Method for producing aromatic compounds

Abstract

To provide a method for efficiently producing an aromatic compound by using a transformed coryneform bacterium.SOLUTION: A method for producing an aromatic compound or a salt thereof includes a step of culturing a modified coryneform bacterium in which a function of a cytochrome bd oxidizing enzyme complex is suppressed or deleted.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing aromatic compounds using modified coryneform bacteria.

Background Art

[0002] In recent years, it has been desired to produce useful aromatic compounds such as gallic acid from glucose, which is an inexpensive raw material, using microorganisms. Microorganisms possess a metabolic pathway (shikimic acid pathway) for biosynthesizing aromatic compounds. That is, phosphoenolpyruvate produced in the glycolysis system combines with erythrose 4-phosphate supplied from the pentose phosphate pathway to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP), which then becomes 3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), and finally shikimic acid. Further, a phosphate group is transferred from adenosine 3-phosphate to shikimic acid to form 3-phosphoshikimic acid, which then becomes chorismic acid via 3-phosphoenolpyruvylshikimic acid. In the shikimic acid pathway, after a carbon six-membered ring is formed, a double bond is formed, and from protocatechuic acid derived from DHS, aromatic compounds such as gallic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), catechol, p-aminobenzoic acid (4-ABA), 4-amino-3-hydroxybenzoic acid (4,3-AHBA), etc. are produced (Figure 1).

[0003] Of these, gallic acid, due to its strong reducing properties, is used as a raw material for photographic developers and blue ink, and esters such as propyl gallate are used as antioxidants for oils and butters. Furthermore, pyrogallol, synthesized by decarboxylation of gallic acid, is used as an electronic material, an organic synthesis reagent, a photographic developer, and a mordant for woolen fabrics, making the efficient production of gallic acid beneficial. In addition, polybenzoxazole (PBO), an engineering plastic with excellent heat resistance and mechanical strength, is used as an insulating film for fiber materials and semiconductor devices (Non-Patent Literature 1), and 4-amino-3-hydroxybenzoic acid (4,3-AHBA) derivatives, which can be used as a raw material for its production, have recently attracted attention. Para-aminobenzoic acid (4-ABA), which can be used as a raw material for UV absorbers, pharmaceuticals, and polymers, has conventionally been chemically synthesized, but requires a great deal of energy, so fermentation production technology, an environmentally friendly process, is desired.

[0004] On the other hand, coryneform bacteria are aerobic bacteria that use oxygen as an electron acceptor for respiration. It is known that there are two types of terminal oxidases in the respiratory chain of coryneform bacteria: cytochrome aa3 oxidase and cytochrome bd oxidase (Non-Patent Literature 2). Conventionally, regarding the relationship between respiratory chain-related enzymes and substance production in Corynebacteria, for example, it has been reported that the productivity of lysine, a TCA cycle derivative, improved in strains in which cytochrome bd-type oxidase was disrupted (Non-Patent Literature 3), and that the productivity of diacetyl improved in strains in which cytochrome bd-type oxidase was overexpressed (Non-Patent Literature 4). Furthermore, Patent Literature 1 discloses a microorganism that has been manipulated to inactivate the respiratory pathway in order to increase the amount of NAD(P)H available to oxidoreductases that require NAD(P)H.

[0005] However, it is completely unknown to date that inhibiting or eliminating the function of cytochrome bd-type oxidase improves the productivity of aromatic compounds. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 2008 / 0293101 [Non-patent literature]

[0007] [Non-Patent Document 1] Hiroki Murase, SENI GAKKAISHI (Textiles and Industry), Vol. 66, No. 6 (2010) [Non-Patent Document 2] Michael Bott et al., Journal of Biotechnology,Vol.104, Issues 1-3,2003,129-153, [Non-Patent Document 3] Armin Kabus et al., American Society for Microbiology Applied and Environmental Microbiology Vol.73, 2006, No.3, 861-868 [Non-Patent Document 4] Cong Chen et al., Journal of Biotechnology Vol.332, 2021, 20-28 [Disclosure of the Invention] [Problems that the invention aims to solve]

[0008] The present invention relates to providing a method for efficiently producing aromatic compounds using transformed Corynebacterium-type bacteria. [Means for solving the problem]

[0009] The inventors have found that in transformants in which the function of bd-type oxidase, one of the terminal oxidases of the respiratory chain of Corynebacterium, is suppressed or deleted, the productivity of aromatic compounds, including gallic acid, para-aminobenzoic acid, and 4-amino-3-hydroxybenzoic acid, is improved.

[0010] In other words, the present invention relates to the following 1) to 2). 1) A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a modified Corynebacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted. 2) Modified Corynebacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, and which have at least the following characteristics: (C), (D), (E), and (F): (C) Polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) Polypeptides required for biosynthesis of para-aminobenzoic acid from colismic acid (F) Polypeptide having para-aminobenzoic acid hydroxylase activity Corynebacteria with enhanced expression of one or more polypeptides selected from the following. [Effects of the Invention]

[0011] According to the present invention, it becomes possible to efficiently produce aromatic compounds such as gallic acid, para-aminobenzoic acid, and 4-amino-3-hydroxybenzoic acid, or their salts, by a fermentation method that has a low environmental impact. [Brief explanation of the drawing]

[0012] [Figure 1]A schematic diagram showing the production pathways of various aromatic compounds when Corynebacterium glutamicum is used as the host. 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, qsuD is a gene encoding quinate / shikimate dehydrogenase, aroE3 is shikimate dehydrogenase, qsuB is a gene encoding dehydroshikimate dehydratase, aroA is a gene encoding 5-enolate pyruvylshikimate-3-phosphate synthase, aroC is a gene encoding colimusylate synthase, aroK is a gene encoding shikimate kinase, pabAB is a gene encoding 4-amino-4-deoxycholismylate synthase, and pabC is a gene encoding 4-amino-4-deoxycholismylate lyase. These are endogenous genes of Corynebacterium glutamicum. pobA* is a gene encoding a polypeptide with 3,4-dihydroxybenzoate hydroxylase activity, which is a mutant of p-hydroxybenzoate hydroxylase (pobA) derived from Corynebacterium glutamicum, and phbh* is a gene encoding a polypeptide with para-aminobenzoate hydroxylase activity, which is a mutant of p-hydroxybenzoate hydroxylase derived from Caulobacter vibrioides, and these are metabolic pathways that are introduced via external gene transfer. In the figure, E4P is erythrose 4-phosphate, PEP is phosphoenolpyruvate, DAHP is 3-deoxy-D-arabino-heptulosone 7-phosphate, DHQ is dehydroquinic acid, DHS is dehydroshikimic acid, SHK is shikimic acid, S3P is shikimic acid-3-phosphate, and EPSP is 5-enoylpyruvinylshikimic acid-3-phosphate. [Modes for carrying out the invention]

[0013] 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 homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12 with Unit size to compare (ktup) being 2.

[0014] In the present invention, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more identity.

[0015] In the present invention, an "amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted" means an amino acid sequence in which 1 to 10 amino acids, preferably 1 to 8 amino acids, more preferably 1 to 5 amino acids, and even more preferably 1 to 3 amino acids are deleted, substituted, added, or inserted. Further, a "nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted" means a nucleotide sequence in which 1 to 30 nucleotides, preferably 1 to 24 nucleotides, more preferably 1 to 15 nucleotides, and even more preferably 1 to 9 nucleotides are deleted, substituted, added, or inserted. In the present invention, the "addition" of an amino acid or a nucleotide includes the addition of an amino acid or a nucleotide to one end and both ends of the sequence.

[0016] In the present invention, an aromatic compound is an organic aromatic compound biosynthesized in coryneform bacteria, specifically, an aromatic compound synthesized via the shikimic acid pathway, preferably an aromatic compound derived from 3-dehydroshikimic acid (DHS) or chorismic acid (Figure 1). Specifically, examples include protocatechuic acid, catechol, gallic acid, phenylalanine, tyrosine, tryptophan, para-hydroxybenzoic acid, 4-aminobenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, 4-amino-3-hydroxybenzoic acid, and the like. Among these, protocatechuic acid derived from DHS; gallic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), catechol derived from protocatechuic acid; para-hydroxybenzoic acid, para-aminobenzoic acid (4-ABA), 4-amino-3-hydroxybenzoic acid (4,3-AHBA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), tyrosine, tryptophan, etc. derived from chorismic acid are preferred, and more preferably, gallic acid, para-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid are included.

