The use according to any one of the preceding claims. <50> Sucrose is used in an amount of preferably 1 wt% or more, more preferably 2 wt% or more, even more preferably 5 wt% or more, even more preferably 20 wt% or more, even more preferably 30 wt% or more and 100 wt% or less, based on the total amount of sugar sources in a medium for culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid. <44> ~ <48> The use according to any one of the preceding claims. <51> The medium contains sugars other than sucrose as a sugar source. <44> ~ <50> The use according to any one of the preceding claims. <52> The content of sugars other than sucrose in the medium is greater than 0 wt% and less than 100 wt%, 1 wt% or more and less than 100 wt%, 5 wt% or more and less than 100 wt%, 10 wt% or more and less than 100 wt%, 30 wt% or more and less than 100 wt%, 50 wt% or more and less than 100 wt%, 60 wt% or more and less than 100 wt%, 70 wt% or more and less than 100 wt%, 90 wt% or more and less than 100 wt%, 95 wt% or more and less than 100 wt%, 98 wt% or more and less than 100 wt%, or 99 wt% or more and less than 100 wt%, <51> Use as described. <53> The sugars other than sucrose are monosaccharides selected from glucose, fructose, mannose, arabinose, xylose and galactose, or sugars (excluding sucrose) that can produce glucose through metabolism. <51> or <52> Use as described. <54> the saccharide capable of producing glucose through metabolism is an oligosaccharide or polysaccharide having a glucose unit, and is a disaccharide selected from cellobiose, lactose, maltose, trehalose, cellobiose, and xylobiose, or a polysaccharide selected from dextrin and soluble starch; <53> Use as described. <55> The sugar other than sucrose is glucose, <53> Use as described. <56> The concentration of the sugar source in the medium is preferably 1 w / v% or more, more preferably 5 w / v% or more. <48> ~ <55> The use according to any one of the preceding claims. <57> The concentration of the sugar source in the medium is preferably 20 w / v% or less, more preferably 15 w / v% or less. <48> ~ <56> The use according to any one of the preceding claims. <58> The concentration of the sugar source in the medium is preferably 1 to 20 w / v%, more preferably 5 to 15 w / v%. <48> ~ <55> The use according to any one of the preceding claims.

