Method for producing aromatic compounds

Abstract

Provided is a method for efficiently producing aromatic compounds by using transformed cells.　This method for producing an aromatic compound or a salt thereof comprises a step for culturing a transformed cell in which the function of the monocarboxylate transporter indicated by (A) or (B) has been suppressed or deleted.　(A) polypeptide comprising the amino acid sequence given by SEQ ID NO: 2; (B) polypeptide having a monocarboxylate transporter activity and comprising an amino acid sequence having at least 90% identity with the amino acid sequence given by SEQ ID NO: 2

Description

Method for producing aromatic compounds

[0001] The present invention relates to a method for producing an aromatic compound using a transformed cell.

[0002] In recent years, there has been a demand for the production of useful aromatic compounds, including gallic acid, using microorganisms from glucose, an inexpensive raw material. Among these, Corynebacterium glutamicum is a useful industrial microorganism that has been used to produce various amino acids and nucleic acids. Recently, the establishment of genetic recombination techniques targeting Corynebacterium bacteria has made it possible to produce a variety of organic compounds, including aromatic amino acids such as tyrosine and tryptophan (Non-Patent Document 1), and aromatic compounds such as gallic acid, 4-hydroxybenzoic acid (Non-Patent Document 1), and 4-aminobenzoic acid (Non-Patent Document 2). Gallic acid, in particular, is used as a raw material for producing photographic developers and blue inks due to its strong reducing properties, and its esters, such as propyl gallate, are used as antioxidants for oils, fats, and butter. Furthermore, pyrogallol, which is synthesized by decarboxylating gallic acid, is used as an electronic material, an organic synthesis reagent, a photographic developer, a mordant for woolen fabrics, etc., and therefore, efficient production of gallic acid is beneficial.

[0003] The shikimate pathway is an important metabolic pathway for the biosynthesis of aromatic compounds in plants and microorganisms. Specifically, phosphoenolpyruvate produced in glycolysis combines with erythrose 4-phosphate supplied by the pentose phosphate pathway to form 3-deoxy-D-arabinopeptulosonic acid 7-phosphate (DAHP), which then undergoes 3-dehydroquinic acid (DHQ) and 3-dehydroshikimic acid (DHS) to form shikimic acid. Furthermore, a phosphate group is transferred from adenosine triphosphate to shikimic acid, which then becomes 3-phosphoshikimic acid, which then becomes 3-phosphoenolpyruvylshikimic acid, which then becomes chorismic acid. In the shikimate pathway, a six-carbon ring is formed, followed by the formation of a double bond. From 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, and L-DOPA are produced (Figure 1).

[0004] [Non-patent document 1] Metab. Eng. 2018. 50:122-141. [Non-patent document 2] Metab. Eng. 2016. 38:322-330.

[0005] The present invention relates to the following: A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a transformed cell in which the function of a monocarboxylic acid transporter shown in (a) or (b) below is suppressed or deleted: (A1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; or (A2) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2 and having monocarboxylic acid transporter activity.

[0006] Schematic diagram showing the production pathways of various aromatic compounds when a coryneform bacterium is used as a host, in which aroG and aroF are 2-dehydro-3-deoxyarabinoheptonate aldolases, aroB is 3-dehydroquinate synthase, aroD and qsuC are dehydroquinate dehydratases, qsuD is quinate / shikimate dehydrogenase, aroE3 is shikimate dehydrogenase, and hfm145 is 3,4-dihydroxybenzoate hydroxylase, qsuB is dehydroshikimate dehydratase, aroA is 5-enolate pyruvylshikimate-3-phosphate synthase, aroC is chorismate synthase, and aroK is shikimate kinase. Detailed Description of the Invention

[0007] The present invention relates to providing a method for efficiently producing aromatic compounds using transformed cells.

[0008] The present inventors have found that the productivity of aromatic compounds such as gallic acid is improved in transformed cells in which the function of MctC, a transporter of monocarboxylic acids such as pyruvate, propionate, and acetate, is suppressed or deleted.

[0009] According to the present invention, it is possible to efficiently produce aromatic compounds such as protocatechuic acid, gallic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, L-DOPA, 4-hydroxybenzoic acid, and 4-aminobenzoic acid, or salts thereof, by a fermentation method that places little strain on the environment.

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

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

[0012] 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 one to ten, preferably one to eight, more preferably one to five, and even more preferably one to three 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 one to 30, preferably one to 24, more preferably one to 15, and even more preferably one to nine 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.

[0013] In the present invention, the term "aromatic compound" refers to an organic aromatic compound biosynthesized in a host cell, specifically an aromatic compound synthesized via the shikimic acid pathway, preferably an aromatic compound derived from 3-dehydroshikimic acid (DHS) or chorismic acid (Figure 1). Specific 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, and 4-amino-3-hydroxybenzoic acid. Of 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-aminobenzoic acid, 4-amino 3-hydroxybenzoic acid, tyrosine, tryptophan, and the like derived from chorismic acid are preferred, and protocatechuic acid, aromatic compounds derived from protocatechuic acid (preferably gallic acid, L-DOPA), 4-hydroxybenzoic acid, 4-amino 3-hydroxybenzoic acid, and more preferably gallic acid are preferred.

