Method for producing aromatic compound

Abstract

To provide a method for producing an aromatic compound with high efficiency using a corynebacterium mutant strain.SOLUTION: Provided is a method for producing an aromatic compound or a salt thereof, the method including a step for culturing a mutant corynebacterium in which the function of a mdh gene represented by (a) or (b) is suppressed. (a) A polynucleotide that comprises a nucleotide sequence having a specific sequence. (b) A polynucleotide that comprises a nucleotide sequence having at least 90% identity with the nucleotide sequence having the specific sequence of (a), and encodes a polypeptide having a malate dehydrogenase activity.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Since Corynebacterium glutamicum was isolated as a glutamic acid-producing bacterium, it has been a useful industrial microorganism used in the production of various amino acids and nucleic acids. In recent years, due to the establishment of gene recombination technology for Corynebacterium glutamicum, various organic compounds such as aromatic amino acids (Non-Patent Document 1) such as tyrosine and tryptophan, gallic acid, 4-hydroxybenzoic acid (Non-Patent Document 1), 4-aminobenzoic acid (Non-Patent Document 2), etc. aromatic compounds can be produced.

[0003] The shikimic acid pathway is an important metabolic pathway for the biosynthesis of aromatic compounds in plants and microorganisms. That is, phosphoenolpyruvate produced in glycolysis combines with erythrose-4-phosphate supplied from the pentose phosphate pathway to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP), and then becomes shikimic acid through 3-dehydroquinic acid (DHQ) and 3-dehydroshikimic acid (DHS). Further, the phosphate group of shikimic acid is transferred from adenosine 3-phosphate to become 3-phosphoshikimic acid, and then becomes chorismic acid through 3-phosphoenolpyruvylshikimic acid. In the shikimic acid pathway, after the formation of a carbon six-membered ring, the formation of a double bond is carried out, 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, L-DOPA, etc., and from chorismic acid, aromatic compounds such as 4-hydroxybenzoic acid, 4-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, etc. are produced (Figure 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] The present invention relates to providing a method for efficiently producing aromatic compounds using a Corynebacterial mutant strain. [Means for solving the problem]

[0006] The inventors have found that in mutant Corynebacterium bacterium in which the function of the cg2613 gene, one of the genes encoding malate dehydrogenase (mdh), is suppressed, the productivity of aromatic compounds, including gallic acid, is improved.

[0007] 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 mutant Corynebacterium in which the function of the mdh gene shown in (a) or (b) below is suppressed. (a) Polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 1 (b) A polynucleotide encoding a polypeptide having malate dehydrogenase activity, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 1. 2) A mutant Corynebacterium in which the function of the mdh gene shown in (a) or (b) below is suppressed. (a) Polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 1 (b) A polynucleotide encoding a polypeptide having malate dehydrogenase activity, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 1. [Effects of the Invention]

[0008] According to the present invention, it becomes possible to efficiently produce aromatic compounds such as gallic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, L-DOPA, and 4-hydroxybenzoic acid, or salts thereof. [Brief explanation of the drawing]

[0009] [Figure 1] A schematic diagram illustrating the production pathways of various aromatic compounds in Corynebacteria. [Modes for carrying out the invention]

[0010] In this invention, the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing the analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver.12 with a unit size to compare (ktup) of 2.

[0011] In the present invention, "at least 90% identity" with respect to the nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0012] In the present invention, "nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted" means a nucleotide sequence in which one to 30, preferably one to 24, more preferably one to 15, even more preferably one to 9, even more preferably one to 6, and even more preferably one to 3 nucleotides are deleted, substituted, added, or inserted. In the present invention, "addition" of amino acids or nucleotides includes the addition of amino acids or nucleotides to one end and both ends of a sequence.

[0013] In this invention, "upstream" and "downstream" with respect to a gene refer to the upstream and downstream regions of the gene's transcription direction. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter in the DNA sense strand, and "upstream of a gene" means the 5' region of the gene in the DNA sense strand.

