Method for producing aromatic compound

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 a modified coryneform bacterium. [Background technology]

[0002] In recent years, there has been a demand for the use of microorganisms to produce useful aromatic compounds, including gallic acid, from glucose, an inexpensive raw material. Microorganisms possess a metabolic pathway for the biosynthesis of aromatic compounds, known as the shikimate pathway. Specifically, phosphoenolpyruvate, produced in glycolysis, combines with erythrose 4-phosphate supplied via the pentose phosphate pathway to form 3-deoxy-D-arabinopeptulosonic acid 7-phosphate (DAHP), which then undergoes the synthesis of shikimate via 3-dehydroquinate (DHQ) and 3-dehydroshikimate (DHS). Furthermore, shikimate undergoes the transfer of a phosphate group from adenosine triphosphate to form 3-phosphoshikimate, which then undergoes the synthesis of 3-phosphoenolpyruvylshikimate and then chorismate. In the shikimate pathway, a six-membered carbon ring is formed, followed by the formation of a double bond. From the protocatechuate derived from DHS, aromatic compounds such as gallic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), catechol, para-aminobenzoic acid (4-ABA), and 4-amino-3-hydroxybenzoic acid (4,3-AHBA) are produced (Figure 1).

[0003] Of these, gallic acid, due to its strong reducing properties, is used as a raw material for producing photographic developers and blue inks, among other things. Furthermore, esters such as propyl gallate are used as antioxidants for oils and butter. Furthermore, pyrogallol, synthesized by decarboxylation of gallic acid, is used in electronic materials, organic synthesis reagents, photographic developers, and woolen textile mordants, making efficient production of gallic acid beneficial. Polybenzoxazole (PBO), an engineering plastic with excellent heat resistance and mechanical strength, is used in textile materials and insulating films for semiconductor devices (Non-Patent Document 1). 4-amino-3-hydroxybenzoic acid (4,3-AHBA), a potential raw material for its production, has also attracted attention in recent years. Furthermore, para-aminobenzoic acid (4-ABA), which can be used as a raw material for UV absorbers, pharmaceuticals, and polymers, has traditionally been synthesized chemically, but the energy required for this process necessitates the development of environmentally friendly fermentation production techniques.

[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 Document 2). Previously, reports have been published regarding the relationship between respiratory chain-related enzymes and substance production in coryneform bacteria, such as improved productivity of lysine, a TCA cycle derivative, in strains in which cytochrome bd oxidase was disrupted (Non-Patent Document 3), and improved productivity of diacetyl in strains in which cytochrome bd oxidase was overexpressed (Non-Patent Document 4). Patent Document 1 also discloses microorganisms engineered to inactivate the respiratory pathway in order to increase the amount of NAD(P)H available for NAD(P)H-requiring oxidoreductases.

[0005] However, it has not been known until now that suppressing or deleting the function of cytochrome bd-type oxidase improves the productivity of aromatic compounds. [Prior art documents] [Patent documents]

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

[0007] [Non-Patent Document 1] Hirotaka 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 [Problem to be solved by the invention]

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

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

[0010] That is, the present invention relates to the following 1) and 2). 1) A method for producing an aromatic compound or a salt thereof, comprising a step of culturing a modified coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted. 2) A modified coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, and which comprises at least the following (C), (D), (E), and (F): (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) A polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate (F) a polypeptide having para-aminobenzoic acid hydroxylase activity A coryneform bacterium in which expression of one or more polypeptides selected from the group consisting of: [Effects of the Invention]

[0011] According to the present invention, aromatic compounds such as gallic acid, para-aminobenzoic acid, and 4-amino-3-hydroxybenzoic acid or salts thereof can be efficiently produced by a fermentation method that places little strain on the environment. [Brief explanation of the drawings]

[0012] [Figure 1]Schematic diagram showing the production pathways of various aromatic compounds when Corynebacterium glutamicum is used as a 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 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. pobA* is a gene encoding a polypeptide with 3,4-dihydroxybenzoate hydroxylase activity, a mutant of pobA from Corynebacterium glutamicum. phbh* is a gene encoding a polypeptide with p-aminobenzoate hydroxylase activity, a mutant of p-hydroxybenzoate hydroxylase from Caulobacter vibrioides. These pathways are mediated by exogenous gene transfer. In the figure, E4P is erythrose 4-phosphate, PEP is phosphoenolpyruvate, DAHP is 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, DHQ is dehydroquinic acid, DHS is dehydroshikimic acid, SHK is shikimic acid, S3P is shikimate-3-phosphate, and EPSP is 5-enoylpyruvinylshikimate-3-phosphate. DETAILED DESCRIPTION OF THE INVENTION

[0013] In the present invention, the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, the identity 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] In the present invention, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence preferably means identity of 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% 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 10, preferably 1 to 8, more preferably 1 to 5, and even more preferably 1 to 3 amino acids have been deleted, substituted, added, or inserted. Furthermore, "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 30, preferably 1 to 24, more preferably 1 to 15, and even more preferably 1 to 9 nucleotides have been deleted, substituted, added, or inserted. In the present invention, "addition" of an amino acid or nucleotide includes addition of an amino acid or nucleotide to one or both ends of a sequence.

[0016] In the present invention, aromatic compounds refer to organic aromatic compounds biosynthesized in coryneform bacteria, specifically aromatic compounds synthesized via the shikimate pathway, preferably aromatic compounds derived from 3-dehydroshikimic acid (DHS) or chorismate (Figure 1). Specific examples include protocatechuic acid, catechol, gallic acid, phenylalanine, tyrosine, tryptophan, parahydroxybenzoic acid, 4-aminobenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, and 4-amino-3-hydroxybenzoic acid. Among these, protocatechuic acid derived from DHS; gallic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), and catechol derived from protocatechuic acid; and parahydroxybenzoic acid, paraaminobenzoic acid (4-ABA), 4-amino-3-hydroxybenzoic acid (4,3-AHBA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), tyrosine, tryptophan, and the like derived from chorismic acid are preferred, with gallic acid, paraaminobenzoic acid, and 4-amino-3-hydroxybenzoic acid being more preferred.

[0017] Examples of the salts of the aromatic compounds include base addition salts, acid addition salts, etc. Examples of the 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 the acid addition salts include mineral acid salts such as hydrochlorides, sulfates, nitrates, and phosphates.