[0014] 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.

[0015] The method for producing an aromatic compound or a salt thereof of the present invention uses a transformed cell in which the function of a monocarboxylic acid transporter shown in (a) or (b) below is suppressed or deleted: (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; or (B) a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2 and having monocarboxylic acid transporter activity. Here, (A) the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 refers to MctC, which is known as a monocarboxylic acid transporter derived from Corynebacterium glutamicum. MctC is a transporter involved in the transport of monocarboxylic acids such as pyruvate, propionate, and acetate.

[0016] In the polypeptide (A), the identity with the amino acid sequence shown in SEQ ID NO: 2 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 include amino acid sequences in which one or several amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 2.

[0017] The presence of monocarboxylic acid transporter activity can be confirmed, for example, by growing a target gene disruptant or enhanced strain in a medium containing monocarboxylic acid as a carbon source, measuring the intracellular monocarboxylic acid concentration or bacterial growth rate, and comparing it with that of the host strain.

[0018] (B) Polypeptides consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 and having monocarboxylic acid transporter activity include, for example, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium sp. Examples include ActP (cation: acetate symporter) derived from SalYVA5 (Corynebacterium SP. SalYVA5), Corynebacterium hadale, Corynebacterium gottingense, Corynebacterium godavarianum, and Corynebacterium senegalense.

[0019] 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 Harbor Laboratory Press, New York, pp. 581-621, 1995). Examples of site-specific mutagenesis techniques include a method using splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), the ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and the Kunkel method (Kunkel, T.A., Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, a 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.

[0020] In the present invention, transformed cells in which the function of a monocarboxylate transporter is suppressed or deleted include cells in which the expression of a monocarboxylate transporter in a host cell is reduced or eliminated, resulting in the suppression or deletion of the function of the protein as a monocarboxylate transporter, preferably cells (mutation-introduced cells) in which the expression of a monocarboxylate transporter is reduced or eliminated compared to a parent cell by introducing a mutation, resulting in the reduction or loss of the function of the protein as a monocarboxylate transporter. In the present invention, "expression" of a protein refers to the production of a translation product from a gene encoding the protein and its localization in a functional state at its site of action. "Reduced or lost expression of a monocarboxylate transporter by introducing a mutation" refers to a state in which the amount of monocarboxylate transporter protein present in a transformed cell is reduced or eliminated, preferably a state in which it is significantly reduced or eliminated compared to that in a parent cell, as a result of modifications at the gene level, transcription level, post-transcriptional regulation level, translation level, or post-translational modification level.

[0021] "Decreased expression of monocarboxylate transporter compared to parent cells" means that the expression level of the monocarboxylate transporter present in the transformed cell is reduced compared to the parent cells; 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 cells, thereby similarly reducing its activity. Most preferably, the expression level of the monocarboxylate transporter is 0%, i.e., the expression of the monocarboxylate transporter is lost. Comparison of the expression levels of the monocarboxylate transporter is performed by measuring the expression level of the polypeptide using well-known immunological techniques such as Western blotting or immunohistochemical staining.

[0022] Specifically, a transformed cell in which expression of the monocarboxylate transporter is reduced or lost can be obtained by suppressing the function of a gene encoding the monocarboxylate transporter in the chromosomal DNA of a host cell. Here, suppression of function may be either complete suppression (inhibition) or incomplete suppression. A gene encoding a monocarboxylate transporter refers to DNA consisting of a transcription region including an ORF and a transcription regulatory region such as a promoter for the gene. In the present invention, preferred examples of genes encoding a monocarboxylate transporter include polynucleotides defined in (a) or (b) below.

[0023] (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 to the nucleotide sequence shown in SEQ ID NO: 1, and encoding a polypeptide having monocarboxylic acid transporter activity. Here, (a) the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 refers to the mctC gene (cg0953) derived from Corynebacterium glutamicum, which encodes a monocarboxylic acid transporter.

[0024] An example of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 is a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence shown in SEQ ID NO: 1. 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.

[0025] The function of such a gene encoding a monocarboxylic acid transporter can be suppressed by introducing a mutation that deletes or inactivates the coding region, non-coding region, or transcription or translation initiation region of the gene encoding the monocarboxylic acid transporter (deletion or inactivation of the gene encoding the monocarboxylic acid transporter), or by suppressing transcription or translation by introducing a polynucleotide that has the activity of degrading the transcription product of the gene encoding the monocarboxylic acid transporter, or a polynucleotide that suppresses translation of the transcription product into protein.