[0014] In this invention, the term "original" used in relation to the function, properties, and characteristics of a cell is used to indicate that the function, properties, or characteristics are inherently present in the cell. In contrast, the term "external" is used to indicate that the function, properties, or characteristics are not inherently present in the cell but have been introduced from outside. For example, an "external" gene or polynucleotide is a gene or polynucleotide introduced into a cell from outside. The external gene or polynucleotide may originate from the same species of organism as the cell into which it was introduced, or from a different species of organism (i.e., a different gene or polynucleotide).

[0015] In this invention, "Coryne-type bacteria" refers to non-motile, aerobic, Gram-positive bacteria belonging to the mycolic acid-containing actinomycetes group that do not have spore-forming ability, and includes a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974). Specifically, examples include Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, and Micrococcus. Examples of Corynebacterium species include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, 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.

[0016] The coryneform bacteria (parent coryneform bacteria) used to produce the mutant coryneform bacteria of the present invention may be wild-type strains, mutant strains thereof, or artificially genetically modified organisms, as long as they have the ability to produce aromatic compounds. For example, they may be knockout strains of genes such as lactate dehydrogenase (LDH) or phosphoenolpyruvate carboxylase. Alternatively, they may be coryneform bacteria modified to acquire or improve the ability to produce aromatic compounds. One method for acquiring or improving the aromatic compound production ability of a microorganism is to incorporate polynucleotides encoding various enzymes for producing the target aromatic compound into the microorganism. In a preferred embodiment, the coryneform bacteria used to produce the mutant coryneform bacteria in which the function of the mdh gene of the present invention is suppressed is a recombinant organism into which an exogenous polynucleotide encoding an enzyme for producing the target aromatic compound or a salt thereof is incorporated. Examples include recombinants having gallic acid production ability by introducing a polynucleotide encoding 3,4-dihydroxybenzoate hydroxylase, recombinants having catechol production ability by introducing a polynucleotide encoding protocatechuic acid decarboxylase, recombinants having L-DOPA production ability by introducing a polynucleotide encoding tyrosine phenol lyase into the recombinant having catechol production ability, recombinants having 2,4-PDCA production ability by introducing a polynucleotide encoding protocatechuic acid-4,5-dioxygenase, recombinants having 2,5-PDCA production ability by introducing a polynucleotide encoding protocatechuic acid-2,3-dioxygenase, and recombinants having 4-hydroxybenzoic acid production ability by introducing a polynucleotide encoding colismic acid lyase.

[0017] In the present invention, the aromatic compound is an organic aromatic compound biosynthesized in the cells of coryneform bacteria, that is, a coryneform bacteria-productive aromatic compound. Specifically, it refers to an aromatic compound synthesized via the shikimic acid pathway, preferably an aromatic compound derived from 3-dehydroshikimic acid (DHS) or chorismic acid (see Figure 1). Specifically, examples include protocatechuic acid, catechol, gallic acid, phenylalanine, L-DOPA, tyrosine, pretyrosine, tryptophan, 4-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, L-DOPA derived from protocatechuic acid; 4-hydroxybenzoic acid, 4-amino 3-hydroxybenzoic acid, tyrosine, tryptophan, etc. derived from chorismic acid are preferred. More preferably, they are protocatechuic acid and aromatic compounds derived from protocatechuic acid, and even more preferably gallic acid.

[0018] 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. Examples of the acid addition salts include mineral acid salts such as hydrochloride, sulfate, nitrate, phosphate, etc.

[0019] In the present invention, the mdh gene is a gene encoding malate dehydrogenase (mdh). Malate dehydrogenase is an oxidoreductase that catalyzes the chemical reaction of oxidizing (or the reverse of) malic acid to oxaloacetic acid. In the present invention, examples of the mdh gene include at least one polynucleotide selected from the group consisting of the following (a) or (b). (a) Polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 1 (b) A polynucleotide encoding a polypeptide having malate dehydrogenase activity, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 1. Here, the polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 1 represents the mdh gene (cg2613) derived from Corynebacterium glutamicum ATCC 13032 strain. Nucleotide sequences having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 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 such nucleotide sequences 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.