[0018] The method for producing an aromatic compound or a salt thereof of the present invention uses a coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted. The "cytochrome bd-type oxidase complex" is an aerobic respiration-related enzyme of coryneform bacteria, and specifically, is one of the terminal oxidases of the respiratory chain. Coryneform bacteria are known to possess 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 subunits I and II, which, based on N-terminal sequence analysis, correspond to CydA and CydB, respectively (Kusumoto et al. 2000, Arch Microbiol 173:390-397). The aa3 oxidase is known to consist of four subunits (CtaD, CtaC, CtaE, and CtaF) and to form a complex with the cytochrome bc1 complex (QcrB, QcrA, and QcrC) (Non-Patent Document 2). The proton export efficiencies of bd oxidase and the bc1-aa3 complex are H + The / O ratios of bd-type oxidase are known to be 2 and 6, respectively, and it has been reported that the efficiency of proton excretion per oxygen molecule is lower in bd-type oxidase, resulting in a lower efficiency of ATP synthesis (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, the cytochrome bd oxidase complex preferably includes a polypeptide complex having cytochrome bd oxidase activity shown in the following (A) or (B): (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 that functions as cytochrome bd-type oxidase subunit I and has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide that functions as cytochrome bd-type oxidase subunit II and has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4. Here, (A) the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 refers to cytochrome bd oxidase subunit I derived from Corynebacterium glutamicum, and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 refers to cytochrome bd oxidase subunit II derived from Corynebacterium glutamicum.

[0020] In the polypeptide (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 having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 or 4 include amino acid sequences in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 2 or 4.

[0021] The presence of cytochrome bd oxidase complex activity can be measured, for example, by referring to the aforementioned non-patent literature (Kusumoto et al. 2000, Arch Microbiol 173:390-397). Specifically, cytochrome bd oxidase is extracted as a membrane protein from bacterial cells, and purified using a column or the like to obtain an enzyme, which can be used to measure the reaction of oxidizing quinol to quinone.

[0022] (B) Polypeptides having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2 and functioning as cytochrome bd-type oxidase subunit I and polypeptides having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4 and functioning as cytochrome bd-type oxidase subunit II include, for example, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium SP. SalYVA5, Corynebacterium hadale, Corynebacterium gottingense, Corynebacterium godavarianum, Corynebacterium Examples include cytochrome bd-type oxidase subunit I and cytochrome bd-type oxidase subunit II derived from Pseudomonas senegalense.

[0023] Methods for introducing mutations such as deletion, substitution, addition, or insertion of amino acids into the amino acid sequence of the above-mentioned polypeptide include, 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; and the method described by Dieffenbach et al. (Cold Spring Harbar Laboratory Press, New York, pp. 581-621, 1995). 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 (Clonetech) and KOD-Plus-Mutagenesis Kit (Toyobo) can also be used.

[0024] In the present invention, modified coryneform bacteria (also referred to as "transformants") in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted 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 deleted, preferably bacterial strains (mutation-introduced strains) in which the expression of the cytochrome bd-type oxidase complex is reduced or lost compared to the parent bacterium due to the introduction of 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 refers to the production of a translation product from a gene encoding the protein and its functional localization at its site of action. "Decreased or lost expression of the cytochrome bd oxidase complex due to the introduction of a mutation" refers to a state in which the amount of cytochrome bd oxidase complex protein present in the transformant is reduced or lost as a result of modifications at the gene level, transcription level, post-transcriptional regulation level, translation level, or post-translational modification level, preferably a state in which the amount is significantly reduced or lost compared to that in the parent bacterium.

[0025] "Decreased expression of cytochrome bd oxidase complex compared to the parent bacterium" means that the expression level of the cytochrome bd oxidase complex present in the transformant is reduced compared to the parent bacterium; more specifically, the expression level of the protein is reduced to typically 50% or less, preferably 20% or less, and more preferably 10% or less compared to the parent bacterium, resulting in a similar reduction in its activity. Most preferably, the expression level of the cytochrome bd oxidase complex is 0%, i.e., expression of the cytochrome bd oxidase complex is lost. The expression levels of the cytochrome bd-type oxidase complex are compared by measuring the expression levels of the polypeptides using well-known immunological techniques such as Western blotting and immunohistochemical staining.

[0026] Specifically, the modified coryneform bacterium in which expression of the cytochrome bd oxidase complex is reduced or eliminated can be obtained by suppressing the function of a gene encoding the cytochrome bd oxidase complex on the chromosomal DNA of the host bacterium, which may be completely or incompletely suppressed. The gene encoding the cytochrome bd-type oxidase complex refers to DNA consisting of a transcription region including an ORF and a transcriptional regulatory region such as the promoter of the gene. In the present invention, the gene encoding the cytochrome bd-type oxidase complex is preferably a polynucleotide of the following (a) or (b):

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

[0028] Examples of nucleotide sequences having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 include nucleotide sequences in which one or several nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. Methods for introducing mutations such as nucleotide deletion, substitution, addition, or insertion into a nucleotide sequence are as described above. The polynucleotide may be in the form of a single strand or a double strand, and may be DNA or RNA. The DNA may be artificial DNA such as cDNA or chemically synthesized DNA.

[0029] The function of such genes encoding cytochrome BD-type oxidase complexes can be suppressed by introducing deletion or inactivation mutations in the coding region, non-coding region, or transcription or translation initiation region of the genes encoding cytochrome BD-type oxidase complexes (deletion or inactivation of the genes encoding cytochrome BD-type oxidase complexes), or by suppressing transcription or translation by introducing polynucleotides that have the activity of degrading the transcription products of genes encoding cytochrome BD-type oxidase complexes, or polynucleotides that suppress translation of the transcription products into proteins.

[0030] In one embodiment, the deletion or inactivation of the gene encoding the 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 mutating 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 of specifically deleting or inactivating the gene encoding the cytochrome bd-type oxidase complex in the genome of a cell, and a method of randomly deleting or inactivating the gene in a bacterial cell, followed by evaluating the expression level or activity of the cytochrome bd-type oxidase complex or performing genetic analysis to select cells with the desired mutation.

[0031] For example, specific deletion or inactivation of the gene encoding the cytochrome bd-type oxidase complex can be achieved by homologous recombination. Specifically, a DNA fragment of the gene encoding the cytochrome bd-type oxidase complex into which an inactivating mutation has been introduced by polynucleotide substitution or insertion, or a DNA fragment containing an 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, and this fragment is then inserted into a parent bacterium to induce homologous recombination with a region of the genome of the parent bacterium containing the gene encoding the cytochrome bd-type oxidase complex, thereby deleting or inactivating the gene encoding the cytochrome bd-type oxidase complex in 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 containing a partial region of the gene encoding the cytochrome bd-type oxidase complex into the parent bacterium and disrupting a partial region of the gene encoding the cytochrome bd-type oxidase complex in the parent bacterial genome by homologous recombination. Methods for randomly deleting or inactivating genes in bacterial cells include introducing DNA fragments into cells in which genes with inactivating mutations have been randomly cloned to induce homologous recombination between the genes and the genome of the cells, and inducing mutations by irradiating cells with ultraviolet light, gamma rays, or the like. Gene inactivating mutations refer to mutations that result in the loss of the original function of the targeted gene, such as silence mutations, missense mutations, nonsense mutations, and frameshift mutations. For example, genes with inactivating mutations do not express proteins or express proteins with impaired original activity.