[0017] Examples of the salt of the aromatic compound include base addition salts, acid addition salts, and the like. Examples of the base addition salts include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, etc., and examples of the acid addition salts include mineral acid salts such as hydrochloride, sulfate, nitrate, phosphate, etc.

[0018] In the method for producing the aromatic compound or its salt of the present invention, a coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted is used. The "cytochrome bd-type oxidase complex" is an enzyme related to aerobic respiration of coryneform bacteria, and specifically, it is one of the terminal oxidases of the respiratory chain. Coryneform bacteria are known to have bd-type oxidase and aa3-type oxidase as terminal oxidases of the respiratory chain. The bd-type oxidase complex has been shown to consist of subunit I and subunit II, and based on the analysis of the N-terminal sequence, it has been shown to correspond to CydA and CydB, respectively (Kusumoto et al. 2000, Arch Microbiol 173:390-397). aa3 oxidase is known to consist of four subunits (CtaD, CtaC, CtaE, CtaF) and is known to form a complex with the cytochrome bc1 complex (QcrB, QcrA, QcrC). (Non-Patent Literature 2) The proton efficiencies of bd oxidase and the bc1-aa3 complex are, respectively, H + It is known that the oxygen / oxygen ratio is 2 and 6, and since the proton efflux efficiency per molecule of oxygen is lower for bd-type oxidases, it has been reported that the final ATP synthesis efficiency is also lower (Matsushita K. (2013) Respiratory Chain and Energy Metabolism of Corynebacterium glutamicum. In: Yukawa H., Inui M. (eds) Corynebacterium glutamicum. Microbiology Monographs, vol 23. Springer, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 978-3-642-29857-8_11).

[0019] In the present invention, preferred cytochrome bd-type oxidase complexes include polypeptide complexes having the cytochrome bd-type oxidase activity shown in (A) or (B) below. (A) A complex consisting of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4. (B) A complex consisting of a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2 and functioning as cytochrome bd-type oxidase subunit I, and a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4 and functioning as cytochrome bd-type oxidase subunit II. Here, (A) the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2 refers to cytochrome bd-type oxidase subunit I derived from Corynebacterium glutamicum, and the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 4 refers to cytochrome bd-type oxidase subunit II derived from Corynebacterium glutamicum.

[0020] In the polypeptide of (A), 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 that have 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 more amino acids are deleted, substituted, added, or inserted from the amino acid sequence shown in SEQ ID NO: 2 or 4.

[0021] The presence of cytochrome bd-type oxidase complex activity can be measured, for example, by referring to the non-patent literature mentioned above (Kusumoto et al. 2000, Arch Microbiol 173:390-397). Specifically, it can be confirmed by extracting cytochrome bd-type oxidase as a membrane protein from bacterial cells, purifying it using a column, and then measuring the reaction of quinol to quinone using the obtained enzyme.

[0022] Polypeptides that consist of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 2 of (B) and function as cytochrome bd-type oxidase subunit I, and polypeptides that consist of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 4 and function as cytochrome bd-type oxidase subunit II include, for example, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium SP.Sa1YVA5, Corynebacterium hadale, Corynebacterium gottingense, Corynebacterium godavarianum, and Corynebacterium Examples include cytochrome bd-type oxidase subunit I and cytochrome bd-type oxidase subunit II, derived from senegalense.

[0023] Methods for introducing mutations such as deletion, substitution, addition, or insertion of amino acids into the amino acid sequence of the above polypeptide include, for example, introducing mutations such as deletion, substitution, addition, or insertion of nucleotides into the nucleotide sequence encoding the amino acid sequence. Methods for introducing mutations into nucleotide sequences include, for example, mutagenesis using chemical mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, and nitrite, or physical mutagens such as ultraviolet light, X-rays, gamma rays, and ion beams, site-directed mutagenesis, and the method described by Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995). Site-directed mutagenesis methods include Splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), ODA method (Hashimoto-Gotoh et al., Gene 152, 271-276, 1995), and Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, there are Site-Directed Mutagenesis System Mutan-SuperExpress Km kits (Takara Bio Inc.) and Transformer TM Commercially available site-directed mutagenesis kits, such as the Site-Directed Mutagenesis Kit (Clonetech) and the KOD-Plus-Mutagenesis Kit (Toyobo), can also be used.

[0024] In the present invention, modified Corynebacterium-type bacteria in which the function of the cytochrome bd-type oxidase complex is suppressed or lost (also referred to as "transformed organisms") include bacterial strains in which the expression of the cytochrome bd-type oxidase complex in the host bacterium is reduced or lost, and the function of the protein as a cytochrome bd-type oxidase complex is suppressed or lost. Preferably, these include bacterial strains (mutagenic strains) in which the expression of the cytochrome bd-type oxidase complex is reduced or lost compared to the parent bacterium by introducing a mutation, and the function of the protein as a cytochrome bd-type oxidase complex is reduced or lost. In the present invention, "expression" of a protein means that a translation product is produced from the gene encoding the protein and is localized to its site of action in a functional state. Reduced or lost expression of cytochrome bd-type oxidase complex by introducing a mutation means that, as a result of modifications at the gene level, transcription level, post-transcriptional regulation level, translation level, or post-translational modification level, the amount of cytochrome bd-type oxidase complex protein present in the transformant is reduced or lost, preferably significantly reduced or lost compared to that in the parent bacterium.

[0025] "Decreased expression of cytochrome bd-type oxidase complex compared to parent bacteria" means that the expression level of cytochrome bd-type oxidase complex in the transformed organism is lower than that of parent bacteria. More specifically, compared to parent bacteria, the expression level of the protein is usually 50% or less, preferably 20% or less, and more preferably 10% or less, and as a result, its activity is also similarly reduced. Most preferably, the expression level of cytochrome bd-type oxidase complex is 0%, i.e., loss of expression of cytochrome bd-type oxidase complex. Furthermore, the comparison of cytochrome bd-type oxidase complex expression levels is performed by measuring the expression level of the polypeptide in question using well-known immunological methods such as Western blotting and immunohistochemistry.

[0026] Modified Corynebacteria in which the expression of the above-mentioned cytochrome bd-type oxidase complex is reduced or lost can be obtained specifically by suppressing the function of the gene encoding the cytochrome bd-type oxidase complex on the chromosomal DNA of the host bacterium. Here, the suppression of function may be either complete suppression (inhibition) or incomplete suppression. A gene encoding a cytochrome bd-type oxidase complex refers to DNA consisting of a transcription region containing an ORF and a transcriptional regulatory region such as the promoter of the gene. In the present invention, the gene encoding a cytochrome bd-type oxidase complex is preferably one of the polynucleotides described in (a) or (b) below.

[0027] (a) A polynucleotide encoding a complex consisting of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 and a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 (b) A polynucleotide encoding a complex consisting of a polynucleotide encoding a polypeptide that functions as cytochrome bd-type oxidase subunit I, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1, and a polynucleotide encoding a polypeptide that functions as cytochrome bd-type oxidase subunit II, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3. Here, (a) the polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 1 refers to the gene cg1301 (cydA) from Corynebacterium glutamicum that encodes cytochrome bd-type oxidase subunit I, and the polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 3 refers to the gene cg1300 (cydB) from Corynebacterium glutamicum that encodes cytochrome bd-type oxidase subunit II.

[0028] 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 more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. The method for introducing mutations such as nucleotide deletion, substitution, addition, or insertion into a nucleotide sequence is as described above. The above 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 chemosynthetic DNA.

[0029] The function of the gene encoding such cytochrome bd-type oxidase complex can be suppressed by introducing a deletion or inactivation mutation into the coding region, non-coding region, or transcription or translation initiation region of the gene encoding the cytochrome bd-type oxidase complex (deletion or inactivation of the gene encoding the cytochrome bd-type oxidase complex), or by introducing a polynucleotide that has the activity to degrade the transcript of the gene encoding the cytochrome bd-type oxidase complex, or a polynucleotide that suppresses the translation of said transcript into protein.