[0075] <59> The microorganism capable of producing 4-amino-3-hydroxybenzoic acid is (A) a microorganism in which expression of a polypeptide having activity of hydroxylating 3-position of 4-aminobenzoic acid is enhanced, or (B) a microorganism in which expression of a polypeptide necessary for biosynthesis of 4-aminobenzoic acid from chorismate is enhanced in addition to (A). <44> ~ <58> The use according to any one of the preceding claims. <60> (A) The polypeptide having 4-aminobenzoic acid 3-hydroxylating activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity to the amino acid sequence shown in SEQ ID NO: 31, and having 4-aminobenzoic acid 3-hydroxylating activity. <59> Use as described. <61> (A) The polypeptide having 4-aminobenzoic acid 3-hydroxylating activity consists of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 31, and is a polypeptide having 4-aminobenzoic acid 3-hydroxylating activity, and is one or more mutant polypeptides selected from a mutant in which position 47 or a position corresponding thereto of the amino acid sequence shown in SEQ ID NO: 31 is leucine, a mutant in which position 106 or a position corresponding thereto is alanine, a mutant in which position 201 or a position corresponding thereto is phenylalanine, a mutant in which position 222 or a position corresponding thereto is phenylalanine, and a mutant in which position 294 or a position corresponding thereto is serine. <59> or <60> Use as described. <62> (A) A microorganism in which the expression of a polypeptide having an activity of hydroxylating 3-position of 4-aminobenzoic acid is enhanced is a microorganism in which the expression of a polynucleotide encoding the polypeptide is enhanced. <59> ~ <61> The use according to any one of the preceding claims. <63> (A) the polynucleotide encoding the polypeptide having the activity of hydroxylating 3-position of 4-aminobenzoic acid is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 36, or a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 36 and encoding the polypeptide having the activity of hydroxylating 3-position of 4-aminobenzoic acid; <62> The method described. <64> (B) The polypeptide necessary for biosynthesis of 4-aminobenzoic acid from chorismate is (B1) a polypeptide having 4-amino-4-deoxychorismate synthase activity or (B2) a polypeptide having 4-amino-4-deoxychorismate lyase activity. <59> ~ <63> The use according to any one of the preceding claims. <65> (B1) The polypeptide having 4-amino-4-deoxychorismate synthase activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 32, and is a polypeptide having 4-amino-4-deoxychorismate synthase activity. <64> Use as described. <66> (B1) A microorganism in which expression of a polypeptide having 4-amino-4-deoxychorismate synthase activity is enhanced is a microorganism in which expression of a polynucleotide encoding the polypeptide is enhanced. <64> or <65> Use as described. <67> (B1) The polynucleotide encoding a polypeptide having 4-amino-4-deoxychorismate synthase activity is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 37, or a polynucleotide having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 37, and encoding a polypeptide having 4-amino-4-deoxychorismate synthase activity. <66> Use as described. <68> (B2) The polypeptide having 4-amino-4-deoxychorismate lyase activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence shown in SEQ ID NO: 33, and is a polypeptide having 4-amino-4-deoxychorismate lyase activity. <64> ~ <67> The use according to any one of the preceding claims. <69> (B2) A microorganism in which expression of a polypeptide having 4-amino-4-deoxychorismate lyase activity is enhanced is a microorganism in which expression of a polynucleotide encoding the polypeptide is enhanced. <64> or <68> Use as described. <70> (B2) The polynucleotide encoding a polypeptide having 4-amino-4-deoxychorismate lyase activity is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 38, or a polynucleotide having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% to the nucleotide sequence shown in SEQ ID NO: 38, and encoding a polypeptide having 4-amino-4-deoxychorismate lyase activity. <69> The method described. <71> The microorganism capable of producing 4-amino-3-hydroxybenzoic acid is (C) a microorganism in which expression of a polypeptide required for the biosynthetic pathway from phosphoenolpyruvate to chorismate is enhanced. <59> ~ <70> The method according to any one of the preceding claims. <72> (C) The polypeptide required for the biosynthetic pathway from phosphoenolpyruvate to chorismate is selected from (C1) a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, (C2) a polypeptide having 3-dehydroquinate synthase activity, (C3) a polypeptide having dehydroquinate dehydratase activity, (C4) a polypeptide having shikimate dehydrogenase activity, (C5) a polypeptide having shikimate kinase activity, (C6) a polypeptide having 5-enolate pyruvylshikimate-3-phosphate synthase activity, and (C7) a polypeptide having chorismate synthase activity. <71> The method described. <73> (C1) The polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence set forth in SEQ ID NO: 34, and a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, or a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 35, or a polypeptide consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the amino acid sequence set forth in SEQ ID NO: 35, and having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, <72> Use as described. <74> (C1) A microorganism in which expression of a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity is enhanced is a microorganism in which expression of a polynucleotide encoding the polypeptide is enhanced. <72> or <73> Use as described. <75> (C1) A polynucleotide encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the nucleotide sequence set forth in SEQ ID NO: 39, and or a polynucleotide encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity, or a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 40, or a polynucleotide consisting of a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% identity to the nucleotide sequence set forth in SEQ ID NO: 40, and encoding a polypeptide having 2-dehydro-3-deoxyarabinoheptonate aldolase activity. <74> Use as described. <76> The microorganism is a microorganism belonging to the genus Corynebacterium, Pantoea, or Escherichia. <44> ~ <75> The use according to any one of the preceding claims. <77> The microorganism is Corynebacterium glutamicum, Pantoea ananatis, or Escherichia coli; <44> ~ <76> The use according to any one of the preceding claims. <78> The 4-amino-3-hydroxybenzoic acid is 4-amino-3-hydroxybenzoic acid represented by the above general formula (1) or a derivative thereof. <44> ~ <77> The use according to any one of the preceding claims. <79> The 4-aminobenzoic acid is 4-aminobenzoic acid represented by the above general formula (2) or a derivative thereof. <45> Use as described. [Example]