[0032] Site-directed mutagenesis is one method for preparing a DNA fragment containing a gene encoding a cytochrome bd-type oxidase complex into which an inactivating mutation has been introduced. Site-directed mutagenesis can be performed using mutagenesis primers containing the nucleotide mutation to be introduced. For example, two pairs of primers containing the nucleotide mutation to be introduced are used to perform PCR using the gene encoding the cytochrome bd-type oxidase complex as a template, to generate DNA fragments by amplifying the upstream and downstream regions of the region containing the gene encoding the cytochrome bd-type oxidase complex. These fragments are then ligated together by SOE-PCR (splicing by overlap extension PCR) (Gene, 1989, 77(1): pp. 61-68), thereby constructing a DNA fragment containing the desired mutation. Alternatively, site-specific mutagenesis can be performed using inverse PCR or annealing (Muramatsu et al., "New Genetic Engineering Handbook, Revised 4th Edition," Yodosha, pp. 82-88), or commercially available site-specific mutagenesis kits such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit.

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

[0034] Well-known techniques can be used to introduce DNA fragments or vectors into host bacteria, such as the calcium phosphate method, electroporation, lipofection, particle gun method, PEG method, etc. For example, methods applicable to coryneform bacteria 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 method (J Bacteriol, 1983, 156:1130-1134).

[0035] Furthermore, examples of polynucleotides that have the activity of degrading the transcript of a gene encoding a cytochrome bd-type oxidase complex, or that suppress translation of the transcript into protein, include polynucleotides that contain a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of a gene encoding a cytochrome bd-type oxidase complex, or a part thereof. Specific examples include antisense RNA for the mRNA of a gene encoding a cytochrome bd-type oxidase complex, siRNA for the mRNA of a gene encoding a cytochrome bd-type oxidase complex, and ribozymes for the mRNA of a gene encoding a 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 confirming 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 bacterium is a coryneform bacterium, including a group of microorganisms defined as coryneform bacteria in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), specifically including bacteria of the genera Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, and Micrococcus. Examples of Corynebacterium bacteria include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium crenatum, Corynebacterium crudilactis, and Corynebacterium callunae. Examples of the Brevibacterium genus include Brevibacterium ammoniagenes. Examples of the genus Arthrobacter include Arthrobacter globiformis. Examples of bacteria of the genus Mycobacterium include Mycobacterium bovis, and examples of bacteria of the genus Micrococcus include Micrococcus freudenreichii, Micrococcus leuteus, Micrococcus ureae, and Micrococcus roseus. Among the coryneform bacteria, the genus Corynebacterium is preferred, and Corynebacterium glutamicum is more preferred. The microbial cells may be wild-type strains, mutant strains thereof, or artificially recombinant strains.

[0038] In the present invention, the coryneform bacterium used as a host may be any bacterium suitable for producing aromatic compounds or their salts, but from the viewpoint of the efficiency of production of aromatic compounds or their salts, particularly aromatic compounds derived from 3-dehydroshikimic acid or 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, or their salts, it is preferable to use a coryneform bacterium with improved 3-dehydroshikimic acid-producing activity and / or chorismic acid-producing activity. Specifically, in order to increase the efficiency of production of compounds derived from 3-dehydroshikimic acid and chorismic acid, it is preferable to use a coryneform bacterium with enhanced expression of one or more polypeptides selected from the following (C), (D), (E), and (F) as a host. (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) A polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate (F) a polypeptide having para-aminobenzoic acid hydroxylase activity

[0039] Therefore, the modified coryneform bacterium of the present invention is preferably a modified coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, and which exhibits at least the following (C), (D), (E), and (F): (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) A polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate (F) a polypeptide having para-aminobenzoic acid hydroxylase activity The coryneform bacterium has enhanced expression of one or more polypeptides selected from the group consisting of:

[0040] Here, the polypeptide (C) having 3,4-dihydroxybenzoate hydroxylase activity is a polypeptide that catalyzes the reaction of producing gallic acid from protocatechuic acid, and examples thereof 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 to 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 SEQ ID NO: 5 is a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity, which is a mutant of parahydroxybenzoate hydroxylase (pobA) derived from Corynebacterium glutamicum (represented as "pobA*" in Figure 1). Furthermore, examples of polypeptides having 3,4-dihydroxybenzoate hydroxylase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 5 of (C2) include polypeptides having 3,4-dihydroxybenzoate hydroxylase activity derived from bacteria of the genus Corynebacterium, specifically 4-hydroxybenzoate-3-monooxygenase derived from Corynebacterium glutamicum, 4-hydroxybenzoate-3-monooxygenase derived from Corynebacterium suranareeae, and 4-hydroxybenzoate-3-monooxygenase derived from Corynebacterium crudilactis.

[0041] The polypeptide (D) having 3-dehydroshikimate dehydratase activity is a polypeptide that catalyzes the reaction of producing protocatechuic acid from 3-dehydroshikimic acid, and examples thereof 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 to the amino acid sequence shown in SEQ ID NO: 6 and having 3-dehydroshikimate dehydratase activity. Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 is 3-dehydroshikimate dehydratase derived from Corynebacterium glutamicum and is known as "qsuB." Furthermore, examples of polypeptides that consist of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6 of (D2) and have 3-dehydroshikimate dehydratase activity include 3-dehydroshikimate dehydratases derived from bacteria of the genus Corynebacterium, and specifically include 3-dehydroshikimate dehydratases derived from Corynebacterium glutamicum and 3-dehydroshikimate dehydratases derived from Corynebacterium thanareae.

[0042] (E) Polypeptides necessary for the biosynthesis of para-aminobenzoic acid from chorismate include (EI) 4-amino-4-deoxychorismate synthase and (EII) 4-amino-4-deoxychorismate lyase. Therefore, enhancing expression of polypeptides necessary for the biosynthesis of para-aminobenzoic acid from chorismate includes enhancing expression of one or more of these polypeptides. Para-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 para-aminobenzoic acid by 4-amino-4-deoxychorismate lyase (PabC). The (EI) 4-amino-4-deoxychorismate synthase is a polypeptide that catalyzes the reaction of producing 4-amino-4-deoxychorismate from chorismate, and examples thereof include (EI-1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, or (EI-2) a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7 and that has 4-amino-4-deoxychorismate synthase activity. The (EII) 4-amino-4-deoxychorismate lyase is a polypeptide that catalyzes the reaction of producing para-aminobenzoic acid from 4-amino-4-deoxychorismate, and examples thereof include (EII-1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8, or (EII-2) a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 8 and that has 4-amino-4-deoxychorismate lyase activity. Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7 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: 8 is 4-amino-4-deoxychorismate lyase derived from Corynebacterium glutamicum and is known as "pabC".