[0026] In one embodiment, the deletion or inactivation of a gene encoding a monocarboxylate transporter can be achieved by removing part or all of the nucleotide sequence of the gene encoding the monocarboxylate transporter from the genome or replacing it with another nucleotide sequence, inserting another polynucleotide fragment into the sequence of the gene encoding the monocarboxylate transporter, or by mutating the transcription or translation initiation region of the gene encoding the monocarboxylate transporter. Preferably, part or all of the nucleotide sequence of the gene encoding the monocarboxylate transporter is deleted or inactivated. More specific examples include a method of specifically deleting or inactivating a gene encoding a monocarboxylate transporter in the genome of a cell, and a method of randomly deleting or inactivating the gene in a cell, followed by evaluating the expression level or activity of the monocarboxylate transporter or performing genetic analysis to select cells having the desired mutation.

[0027] Specific deletion or inactivation of a gene encoding a monocarboxylate transporter can be achieved, for example, by homologous recombination. Specifically, a DNA fragment of a gene encoding a monocarboxylate transporter 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 a monocarboxylate transporter but not the gene encoding a monocarboxylate transporter, can be constructed, and the gene encoding a monocarboxylate transporter in the genome can be deleted or inactivated by incorporating this into a parent cell and causing homologous recombination with a region of the genome of the parent cell that contains the gene encoding a monocarboxylate transporter. Alternatively, a recombinant vector (e.g., a plasmid) containing a DNA fragment containing a partial region of the gene encoding a monocarboxylate transporter can be incorporated into a parent cell, and a partial region of the gene encoding a monocarboxylate transporter in the genome of the parent cell can be disrupted by homologous recombination, thereby inactivating the gene encoding a monocarboxylate transporter. Methods for randomly deleting or inactivating a gene in a cell include introducing a DNA fragment into a cell in which an inactivating mutation has been introduced, randomly cloned, and causing homologous recombination between the gene and the genome of the cell, and irradiating the cell with ultraviolet light, gamma rays, or the like to induce mutations. An inactivating mutation of a gene refers to a mutation that causes the target gene to lose its original function due to a silence mutation, missense mutation, nonsense mutation, frameshift mutation, etc. For example, a gene into which an inactivating mutation has been introduced will not express a protein, or will express a protein with impaired original activity.

[0028] Methods for preparing a DNA fragment containing a gene encoding a monocarboxylate transporter into which an inactivating mutation has been introduced include site-directed mutagenesis. 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 monocarboxylate transporter as a template, to prepare DNA fragments by amplifying the upstream and downstream regions of a region containing the gene encoding the monocarboxylate transporter, and then these fragments are 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, for site-specific mutagenesis, inverse PCR or annealing may be used (Muramatsu et al., eds., "New Genetic Engineering Handbook, 4th Revised 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 may be used.

[0029] 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 monocarboxylic acid transporter used as a template may be prepared from a host cell by a conventional method, or may be chemically synthesized.

[0030] To introduce a DNA fragment or a vector into a host cell, well-known techniques can be applied, 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).

[0031] Furthermore, examples of polynucleotides that have the activity of degrading the transcription product of a gene encoding a monocarboxylic acid transporter, or polynucleotides that suppress translation of the transcription product into protein, include polynucleotides that contain a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of the gene encoding a monocarboxylic acid transporter, or a part thereof. Specific examples include antisense RNA for the mRNA of the gene encoding a monocarboxylic acid transporter, siRNA for the mRNA of the gene encoding a monocarboxylic acid transporter, and ribozymes for the mRNA of the gene encoding a monocarboxylic acid transporter.

[0032] Cells in which the function of a gene encoding a monocarboxylate transporter is suppressed can be selected by confirming the genome sequence, or cells in which the function of a gene encoding a monocarboxylate transporter is suppressed can be selected using the expression level or activity of the monocarboxylate transporter as an indicator.

[0033] In the present invention, the host cell may be any cell suitable for producing an aromatic compound or a salt thereof, and may be any of a microbial cell, a plant cell, and an animal cell, but is preferably a microbial cell. From the viewpoint of the production efficiency of an aromatic compound or a salt thereof, particularly an aromatic compound derived from 3-dehydroshikimic acid, such as protocatechuic acid, gallic acid, shikimic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, L-DOPA, chorismic acid, 4-hydroxybenzoic acid, 4-aminobenzoic acid, and 4-amino-3-hydroxybenzoic acid, or a salt thereof, it is more preferable to use a microbial cell with improved 3-dehydroshikimic acid production activity as the host cell.

[0034] Microbial cells that can be used include Escherichia coli, Bacillus subtilis, actinomycetes, Pseudomonas bacteria, Streptococcus bacteria, Lactobacillus bacteria, fungi (such as Neurospora, Aspergillus, and Trichoderma), and yeasts (such as Saccharomyces, Kluyveromyces, Schizosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Pichia, and Candida), but are preferably prokaryotic microbial cells, more preferably gram-positive bacteria, and actinomycetes are preferred.