[0020] In the present invention, suppression of the function of the mdh gene includes not only suppression of mdh gene expression but also suppression of the activity of the protein encoded by the mdh gene, and may be either complete suppression (inhibition) or incomplete suppression of function. Such suppression of the function of the mdh gene can be achieved by introducing a mutation that causes deletion or inactivation of the coding region, non-coding region, or transcription or translation initiation region of the mdh gene (deletion or inactivation of the mdh gene), or by suppressing transcription or translation by introducing a polynucleotide that has the activity to degrade the transcript of the mdh gene, or a polynucleotide that suppresses the translation of said transcript into protein. In one embodiment, deletion or inactivation of the mdh gene can be achieved by removing part or all of the nucleotide sequence of the mdh gene from the genome or replacing it with another nucleotide sequence, inserting another polynucleotide fragment into the sequence of the mdh gene, or introducing a mutation into the transcription or translation initiation region of the mdh gene. Preferably, part or all of the nucleotide sequence of the mdh gene is deleted or inactivated. More specific examples include a method for specifically deleting or inactivating the mdh gene on the genome of a cell, and a method for selecting cells with the desired mutation after introducing a random deletion or inactivation mutation into the gene in a cell and then evaluating the expression level or activity of the mdh protein or performing genetic analysis.

[0021] Specific deletion or inactivation of the mdh gene can be achieved, for example, by homologous recombination. Specifically, a DNA fragment of the mdh gene into which an inactivating mutation has been introduced by base substitution or base insertion, or a DNA fragment containing the outer region of the mdh gene but not the mdh gene itself, can be constructed. This fragment can then be incorporated into the cells of a parent microorganism (Coryneform bacteria) to induce homologous recombination with the region of the parent microorganism's genome containing the mdh gene, thereby deleting or inactivating the mdh gene on the genome. Alternatively, a recombinant vector (such as a plasmid) containing a DNA fragment with a portion of the mdh gene's region can be incorporated into the parent microorganism's cells, and the mdh gene can be inactivated by homologous recombination that isolates a portion of the mdh gene on the parent microorganism's genome. Methods for randomly deleting or inactivating genes in cells include introducing DNA fragments obtained by randomly cloning a gene into which an inactivating mutation has been introduced into cells and inducing homologous recombination with the gene on the cell's genome, or inducing mutations by irradiating cells with ultraviolet light, gamma rays, etc. Gene inactivating mutations 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 the protein, or will express a protein with impaired original activity.

[0022] One method for constructing DNA fragments containing an inactivating mutation in the mdh gene is site-directed mutagenesis (SMU). SMU can be performed using mutation primers containing the nucleotide mutation to be introduced. For example, by PCR using the mdh gene as a template with two sets of primers containing the nucleotide mutation to be introduced, DNA fragments are created by amplifying the upstream and downstream regions of the mdh gene, respectively. These fragments are then ligated together using 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 also be performed using inverse PCR or annealing methods (Muramatsu et al., "Revised 4th Edition New Gene Engineering Handbook," Yodosha, p82-88), 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.

[0023] Mutation primers can be prepared by well-known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). The mdh gene used as a template may be prepared by conventional methods from the aforementioned Corynebacterium, or it may be chemically synthesized.

[0024] To introduce DNA fragments or vectors into microbial cells, 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).

[0025] Furthermore, polynucleotides that have the activity to degrade the transcript of the mdh gene, 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 mdh gene mRNA. Specifically, these include antisense RNA for the mdh gene mRNA, siRNA for the mdh gene mRNA, and ribozymes for the mdh gene mRNA.

[0026] Cells in which the function of the mdh gene is suppressed can be selected by examining their genome sequence. Alternatively, cells in which the function of the mdh gene is suppressed can be selected using the expression level or activity of the mdh protein as an indicator.

[0027] A mutant Corynebacterium in which the function of the thus constructed mdh gene is suppressed can be cultured, preferably in the presence of sugars, and the target aromatic compound or its salt can be recovered to produce the aromatic compound or its salt.

[0028] 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.

[0029] The culture medium for culturing the mutant Corynebacterium can 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 mutant Corynebacterium 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 30 w / v%, more preferably 2 to 20 w / v%, and even more preferably 2 to 10 w / v%.

[0030] As nitrogen sources, for example, peptone, meat extract, yeast extract, casein hydrolysate, soybean meal alkali extract, corn steep liquor, 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.

[0031] 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.

[0032] Examples of culture media for Corynebacteria include LB medium, A medium [J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)], BT medium [J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)], and CGXII medium [J. Bacteriol. 175:5595-5603 (1993)]. These media should be used with sugar concentrations within the above range.