[0030] In one embodiment, deletion or inactivation of a gene encoding a cytochrome bd-type oxidase complex can be achieved by removing part or all of the nucleotide sequence of the gene encoding the cytochrome bd-type oxidase complex from the genome or replacing it with another nucleotide sequence, inserting another polynucleotide fragment into the sequence of the gene encoding the cytochrome bd-type oxidase complex, or introducing a mutation into the transcription or translation initiation region of the gene encoding the cytochrome bd-type oxidase complex. Preferably, part or all of the nucleotide sequence of the gene encoding the cytochrome bd-type oxidase complex is deleted or inactivated. More specific examples include a method for specifically deleting or inactivating a gene encoding a cytochrome bd-type oxidase complex on the genome of a cell, and a method for selecting cells with the desired mutation by introducing a random deletion or inactivation mutation into the gene in bacterial cells, and then evaluating the expression level and activity of the cytochrome bd-type oxidase complex or performing genetic analysis.

[0031] One method for the specific deletion or inactivation of the gene encoding the cytochrome bd-type oxidase complex is homologous recombination. Specifically, a DNA fragment of the gene encoding the cytochrome bd-type oxidase complex, or a DNA fragment containing the outer region of the gene encoding the cytochrome bd-type oxidase complex but not the gene encoding the cytochrome bd-type oxidase complex, is constructed by introducing an inactivating mutation through polynucleotide substitution or insertion, and this is incorporated into the parent bacterium to induce homologous recombination with the region of the parent bacterium's genome containing the gene encoding the cytochrome bd-type oxidase complex. This makes it possible to delete or inactivate the gene encoding the cytochrome bd-type oxidase complex on the genome. Alternatively, it is possible to inactivate the gene encoding the cytochrome bd-type oxidase complex by incorporating a recombinant vector (such as a plasmid) containing a DNA fragment that includes a portion of the gene encoding the cytochrome bd-type oxidase complex into the parent bacterium, thereby disrupting the portion of the gene encoding the cytochrome bd-type oxidase complex in the parent bacterium's genome through homologous recombination. Methods for randomly deleting or inactivating genes in bacterial cells include introducing randomly cloned DNA fragments containing inactivating mutations into cells and inducing homologous recombination with genes on the cell's genome, and inducing mutations by irradiating cells with ultraviolet light, gamma rays, etc. Inactivating mutations in genes refer to mutations such as silence mutations, missense mutations, nonsense mutations, and frameshift mutations that cause the target gene to lose its original function. For example, a gene into which an inactivating mutation has been introduced will either not express a protein or will express a protein with impaired original activity.

[0032] One method for constructing a DNA fragment containing a gene encoding a cytochrome bd-type oxidase complex into which an inactivating mutation has been introduced is site-directed mutagenesis (SMU). Site-directed mutagenesis can be performed using mutation primers containing the nucleotide mutation to be introduced. For example, by PCR using a gene encoding a cytochrome bd-type oxidase complex as a template with two sets of primers containing the nucleotide mutation to be introduced, DNA fragments can be prepared by amplifying the upstream and downstream regions of the gene encoding the cytochrome bd-type oxidase complex, respectively. These fragments can then be ligated together by SOE-PCR (splicing by overlap extension PCR) (Gene, 1989, 77(1): p61-68) to construct a DNA fragment containing the desired mutation. Alternatively, site-directed mutagenesis can be performed using methods such as inverse PCR or annealing (Muramatsu et al., "Revised 4th Edition New Genetic Engineering Handbook," Yodosha, pp. 82-88), or by using commercially available site-directed mutagenesis kits such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit.

[0033] Mutation primers can be prepared by well-known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). The gene encoding the cytochrome bd-type oxidase complex used as a template may be prepared from host bacteria by conventional methods or chemically synthesized.

[0034] To introduce DNA fragments or vectors into host bacteria, well-known techniques such as the calcium phosphate method, electroporation, lipofection, particle gun method, and PEG method can be applied. For example, methods applicable to Corynebacteria include competent cell transformation (J Bacteriol, 1967, 93:1925-1937), electroporation (FEMS Microbiol Lett, 1990, 55:135-138), protoplast transformation (Mol Gen Genet, 1979, 168:111-115), and Tris-PEG (J Bacteriol, 1983, 156:1130-1134).

[0035] Furthermore, polynucleotides that have the activity to degrade the transcript of a gene encoding a cytochrome bd-type oxidase complex, or polynucleotides that suppress the translation of said transcript into protein, include polynucleotides that contain a nucleotide sequence or a part thereof that is complementary or substantially complementary to the nucleotide sequence of the mRNA of the gene encoding the cytochrome bd-type oxidase complex. Specifically, these include antisense RNA for the mRNA of the gene encoding the cytochrome bd-type oxidase complex, siRNA for the mRNA of the gene encoding the cytochrome bd-type oxidase complex, and ribozymes for the mRNA of the gene encoding the cytochrome bd-type oxidase complex.

[0036] Bacteria in which the function of the gene encoding the cytochrome bd-type oxidase complex is suppressed can be selected by examining their genome sequence. Alternatively, bacteria in which the function of the gene encoding the cytochrome bd-type oxidase complex is suppressed can be selected using the expression level or activity of the cytochrome bd-type oxidase complex as an indicator.

[0037] In the present invention, the host bacteria are coryneform bacteria. Examples of coryneform bacteria include a group of microorganisms defined as coryneform bacteria in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), specifically including Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, and Micrococcus. Examples of Corynebacterium species include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium crenatum, Corynebacterium crudilactis, and Corynebacterium callunae. Examples of bacteria belonging to the genus Brevibacterium include Brevibacterium ammoniagenes. Examples of Arthrobacter species include Arthrobacter globiformis. Examples of Mycobacterium species include Mycobacterium bovis, while examples of Micrococcus species include Micrococcus freudenreichii, Micrococcus leuteus, Micrococcus ureae, and Micrococcus roseus. Among coryneform bacteria, those of the genus Corynebacterium are preferred, and more preferably Corynebacterium glutamicum. The microbial cells mentioned above may be wild-type, mutant, or genetically modified.

[0038] In the present invention, the Corynebacterium used as a host can be any Corynebacterium suitable for the production of aromatic compounds or their salts. However, from the viewpoint of the production efficiency of aromatic compounds or their salts, particularly the production efficiency of aromatic compounds or their salts derived from 3-dehydrosikimic acid and chorismic acid such as protocatechuic acid, gallic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, parahydroxybenzoic acid, paraaminobenzoic acid, and 4-amino-3-hydroxybenzoic acid, it is preferable to use Corynebacterium with enhanced 3-dehydrosikimic acid production activity and, more specifically, chorismic acid production activity. Specifically, from the viewpoint of increasing the production efficiency of compounds derived from 3-dehydrosikimic acid and chorismic acid, it is preferable to use Corynebacterium with enhanced expression of one or more polypeptides selected from (C), (D), (E), and (F) below as a host. (C) Polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) Polypeptides required for biosynthesis of para-aminobenzoic acid from colismic acid (F) Polypeptide having para-aminobenzoic acid hydroxylase activity

[0039] Therefore, the modified coryneform bacteria of the present invention are preferably modified coryneform bacteria in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, and which possess at least the following (C), (D), (E), and (F): (C) Polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) Polypeptides required for biosynthesis of para-aminobenzoic acid from colismic acid (F) Polypeptide having para-aminobenzoic acid hydroxylase activity It is a Corynebacterium in which the expression of one or more polypeptides selected from is enhanced.

[0040] Here, the polypeptide having 3,4-dihydroxybenzoate hydroxylase activity in (C) is a polypeptide that catalyzes the reaction to produce gallic acid from protocatechuic acid, and examples include (C1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5, or (C2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 5 and having 3,4-dihydroxybenzoate hydroxylase activity. Here, the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 5 is a polypeptide with 3,4-dihydroxybenzoate hydroxylase activity, which is a variant of p-hydroxybenzoate hydroxylase (pobA) derived from Corynebacterium glutamicum (indicated as "pobA*" in Figure 1). Furthermore, polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 5 of (C2) and possessing 3,4-dihydroxybenzoate hydroxylase activity include, for example, polypeptides possessing 3,4-dihydroxybenzoate hydroxylase activity derived from bacteria of the genus Corynebacterium. Specifically, these include 4-hydroxybenzoate-3-monooxygenase from Corynebacterium glutamicum, 4-hydroxybenzoate-3-monooxygenase from Corynebacterium suranareeae, and 4-hydroxybenzoate-3-monooxygenase from Corynebacterium crudilactis.