[0035] As actinomycetes, a group of microorganisms defined as coryneform bacteria (Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974)) is preferred, and specific examples include bacteria of the genus Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, and Micrococcus. Examples of bacteria of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium callunae, etc. Examples of bacteria of the genus Brevibacterium include Brevibacterium ammoniagenes, etc. Examples of the genus Arthrobacter include Arthrobacter globiformis, etc. Examples of the genus Mycobacterium include Mycobacterium bovis, etc. Examples of the genus Micrococcus include Micrococcus freudenreichii, Micrococcus leuteus, Micrococcus ureae, Micrococcus roseus, etc. Among the coryneform bacteria, preferred are the genus Corynebacterium, and more preferred is Corynebacterium glutamicum.The microbial cells may be wild-type strains, mutant strains thereof, or artificially recombinant strains.

[0036] Microbial cells with improved 3-dehydroshikimic acid production activity include microbial cells in which genes necessary for producing 3-dehydroshikimic acid have been enhanced. Specifically, these include microbial cells that have been subjected to one or more of the following genetic manipulations (i), (ii), (iii), and (iv), preferably two or more of (i), (ii), (iii), and (iv), more preferably three or more of (i), (ii), (iii), and (iv), and even more preferably all of (i), (ii), (iii), and (iv). Here, gene enhancement includes introducing a specific gene in an expressible state, introducing a mutation into a specific gene or the regulatory region of the gene, etc. (i) Enhancement of one or more genes selected from the group consisting of a dehydroshikimate dehydratase gene, a dehydroquinate dehydratase gene, a quinate dehydrogenase gene, and a shikimate dehydrogenase gene. (ii) enhancement of one or more genes selected from the group of genes involved in the shikimic acid synthesis pathway consisting of the 2-dehydro-3-deoxyarabinoheptonate aldolase gene, the 3-dehydroquinate synthase gene, and the shikimate dehydrogenase gene; (iii) enhancement of one or more genes selected from the group of genes involved in the pentose phosphate pathway consisting of the glucose-6-phosphate dehydrogenase gene, the 6-phosphogluconolactonase gene, the phosphogluconate dehydrogenase gene, the ribose-5-phosphate isomerase gene, the ribulose-5-phosphate-3-epimerase gene, the transketolase gene, and the transaldolase gene; (iv) enhancement of a gene encoding a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity.

[0037] The transformed cells thus prepared are cultured, the productivity of the aromatic compound or a salt thereof is evaluated, and an appropriate transformed cell is selected, thereby obtaining a useful cell that produces an aromatic compound or a salt thereof. The product can be measured according to the method described in the Reference Examples below.

[0038] The method for producing an aromatic compound or a salt thereof of the present invention is carried out by culturing the above-described transformed cells, preferably in the presence of a sugar, and recovering the target aromatic compound or a salt thereof. As the sugar, glucose is preferred, 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 having glucose units, such as disaccharides such as cellobiose, sucrose (cane sugar), 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. In addition, inedible agricultural waste such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw, etc.), bagasse, corn stover, etc., energy crops such as switchgrass, napier grass, and miscanthus, wood chips, waste paper, etc., can be saccharified using a saccharifying enzyme or the like to produce a saccharified liquid containing multiple sugars such as glucose.

[0039] The medium for culturing the transformed cells 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 allows efficient cultivation of the transformed cells of the present invention. The carbon source may be any of the sugars listed above or molasses or a saccharified solution containing such sugars. In addition to the sugars listed above, other carbon sources may also be used: 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. The carbon source may be used alone or in combination of two or more. The concentration of the sugars used as 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%.

[0040] 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.

[0041] 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.

[0042] 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 [Japanese Patent No. 6322576]. These media may be used with the sugar concentration within the above range.

[0043] 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.

[0044] The culture 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, more preferably 24 to 72 hours, with stirring or shaking as needed. Antibiotics such as ampicillin or kanamycin may be added to the medium during culture as needed. Culture may be performed in a batch, fed-batch, or continuous manner. Among these, the batch method is preferred. Culture or reaction may be performed under aerobic or reducing conditions, but aerobic conditions are preferred. When reaction or culture is performed under aerobic conditions, it is preferable to perform the culture under conditions that suppress excessive growth of the transformant, from the viewpoint of the production efficiency of the aromatic compound or its salt. When the aromatic compound is susceptible to oxidation, culture is preferably performed under conditions with a low dissolved oxygen concentration. For example, in the production of gallic acid, the dissolved oxygen concentration is preferably 0.1 to 3 ppm, more preferably 0.1 to 1 ppm.

[0045] 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.