[0033] Prior to the reaction or culture involving sugars, it is preferable to culture the mutant Corynebacterium in the same medium under aerobic conditions at a temperature of approximately 25-38°C for approximately 12-48 hours to allow it to grow.

[0034] 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 in batch, fed-batch, or continuous manner. 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 a reaction or culture under aerobic conditions, it is preferable to do so under conditions that suppress the excessive growth of mutant Corynebacterium, in order to improve the efficiency of producing the target substance. If the target substance is easily oxidized, it is preferable to perform the culture under conditions with a low dissolved oxygen concentration to prevent oxidation of the target substance. Specifically, a dissolved oxygen concentration of 0.1 to 3 ppm is preferred, and 0.1 to 1 ppm is more preferred.

[0035] 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.

[0036] 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 mutant Corynebacterium in which the function of the mdh gene shown in (a) or (b) below is suppressed. (a) Polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 1 (b) A polynucleotide encoding a polypeptide having malate dehydrogenase activity, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 1. <2> The aromatic compound is protocatechuic acid, gallic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, L-DOPA, 4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid, tyrosine, tryptophan, or a salt thereof, preferably gallic acid, protocatechuic acid, catechol, L-DOPA, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, or a salt thereof. <1> The method. <3> The aromatic compound is gallic acid or a salt thereof. <1> The method. <4> Suppression of the function of the mdh gene is either suppression of mdh gene expression or suppression of the activity of the protein encoded by the mdh gene. <1> ~ <3> One of the following methods. <5> The function of the mdh gene is suppressed by deletion or inactivation of the mdh gene. <1> ~ <3> One of the following methods. <6> Corynebacterium-type bacteria include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanoritycum, or Corynebacterium carnae. <1> ~ <5> One of the following methods. <7> Corynebacterium glutamicum is a type of coryneform bacterium. <1> ~ <5> One of the following methods. <8> It is cultured in a medium containing sugars as a carbon source. <1> ~ <7> One of the following methods. <9> The process includes recovering an aromatic compound or a salt thereof from the culture, <1> ~ <8> One of the following methods. <10> A mutant Corynebacterium in which the function of the mdh gene, as shown in (a) or (b) below, is suppressed. (a) Polynucleotide consisting of the nucleotide sequence shown in Sequence ID No. 1 (b) A polynucleotide encoding a polypeptide having malate dehydrogenase activity, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 1. [Examples]

[0037] 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.

[0038] (1) Preparation of gallic acid-producing bacteria 1) Construction of a plasmid for replacing the cg0620 gene region with a polypeptide gene possessing 3,4-dihydroxybenzoate hydroxylase activity. The base numbers shown in the following examples are the base numbers of the genome sequence of strain ATCC13032. This genome sequence information was obtained from the NCBI GB database under accession number NC_006958. For PCR, we used PrimeSTAR Max DNA Polymerase (TaKaRa).

[0039] The genomic DNA of strain ATCC13032 (=NBRC 12168) was amplified using primers OT20 and OT21 to obtain the 5' DNA fragment of the cg0620 gene region. The genomic DNA was also amplified using primers OT23 and OT24 to obtain the 3' DNA fragment of the cg0620 gene region. Furthermore, a DNA fragment (OT25) containing the promoter of the tuf gene (cg0587) present in Corynebacterium glutamicum strain ATCC13032 (hereinafter referred to as the tu promoter) was synthesized artificially. This was amplified using primers OT26 and OT27 to obtain the promoter region DNA fragment. Additionally, two types of DNA fragments (SEQ ID NOs. 2 and 3) containing a polypeptide gene with 3,4-dihydroxybenzoate hydroxylase activity (hereinafter abbreviated as hfm145VF) were synthesized artificially. Each DNA fragment was used as a template and amplified using two types of DNA primers (OT30 and OT31, and OT32 and OT33) to obtain two types of DNA fragments. In addition, the vector fragment was amplified using pHKPsacB1 (described in Patent Document 1) as a template with primers OT34 and OT35. The obtained PCR products were treated with DpnI (Takara Bio). Each DNA fragment was purified from the six obtained PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt was constructed by ligation using In-Fusion HD Cloning Kit (Takara Bio). The obtained plasmid solution was used to transform ECOS Competent E. coli DH5α strain (Nippon Gene Co., Ltd.), and the cell suspension was spread on LB agar medium containing kanamycin and left to stand overnight at 37°C. Transformants containing the plasmid were inoculated into 2 mL of LB liquid medium containing kanamycin and incubated overnight at 37°C. Plasmid purification was performed from this culture medium using NucleoSpin Plasmid EasyPure (TaKaRa), yielding pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt.