[0043] The polypeptide (F) having para-aminobenzoate hydroxylase activity is a polypeptide that catalyzes the reaction of producing 4-amino-3-hydroxybenzoic acid from para-aminobenzoic acid, and examples thereof 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 to the amino acid sequence shown in SEQ ID NO: 9 and having para-aminobenzoate hydroxylase activity. Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 is a polypeptide having para-aminobenzoate hydroxylase activity, which is a mutant of para-hydroxybenzoate hydroxylase derived from Caulobacter vibrioides (represented as "phbh*" in Figure 1). Furthermore, (F2) examples of polypeptides having para-aminobenzoate hydroxylase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9 include para-hydroxybenzoate hydroxylases derived from bacteria of the genus Caulobacter, and specifically include para-hydroxybenzoate hydroxylases derived from Caulobacter rhizosphaerae and Caulobacter sp.

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

[0045] Furthermore, in order to improve 3-dehydroshikimic acid production or chorismic acid production activity, it is preferable to use a coryneform bacterium used as a host in the present invention that further enhances the expression of, for example, the polypeptides shown in (G) to (O) below. (G) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity; (H) a polypeptide having 3-dehydroquinate synthase activity; (I) a polypeptide having 3-dehydroquinate dehydratase activity, (J) a polypeptide having 5-shikimate dehydrogenase activity; (K) a polypeptide having shikimate kinase activity; (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyltransferase activity, (M) a polypeptide having chorismate synthase activity; (N) a polypeptide having pyruvate phosphate dikinase activity; (O) Enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase.

[0046] Specifically, the host coryneform bacterium in which expression of the above-mentioned polypeptide is enhanced is a coryneform bacterium into which a polynucleotide necessary for expression of the polypeptide has been introduced so as to be expressible, and the polypeptide may be either exogenous or endogenous to the bacterium. Examples include a coryneform bacterium into which the polynucleotide has been introduced so as to be expressible, and a coryneform bacterium in which the level of expression of the polynucleotide has been enhanced. Specific examples include a coryneform bacterium into which a vector or DNA fragment containing the polynucleotide and a control region operably linked thereto has been introduced, and a coryneform bacterium in which the control region of the polynucleotide has been replaced with a strong control region. Examples of strong regulatory regions include known high expression promoters such as the T7 promoter, lac promoter, tac promoter, trp promoter, tu promoter, and gap promoter, 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.

[0047] The modified coryneform bacterium thus prepared is cultured, the productivity of an aromatic compound or a salt thereof is evaluated, and an appropriate transformant is selected, thereby obtaining a useful bacterium that produces an aromatic compound or a salt thereof. The product can be measured according to the method described in the Examples below.

[0048] The method for producing an aromatic compound or a salt thereof of the present invention is carried out by culturing the above-mentioned modified coryneform bacterium, preferably in the presence of sugars, and recovering the target aromatic compound or a salt thereof. Although glucose is preferred as a sugar, other sugars that can produce glucose through metabolism, such as monosaccharides like fructose, mannose, arabinose, xylose, and galactose, can also be used. Such sugars include oligosaccharides or polysaccharides having glucose units, such as disaccharides like cellobiose, sucrose (cane sugar), lactose, maltose, trehalose, cellobiose, and xylobiose; and polysaccharides like 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.

[0049] The medium for culturing the transformant 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 coryneform bacterium of the present invention. As the carbon source, the above-mentioned sugars or molasses or saccharified solution containing them can be used, but in addition to the above-mentioned sugars, sugar alcohols such as mannitol, sorbitol, xylitol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; alcohols such as ethanol and propanol; hydrocarbons such as normal paraffin, etc. The carbon source can be used alone or in combination of two or more. The concentration of the sugars, which are 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] 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.

[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, if necessary, vitamins, antifoaming agents, etc. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.

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

[0053] Prior to the reaction or culture containing sugars, the transformant is preferably cultured and grown in the same medium under aerobic conditions at a temperature of about 25 to 38°C for about 12 to 48 hours.

[0054] The culture temperature or reaction temperature is preferably 15 to 45°C, more preferably 25 to 37°C. The culture or reaction time is 24 to 168 hours, preferably 24 to 96 hours, and more preferably 24 to 72 hours, and can be performed with stirring or shaking as necessary. During culture, antibiotics such as ampicillin or kanamycin may be added to the medium 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 the transformant, in terms of the efficiency of production of the aromatic compound or a salt thereof.

[0055] The method for recovering and purifying the aromatic compound or a salt thereof from the culture is not particularly limited. That is, the method can be carried out by combining well-known methods such as ion exchange resin methods, precipitation methods, crystallization methods, recrystallization methods, concentration methods, and the like. For example, the aromatic compound or a salt thereof can be obtained by removing the bacterial cells by centrifugation or the like, removing ionic substances with cation and anion exchange resins, and concentrating the mixture. The aromatic compound or a salt thereof accumulated in the culture may be used as is without isolation.