[0046] In addition to the above-described embodiments, 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 transformed cell in which the function of a monocarboxylic acid transporter shown in (A) or (B) below is suppressed or deleted: (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; (B) a polypeptide consisting of an amino acid sequence at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2 and having monocarboxylic acid transporter activity. <2> The method described in <1>, in which the transformed cell in which the function of a monocarboxylic acid transporter is suppressed or deleted is obtained by suppressing the function of a gene encoding a monocarboxylic acid transporter in the chromosomal DNA of a host cell, and the gene encoding the monocarboxylic acid transporter is a polynucleotide shown in (a) or (b) below: (a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1; or (b) a polynucleotide consisting of a nucleotide sequence at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide having monocarboxylic acid transporter activity. <3> The method described in <1> or <2>, in which a microbial cell in which 3-dehydroshikimic acid-producing activity is improved is used as a host. <4> The method according to <3>, wherein the microbial cell having improved 3-dehydroshikimic acid-producing activity is a microbial cell that has been subjected to one or more of the following genetic manipulations (i), (ii), (iii), and (iv): (i) enhancement of one or more genes selected from a dehydroshikimate dehydratase gene, a dehydroquinate dehydratase gene, a quinate dehydrogenase gene, and a shikimate dehydrogenase gene, or (ii) enhancement of one or more genes selected from a group of genes involved in the shikimic acid synthesis pathway consisting of a 2-dehydro-3-deoxyarabinoheptonate aldolase gene, a 3-dehydroquinate synthase gene, and a shikimate dehydrogenase gene.(iii) Enhancement of one or more genes selected from the group of genes involved in the pentose phosphate pathway consisting of a glucose-6-phosphate dehydrogenase gene, a 6-phosphogluconolactonase gene, a phosphogluconate dehydrogenase gene, a ribose-5-phosphate isomerase gene, a ribulose-5-phosphate-3-epimerase gene, a transketolase gene, and a transaldolase gene. (iv) Enhancement of a gene encoding a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity. <5> The method according to <4>, wherein the microbial cell having improved 3-dehydroshikimic acid-producing activity is a microbial cell that has been subjected to genetic manipulations of two or more of (i), (ii), (iii), and (iv). <6> The method according to <4>, wherein the microbial cell having improved 3-dehydroshikimic acid-producing activity is a microbial cell that has been subjected to genetic manipulations of three or more of (i), (ii), (iii), and (iv). <7> The method according to <4>, wherein the microbial cells having improved 3-dehydroshikimic acid-producing activity are microbial cells that have been subjected to the genetic manipulations (i), (ii), (iii), and (iv) described above. <8> The method according to any one of <3> to <7>, wherein the microbial cells are coryneform bacteria. <9> The method according to <8>, wherein the coryneform bacteria are bacteria of the genus Corynebacterium. <10> The method according to <9>, wherein the Corynebacterium bacteria are Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerans, Corynebacterium alkanolyticum, Corynebacterium callnae, Corynebacterium crenatum, or Corynebacterium crudilactis. <11> The method according to <9>, wherein the Corynebacterium bacteria are Corynebacterium glutamicum. <12> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is an aromatic compound or a salt thereof derived from 3-dehydroshikimic acid.<13> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is gallic acid, protocatechuic acid, catechol, L-DOPA, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, 4-hydroxybenzoic acid, 4-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof. <14> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is gallic acid, protocatechuic acid, L-DOPA, 4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof. <15> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is gallic acid, protocatechuic acid, or a salt thereof. <16> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is gallic acid or a salt thereof. <17> The method according to any one of <1> to <16>, wherein the transformed cell is cultured in the presence of a sugar.

[0047] 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.

[0048] (1) Preparation of gallic acid-producing bacteria 1) Construction of a plasmid for replacing the cg0620 gene region with a polypeptide gene having 3,4-dihydroxybenzoate hydroxylase activity The base numbers shown in the following examples are those of the genome sequence of the ATCC13032 strain, and this genome sequence information was obtained from the NCBI GB database under accession number NC_006958. PrimeSTAR Max DNA Polymerase (TaKaRa) was used as the PCR enzyme.

[0049] Using the genomic DNA of the ATCC13032 strain (NBRC 12168 strain) as a template, amplification was performed with primers OT20 and OT21 to obtain a DNA fragment on the 5' side of the cg0620 gene region. Furthermore, amplification was performed with primers OT23 and OT24 using the genomic DNA as a template to obtain a DNA fragment on the 3' side of the cg0620 gene region. Furthermore, a DNA fragment (OT25) containing the promoter (hereinafter referred to as the tu promoter) of the tuf gene (cg0587) contained in the Corynebacterium glutamicum ATCC13032 strain was prepared by artificial gene synthesis. This was amplified using primers OT26 and OT27 to obtain a DNA fragment of the promoter region. Furthermore, two types of DNA fragments (SEQ ID NOs: 3 and 4) containing a polypeptide gene having 3,4-dihydroxybenzoate hydroxylase activity (hereinafter abbreviated as hfm145VF) were prepared by artificial gene synthesis. Each DNA fragment was used as a template and amplified with two DNA primers (OT30 and OT31, and OT32 and OT33), yielding two types of DNA fragments. Furthermore, a vector fragment was amplified using primers OT34 and OT35 with pHKPsacB1 as a template. The resulting PCR product was treated with DpnI (Takara Bio). Each DNA fragment was purified from the six PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio), and ligated using In-Fusion HD Cloning Kit (Takara Bio) to produce the plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt. The resulting plasmid solution was used in ECOS Competent E. The transformant was transformed into E. coli DH5α strain (Nippon Gene), and the cell suspension was spread on LB agar medium containing kanamycin and allowed to stand overnight at 37°C. The transformant carrying the plasmid was inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. The plasmid was purified from this culture using NucleoSpin Plasmid EasyPure (TaKaRa) to obtain pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt.