[0040] 2) Creation of polypeptide gene transfection strains possessing 3,4-dihydroxybenzoate hydroxylase activity Using a transformation method by electroporation (Bio-rad), the plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt described above was introduced into the CY44 strain (the CY44 strain is the tkt strain described in Reference Example 14 of Japanese Patent No. 6322576, in which the expression of the tkt gene is enhanced by controlling the transcription of the tkt gene with the tu promoter. Furthermore, the transcription of the qsuB gene and the vanR gene can be induced by the addition of benzoic acid. In addition, the aroE3 gene is controlled by the VanR repressor.) and selected for kanamycin resistance to obtain the KC148sr strain. Analysis of the KC148sr strain using PCR with primers OT20 and OT36 (Sapphire Amp (Takara Bio)) yielded the expected results, confirming that the KC148sr strain is a one-time crossover homologous recombinant in which the plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt is introduced into the cg0620 gene region. The KC148 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 KC148 strain. PCR using primers OT36 and OT37 (Sapphire Amp (Takara Bio)) confirmed that the KC148 strain is a double-crossover homologous recombinant in which the Ptu-hfm145VF-hfm145VFop gene is introduced into the cg0620 gene region, as expected.

[0041] (2) Creation of MDH-disrupted strains 1) Creation of a plasmid to disrupt the mdh(cg2613) gene PrimeSTAR Max DNA Polymerase (TaKaRa) was used as the enzyme for PCR. Using pHKPsacB1 (described in Japanese Patent Publication No. 6322576) as a template, the vector fragment was amplified with primers pHKPsacB-F2 and pHKPsacB-R2. Using the genomic DNA of strain ATCC13032 (=NBRC strain 12168) as a template, the 5' DNA fragment of the cg2613 gene was amplified with primers 2613-up-F and 2613-up-R, and the 3' DNA fragment of the cg2613 gene was amplified using the genomic DNA as a template with primers 2613-down-F and 2613-down-R. The obtained PCR products were treated with DpnI (Takara Bio). The three obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and then ligated using the In-Fusion HD cloning kit (clontech) to produce pHKBsacB-Δmdh. The obtained plasmid solution was used to transform ECOS Competent E. coli DH5α strain (Nippon Gene), and the cell suspension was spread onto LB agar medium containing kanamycin and incubated overnight at 37°C. The resulting colonies were used as templates, and colony PCR was performed using Sapphire Amp (TaKaRa) as the enzyme. The primers used were 2613-up-F and 2613-down-R to confirm the introduction of the target DNA fragment. Transformants containing plasmids in which gene introduction was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and incubated overnight at 37°C. Plasmid purification was performed using NucleoSpin Plasmid EasyPure (TaKaRa Co.) from this culture medium to obtain pHKBsacB-Δmdh.

[0042] 2) Obtaining a knockout strain of the mdh gene (cg2613) Using the plasmid pHKBsacB-Δmdh obtained above, KC148 was transformed by electroporation (Bio-rad). By selecting for kanamycin resistance, KC148Δmdh-sr was obtained. Using the obtained colonies as a template, PCR (Sapphire Amp) was performed using primers sacB-1 and 2613-up1500, and the expected results were obtained, confirming that the plasmid pHKBsacB-Δmdh was introduced into the cg2613 gene region by one crossover homologous recombination. KC148Δmdh-sr was cultured in 1 mL of LB liquid medium for 24 hours, and a portion of the culture medium was smeared on LB saturates containing 20% ​​sucrose to obtain the KC148Δmdh strain. Colony PCR (Sapphire Amp) using primers 2613-coloP-F and 2613-coloP-R confirmed the deletion of the mdh gene (cg2613) by two cross-recombination homologous recombinations. Additionally, deletions of the kanamycin resistance gene and the sacB gene were also confirmed.