[0076] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0077] Example 1 Preparation of 4-amino-3-hydroxybenzoic acid (1) Introduction of the plasmid into the host cell Using pKCG1_PtufT1_HFM122_M106A_T294S (see JP 2024-14569 A), Corynebacterium glutamicum KC617 strain (preparation method described below) was transformed by electroporation (ELEPO21, Nepa Gene Co., Ltd.). The resulting transformed cell solution was spread on LBKm agar medium and allowed to stand at 30°C for 2 days, and the resulting colonies were used as transformed strains.

[0078] (2) Cultivation of transformed strains The transformed strains obtained above were precultured (30°C) in CGTG15 medium (containing 50 μg / mL kanamycin sulfate) shown in Table 7. Glucose (Glc) and sucrose (Suc) were added to the CGT1 medium (Table 8) to the concentrations indicated below to prepare the medium. Six mL of the prepared medium was inoculated and cultured at 30°C for 24 hours. The culture was then diluted appropriately with dilute sulfuric acid, centrifuged to remove the bacterial cells, and the supernatant was collected. The concentrations of 4-aminobenzoic acid (4-ABA) and 4-amino-3-hydroxybenzoic acid (4,3-AHBA) in the supernatant were quantified. The absorbance of the supernatant at 470 nm (Ab470) was also measured. The results are shown in Tables 1 to 6.

[0079] (2-1) The total sugar concentration was set to 100 g / L, and the weight ratio of Glc / Suc was changed as appropriate. The first study was conducted with concentrations of 100 / 0, 90 / 10, 70 / 30, 60 / 40, and 50 / 50. The results are shown in Tables 1 and 2. It was shown that the addition of sucrose improved the conversion rate (%) of 4-aminobenzoic acid (4-ABA) to 4-amino-3-hydroxybenzoic acid (4,3-AHBA) (4,3-AHBA / (4-ABA + 4,3-AHBA) × 100) and reduced the color.

[0080] [Table 1]

[0081] [Table 2]

[0082] (2-2) The experiments were carried out with Glc / Suc ratios of 100 / 0, 95 / 5, 70 / 30, 30 / 70, 10 / 90, 5 / 95, and 0 / 100. The results are shown in Tables 3 and 4. It was shown that the conversion rate improved and color reduction was possible even when 50% or more sucrose was added.

[0083] [Table 3]

[0084] [Table 4]

[0085] (2-3) The experiments were carried out with Glc / Suc ratios of 100 / 0, 99 / 1, 98 / 2, 97 / 3, and 95 / 5. The results are shown in Tables 5 and 6. It was shown that the conversion rate improved even with the addition of 1% sucrose, and that color reduction was possible.

[0086] [Table 5]

[0087] [Table 6]

[0088] [Table 7]

[0089] [Table 8]

[0090] <Analysis conditions> The collected supernatant was subjected to removal of insoluble matter using an AcroPrep 96 filter plate (0.2 μm GHP membrane, Nippon Pall). The reaction mixture was then subjected to HPLC. The HPLC system used was a Chromaster (Hitachi High-Tech Science). Analysis of 4,3-AHBA and 4-ABA was performed using an L-column ODS (4.6 mm ID × 150 mm, Chemicals Evaluation and Research Institute, Japan) with gradient elution at a flow rate of 1.0 mL / min and a column temperature of 40°C, using 0.1 M potassium dihydrogen phosphate in 0.1% phosphoric acid as eluent A and 70% methanol as eluent B. 4,3-AHBA and 4-ABA were detected using a UV detector (detection wavelength 280 nm). Concentration calibration curves were prepared using standard samples [4,3-AHBA (Tokyo Chemical Industry Co., Ltd.) and 4-ABA (Tokyo Chemical Industry Co., Ltd.)], and quantification was performed based on the calibration curves. Ab470 was measured using a spectrophotometer. After the culture medium was centrifuged to remove the bacterial cells, it was diluted appropriately with deionized water and then measured.