[0056] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A method for producing an aromatic compound or a salt thereof, comprising a step of culturing a modified coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted. <2> The modified coryneform bacterium is a bacterial strain in which expression of the cytochrome bd-type oxidase complex is reduced or lost compared to the parent bacterium. <1> How to do it. <3> The cytochrome bd 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 having at least 90% identity with 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 having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as cytochrome bd-type oxidase subunit II; A polypeptide complex having cytochrome bd-type oxidase activity represented by <1> or <2> How to do it. <4> The modified coryneform bacterium is a bacterial strain in which a part or all of the nucleotide sequence of the following polynucleotide (a) or (b) has been deleted or inactivated: <2> or <3> method. (a) a polynucleotide encoding a complex consisting 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 consisting of a polynucleotide having a nucleotide sequence at least 90% identical to 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 a nucleotide sequence at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a polypeptide that functions as cytochrome bd-type oxidase subunit II. <5> As a host, at least the following (C), (D), (E) and (F): (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) A polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate (F) a polypeptide having para-aminobenzoic acid hydroxylase activity A coryneform bacterium in which expression of one or more polypeptides selected from the following is enhanced is used. <1> ~ <4> Either way. <6> (C) The polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is (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 to the amino acid sequence shown in SEQ ID NO: 5 and having 3,4-dihydroxybenzoate hydroxylase activity. <5> How to do it. <7> (D) The polypeptide having 3-dehydroshikimate 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 to the amino acid sequence shown in SEQ ID NO: 6 and having 3-dehydroshikimate dehydratase activity. <5> How to do it. <8> (E) the polypeptide necessary for biosynthesis of para-aminobenzoic acid from chorismate is one or more selected from (EI) 4-amino-4-deoxychorismate synthase and (EII) 4-amino-4-deoxychorismate lyase; <5> How to do it. <9> (EI) the 4-amino-4-deoxychorismate synthase is (EI-1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, or (EI-2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 7 and having 4-amino-4-deoxychorismate synthase activity, and (EII) the 4-amino-4-deoxychorismate lyase is (EII-1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8, or (EII-2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 8 and having 4-amino-4-deoxychorismate lyase activity. <8> How to do it. <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 to the amino acid sequence shown in SEQ ID NO: 9 and having para-aminobenzoic acid hydroxylase activity. <5> How to do it. <11> As hosts, the following (G) to (O): (G) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity; (H) a polypeptide having 3-dehydroquinate synthase activity; (I) a polypeptide having 3-dehydroquinate dehydratase activity, (J) a polypeptide having 5-shikimate dehydrogenase activity; (K) a polypeptide having shikimate kinase activity; (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyltransferase activity, (M) a polypeptide having chorismate synthase activity; (N) a polypeptide having pyruvate phosphate dikinase activity; (O) a group of enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase; A coryneform bacterium in which expression of one or more polypeptides selected from the following is enhanced is used. <5> ~ <10> Either way. <12> The aromatic compound or its salt 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; <1> ~ <11> Either way. <13> The aromatic compound or its salt is gallic acid, protocatechuic acid, parahydroxybenzoic acid, paraaminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof; <1> ~ <11> Either way. <14> The aromatic compound or its salt is gallic acid, para-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof; <1> ~ <11> Either way. <15> The coryneform bacteria are bacteria of the genus Corynebacterium, <1> ~ <14> Either way. <16> the bacterium of the genus Corynebacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerans, Corynebacterium alkanolyticum, Corynebacterium callunae, Corynebacterium crenatum, or Corynebacterium cruzilactis; <1> ~ <14> Either way. <17> The Corynebacterium bacterium is Corynebacterium glutamicum. <1> ~ <14> Either way.

[0057] <18> A modified coryneform bacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted, comprising at least the following (C), (D), (E), and (F): (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) A polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate (F) a polypeptide having para-aminobenzoic acid hydroxylase activity A coryneform bacterium in which expression of one or more polypeptides selected from the group consisting of: <19> The cytochrome bd 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 having at least 90% identity with 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 having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as cytochrome bd-type oxidase subunit II; A polypeptide complex having cytochrome bd-type oxidase activity represented by <18> Corynebacterium species. <20> A polynucleotide comprising the following polynucleotide (a) or (b) in which a part or all of the nucleotide sequence is deleted or inactivated: <18> or <19> Corynebacterium species. (a) a polynucleotide encoding a complex consisting 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 consisting of a polynucleotide having a nucleotide sequence at least 90% identical to 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 a nucleotide sequence at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a polypeptide that functions as cytochrome bd-type oxidase subunit II. <21> (C) The polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is (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 to the amino acid sequence shown in SEQ ID NO: 5 and having 3,4-dihydroxybenzoate hydroxylase activity. <18> ~ <20> Any of the coryneform bacteria. <22> (D) The polypeptide having 3-dehydroshikimate 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 to the amino acid sequence shown in SEQ ID NO: 6 and having 3-dehydroshikimate dehydratase activity. <18> ~ <20> Any of the coryneform bacteria. <23> (E) the polypeptide necessary for biosynthesis of para-aminobenzoic acid from chorismate is one or more selected from (EI) 4-amino-4-deoxychorismate synthase and (EII) 4-amino-4-deoxychorismate lyase; <18> ~ <20> Any of the coryneform bacteria. <23> (EI) the 4-amino-4-deoxychorismate synthase is (EI-1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, or (EI-2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 7 and having 4-amino-4-deoxychorismate synthase activity, and (EII) the 4-amino-4-deoxychorismate lyase is (EII-1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8, or (EII-2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 8 and having 4-amino-4-deoxychorismate lyase activity. <22> Corynebacterium species. <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 to the amino acid sequence shown in SEQ ID NO: 9 and having para-aminobenzoic acid hydroxylase activity. <18> ~ <20> Any of the coryneform bacteria. <25> In addition, (G) to (O) below: (G) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity; (H) a polypeptide having 3-dehydroquinate synthase activity; (I) a polypeptide having 3-dehydroquinate dehydratase activity, (J) a polypeptide having 5-shikimate dehydrogenase activity; (K) a polypeptide having shikimate kinase activity; (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyltransferase activity, (M) a polypeptide having chorismate synthase activity; (N) a polypeptide having pyruvate phosphate dikinase activity; (O) a group of enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase; The expression of one or more polypeptides selected from the group consisting of: <18> ~ <24> Any of the coryneform bacteria. [Example]

[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 improved chorismate production activity> 1) Construction of a plasmid incorporating the tu promoter into the cg1829(aroC) promoter region The 5' upstream region of the cg1829 gene (SEQ ID NO: 10) was amplified by PCR using two 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 DNA primers (SEQ ID NOs: 14 and 15) to obtain DNA fragments. A DNA fragment containing the tu promoter (SEQ ID NO: 16) was amplified by PCR using the genome of the ATCC13032 strain as a template and two DNA primers (SEQ ID NOs: 17 and 18) to obtain a DNA fragment. Furthermore, a PCR amplification was performed using pHKPsacB1 (see Patent Application No. 2014-523757) as a template and two DNA primers (SEQ ID NOs: 19 and 20), and the resulting PCR product was 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_Ptu-aroC.

[0060] 2) Construction of a strain in which the tu promoter was introduced into the cg1829(aroC) promoter region The KC367sr strain was obtained by introducing the above-mentioned plasmid pHKPsacB_Ptu-aroC into the KC315 strain (Patent Application No. 2021-201877) using electroporation transformation and selecting for kanamycin resistance. The KC367sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 11 and 21. The expected results were obtained, confirming that the KC367sr strain is a single-crossover homologous recombinant into which the pHKPsacB_Ptu-aroC plasmid was introduced. The KC367sr 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 KC367 strain. PCR (Sapphire Amp (Takara Bio)) using primers SEQ ID NOs: 21 and 22 confirmed that the KC367 strain was a double-crossover homologous recombinant with the tu promoter inserted into the cg1829 (aroC) promoter region, as expected.