[0043] [Table 1]

[0044] (3) Evaluation of gallic acid productivity KC148 and KC148Δmdh strains were streaked onto LB plates and cultured at 30°C for 2 days. Sodium benzoate was added to CGXII medium shown in Table 2 to a final concentration of 1 mM, and 1.5 mL was placed in a round-bottom tube (Eiken Chemical). Colonies of KC148 and KC148Δmdh were inoculated, and cultured with shaking at 32°C and 200 rpm for 24 hours to evaluate gallic acid productivity. The culture solution was diluted with dilute sulfuric acid as appropriate, and the cells were removed by centrifugation, after which the supernatant was collected. The glucose concentration and gallic acid concentration in the supernatant were quantified. The results are shown in Table 3. Compared with the KC148 strain, the gallic acid yield relative to consumed sugar was 1.4 times higher in the KC148Δmdh strain.

[0045] [Table 2]

[0046] [Table 3]

[0047] Reference Example 1: Determination of gallic acid, para-aminobenzoic acid, and glucose 1)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 and para-aminobenzoic 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 wavelengths 210 and 280 nm) was used to detect gallic acid and para-aminobenzoic 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.

[0048] Comparative Example (1) Extraction of genes possessing malate dehydrogenase (mdh) activity from Corynebacteria Genes with MDH activity other than cg2613 were extracted from the database (KEGG: Kyoto Encyclopedia of Genes and Genomes, https: / / www.genome.jp / kegg-bin / show_organism?org=T00244), and cg763 (sequence number 28) and cg2192 (sequence number 29) were obtained.

[0049] (2) Creation of cg763 disruption strain 1) Creation of a plasmid to disrupt the cg763 gene The procedure was carried out in the same manner as in Example (2)1). Specifically, primers 763-up-F and 763-up-R were used to amplify the 5' DNA fragment of the cg763 gene, and primers 763-down-F and 763-down-R were used to amplify the 3' DNA fragment of the cg2613 gene. Plasmid pHKBsacB-Δcg763 was constructed using these two fragments and the vector fragment described in Example (2)1). For colony PCR, primers 763-up-F and 763-down-R were used.

[0050] 2) Obtaining a knockout strain of the cg763 gene The plasmid obtained in (2)1) was used and the procedure was carried out in the same manner as in Example (2)2). For PCR confirmation of the KC148Δcg763-sr strain, primers sacB-1 and 763-up1500 were used. For PCR confirmation of the KC148Δcg763 strain, primers 763-coloP-F and 763-coloP-R were used.

[0051] (3) Creation of cg2192 disruption strain 1) Creation of a plasmid to disrupt the cg2192 gene The procedure was carried out in the same manner as in Example (2)1). Specifically, primers 2192-up-F and 2192-up-R were used to amplify the 5' DNA fragment of the cg2192 gene, and primers 2192-down-F and 2192-down-R were used to amplify the 3' DNA fragment of the cg2192 gene. Plasmid pHKBsacB-Δcg2192 was constructed using these two fragments and the vector fragment described in Example (2)1). For colony PCR, primers 2192-up-F and 2192-down-R were used.

[0052] 2) Obtaining a knockout strain of the cg2192 gene (3)1) The plasmid obtained in (3)1) was used and the procedure was carried out in the same manner as in Example (2)2). For PCR confirmation of the KC148Δcg2192-sr strain, primers sacB-1 and 2192-up1500 were used. For PCR confirmation of the KC148Δcg2192 strain, primers 2192-coloP-F and 2192-coloP-R were used.

[0053] [Table 4]

[0054] (4) Evaluation of gallic acid productivity The KC148, KC148Δ763, and KC148Δ2192 strains were evaluated using the same method as in Example (3). The results are shown in Table 5. Compared to the KC148 strain, the gallic acid yield relative to consumed sugar decreased to 0.91 times in the KC148Δ763 strain and to 0.71 times in the KC148Δ2192 strain.