[0050] 2) Construction of a strain carrying a polypeptide gene having 3,4-dihydroxybenzoate hydroxylase activity Using a transformation method by electroporation (Bio-rad), the above-mentioned plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt was introduced into the CY44 strain (the CY44 strain is the tkt strain described in Reference Example 14 of Japanese Patent No. 6,322,576, in which expression is enhanced by controlling the transcription of the transketolase (sometimes referred to as tkt) gene with the tu promoter. Furthermore, the transcription of the dehydroshikimate dehydratase gene (sometimes referred to as qsuB) and the vanR (cg2615) gene can be induced by the addition of benzoic acid. Furthermore, the shikimate dehydrogenase (sometimes referred to as aroE3) gene is controlled by the VanR repressor), and the KC148sr strain was obtained by selecting for kanamycin resistance. Strain KC148sr was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers OT20 and OT36. The expected results were obtained, confirming that strain KC148sr is a single-crossover homologous recombinant in which the plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt was introduced into the cg0620 gene region. Strain KC148sr 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 smeared onto LB agar medium containing 20% ​​sucrose to obtain strain KC148. By PCR (Sapphire Amp (Takara Bio)) using primers OT36 and OT37, it was confirmed that the KC148 strain was a double-crossover homologous recombinant in which the Ptu-hfm145VF-hfm145VFop gene had been introduced into the cg0620 gene region, as expected.

[0051] (2) Construction of lactate dehydrogenase (sometimes called ldh) disruptant strain 1) Construction of a plasmid for disrupting the ldh (cg3219) gene PrimeSTAR Max DNA Polymerase (TaKaRa) was used as the PCR enzyme. Using pHKPsacB1 (described in Japanese Patent No. 6322576) as a template, a vector fragment was amplified with primers pHKPsacB-F2 and pHKPsacB-R2. Using genomic DNA of the ATCC13032 strain (=NBRC 12168 strain) as a template, the 5' DNA fragment of the cg3219 gene was amplified with primers 3219-up-F and 3219-up-R, and the 3' DNA fragment of the cg3219 gene was amplified with primers 3219-down-F and 3219-down-R using genomic DNA as a template. The resulting PCR product was treated with DpnI (Takara Bio). After purifying each DNA fragment using NucleoSpin Gel and PCR Clean-up (Takara Bio) for the three resulting PCR products, they were ligated using the In-Fusion HD cloning kit (Clontech) to produce pHKBsacB-Δldh. The resulting plasmid solution was used to transform ECOS Competent E. coli DH5α strain (Nippon Gene), and the cell syrup was spread on LB agar medium containing kanamycin and left to stand overnight at 37 ° C. Using the resulting colonies as templates, colony PCR was performed using Sapphire Amp (TaKaRa) as the enzyme. Introduction of the target DNA fragment was confirmed using primers 3219-up-F and 3219-down-R. Transformants carrying the plasmid, in which gene introduction was confirmed, were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37 ° C. The plasmid was purified from this culture using NucleoSpin Plasmid EasyPure (TaKaRa), and pHKBsacB-Δldh was obtained.

[0052] 2) Obtaining a strain with the ldh gene (cg3219) disrupted. The plasmid pHKBsacB-Δldh obtained above was transformed into KC148 by electroporation (Bio-Rad). KC148Δldh-sr was obtained by selecting for kanamycin resistance. PCR (Sapphire Amp) was performed using the resulting colonies as templates and primers sacB-1 and 3219-up1500. The expected results confirmed that the pHKBsacB-Δldh plasmid had been introduced into the cg3219 gene region by single-crossover homologous recombination. KC148Δldh-sr was cultured in 1 mL of LB liquid medium for 24 hours, and a portion of the culture was smeared on LB agar medium containing 20% ​​sucrose to obtain the KC148Δldh strain. Colony PCR (Sapphire Amp) using primers 3219-coloP-F and 3219-coloP-R confirmed that the ldh gene (cg3219) had been deleted by double-crossover homologous recombination. Additionally, the kanamycin resistance gene and sacB gene were also deleted.