[0061] 3) Construction of a plasmid incorporating the tu promoter into the cg1774(tkt) promoter region The 5' upstream region of the cg1774 gene region (SEQ ID NO: 23) was amplified by PCR using two 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 DNA primers (SEQ ID NOs: 27 and 28) to obtain DNA fragments. A DNA fragment containing the tu promoter (SEQ ID NO: 16) was amplified by PCR using the genome of the ATCC13032 strain as a template and two DNA primers (SEQ ID NOs: 17 and 18) to obtain a DNA fragment. A PCR amplification was also performed using pHKPsacB1 as a template and two DNA primers (SEQ ID NOs: 19 and 20), and the resulting PCR product was 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_Ptu-tkt.

[0062] 4) Construction of a strain in which the tu promoter was introduced into the cg1774(tkt) promoter region The KC376sr strain was isolated by electroporation transformation using the pHKPsacB_Ptu-tkt plasmid and selection for kanamycin resistance. Analysis of the KC376sr strain by PCR (Sapphire Amp, Takara Bio) using primers with sequence numbers 24 and 29 yielded the expected results, confirming that the KC376sr strain was a single-crossover homologous recombinant in which the pHKPsacB_Ptu-tkt plasmid had been introduced into the promoter region of cg1774. The KC376sr 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 KC376 strain. PCR (Sapphire Amp (Takara Bio)) using primers SEQ ID NOs: 29 and 30 confirmed that the KC376 strain was a double-crossover homologous recombinant in which the tu promoter had been introduced into the cg1774(tkt) promoter region, as expected.

[0063] 5) Construction of a plasmid incorporating the tu promoter into the cg0644(ppsA) promoter region The 5' upstream region of the cg0644 gene region (SEQ ID NO: 31) was amplified by PCR using two 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 DNA primers (SEQ ID NOs: 35 and 36) to obtain DNA fragments. A DNA fragment containing the tu promoter (SEQ ID NO: 16) was amplified by PCR using the genome of the ATCC13032 strain as a template and two DNA primers (SEQ ID NOs: 17 and 18) to obtain a DNA fragment. A PCR amplification was also performed using pHKPsacB1 as a template and two DNA primers (SEQ ID NOs: 19 and 20), and the resulting PCR product was 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_Ptu-ppsA.

[0064] 6) Construction of a strain in which the tu promoter was introduced into the cg0644(ppsA) promoter region The KC408sr strain was isolated by electroporation transformation using the pHKPsacB_Ptu-ppsA plasmid and selection for kanamycin resistance. Analysis of the KC408sr strain by PCR (Sapphire Amp, Takara Bio) using primers with sequence numbers 32 and 37 yielded the expected results, confirming that the KC408sr strain was a single-crossover homologous recombinant containing the pHKPsacB_Ptu-tkt plasmid. The KC408sr 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 KC408 strain. PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 37 and 38 confirmed that the KC408 strain was a double-crossover homologous recombinant in which the tu promoter had been introduced into the cg0644(tkt) promoter region, as expected.

[0065] <Productivity evaluation of 4-aminobenzoic acid (4-ABA)> (1) Construction of 4-ABA-producing strains 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 region (SEQ ID NO: 39) was amplified by PCR using two 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 DNA primers (SEQ ID NOs: 43 and 44) ​​to obtain DNA fragments. A DNA fragment containing the cg2875 gene promoter (SEQ ID NO: 45) was amplified by PCR using the genome of the ATCC13032 strain as a template and two DNA primers (SEQ ID NOs: 46 and 47) to obtain a DNA fragment. Furthermore, PCR was performed using pHKPsacB1 as a template and two DNA primers (SEQ ID NOs: 19 and 20), and the resulting PCR product was 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_Pcg2875-pabAB.

[0066] 2) Construction of a strain in which the promoter of the cg2875 gene was introduced into the cg1134(pabAB) promoter region The KC412sr strain was isolated by electroporation transformation using the aforementioned plasmid pHKPsacB_Pcg2875-pabAB and selection for kanamycin resistance. Analysis of the KC412sr strain by PCR (Sapphire Amp (Takara Bio)) using primers with SEQ ID NOs: 40 and 48 yielded the expected results, confirming that the KC412sr strain was a single-crossover homologous recombinant containing the pHKPsacB_Pcg2875-pabAB plasmid. The KC412sr 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 KC412 strain. PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 48 and 49 confirmed that the KC412 strain was a double-crossover homologous recombinant in which the cg2875 gene promoter had been introduced into the cg1134 (pabAB) promoter region, as expected.

[0067] (2) Construction of a 4-ABA-producing cydAB disruptant 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 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 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 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 PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated using the In-Fusion HD Cloning Kit (Takara Bio) to generate the plasmid pHKPsacB_ΔcydAB.

[0068] 2) Creation of a strain lacking the cg1300-1301 (cydAB) gene region The KC418sr strain was isolated by electroporation transformation using the pHKPsacB_ΔcydAB plasmid and selection for kanamycin resistance. PCR analysis of the KC418sr strain using primers SEQ ID NOs: 51 and 56 (Sapphire Amp, Takara Bio) yielded the expected results, confirming that the KC418sr strain was a single-crossover homologous recombinant containing the pHKPsacB_ΔcydAB plasmid. The KC418sr 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 KC418 strain. PCR (Sapphire Amp (Takara Bio)) using primers SEQ ID NOs: 56 and 57 confirmed that the KC418 strain was a double-crossover homologous recombinant in which the cg1300-1301 (cydAB) gene region had been deleted, as expected.

[0069] (3) Construction of a 4-ABA-producing ctaCF disruptant 1) Construction of a plasmid to delete the cg2408-2409 (ctaCF) gene region The 5' upstream region of the cg2408 gene (SEQ ID NO: 58) was amplified by PCR using two DNA primers (SEQ ID NOs: 59 and 60), and the 3' upstream region of the cg2409 gene (nucleotide number 61) was amplified by PCR using two DNA primers (SEQ ID NOs: 62 and 63) to obtain DNA fragments. Furthermore, PCR was performed using pHKPsacB1 as a template and two DNA primers (SEQ ID NOs: 19 and 20), and the resulting PCR products were treated with DpnI (Takara Bio). Each DNA fragment was purified from the three PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated using the In-Fusion HD Cloning Kit (Takara Bio) to generate the plasmid pHKPsacB_ΔctaCF.