[0041] The polypeptide having 3-dehydroshikimic acid dehydratase activity of (D) is a polypeptide that catalyzes the reaction to produce protocatechuic acid from 3-dehydroshikimic acid, and examples include (D1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or (D2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having 3-dehydroshikimic acid dehydratase activity. Here, the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 6 is 3-dehydroshikimic acid dehydratase derived from Corynebacterium glutamicum, and is known as "qsuB". Furthermore, polypeptides that consist of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 6 of (D2) and possessing 3-dehydroshikimate dehydratase activity include, for example, 3-dehydroshikimate dehydratase derived from bacteria of the genus Corynebacterium. Specifically, these include 3-dehydroshikimate dehydratase derived from Corynebacterium glutamicum and 3-dehydroshikimate dehydratase derived from Corynebacterium suranareae.

[0042] Polypeptides necessary for the biosynthesis of para-aminobenzoic acid from chorismic acid (E) include (EI) 4-amino-4-deoxychorismate synthase and (EII) 4-amino-4-deoxychorismate lyase. Therefore, enhancing the expression of polypeptides necessary for the biosynthesis of para-aminobenzoic acid from chorismic acid can be achieved by enhancing the expression of one or more of these polypeptides. Para-aminobenzoic acid is produced from chorismic acid via 4-amino-4-deoxycholismic acid. The conversion from chorismic acid to 4-amino-4-deoxycholismic acid involves para-aminobenzoic acid synthetase component II (PabA) and para-aminobenzoic acid synthetase component I (PabB), while 4-amino-4-deoxycholismic acid is converted to para-aminobenzoic acid by 4-amino-4-deoxycholismic acid lyase (PabC). The above (EI) 4-amino-4-deoxycholismic acid synthase is a polypeptide that catalyzes the reaction to produce 4-amino-4-deoxycholismic acid from colismic acid. Examples include a polypeptide consisting of the amino acid sequence shown in (EI-1) Sequence ID No. 7, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in (EI-2) Sequence ID No. 7, and having 4-amino-4-deoxycholismic acid synthase activity. The above (EII) 4-amino-4-deoxycholismic acid lyase is a polypeptide that catalyzes the reaction to produce para-aminobenzoic acid from 4-amino-4-deoxycholismic acid. Examples include a polypeptide consisting of the amino acid sequence shown in (EII-1) Sequence ID No. 8, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in (EII-2) Sequence ID No. 8, and having 4-amino-4-deoxycholismic acid lyase activity. Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7 is 4-amino-4-deoxycholismic acid synthase derived from Corynebacterium glutamicum and is known as "pabAB," and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8 is 4-amino-4-deoxycholismic acid lyase derived from Corynebacterium glutamicum and is known as "pabC."

[0043] Polypeptides having the para-aminobenzoic acid hydroxylase activity of (F) are polypeptides that catalyze the reaction to produce 4-amino-3-hydroxybenzoic acid from para-aminobenzoic acid. Examples include (F1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, or (F2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9, and having para-aminobenzoic acid hydroxylase activity. Here, the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 9 is a polypeptide possessing para-aminobenzoic acid hydroxylase activity, which is a variant of para-hydroxybenzoic acid hydroxylase derived from Caulobacter vibrioides (denoted as "phbh*" in Figure 1). Furthermore, polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in (F2) Sequence ID No. 9 and possessing para-aminobenzoate hydroxylase activity include, for example, para-hydroxybenzoate hydroxylase derived from bacteria of the genus Caulobacter, specifically, para-hydroxybenzoate hydroxylase derived from Caulobacter rhizosphaerae and para-hydroxybenzoate hydroxylase derived from Caulobacter sp.

[0044] Of these, in the production of gallic acid, it is preferable to use a host in which the expression of a polypeptide having (C)3,4-dihydroxybenzoate hydroxylase activity is enhanced, and it is even more preferable to use a host in which the expression of a polypeptide having (D)dehydroshikimic acid dehydratase activity is enhanced. Furthermore, in the production of para-aminobenzoic acid, it is preferable to use a host in which the polypeptide necessary for the biosynthesis of para-aminobenzoic acid from (E)cholismic acid is enhanced in expression. In the production of 4-amino-3-hydroxybenzoic acid, it is preferable to use a host in which the polypeptide having (F)3,4-dihydroxybenzoic acid hydroxylase activity is enhanced in expression. Moreover, it is even more preferable to use a host in which the polypeptide necessary for the biosynthesis of para-aminobenzoic acid from (E)cholismic acid is enhanced in expression.

[0045] Furthermore, in order to improve the 3-dehydroshikimic acid production or chorismic acid production activity, it is preferable to use a Corynebacterium in which the expression of polypeptides shown in (G) to (O) below is enhanced as the Corynebacterium used as the host in the present invention. (G) Polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity, Polypeptides having (H)3-dehydroquinate synthase activity, (I) Polypeptides having 3-dehydroquinate dehydratase activity, (J) Polypeptides having 5-shikimate dehydrogenase activity, (K) Polypeptides having shikimic acid kinase activity, (L)3-phosphosikimate-1-carboxyvinyltransferase activity-containing polypeptide, (M) polypeptide having colismyate synthase activity, (N) Polypeptides having pyruvate phosphate dikinase activity, (O) Enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactose, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase.

[0046] Specifically, the host coryneform bacteria in which the expression of the above polypeptide is enhanced are coryneform bacteria into which the polynucleotide necessary for the expression of the polypeptide has been introduced to enable expression, and the polypeptide may be foreign or naturally present in the bacterium. Examples include coryneform bacteria into which the polynucleotide has been introduced to enable expression, and coryneform bacteria in which the degree of expression of the polynucleotide has been enhanced. Specifically, examples include coryneform bacteria into which a vector or DNA fragment containing the polynucleotide and a regulatory region operably linked thereto has been introduced, and coryneform bacteria in which the regulatory region of the polynucleotide has been replaced with a strongly regulatory region. Examples of strongly regulatory regions include, but are not limited to, well-known high-expression promoters such as the T7 promoter, lac promoter, tac promoter, trp promoter, tu promoter, and gap promoter. Furthermore, prokaryotic induction promoters can be used as strongly regulatory regions. Examples include 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 nagI (cg3351) gene promoter, which is induced by the addition of 3-hydroxybenzoic acid; the benA (cg2637) gene promoter (hereinafter abbreviated as Pben), which is induced by the addition of benzoic acid; or the cg2118 gene promoter or ptsS (cg2925) gene promoter, which are induced by the addition of either fructose or sucrose. However, the examples are not limited to these. Methods for replacing the regulatory regions of polynucleotides present on the genome of host cells with strongly regulatory regions include introducing a DNA fragment containing the strongly regulatory region and the polynucleotide sequence of a selection marker into host cells and selecting cells transformed by homologous or non-homologous recombination.

[0047] Thus, by culturing the modified Corynebacterium strains, evaluating the productivity of aromatic compounds or their salts, and selecting appropriate transformants, useful aromatic compound or salt-producing bacteria can be obtained. The product can be measured according to the method described in the examples below.

[0048] The present invention's method for producing aromatic compounds or salts thereof is carried out by culturing the modified Corynebacterium bacterium described above, preferably in the presence of sugars, and recovering the target aromatic compound or salt thereof. Glucose is preferred as the sugar, but monosaccharides such as fructose, mannose, arabinose, xylose, and galactose, as well as sugars that can produce glucose through metabolism, can also be used. Such sugars include oligosaccharides or polysaccharides that have glucose units, and examples include disaccharides such as cellobiose, sucrose, lactose, maltose, trehalose, cellobiose, and xylobiose; and polysaccharides such as dextrin or soluble starch. Furthermore, molasses can also be used as a raw material containing these raw material compounds, for example. In addition, saccharified liquids containing multiple sugars such as glucose can be used, obtained by saccharifying non-edible agricultural waste such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw, etc.), bagasse, corn stover, energy crops such as switchgrass, napier grass, miscanthus, wood chips, waste paper, etc., with saccharifying enzymes.

[0049] The culture medium for culturing the transformants may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc., and is capable of efficiently culturing the modified Corynebacterium-type bacteria of the present invention. As a carbon source, the above-mentioned sugars or molasses or saccharified liquids containing them can be used. In addition to the above-mentioned sugars, sugar alcohols such as mannitol, sorbitol, xylitol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; alcohols such as ethanol and propanol; and hydrocarbons such as normal paraffin can also be used. The carbon source can be used individually or in mixtures of two or more types. The concentration of sugars, which are the raw material compounds, in the culture medium is preferably 1 to 20 w / v%, more preferably 2 to 10 w / v%, and even more preferably 2 to 5 w / v%.

[0050] As nitrogen sources, for example, peptone, meat extract, yeast extract, casein hydrolysate, soybean meal alkali extract, 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, potassium nitrate, etc. can be used.