[0053] (3) Preparation of mctC-disrupted strain 1) Preparation of a plasmid for disrupting the mctC (cg0953) gene PrimeSTAR Max DNA Polymerase (TaKaRa) was used as the PCR enzyme. Using pHKPsacB1 (described in Japanese Patent Publication No. 6322576) as a template, a vector fragment was amplified with primers pHKPsacB-F2 and pHKPsacB-R2. Using genomic DNA of the ATCC13032 strain (NBRC 12168 strain) as a template, a DNA fragment on the 5' side of the cg0953 gene was amplified with primers ocJK197 and ocJK198, and a DNA fragment on the 3' side of the cg0953 gene was amplified with primers ocJK199 and ocJK200 using genomic DNA as a template. The resulting PCR product was treated with DpnI (Takara Bio). Each DNA fragment was purified from the three resulting PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio), and then ligated using an In-Fusion HD cloning kit (Clontech) to produce pHKBsacB-ΔmctC. The resulting plasmid solution was used to transform ECOS Competent E. coli DH5α strain (Nippon Gene), and the cell solution was spread on LB agar medium containing kanamycin and left to stand overnight at 37 ° C. Using the resulting colonies as templates, colony PCR was performed using Sapphire Amp (TaKaRa) as the enzyme. Introduction of the target DNA fragment was confirmed using primers ocJK216 and ocJK217. The transformant carrying the plasmid for which gene introduction was confirmed was inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37° C. The plasmid was purified from this culture using NucleoSpin Plasmid EasyPure (TaKaRa) to obtain pHKBsacB-ΔmctC.

[0054] 2) Obtaining an mctC-disrupted strain The KC148Δldh strain was transformed with the pHKBsacB-ΔmctC plasmid obtained above by electroporation (Bio-Rad). KC148ΔldhΔmctC-sr was obtained by selecting for kanamycin resistance. PCR (Sapphire Amp) was performed using the resulting colonies as templates and primers ocJK217 and ocJK224. The expected results confirmed that the pHKBsacB-ΔmctC plasmid had been introduced into the cg0953 gene region by single-crossover homologous recombination. KC148ΔldhΔmctC-sr was cultured in 1 mL of LB liquid medium for 24 hours, and a portion of the culture was smeared on LB agar medium containing 20% ​​sucrose to obtain the KC148ΔldhΔmctC strain. Colony PCR (Sapphire Amp) using primers ocJK491 and ocJK492 confirmed that the mctC gene (cg0953) had been deleted by double-crossover homologous recombination, along with the kanamycin resistance gene and sacB gene.

[0055]

[0056] (4) Evaluation of Aromatic Compound Productivity The KC148Δldh strain and the KC148ΔldhΔmctC strain were streaked on LB plates and cultured at 30°C for 3 days. The bacterial cells grown on the plates were inoculated into a round-bottomed spitzer (Eiken Chemical) containing 4 mL of LB medium and subjected to shaking culture (preculture) at 30°C and 200 rpm for 24 hours. Sodium benzoate was added to the CGXII medium shown in Table 2 to a final concentration of 1 mM, and 100 mL was placed in a Bio Jr. 8 (Able Co., Ltd.) fermenter. 1 mL of the preculture was inoculated and cultured with shaking for 18 hours at 32°C, 700 rpm, and an aeration rate of 100 mL / min to evaluate aromatic compound productivity. The culture solution was appropriately diluted with dilute sulfuric acid, the bacterial cells were removed by centrifugation, and the supernatant was collected. The concentrations of gallic acid and protocatechuic acid in the supernatant were quantified. The results are shown in Table 3. The gallic acid concentration was 2.3 times higher in the KC148ΔldhΔmctC strain than in the KC148Δldh strain, confirming the effect of improving aromatic compound productivity.

[0057]

[0058]

[0059] Reference Example 1: Quantification of Gallic Acid The collected supernatant was subjected to removal of insoluble matter using an AcroPrep 96 filter plate (0.2 μm GHP membrane, Nippon Pall), and the reaction solution was subjected to HPLC. A Chromaster (Hitachi High-Tech Science) was used as the HPLC device. An L-column ODS (4.6 mm ID x 150 mm, Chemicals Evaluation and Research Institute, Japan) was used, and 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. A UV detector (detection wavelength 210) was used for detection.

Claims

1. A method for producing an aromatic compound or a salt thereof, comprising a step of culturing a transformed cell in which the function of a monocarboxylic acid transporter represented by (A) or (B) below is suppressed or deleted, The method, wherein the productivity of the aromatic compound or a salt thereof by the transformed cell is improved compared to that of the cell before transformation. (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (B) A polypeptide having monocarboxylic acid transporter activity, which comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

2.

2. The method described in claim 1, further comprising a step of recovering the aromatic compound or its salt.

3. A method for improving the productivity of an aromatic compound or a salt thereof, comprising the step of culturing a transformed cell in which the function of a monocarboxylic acid transporter shown in (A) or (B) below is suppressed or deleted. (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (B) A polypeptide having monocarboxylic acid transporter activity, which comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

2.