[0091] Reference Example 1: Construction of Corynebacterium glutamicum KC617 strain 1) Preparation of pKCG1_PtufT1-aroGec_D146N pKCG1_PtufT1 (see Patent Application No. 2022-117492) was used as a template for PCR amplification using two DNA primers (SEQ ID NOs: 1 and 2). The resulting PCR product was treated with DpnI (Takara Bio). The aroG gene from Escherichia coli was mutated from D to N at amino acid position 146 and codon-optimized for Corynebacterium glutamicum (SEQ ID NO: 3, Eurofin Genomics). This gene fragment was synthesized and amplified by PCR using two DNA primers (SEQ ID NOs: 4 and 5). The resulting two PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated using the In-Fusion HD Cloning Kit (Takara Bio) to create the plasmid pKCG1_PtufT1-aroGec_D146N.

[0092] 2) Construction of a plasmid for introducing the aroGec_D146N gene linked to the tuf gene promoter and rrnB terminator into the cg2567 gene region Using the genome of the ATCC13032 strain as a template, the 5' upstream region of the cg2567 gene (SEQ ID NO: 6) was amplified by PCR using two DNA primers (SEQ ID NO: 7 and 8), and the 3' region of the cg2567 gene (SEQ ID NO: 9) was amplified by PCR using two DNA primers (SEQ ID NO: 10 and 11) to obtain DNA fragments. The aroGec_D146N gene fused to the tuf promoter and rrnB terminator was amplified by PCR using pKCG1_PtufT1_Ptu-aroGec_D146N as a template and two DNA primers (SEQ ID NO: 12 and 13) to obtain DNA fragments. Furthermore, pHKPsacB1 (see Japanese Patent Publication No. 6322576) was amplified by PCR using two DNA primers (SEQ ID NO: 14 and 15), and the resulting PCR products were treated with DpnI (Takara Bio). The four PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) to separate the DNA fragments, and then ligated using the In-Fusion HD Cloning Kit (Takara Bio) to create the plasmid pHKPsacB_Δcg2567::Ptu-aroGec_D146N_OPT.

[0093] 3) Construction of a strain in which the aroGec_D146N gene linked to the tuf gene promoter and rrnB terminator was introduced into the cg2567 gene region. The KC594sr strain was obtained by introducing the above-mentioned plasmid pHKPsacB_Δcg2567::Ptu-aroGec_D146N_OPT into the KC551 strain (see Patent Application No. 2022-117492) using electroporation transformation and selecting for kanamycin resistance. The KC594sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 7 and 16. The expected results were obtained, confirming that the KC594sr strain is a single-crossover homologous recombinant into which the plasmid pHKPsacB_Δcg2567::Ptu-aroGec_D146N_OPT had been introduced. The KC594sr 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 spread onto LB agar medium containing 20% ​​sucrose to obtain the KC594 strain. PCR (Sapphire Amp (Takara Bio)) using primers SEQ ID NOs: 16 and 17 confirmed that the KC594 strain was a double-crossover homologous recombinant, carrying the aroGec_D146N gene linked to the tuf gene promoter in the cg2567 gene region, as expected.

[0094] 4) Preparation of pKCG1_PtufT1-aroF_P155L pKCG1_PtufT1 was amplified by PCR using two DNA primers (SEQ ID NOs: 1 and 2) as a template, and the resulting PCR product was treated with DpnI (Takara Bio). The aroF gene fragment (SEQ ID NO: 18) was also amplified by PCR using the genome of the ATCC13032 strain as a template and two DNA primers (SEQ ID NOs: 19 and 20) to obtain a DNA fragment. Each of the resulting PCR products was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated using the In-Fusion HD Cloning Kit (Takara Bio) to produce the plasmid pKCG1_PtufT1-aroF. pKCG1_PtufT1-aroF was amplified by PCR using two DNA primers (SEQ ID NOs: 21 and 22) as a template, and the resulting PCR product was treated with DpnI (Takara Bio). The resulting PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) to separate the DNA fragments, which were then ligated using the In-Fusion HD Cloning Kit (Takara Bio) to create the plasmid pKCG1_PtufT1-aroF_P155L.