[0051] 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, vitamins and antifoaming agents can be added as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0052] Examples of culture media for Corynebacteria include Medium A [J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)], Medium BT [J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)], and Medium CGXII [Patent No. 6322576]. These media should be used with sugar concentrations within the above range.

[0053] Prior to reactions or cultures involving sugars, it is preferable to culture the transformants in the same medium under aerobic conditions at a temperature of approximately 25-38°C for approximately 12-48 hours to allow them to grow.

[0054] The culture temperature or reaction temperature is preferably 15 to 45°C, and more preferably 25 to 37°C. Furthermore, the incubation or reaction time can be 24 to 168 hours, preferably 24 to 96 hours, more preferably 24 to 72 hours, while stirring or shaking as necessary. During incubation, antibiotics such as ampicillin or kanamycin may be added to the culture medium as needed. The culture can be carried out using a batch, fed-batch, or continuous method. Of these, the batch method is preferred. The culture or reaction may be carried out under aerobic or reducing conditions, but it is preferable to carry it out under aerobic conditions. When carrying out the reaction or culture under aerobic conditions, it is preferable to do so under conditions that suppress excessive growth of the transformants, from the viewpoint of the efficiency of producing aromatic compounds or their salts.

[0055] The method for recovering and purifying aromatic compounds or their salts from cultures is not particularly limited. That is, it 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, after removing microbial cells by centrifugation or the like, aromatic compounds or their salts can be obtained by removing ionic substances with cation and anion exchange resins and then concentrating the mixture. Aromatic compounds or their salts accumulated in the culture may be used directly without isolation.

[0056] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a modified Corynebacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted. <2> Modified Corynebacteria are bacterial strains in which the expression of the cytochrome bd-type oxidase complex is reduced or lost compared to the parent bacterium. <1> The method. <3> The cytochrome bd-type oxidase complex is either (A) or (B) below: (A) A complex composed of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, (B) A complex consisting of a polypeptide having at least 90% identity with the amino acid sequence shown in Sequence ID No. 2 and functioning as cytochrome bd-type oxidase subunit I, and a polypeptide having at least 90% identity with the amino acid sequence shown in Sequence ID No. 4 and functioning as cytochrome bd-type oxidase subunit II, This is a polypeptide complex having cytochrome bd-type oxidase activity as shown by <1> or <2> The method. <4> A modified Corynebacterium strain is a bacterial strain obtained by deleting or inactivating part or all of the nucleotide sequence of the polynucleotide described in (a) or (b) below. <2> or <3> method. (a) A polynucleotide encoding a complex consisting of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 and a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 (b) A polynucleotide encoding a complex consisting of a polynucleotide encoding a polypeptide that functions as cytochrome bd-type oxidase subunit I, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1, and a polynucleotide encoding a polypeptide that functions as cytochrome bd-type oxidase subunit II, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3. <5> As hosts, at least the following (C), (D), (E), and (F): (C) Polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) Polypeptides required for biosynthesis of para-aminobenzoic acid from colismic acid (F) Polypeptide having para-aminobenzoic acid hydroxylase activity Using Corynebacterium bacterium in which the expression of one or more polypeptides selected from the above is enhanced, <1> ~ <4> One of the following methods. <6> (C) A polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is a polypeptide consisting of (C1) the amino acid sequence shown in SEQ ID NO: 5, or (C2) an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 5, and having 3,4-dihydroxybenzoate hydroxylase activity. <5> The method. <7> (D) A polypeptide having 3-dehydroshikimic acid dehydratase activity is (D1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or (D2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6, and having 3-dehydroshikimic acid dehydratase activity. <5> The method. <8> (E) One or more polypeptides selected from (EI) 4-amino-4-deoxycholismic acid synthase and (EII) 4-amino-4-deoxycholismic acid lyase are required for the biosynthesis of para-aminobenzoic acid from colismic acid. <5> The method. <9> (EI)4-amino-4-deoxycholismic acid synthase is a polypeptide consisting of the amino acid sequence shown in (EI-1)Sequence ID 7, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in (EI-2)Sequence ID 7, and having 4-amino-4-deoxycholismic acid synthase activity; (EII)4-amino-4-deoxycholismic acid lyase is a polypeptide consisting of the amino acid sequence shown in (EII-1)Sequence ID 8, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in (EII-2)Sequence ID 8, and having 4-amino-4-deoxycholismic acid lyase activity. <8> The method. <10> (F) The polypeptide having para-aminobenzoic acid hydroxylase activity is (F1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, or (F2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9, and having para-aminobenzoic acid hydroxylase activity. <5> The method. <11> As a host, further (G)~(O): (G) Polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity, Polypeptides having (H)3-dehydroquinate synthase activity, (I) Polypeptides having 3-dehydroquinate dehydratase activity, (J) Polypeptides having 5-shikimate dehydrogenase activity, (K) Polypeptides having shikimic acid kinase activity, (L)3-phosphosikimate-1-carboxyvinyltransferase activity-containing polypeptide, (M) polypeptide having colismyate synthase activity, (N) Polypeptides having pyruvate phosphate dikinase activity, (O) Enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactose, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase. Using Corynebacterium bacterium in which the expression of one or more polypeptides selected from the above is enhanced, <5> ~ <10> One of the following methods. <12> The aromatic compound or its salt is gallic acid, protocatechuic acid, catechol, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, para-hydroxybenzoic acid, para-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof. <1> ~ <11> One of the following methods. <13> The aromatic compound or its salt is gallic acid, protocatechuic acid, para-hydroxybenzoic acid, para-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof. <1> ~ <11> One of the following methods. <14> The aromatic compound or its salt is gallic acid, para-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof. <1> ~ <11> One of the following methods. <15> Corynebacteria are bacteria of the genus Corynebacterium. <1> ~ <14> One of the following methods. <16> The bacteria of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanoricum, Corynebacterium carnae, Corynebacterium crenatum, or Corynebacterium crudylactis. <1> ~ <14> One of the following methods. <17> Corynebacterium glutamicum is a bacterium of the genus Corynebacterium. <1> ~ <14> One of the following methods.

[0057] <18> Modified Corynebacteria in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, and which meet at least the following conditions (C), (D), (E), and (F): (C) Polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) Polypeptides required for biosynthesis of para-aminobenzoic acid from colismic acid (F) Polypeptide having para-aminobenzoic acid hydroxylase activity Corynebacteria with enhanced expression of one or more polypeptides selected from the following. <19> The cytochrome bd-type oxidase complex is either (A) or (B) below: (A) A complex composed of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, (B) A complex consisting of a polypeptide having at least 90% identity with the amino acid sequence shown in Sequence ID No. 2 and functioning as cytochrome bd-type oxidase subunit I, and a polypeptide having at least 90% identity with the amino acid sequence shown in Sequence ID No. 4 and functioning as cytochrome bd-type oxidase subunit II, This is a polypeptide complex having cytochrome bd-type oxidase activity as shown by <18> Corynebacteria. <20> The following polynucleotides are obtained by deleting or inactivating part or all of the nucleotide sequence of (a) or (b): <18> or <19> Corynebacteria. (a) A polynucleotide encoding a complex consisting of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 and a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 (b) A polynucleotide encoding a complex consisting of a polynucleotide encoding a polypeptide that functions as cytochrome bd-type oxidase subunit I, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1, and a polynucleotide encoding a polypeptide that functions as cytochrome bd-type oxidase subunit II, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3. <21> (C) A polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is a polypeptide consisting of (C1) the amino acid sequence shown in SEQ ID NO: 5, or (C2) an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 5, and having 3,4-dihydroxybenzoate hydroxylase activity. <18> ~ <20> One of the following Corynebacteria. <22> (D) A polypeptide having 3-dehydroshikimic acid dehydratase activity is (D1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or (D2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6, and having 3-dehydroshikimic acid dehydratase activity. <18> ~ <20> One of the following Corynebacteria. <23> (E) One or more polypeptides selected from (EI) 4-amino-4-deoxycholismic acid synthase and (EII) 4-amino-4-deoxycholismic acid lyase are required for the biosynthesis of para-aminobenzoic acid from colismic acid. <18> ~ <20> One of the following Corynebacteria. <23> (EI)4-amino-4-deoxycholismic acid synthase is a polypeptide consisting of the amino acid sequence shown in (EI-1)Sequence ID 7, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in (EI-2)Sequence ID 7, and having 4-amino-4-deoxycholismic acid synthase activity; (EII)4-amino-4-deoxycholismic acid lyase is a polypeptide consisting of the amino acid sequence shown in (EII-1)Sequence ID 8, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in (EII-2)Sequence ID 8, and having 4-amino-4-deoxycholismic acid lyase activity. <22> Corynebacteria. <24> (F) The polypeptide having para-aminobenzoic acid hydroxylase activity is (F1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, or (F2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9, and having para-aminobenzoic acid hydroxylase activity. <18> ~ <20> One of the following Corynebacteria. <25> Furthermore, (G)~(O) below: (G) Polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity, Polypeptides having (H)3-dehydroquinate synthase activity, (I) Polypeptides having 3-dehydroquinate dehydratase activity, (J) Polypeptides having 5-shikimate dehydrogenase activity, (K) Polypeptides having shikimic acid kinase activity, (L)3-phosphosikimate-1-carboxyvinyltransferase activity-containing polypeptide, (M) polypeptide having colismyate synthase activity, (N) Polypeptides having pyruvate phosphate dikinase activity, (O) Enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactose, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase. The expression of one or more polypeptides selected from was enhanced. <18> ~ <24> One of the following Corynebacteria. [Examples]

[0058] 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.