4. A method described in claim 1 or 3, wherein the transformed cell in which the function of the monocarboxylic acid transporter is suppressed or deleted is a transformed cell in which the function of a gene encoding the monocarboxylic acid transporter on the chromosomal DNA of the host cell is suppressed, and the gene encoding the monocarboxylic acid transporter is a polynucleotide of (a) or (b) below. (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 to the nucleotide sequence shown in SEQ ID NO: 1, and encoding a polypeptide having monocarboxylic acid transporter activity.

5. The method according to claim 1 or 3, wherein a microbial cell having improved 3-dehydroshikimic acid producing activity is used as the host.

6. The method according to claim 5, wherein the microbial cells having improved 3-dehydroshikimic acid-producing activity are microbial cells that have been subjected to any one or more of the following genetic manipulations (i), (ii), (iii), and (iv): (i) Enhancement of one or more genes selected from the group consisting of a dehydroshikimate dehydratase gene, a dehydroquinate dehydratase gene, a quinate dehydrogenase gene, and a shikimate dehydrogenase gene. (ii) Enhancement of one or more genes selected from the group of genes involved in the shikimate synthesis pathway consisting of the 2-dehydro-3-deoxyarabinoheptonate aldolase gene, the 3-dehydroquinate synthase gene, and the shikimate dehydrogenase gene. (iii) Enhancement of one or more genes selected from the group of genes involved in the pentose phosphate pathway consisting of glucose-6-phosphate dehydrogenase gene, 6-phosphogluconolactonase gene, phosphogluconate dehydrogenase gene, ribose-5-phosphate isomerase gene, ribulose-5-phosphate-3-epimerase gene, transketolase gene, and transaldolase gene. (iv) Enrichment of genes encoding polypeptides having 3,4-dihydroxybenzoate hydroxylase activity.

7. The method described in claim 6, wherein the microbial cells with improved 3-dehydroshikimic acid production activity are microbial cells that have been subjected to genetic manipulation of three or more of (i), (ii), (iii), and (iv).

8. The method described in claim 6, wherein the microbial cells with improved 3-dehydroshikimic acid production activity are microbial cells that have been subjected to the genetic manipulations of (i), (ii), (iii), and (iv).

9. The method of claim 5, wherein the microbial cells are coryneform bacteria.

10. The method according to claim 9, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.

11. 4. The method according to claim 1, wherein the aromatic compound or a salt thereof is an aromatic compound or a salt thereof derived from 3-dehydroshikimic acid.

12. The method according to claim 1 or 3, wherein the aromatic compound or a salt thereof is gallic acid, protocatechuic acid, catechol, L-DOPA, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, 4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

13. The method according to claim 1 or 3, wherein the transformed cells are cultured in the presence of sugars.

14. The method of claim 13, wherein the sugar is selected from glucose, fructose, mannose, arabinose, xylose, galactose, or a sugar that can be metabolized to produce glucose.

15. A method according to claim 1 or 3, wherein the transformed cells are cultured under aerobic conditions.

16. A method for producing an aromatic compound or a salt thereof, comprising a step of culturing a transformed cell in which the function of a monocarboxylic acid transporter shown in (A) or (B) below is suppressed or deleted, wherein the method uses a microbial cell with improved 3-dehydroshikimic acid production activity as a host. (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (B) A polypeptide having monocarboxylic acid transporter activity, which comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

2.

17. The method described in claim 16, wherein the microbial cells with improved 3-dehydroshikimic acid production activity are microbial cells that have been subjected to one or more genetic manipulations of the following (i), (ii), (iii) and (iv): (i) Enhancement of one or more genes selected from the group consisting of a dehydroshikimate dehydratase gene, a dehydroquinate dehydratase gene, a quinate dehydrogenase gene, and a shikimate dehydrogenase gene. (ii) Enhancement of one or more genes selected from the group of genes involved in the shikimate synthesis pathway consisting of the 2-dehydro-3-deoxyarabinoheptonate aldolase gene, the 3-dehydroquinate synthase gene, and the shikimate dehydrogenase gene. (iii) Enhancement of one or more genes selected from the group of genes involved in the pentose phosphate pathway consisting of glucose-6-phosphate dehydrogenase gene, 6-phosphogluconolactonase gene, phosphogluconate dehydrogenase gene, ribose-5-phosphate isomerase gene, ribulose-5-phosphate-3-epimerase gene, transketolase gene, and transaldolase gene. (iv) Enrichment of genes encoding polypeptides having 3,4-dihydroxybenzoate hydroxylase activity.

18. A method according to claim 16 or 17, wherein the microbial cells are coryneform bacteria.

19. The method described in claim 18, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.

20. The method according to claim 16 or 17, wherein the aromatic compound or its salt is an aromatic compound or its salt derived from 3-dehydroshikimic acid.

21. The method described in claim 16 or 17, wherein the aromatic compound or its salt is gallic acid, protocatechuic acid, catechol, L-DOPA, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, 4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.