[0055] [Table 5]

Claims

**Claim 1** A method for improving the productivity of an aromatic compound or a salt thereof in a coryneform bacterium having the ability to produce an aromatic compound, which comprises a step of suppressing the function of the mdh gene represented by the following (a) or (b). (a) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 (b) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide having malate dehydrogenase activity **Claim 2** A method for producing a coryneform bacterium with improved productivity of an aromatic compound or a salt thereof, which comprises a step of suppressing the function of the mdh gene represented by the following (a) or (b) in a coryneform bacterium having the ability to produce an aromatic compound. (a) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 (b) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide having malate dehydrogenase activity **Claim 3** A method for producing an aromatic compound or a salt thereof, which comprises a step of culturing a mutant coryneform bacterium having the ability to produce an aromatic compound, in which the function of the mdh gene represented by the following (a) or (b) is suppressed. (a) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 (b) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide having malate dehydrogenase activity **Claim 4** The method according to any one of claims 1 to 3, wherein the aromatic compound is gallic acid, protocatechuic acid, catechol, L-DOPA, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid or a salt thereof. **Claim 5** The method according to claim 4, wherein the aromatic compound is gallic acid or a salt thereof. **Claim 6** The method according to any one of claims 1 to 3, wherein the suppression of the function of the mdh gene is suppression of the expression of the mdh gene or suppression of the activity of the protein encoded by the mdh gene. **Claim 7** The method according to any one of claims 1 to 3, wherein the suppression of the function of the mdh gene is achieved by deletion or inactivation of the mdh gene. **Claim 8** The method according to any one of claims 1 to 3, wherein the coryneform bacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerans, Corynebacterium alkanolyticum or Corynebacterium carnae.

9. The method according to any one of claims 1 to 3, wherein the coryneform bacterium is Corynebacterium glutamicum.

10. A recombinant coryneform bacterium having the ability to produce gallic acid, into which a polynucleotide encoding 3,4-dihydroxybenzoic acid hydroxylase has been introduced, a recombinant coryneform bacterium having the ability to produce catechol, into which a polynucleotide encoding protocatechuate decarboxylase has been introduced, a recombinant coryneform bacterium having the ability to produce L-DOPA, into which a polynucleotide encoding tyrosine phenol lyase has been introduced into the recombinant coryneform bacterium having the ability to produce catechol, a recombinant coryneform bacterium having the ability to produce 2,4-PDCA, into which a polynucleotide encoding protocatechuate-4,5-dioxygenase has been introduced, a recombinant coryneform bacterium having the ability to produce 2,5-PDCA, into which a polynucleotide encoding protocatechuate-2,3-dioxygenase has been introduced, a recombinant coryneform bacterium having the ability to produce 4-hydroxybenzoic acid, into which a polynucleotide encoding chorismic acid lyase has been introduced, which is selected from the group consisting of the recombinant coryneform bacteria according to any one of claims 1 to 3.

11. The method according to claim 3, wherein the method is carried out by culturing in a medium containing saccharides as a carbon source.

12. The method according to claim 3, which comprises a step of recovering an aromatic compound or a salt thereof from the culture.

13. A mutant coryneform bacterium having the ability to produce an aromatic compound, in which the function of the mdh gene represented by the following (a) or (b) is suppressed: A recombinant coryneform bacterium having the ability to produce gallic acid, into which a polynucleotide encoding 3,4-dihydroxybenzoic acid hydroxylase has been introduced, a recombinant coryneform bacterium having the ability to produce catechol, into which a polynucleotide encoding protocatechuate decarboxylase has been introduced, a recombinant coryneform bacterium having the ability to produce L-DOPA, into which a polynucleotide encoding tyrosine phenol lyase has been introduced into the recombinant coryneform bacterium having the ability to produce catechol, a recombinant coryneform bacterium having the ability to produce 2,4-PDCA, into which a polynucleotide encoding protocatechuate-4,5-dioxygenase has been introduced, a recombinant coryneform bacterium having the ability to produce 2,5-PDCA, into which a polynucleotide encoding protocatechuate-2,3-dioxygenase has been introduced, and a recombinant coryneform bacterium having the ability to produce 4-hydroxybenzoic acid, into which a polynucleotide encoding chorismic acid lyase has been introduced, selected from the group consisting of mutant coryneform bacteria Mutant coryneform bacterium a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide having malate dehydrogenase activity

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