[0059] <Creation of strains with enhanced colismic acid production activity> 1) Construction of a plasmid in which the tu promoter is introduced into the cg1829(aroC) promoter region. The 5' upstream region of the cg1829 gene (SEQ ID NO: 10) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 11 and 12), and the 5' region of the cg1829 gene ORF (SEQ ID NO: 13) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 14 and 15) to obtain DNA fragments. In addition, a DNA fragment containing the tu promoter (SEQ ID NO: 16) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 17 and 18) with the genome of strain ATCC13032 as a template to obtain DNA fragments. Furthermore, pHKPsacB1 (see Japanese Patent Application No. 2014-523757) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 19 and 20), and the obtained PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Ptu-aroC was constructed by ligating them using the In-Fusion HD Cloning Kit (Takara Bio).

[0060] 2) Creation of a strain by introducing the tu promoter into the promoter region of cg1829(aroC) Using an electroporation transformation method, the plasmid pHKPsacB_Ptu-aroC described above was introduced into strain KC315 (Japanese Patent Application No. 2021-201877), and strain KC367sr was obtained by selecting for kanamycin resistance. Analysis of strain KC367sr using PCR with primers SEQ ID NOs. 11 and 21 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that strain KC367sr is a once-cross-recombinant homologous recombinant into which the plasmid pHKPsacB_Ptu-aroC has been introduced. The KC367sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture solution was streaked onto LB agar containing 20% ​​sucrose to obtain the KC367 strain. PCR using primers SEQ ID NOs. 21 and 22 (Sapphire Amp (Takara Bio)) confirmed that the KC367 strain is a double-crossover homologous recombinant with the tu promoter introduced into the cg1829(aroC) promoter region, as expected.

[0061] 3) Construction of a plasmid in which the TU promoter is introduced into the CG1774(TKT) promoter region. The 5' upstream region of the cg1774 gene (SEQ ID NO: 23) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 24 and 25), and the 5' region of the cg1774 gene ORF (nucleotide number 26) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 27 and 28) to obtain DNA fragments. In addition, a DNA fragment containing the tu promoter (SEQ ID NO: 16) was amplified by PCR using the genome of strain ATCC13032 as a template and two types of DNA primers (SEQ ID NOs: 17 and 18) to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified by PCR using two types of DNA primers (SEQ ID NOs: 19 and 20), and the obtained PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Ptu-tkt was constructed by ligating them using the In-Fusion HD Cloning Kit (Takara Bio).

[0062] 4) Creation of a strain by introducing the TU promoter into the promoter region of cg1774(tkt) Using electroporation-based transformation, the plasmid pHKPsacB_Ptu-tkt described above was introduced into the KC367 strain, and the KC376sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC376sr strain using PCR with primers SEQ ID NOs. 24 and 29 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC376sr strain is a once-crossover homologous recombinant in which the plasmid pHKPsacB_Ptu-tkt was introduced into the promoter region of cg1774. The KC376sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture solution was streaked onto LB agar containing 20% ​​sucrose to obtain the KC376 strain. PCR using primers SEQ ID NOs. 29 and 30 (Sapphire Amp (Takara Bio)) confirmed that the KC376 strain is a double-crossover homologous recombinant with the tu promoter introduced into the cg1774(tkt) promoter region, as expected.

[0063] 5) Construction of a plasmid in which the tu promoter is introduced into the cg0644(ppsA) promoter region. The 5' upstream region of the cg0644 gene (SEQ ID NO: 31) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 32 and 33), and the 5' region of the cg0644 gene ORF (nucleotide number 34) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 35 and 36) to obtain DNA fragments. In addition, a DNA fragment containing the tu promoter (SEQ ID NO: 16) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 17 and 18) with the genome of strain ATCC13032 as a template to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified by PCR using two types of DNA primers (SEQ ID NOs: 19 and 20), and the obtained PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Ptu-ppsA was constructed by ligating them using the In-Fusion HD Cloning Kit (Takara Bio).

[0064] 6) Creation of a strain by introducing the tu promoter into the promoter region of cg0644(ppsA) Using electroporation for transformation, the plasmid pHKPsacB_Ptu-ppsA described above was introduced into the KC376 strain, and the KC408sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC408sr strain using PCR with primers SEQ ID NOs. 32 and 37 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC408sr strain is a once-cross-recombinant homologous recombinant with the plasmid pHKPsacB_Ptu-tkt introduced. The KC408 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 medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC408 strain. PCR using primers SEQ ID NOs. 37 and 38 (Sapphire Amp (Takara Bio)) confirmed that the KC408 strain is a double-crossover homologous recombinant with the tu promoter introduced into the cg0644(tkt) promoter region, as expected.

[0065] <Evaluation of the productivity of 4-aminobenzoic acid (4-ABA)> (1) Preparation of 4-ABA producing strain 1) Construction of a plasmid for introducing the promoter of the cg2875 gene into the cg1134(pabAB) promoter region. The 5' upstream region of the cg1134 gene (SEQ ID NO: 39) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 40 and 41), and the 5' region of the cg1134 gene ORF (SEQ ID NO: 42) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 43 and 44) ​​to obtain DNA fragments. In addition, a DNA fragment containing the promoter of the cg2875 gene (SEQ ID NO: 45) was amplified by PCR using the genome of strain ATCC13032 as a template and two types of DNA primers (SEQ ID NOs: 46 and 47) to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified by PCR using two types of DNA primers (SEQ ID NOs: 19 and 20), and the obtained PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Pcg2875-pabAB was constructed by ligating them using the In-Fusion HD Cloning Kit (Takara Bio).

[0066] 2) Creation of a strain by introducing the promoter of the cg2875 gene into the cg1134(pabAB) promoter region. Using electroporation for transformation, the plasmid pHKPsacB_Pcg2875-pabAB was introduced into the KC408 strain, and the KC412sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC412sr strain using PCR with primers SEQ ID NOs. 40 and 48 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC412sr strain is a once-cross-recombinant homologous recombinant into which the plasmid pHKPsacB_Pcg2875-pabAB has been introduced. The KC412 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 medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC412 strain. PCR using primers SEQ ID NOs. 48 and 49 (Sapphire Amp (Takara Bio)) confirmed that the KC412 strain is a double-crossover homologous recombinant with the cg2875 gene promoter introduced into the cg1134 (pabAB) promoter region, as expected.

[0067] (2) Creation of a 4-ABA-producing cydAB disruption strain 1) Construction of a plasmid to delete the cg1300-1301 (cydAB) gene region. The 5' upstream region of the cg1300 gene (SEQ ID NO: 50) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 51 and 52), and the 3' downstream region of the cg1301 gene (nucleotide number 53) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 54 and 55) to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified by PCR using two types of DNA primers (SEQ ID NOs: 19 and 20). The resulting PCR products were treated with DpnI (Takara Bio). Each DNA fragment was purified from the three obtained PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_ΔcydAB was constructed by ligation using the In-Fusion HD Cloning Kit (Takara Bio).