[0070] 2) Creation of a strain lacking the cg2408-2409 (ctaCF) gene region The KC469sr strain was isolated by electroporation transformation using the pHKPsacB_ΔctaCF plasmid and selection for kanamycin resistance. Analysis of the KC469sr strain by PCR (Sapphire Amp, Takara Bio) using primers with sequence numbers 59 and 64 yielded the expected results, confirming that the KC469sr strain was a single-crossover homologous recombinant containing the pHKPsacB_ΔctaCF plasmid. The KC469sr 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 KC469 strain. PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 64 and 65 confirmed that the KC469 strain was a double-crossover homologous recombinant in which the cg2408-2409 (ctaCF) gene region had been deleted, as expected.

[0071] (4) Cultivation of transformed strains 1) The transformed strain obtained above was streaked onto an LB plate and cultured at 30°C for 3 days. The cells grown on the plate were inoculated into 6 mL of CGTG15 medium (Table 4) and cultured with shaking at 30°C and 250 rpm for 24 hours (preculture). 60 mL of CGTG15 medium (excluding urea) was placed in a Bio Jr. 8 (Able Co., Ltd.) fermenter. 1.2 mL of the preculture was inoculated and cultured with shaking at 32°C, pH 6.9, 700 rpm, and 60 mL / min for 30 hours. The aromatic compound productivity was evaluated. The culture was appropriately diluted with dilute sulfuric acid, centrifuged to remove the cells, and the supernatant was collected. The concentrations in the supernatant were quantified. The results are shown in the table.

[0072] 2) Results In comparison, the 4-ABA concentration was confirmed to be increased in the KC418 strain, indicating that disruption of the cydAB gene had a positive effect on production, while the 4-ABA concentration was confirmed to be decreased in the KC469 strain, indicating that disruption of the ctaCF gene had a negative effect on production (Table 1-1, Table 1-2).

[0073] [Table 1-1]

[0074] [Table 1-2]

[0075] <Productivity evaluation of 4-amino-3-hydroxybenzoic acid (4,3-AHBA)> (1) Creation of 4,3-AHBA-producing bacteria 1) Construction of a plasmid for introducing the HFM122 mutant gene linked to the promoter of the cg2875 gene into the cg2484 gene region pECsf_gapS_pabABC_tuD_HFM122 (see Patent Application No. 2019-203523) was used as a template for PCR amplification using two DNA primers (SEQ ID NOs: 63 and 64). 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 amplified by PCR using two DNA primers (SEQ ID NOs: 70 and 71, and 72 and 73). The resulting DNA fragments 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 plasmids pECsf_gapS_pabABC_tuD_HFM122LA_OPT1 and HFM122LA_OPT2. The 5' upstream region of the cg2484 gene region (SEQ ID NO: 74) was amplified by PCR using two DNA primers (SEQ ID NOs: 75 and 76), and the 3' region of the cg2484 gene region (SEQ ID NO: 77) was amplified by PCR using two DNA primers (SEQ ID NOs: 78 and 79) to obtain DNA fragments. A DNA fragment containing the cg2875 gene promoter (SEQ ID NO: 45) was also amplified by PCR using the genome of the ATCC13032 strain as a template and two DNA primers (SEQ ID NOs: 46 and 47). The plasmids pECsf_gapS_pabABC_tuD_HFM122LA_OPT1-HFM122LA_OPT2 were also amplified by PCR using two DNA primers (SEQ ID NOs: 69 and 80), and the resulting PCR products were treated with DpnI (Takara Bio). Additionally, pHKPsacB1 was used as a template for PCR amplification using two DNA primers (SEQ ID NOs: 19 and 20), and the resulting PCR products were treated with DpnI (Takara Bio). The five PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the resulting DNA fragments were ligated using the In-Fusion HD Cloning Kit (Takara Bio) to generate the plasmids pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2.

[0076] 2) Construction of a strain carrying the HFM122 mutant gene linked to the promoter of the cg2875 gene in the cg2484 gene region. The KC417sr strain was isolated by electroporation transformation using the aforementioned plasmid pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2 and selection for kanamycin resistance. Analysis of the KC417sr strain by PCR (Sapphire Amp (Takara Bio)) using primers 67 and 81 yielded the expected results, confirming that the KC417sr strain was a single-crossover homologous recombinant containing the pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2 plasmid. The KC417sr 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 KC417 strain. PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 81 and 82 confirmed that the KC417 strain was a double-crossover homologous recombinant, in which the HFM122 mutant gene linked to the cg2875 gene promoter had been introduced into the cg2484 gene region, as expected.

[0077] (2) Construction of a 4,3-AHBA-producing cydAB disruptant The KC427sr strain was isolated by electroporation transformation using the pHKPsacB_ΔcydAB plasmid and selection for kanamycin resistance. Analysis of the KC427sr strain by PCR (Sapphire Amp, Takara Bio) using primers with sequence numbers 51 and 56 yielded the expected results, confirming that the KC427sr strain was a single-crossover homologous recombinant containing the pHKPsacB_ΔcydAB plasmid. The KC427sr 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 KC427 strain. PCR (Sapphire Amp (Takara Bio)) using primers SEQ ID NOs: 56 and 57 confirmed that the KC427 strain was a double-crossover homologous recombinant in which the cg1300-1301 (cydAB) gene region had been deleted, as expected.

[0078] (3) Construction of a 4,3-AHBA-producing ctaCF-disrupted strain The KC470sr strain was isolated by electroporation transformation using the pHKPsacB_ΔctaCF plasmid and selection for kanamycin resistance. Analysis of the KC470sr strain by PCR (Sapphire Amp, Takara Bio) using primers with sequence numbers 59 and 64 yielded the expected results, confirming that the KC470sr strain was a single-crossover homologous recombinant containing the pHKPsacB_ΔctaCF plasmid. The KC470sr 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 KC470 strain. PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 64 and 65 confirmed that the KC470 strain was a double-crossover homologous recombinant in which the cg2408-2409 (ctaCF) gene region had been deleted, as expected.

[0079] (4) Cultivation of transformed strains 1) The transformed strain obtained above was streaked onto an LB plate and cultured at 30°C for 3 days. The bacterial cells grown on the plate were inoculated into 6 mL of CGTG15 medium and cultured with shaking at 30°C and 250 rpm for 24 hours (preculture). 60 mL of CGTG15 medium (excluding urea) was placed in a Bio Jr. 8 (Able Co., Ltd.) culture vessel. 1.2 mL of the preculture was inoculated and cultured with shaking at 32°C, pH 6.9, 700 rpm, and 60 mL / min for 30 hours. The aromatic compound productivity was evaluated. The culture was appropriately diluted with dilute sulfuric acid, the bacterial 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 4,3-AHBA concentration was increased in KC427, indicating that disruption of the cydAB gene had a positive effect on production, while the 4,3-AHBA concentration was decreased in KC470, indicating that disruption of the ctaCF gene had a negative effect on production.