[0095] 5) Construction of a plasmid for introducing the aroF_P155L gene linked to the tuf promoter and rrnB terminator into the cg2088 gene region Using the genome of the ATCC13032 strain as a template, the 5' upstream region of the cg2088 gene region (SEQ ID NO: 23) was amplified by PCR using two DNA primers (SEQ ID NOs: 24 and 25), and the 3' upstream region of the cg2088 gene region (SEQ ID NO: 26) was amplified by PCR using two DNA primers (SEQ ID NOs: 27 and 28) to obtain DNA fragments. The aroF_P155L gene linked to the tuf promoter and rrnB terminator was amplified by PCR using pKCG1_PtufT1-aroF_P155L as a template and two DNA primers (SEQ ID NOs: 12 and 13) to obtain DNA fragments. Furthermore, PCR was performed using pHKPsacB1 as a template and two DNA primers (SEQ ID NOs: 14 and 15), and the resulting PCR products were treated with DpnI (Takara Bio). The four PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) to separate the DNA fragments, and then ligated using the In-Fusion HD Cloning Kit (Takara Bio) to create the plasmid pHKPsacB_Δcg2088::Ptu-aroF_P155L.

[0096] 6) Construction of a strain in which the aroF_P155L gene linked to the tuf gene promoter and rrnB terminator was introduced into the cg2088 gene region. The KC617sr strain was isolated by electroporation transformation using the aforementioned plasmid pHKPsacB_Δcg2088::Ptu-aroF_P155L and selection for kanamycin resistance. Analysis of the KC617sr strain by PCR (Sapphire Amp (Takara Bio)) using primers sequence numbers 25 and 30 yielded the expected results, confirming that the KC617sr strain was a single-crossover homologous recombinant containing the pHKPsacB_Δcg2088::Ptu-aroF_P155L plasmid. The KC617sr 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 spread onto LB agar medium containing 20% ​​sucrose to obtain the KC617 strain. PCR (Sapphire Amp (Takara Bio)) using primers SEQ ID NOs: 29 and 30 confirmed that the KC617 strain was a double-crossover homologous recombinant, in which the aroF_P155L gene linked to the tuf gene promoter had been introduced into the cg2088 gene region, as expected.

Claims

1. A method for producing 4-amino-3-hydroxybenzoic acids or salts thereof, comprising the step of culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source.

2. A method for improving the conversion rate of 4-aminobenzoic acids or salts thereof to 4-amino-3-hydroxybenzoic acids or salts thereof, comprising a step of culturing a microorganism capable of producing 4-amino-3-hydroxybenzoic acid in a medium containing sucrose as a sugar source.

3. A method for inhibiting discoloration of a culture of a microorganism capable of producing 4-amino-3-hydroxybenzoic acid, comprising the step of culturing the microorganism in a medium containing sucrose as a sugar source.

4. The method according to any one of claims 1 to 3, wherein the sucrose content in the medium is 1 to 100 wt % based on the total amount of sugar sources in the medium.

5. The method according to any one of claims 1 to 3, wherein the microorganism is a microorganism belonging to the genus Corynebacterium, Pantoea, or Escherichia.

6. 4. The method according to claim 1, wherein the microorganism is Corynebacterium glutamicum, Pantoea ananatis, or Escherichia coli.

7. The 4-amino-3-hydroxybenzoic acid is represented by the following general formula (1): 【Chemistry 1】 [In the formula, R １ represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group; R ２ represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group; X １ and X ２ represents a hydrogen atom or a hydroxy group, and at least one of them represents a hydroxy group.

3. The method according to claim 1, wherein the compound is 4-amino-3-hydroxybenzoic acid or a derivative thereof represented by the formula:

8. The 4-aminobenzoic acid is represented by the following general formula (2): 【Chemistry 2】 [In the formula, R １ represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group; R ２ represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group.

3. The method according to claim 2, wherein the 4-aminobenzoic acid is 4-aminobenzoic acid or a derivative thereof represented by the formula:

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