[0068] 2) Creation of a strain lacking the cg1300-1301 (cydAB) gene region. The plasmid pHKPsacB_ΔcydAB described above was introduced into the KC412 strain using electroporation, and the KC418sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC418sr strain using PCR with primers SEQ ID NOs. 51 and 56 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC418sr strain is a once-cross-recombinant homologous recombinant with the plasmid pHKPsacB_ΔcydAB introduced. The KC418 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 medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC418 strain. PCR using primers SEQ ID NOs. 56 and 57 (Sapphire Amp (Takara Bio)) confirmed that the KC418 strain was a double-crossover homologous recombinant with a deletion in the cg1300-1301 (cydAB) gene region, as expected.

[0069] (3) Creation of a 4-ABA-producing ctaCF disruption strain 1) Construction of a plasmid to delete the cg2408-2409 (ctaCF) gene region. The 5' upstream region of the cg2408 gene (sequence number 58) was amplified by PCR using two types of DNA primers (sequence numbers 59 and 60), and the 3' region of the cg2409 gene (base number 61) was amplified by PCR using two types of DNA primers (sequence numbers 62 and 63) to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified by PCR using two types of DNA primers (sequence numbers 19 and 20). The resulting PCR products were treated with DpnI (Takara Bio). Each DNA fragment was purified from the three obtained PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_ΔctaCF was constructed by ligation using the In-Fusion HD Cloning Kit (Takara Bio).

[0070] 2) Creation of a strain lacking the cg2408-2409 (ctaCF) gene region. The plasmid pHKPsacB_ΔctaCF described above was introduced into the KC412 strain using electroporation, and the KC469sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC469sr strain using PCR with primers SEQ ID NOs. 59 and 64 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC469sr strain is a once-cross-recombinant homologous recombinant with the plasmid pHKPsacB_ΔctaCF introduced. The KC469sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC469 strain. PCR using primers for SEQ ID NOs. 64 and 65 (Sapphire Amp (Takara Bio)) confirmed that the KC469 strain is a double-crossover homologous recombinant with a deletion in the cg2408-2409 (ctaCF) gene region, as expected.

[0071] (4) Culture of transformed strains 1) The transformed strains obtained above were streaked onto LB plates and cultured at 30°C for 3 days. The cells grown on the plates were inoculated into 6 mL of CGTG15 medium (Table 4) and cultured with shaking at 30°C and 250 rpm for 24 hours (pre-culture). 60 mL of CGTG15 medium (with urea removed from its components) was placed in a Bio Jr.8 culture tank (Able Co., Ltd.). 1.2 mL of the pre-culture solution was inoculated and cultured with shaking at 32°C, pH 6.9, 700 rpm, and an aeration rate of 60 mL / min for 30 hours, and the productivity of aromatic compounds was evaluated. The culture solution was appropriately diluted with dilute sulfuric acid, the cells were removed by centrifugation, and the supernatant was collected. The concentration in the supernatant was quantified. The results are shown in the table.

[0072] 2) Results In comparison, the KC418 strain showed an increased 4-ABA concentration, indicating that disrupting the cydAB gene has a positive effect on production. Conversely, the KC469 strain showed a decreased 4-ABA concentration, indicating that disrupting the ctaCF gene has a negative effect on production (Tables 1-1 and 1-2).

[0073] [Table 1-1]

[0074] [Table 1-2]

[0075] <Evaluation of the productivity of 4-amino-3-hydroxybenzoic acid (4,3-AHBA)> (1) Preparation of 4,3-AHBA-producing bacteria 1) Construction of a plasmid for introducing the HFM122 mutant gene, in which the cg2875 gene promoter is linked to the cg2484 gene region. pECsf_gapS_pabABC_tuD_HFM122 (see Japanese Patent Application No. 2019-203523) was amplified by PCR using two types of DNA primers (SEQ ID NOs. 63 and 64) as a template, and the resulting PCR product was treated with DpnI (Takara Bio). Two fragments of the HFM122 mutant gene (SEQ ID NOs. 68 and 69) were synthesized (Eurofin Genomics and GenScript), and each was amplified by PCR using two types of DNA primers (SEQ ID NOs. 70 and 71 and 72 and 73) to obtain DNA fragments. Each of the three obtained PCR products was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmids pECsf_gapS_pabABC_tuD_HFM122LA_OPT1-HFM122LA_OPT2 were constructed by ligation using the In-Fusion HD Cloning Kit (Takara Bio). The 5' upstream region of the cg2484 gene (SEQ ID NO: 74) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 75 and 76), and the 3' region of the cg2484 gene (SEQ ID NO: 77) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 78 and 79) to obtain DNA fragments. In addition, a DNA fragment containing the promoter of the cg2875 gene (SEQ ID NO: 45) was amplified by PCR using two types of DNA primers (SEQ ID NOs: 46 and 47) with the genome of strain ATCC13032 as a template to obtain DNA fragments. Furthermore, the plasmid pECsf_gapS_pabABC_tuD_HFM122LA_OPT1-HFM122LA_OPT2 was amplified by PCR using two types of DNA primers (SEQ ID NOs: 69 and 80) as a template, and the obtained PCR product was treated with DpnI (Takara Bio). Furthermore, pHKPsacB1 was used as a template and amplified by PCR using two types of DNA primers (SEQ ID NOs. 19 and 20). The resulting PCR products were then treated with DpnI (Takara Bio). The five resulting PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmids pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2 were constructed by ligation using the In-Fusion HD Cloning Kit (Takara Bio).

[0076] 2) Creation of a strain by introducing the HFM122 mutant gene, in which the cg2875 gene promoter is linked to the cg2484 gene region. Using electroporation for transformation, the plasmid pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2 was introduced into the KC412 strain, and the KC417sr strain was obtained by selecting based on kanamycin resistance. Analysis of the KC417sr strain by PCR using primers SEQ ID NOs. 67 and 81 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC417sr strain is a once-crossover homologous recombinant with the plasmid pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2 introduced. The KC417 strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC417 strain. PCR using primers SEQ ID NOs. 81 and 82 (Sapphire Amp (Takara Bio)) confirmed that the KC417 strain is a double-crossover homologous recombinant with the HFM122 mutant gene, which has been introduced into the cg2484 gene region with the promoter of the cg2875 gene linked to it, as expected.

[0077] (2) Preparation of a 4,3-AHBA-producing cydAB disruption strain The plasmid pHKPsacB_ΔcydAB described above was introduced into the KC417 strain using electroporation, and the KC427sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC427sr strain using PCR with primers SEQ ID NOs. 51 and 56 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC427sr strain is a once-cross-recombinant homologous recombinant with the plasmid pHKPsacB_ΔcydAB introduced. The KC427 strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC427 strain. PCR using primers SEQ ID NOs. 56 and 57 (Sapphire Amp (Takara Bio)) confirmed that the KC427 strain was, as expected, a double-crossover homologous recombinant with a deletion in the cg1300-1301 (cydAB) gene region.

[0078] (3) Preparation of a 4,3-AHBA-producing ctaCF disruption strain The plasmid pHKPsacB_ΔctaCF described above was introduced into the KC417 strain using electroporation, and the KC470sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC470sr strain using PCR with primers SEQ ID NOs. 59 and 64 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC470sr strain is a once-cross-recombinant homologous recombinant with the plasmid pHKPsacB_ΔctaCF introduced. The KC470sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar medium containing 20% ​​sucrose to obtain the KC470 strain. PCR using primers SEQ ID NOs. 64 and 65 (Sapphire Amp (Takara Bio)) confirmed that the KC470 strain was, as expected, a double-crossover homologous recombinant with a deletion in the cg2408-2409 (ctaCF) gene region.

[0079] (4) Culture of transformed strains 1) The transformed strains obtained above were streaked onto LB plates and cultured at 30°C for 3 days. The cells grown on the plates were inoculated into 6 mL of CGTG15 medium and cultured with shaking at 30°C and 250 rpm for 24 hours (pre-culture). 60 mL of CGTG15 medium (with urea removed from its components) was placed in a Bio Jr.8 culture tank (Able Co., Ltd.). 1.2 mL of the pre-culture solution was inoculated and cultured with shaking at 32°C, pH 6.9, 700 rpm, and an aeration rate of 60 mL / min for 30 hours, and the productivity of aromatic compounds was evaluated. The culture solution was appropriately diluted with dilute sulfuric acid, the cells were removed by centrifugation, and the supernatant was collected. The concentration in the supernatant was quantified. The results are shown in the table.

[0080] 2) Results In comparison, the KC427 strain showed an increased 4,3-AHBA concentration, indicating that disrupting the cydAB gene has a positive effect on production. Conversely, the KC470 strain showed a decreased 4,3-AHBA concentration, indicating that disrupting the ctaCF gene has a negative effect on production.