[0081] [Table 2-1]

[0082] [Table 2-2]

[0083] <Productivity evaluation of gallic acid (GAL)> (1) Construction of a GAL-producing cydAB disruptant The KC201sr strain was obtained by introducing the above-mentioned plasmid pHKPsacB_ΔcydAB into the KC148 strain (Patent Application No. 2020-94649) using electroporation transformation and selecting for kanamycin resistance. The KC201sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 51 and 56. The expected results were obtained, confirming that the KC201sr strain is a single-crossover homologous recombinant into which the pHKPsacB_ΔcydAB plasmid was introduced. The KC201sr 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 KC201 strain. PCR (Sapphire Amp (Takara Bio)) using primers SEQ ID NOs: 56 and 57 confirmed that the KC201 strain was a double-crossover homologous recombinant in which the cg1300-1301 (cydAB) gene region had been deleted, as expected.

[0084] (2) Construction of a GAL-producing ctaCF disruptant The KC468sr strain was isolated by electroporation transformation using the pHKPsacB_ΔctaCF plasmid and selection for kanamycin resistance. Analysis of the KC468sr strain by PCR (Sapphire Amp, Takara Bio) using primers with sequence numbers 59 and 64 yielded the expected results, confirming that the KC468sr strain was a single-crossover homologous recombinant containing the pHKPsacB_ΔctaCF plasmid. The KC468sr 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 KC468 strain. PCR (Sapphire Amp (Takara Bio)) using primers with sequence numbers 64 and 65 confirmed that the KC468 strain was a double-crossover homologous recombinant in which the cg2408-2409 (ctaCF) gene region had been deleted, as expected.

[0085] (3) Cultivation of transformed strains 1) The transformed strain obtained above was streaked onto an LB plate and cultured at 30°C for 3 days. The cells grown on the plate were inoculated into 6 mL of CGTG15 medium and cultured with shaking at 30°C and 250 rpm for 24 hours (preculture). Sodium benzoate was added to CGTG15 medium (excluding urea) to a final concentration of 1 mM, and 60 mL was placed in a Bio Jr. 8 (Able Co., Ltd.) fermenter. 1.2 mL of the preculture was inoculated and cultured with shaking at 32°C, pH 6.9, 600 rpm, and 60 mL / min for 30 hours. GAL productivity was evaluated. The culture was diluted appropriately with dilute sulfuric acid, centrifuged to remove the cells, and the supernatant was collected. The concentration in the supernatant was quantified. The results are shown in the table.

[0086] 2) Results The GAL concentration was increased in KC201, indicating that disruption of the cydAB gene had a positive effect on production, while the GAL concentration was decreased in KC468, indicating that disruption of the ctaCF gene had a negative effect on production.

[0087] [Table 3-1]

[0088] [Table 3-2]

[0089] [Table 4]

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

Claims

1. A method for producing an aromatic compound or a salt thereof, comprising a step of culturing a modified coryneform bacterium in which the function of cytochrome bd-type oxidase complex is suppressed or deleted, wherein the aromatic compound or a 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 oxidase complex is the following (A) or (B): (A) a complex consisting of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide having the amino acid sequence shown in SEQ ID NO: 4; (B) a complex comprising a polypeptide having an amino acid sequence having at least 90% identity with 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 having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as cytochrome bd-type oxidase subunit II; The method according to claim 1, wherein the polypeptide complex has cytochrome bd oxidase activity and is represented by the formula:

3. A method for producing an aromatic compound or a salt thereof, comprising a step of culturing a modified coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, As the host, at least the following (C), (D), (E) and (F): (C) A polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity. (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) A polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate (F) a polypeptide having para-aminobenzoic acid hydroxylase activity A method for producing a coryneform bacterium comprising:

4. A method for producing an aromatic compound or a salt thereof, comprising a step of culturing a modified coryneform bacterium in which the function of the cytochrome bd-type oxidase complex is suppressed or deleted, As the host, the following (G) to (O): (G) a polypeptide having 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity; (H) a polypeptide having 3-dehydroquinate synthase activity; (I) a polypeptide having 3-dehydroquinate dehydratase activity, (J) a polypeptide having 5-shikimate dehydrogenase activity; (K) a polypeptide having shikimate kinase activity; (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyltransferase activity, (M) a polypeptide having chorismate synthase activity; (N) a polypeptide having pyruvate phosphate dikinase activity; (O) enzymes involved in the pentose phosphate pathway: one or more selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase; A method for producing a coryneform bacterium comprising:

5. The method according to claim 3, wherein the aromatic compound or a 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.

6. The method according to claim 4, wherein the aromatic compound or a 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.

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

8. The method of 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 of claim 3, wherein the aromatic compound or its salt 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 efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerans, Corynebacterium alkanolyticum, Corynebacterium callunae, Corynebacterium crenatum or Corynebacterium cruzilactis.

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

13. A modified coryneform bacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted, comprising at least the following (C), (D), (E), and (F): (C) A polypeptide having 3,4-dihydroxybenzoic acid hydroxylase activity. (D) Polypeptide having 3-dehydroshikimate dehydratase activity (E) A polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate (F) a polypeptide having para-aminobenzoic acid hydroxylase activity A coryneform bacterium in which expression of one or more polypeptides selected from the group consisting of the above is enhanced.

14. The cytochrome bd oxidase complex is the following (A) or (B): (A) a complex consisting of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide having the amino acid sequence shown in SEQ ID NO: 4; (B) a complex comprising a polypeptide having an amino acid sequence having at least 90% identity with 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 having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as cytochrome bd-type oxidase subunit II; The coryneform bacterium according to claim 13, which is a polypeptide complex having cytochrome bd-type oxidase activity represented by the formula:

15. A coryneform bacterium according to claim 13, in which a part or all of the nucleotide sequence of the following polynucleotide (a) or (b) has been deleted or inactivated: (a) a polynucleotide encoding a complex consisting of a polynucleotide having a nucleotide sequence shown in SEQ ID NO:1 and a polynucleotide having a nucleotide sequence shown in SEQ ID NO:3; (b) a polynucleotide encoding a complex consisting of a polynucleotide having a nucleotide sequence at least 90% identical to 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 a nucleotide sequence at least 90% identical to 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 described in claim 13, which is a bacterium of the genus Corynebacterium.

17. The coryneform bacterium described in claim 16, wherein the Corynebacterium genus bacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerans, Corynebacterium alkanolyticum, Corynebacterium callnae, Corynebacterium crenatum or Corynebacterium cruzilactis.

18. The coryneform bacterium described in claim 16, wherein the Corynebacterium genus bacterium is Corynebacterium glutamicum.