[0081] [Table 2-1]

[0082] [Table 2-2]

[0083] <Evaluation of gallic acid (GAL) productivity> (1) Creation of a GAL-producing cydAB disruption strain Using electroporation for transformation, the plasmid pHKPsacB_ΔcydAB described above was introduced into strain KC148 (Japanese Patent Application No. 2020-94649), and strain KC201sr was obtained by selecting for kanamycin resistance. Analysis of strain KC201sr using PCR with primers SEQ ID NOs. 51 and 56 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that strain KC201sr is a once-cross-reacting homologous recombinant with the plasmid pHKPsacB_ΔcydAB introduced. The KC201 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 medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC201 strain. PCR using primers SEQ ID NOs. 56 and 57 (Sapphire Amp (Takara Bio)) confirmed that the KC201 strain was, as expected, a double-crossover homologous recombinant with a deletion in the cg1300-1301 (cydAB) gene region.

[0084] (2) Creation of GAL-producing ctaCF disruption strain The plasmid pHKPsacB_ΔctaCF described above was introduced into the KC148 strain using electroporation, and the KC468sr strain was obtained by selecting for kanamycin resistance. Analysis of the KC468sr strain using PCR with primers SEQ ID NOs. 59 and 64 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC468sr strain is a once-cross-recombinant homologous recombinant with the plasmid pHKPsacB_ΔctaCF introduced. The KC468sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC468 strain. PCR using primers SEQ ID NOs. 64 and 65 (Sapphire Amp (Takara Bio)) confirmed that the KC468 strain is a double-crossover homologous recombinant with a deletion in the cg2408-2409 (ctaCF) gene region, as expected.

[0085] (3) Culture of transformed strains 1) The transformed strains obtained above were streaked onto LB plates and cultured at 30°C for 3 days. The cells grown on the plates were inoculated into 6 mL of CGTG15 medium and cultured with shaking at 30°C and 250 rpm for 24 hours (pre-culture). Sodium benzoate was added to CGTG15 medium (with urea removed from its components) to a final concentration of 1 mM, and 60 mL was placed in a Bio Jr.8 culture tank (Able Co., Ltd.). 1.2 mL of the pre-culture solution was inoculated and cultured with shaking at 32°C, pH 6.9, 600 rpm, and aeration rate of 60 mL / min for 30 hours, and the productivity of GAL was evaluated. The culture solution was appropriately diluted with dilute sulfuric acid, the cells were removed by centrifugation, and the supernatant was collected. The concentration in the supernatant was quantified. The results are shown in the table.

[0086] 2) Results In comparison, the KC201 strain showed an increase in GAL concentration, indicating that disrupting the cydAB gene has a positive effect on production. Conversely, the KC468 strain showed a decrease in GAL concentration, indicating that disrupting the ctaCF gene has a negative effect on production.

[0087] [Table 3-1]

[0088] [Table 3-2]

[0089] [Table 4]

[0090] (4)Analysis conditions The recovered supernatant was subjected to HPLC to remove insoluble matter using an AcroPrep 96 filter plate (0.2 μm GHP membrane, Nippon Pall), and the reaction mixture was subjected to HPLC. A Chromaster HPLC system (Hitachi High-Tech Science) was used for the analysis of gallic acid. An L-column ODS (4.6 mm ID × 150 mm, Chemicals Evaluation and Research Institute) was used, with eluent A being a 0.1% phosphoric acid solution of 0.1 M potassium dihydrogen phosphate and eluent B being 70% methanol. Gradient elution was performed at a flow rate of 1.0 mL / min and a column temperature of 40°C. A UV detector (detection wavelength 280 nm) was used to detect gallic acid. For glucose analysis, an ICsep ION-300 (7.8 mm × 300 mm, Tokyo Chemical Industry Co., Ltd.) was used, with a 37 mM sulfuric acid solution as the eluent, and detection was performed under conditions of a flow rate of 0.5 mL / min and a column temperature of 50°C. Radioisotopes (RIs) were used for glucose detection.

Claims

1. A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a modified Corynebacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted, wherein the aromatic compound or salt thereof is gallic acid, protocatechuic acid, catechol, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, parahydroxybenzoic acid, paraaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

2. The cytochrome bd-type oxidase complex is either (A) or (B) below: (A) A complex composed of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (B) A complex comprising a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 2 and functioning as cytochrome bd-type oxidase subunit I, and a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 4 and functioning as cytochrome bd-type oxidase subunit II, The method according to claim 1, wherein the polypeptide complex has the cytochrome bd-type oxidase activity shown by .

3. A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a modified Corynebacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted, As hosts, at least the following (C), (D), (E), and (F): (C) Polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimic acid dehydratase activity (E) Polypeptides necessary for biosynthesis of para-aminobenzoic acid from colismic acid (F) Polypeptide having para-aminobenzoic acid hydroxylase activity A method using Corynebacterium bacterium in which the expression of one or more polypeptides selected from the above is enhanced.

4. A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a modified Corynebacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted, As hosts, (G) to (O) below: (G) Polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity, (H) Polypeptide having 3-dehydroquinate synthase activity, (I) Polypeptides having 3-dehydroquinate dehydratase activity, (J) Polypeptides having 5-shikimic acid dehydrogenase activity, (K) Polypeptide having shikimic acid kinase activity, (L) Polypeptide having 3-phosphosikimic acid-1-carboxyvinyltransferase activity, (M) Polypeptide having colismic acid synthase activity, (N) Polypeptide having pyruvate phosphate dikinase activity, (O) Enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactose, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase. A method using Corynebacterium bacterium in which the expression of one or more polypeptides selected from the above is enhanced.

5. The method according to claim 3, wherein the aromatic compound or salt thereof is gallic acid, protocatechuic acid, catechol, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, para-hydroxybenzoic acid, para-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

6. The method according to claim 4, wherein the aromatic compound or salt thereof is gallic acid, protocatechuic acid, catechol, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, para-hydroxybenzoic acid, para-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

7. The method according to claim 1, wherein the aromatic compound or salt thereof is gallic acid, protocatechuic acid, parahydroxybenzoic acid, paraaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

8. The method according to claim 1, wherein the aromatic compound or a salt thereof is gallic acid, 4-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

9. The method according to claim 3, wherein the aromatic compound or salt thereof is gallic acid, 4-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

10. The method according to claim 1, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.

11. The method according to claim 10, wherein the bacterium of the genus Corynebacterium is Corynebacterium glutamicum, Corynebacterium efficiency, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanoritycum, Corynebacterium carnae, Corynebacterium crenatum, or Corynebacterium crudylactis.

12. The method according to claim 10, wherein the bacterium of the genus Corynebacterium is Corynebacterium glutamicum.

13. Modified Corynebacteria in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, and which have at least the following (C), (D), (E), and (F): (C) Polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimic acid dehydratase activity (E) Polypeptides necessary for biosynthesis of para-aminobenzoic acid from colismic acid (F) Polypeptide having para-aminobenzoic acid hydroxylase activity Corynebacteria with enhanced expression of one or more polypeptides selected from the following.

14. The cytochrome bd-type oxidase complex is (A) or (B) below: (A) A complex composed of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (B) A complex comprising a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 2 and functioning as cytochrome bd-type oxidase subunit I, and a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 4 and functioning as cytochrome bd-type oxidase subunit II, The Corynebacter bacterium according to claim 13, which is a polypeptide complex having cytochrome bd-type oxidase activity as shown by [formula].

15. The Corynebacterium bacterium according to claim 13, wherein part or all of the nucleotide sequence of the polynucleotide of (a) or (b) below is deleted or inactivated. (a) A polynucleotide encoding a complex composed of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 (b) A polynucleotide encoding a complex comprising a polynucleotide having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide that functions as cytochrome bd-type oxidase subunit I, and a polynucleotide having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and encoding a polypeptide that functions as cytochrome bd-type oxidase subunit II.

16. The coryneform bacterium according to claim 13, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.

17. The Corynebacterium bacterium according to claim 16, wherein the Corynebacterium bacterium is Corynebacterium glutamicum, Corynebacterium efficiency, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanoritycum, Corynebacterium carnae, Corynebacterium crenatum, or Corynebacterium crudylactis.

18. The Corynebacterium bacterium according to claim 16, wherein the Corynebacterium bacterium is Corynebacterium glutamicum.

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