WO2023106352A1 - Method for producing aromatic compounds - Google Patents

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 compound

The present invention relates to a method for producing aromatic compounds using transformed cells.

In recent years, it has been desired to use microorganisms to produce useful aromatic compounds, including gallic acid, from glucose, which is an inexpensive raw material. Among them, corynebacterium glutamicum is a useful industrial microorganism that has been used for the production of various amino acids and nucleic acids. Various organic compounds such as aromatic amino acids (Non-Patent Document 1), gallic acid, 4-hydroxybenzoic acid (Non-Patent Document 1), 4-aminobenzoic acid (Non-Patent Document 2) and other aromatic compounds such as is available for production. Among them, gallic acid has a strong reducing property, so it is used as a raw material for producing photographic developers and blue inks, and esters such as propyl gallate are used as antioxidants for oils and butters. . 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. Therefore, efficient production of gallic acid is beneficial. .

The shikimate pathway is an important metabolic pathway for the biosynthesis of aromatic compounds by 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-arabinopeptulosonic acid 7-phosphate (DAHP). , 3-dehydroquinic acid (DHQ) and 3-dehydroshikimic acid (DHS) to shikimic acid. Furthermore, shikimic acid undergoes transfer of the phosphate group from adenosine triphosphate to become 3-phosphoshikimic acid, and then to chorismic acid via 3-phosphoeno-lpyruvylshikimic acid. In the shikimate pathway, a 6-carbon ring is formed followed by double bond formation, and from protocatechuic acid derived from DHS, gallic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA) , 2,5-pyridinedicarboxylic acid (2,5-PDCA), catechol, L-DOPA and other aromatic compounds are produced (Fig. 1).

[Non-Patent Document 1] Metab. Eng. 2018. 50:122-141.
[Non-Patent Document 2] Metab. Eng. 2016. 38:322-330.

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 by SEQ ID NO: 2 (A2) A polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown by SEQ ID NO: 2 and having monocarboxylic acid transporter activity

Schematic diagram showing production pathways of various aromatic compounds when coryneform bacteria are used as hosts. In the figure, aroG and aroF are 2-dehydro-3-deoxyarabinoheptonate aldolase, aroB is 3-dehydroquinate synthase, aroD and qsuC are dehydroquinate dehydratase, and 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

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

The present inventors have investigated the productivity of aromatic compounds such as gallic acid in transformed cells in which the function of MctC, a transporter of monocarboxylic acids such as pyruvate, propionic acid and acetic acid, is suppressed or deleted. was found to improve.

According to the present invention, protocatechuic acid, gallic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, L-DOPA, 4-hydroxybenzoic acid, 4- It becomes possible to efficiently produce an aromatic compound such as aminobenzoic acid or a salt thereof.

In the present invention, the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, genetic information processing software GENETYX Ver. 12 homology analysis (Search homology) program is used, unit size to compare (ktup) is set to 2, and analysis is performed.

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

In the present invention, "an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted" means 1 or more and 10 or less, preferably 1 or more and 8 or less, more preferably 1 It refers to an amino acid sequence in which 5 or less, more preferably 1 or more and 3 or less amino acids are deleted, substituted, added, or inserted. In addition, "a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted" means 1 or more and 30 or less, preferably 1 or more and 24 or less, more preferably 1 or more and 15 It refers to a nucleotide sequence in which no more than 1, more preferably from 1 to 9 nucleotides have been deleted, substituted, added or inserted. In the present invention, "addition" of amino acids or nucleotides includes addition of amino acids or nucleotides to one and both termini of a sequence.

In the present invention, the aromatic compound is an organic aromatic compound biosynthesized in the host cell, specifically an aromatic compound synthesized via the shikimic acid pathway, preferably 3-dehydroshikimic acid (DHS) and aromatic compounds derived from chorismate (Fig. 1).
Specifically, 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 them, protocatechuic acid derived from DHS; gallic acid derived from protocatechuic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), Catechol, L-DOPA; 4-hydroxybenzoic acid, 4-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid derived from chorismic acid, tyrosine, tryptophan, etc. are preferred, more preferably protocatechuic acid, protocatechuic acid They are derived aromatic compounds (preferably gallic acid, L-DOPA), 4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid, and more preferably gallic acid.

Examples of the salt of the aromatic compound include base addition salts and acid addition salts. Examples of base addition salts include salts with alkali metals such as sodium and potassium, and salts with alkaline earth metals such as calcium and magnesium. Examples of acid addition salts include hydrochlorides, sulfates, Mineral acid salts such as nitrates and phosphates are included.

In the method for producing an aromatic compound or a salt thereof of the present invention, a transformed cell in which the function of the monocarboxylic acid transporter shown in (a) or (b) below is suppressed or deleted is used.
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; (B) a polypeptide 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 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. The MctC is a transporter involved in the transport of monocarboxylic acids such as pyruvate, propionic acid and acetic acid.

In the polypeptide of (A), the identity with the amino acid sequence shown in SEQ ID NO: 2 is preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, still more preferably 98% or more, In addition, it is 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 deletion, substitution, addition, or Inserted amino acid sequences are included.

Having monocarboxylic acid transporter activity means that, for example, when a target gene-disrupted strain or enhanced strain is grown in a medium containing monocarboxylic acid as a carbon source, the concentration of monocarboxylic acid in the cells or the growth rate of the cells increases. can be determined by measuring and comparing with the host strain.

Polypeptides consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 of (B) and having monocarboxylic acid transporter activity include, for example, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium SP. Sa1YVA5 (Corynebacterium SP. Sa1YVA5), Corynebacterium hadale, Corynebacterium gottingense, Corynebacterium godavarianum, Corynebacterium senegalense-derived ActP (cation: acetic acid symporter) and the like.

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

In the present invention, the transformed cells in which the function of the monocarboxylic acid transporter is suppressed or deleted include the expression of the monocarboxylic acid transporter in the host cell is reduced or lost, and the protein as the monocarboxylic acid transporter is Cells in which the function is suppressed or deleted, preferably, the expression of the monocarboxylic acid transporter is reduced or lost compared to the parent cell by introducing a mutation, and the function of the protein as a monocarboxylic acid transporter is reduced. Reduced or lost cells (mutagenized cells) are included.
In the present invention, the term "expression" of a protein means that a translation product is produced from a gene encoding the protein and localized at its site of action in a functional state. Reduction or loss of monocarboxylic acid transporter expression by introducing a mutation means that modification at the gene level, transcription level, post-transcriptional regulation level, translation level, or post-translational modification level results in transformed cells It means a state in which the amount of monocarboxylic acid transporter protein present in the cell is reduced or lost, preferably a state in which it is significantly reduced or lost compared to that in the parent cell.

"The expression of the monocarboxylic acid transporter is decreased compared to the parent cell" means that the expression level of the monocarboxylic acid transporter present in the transformed cell is decreased compared to the parent cell. The expression level of the protein is usually reduced to 50% or less, preferably 20% or less, more preferably 10% or less compared to the parent cell, and the activity thereof is also similarly reduced. means Most preferred is 0% monocarboxylic acid transporter expression, ie, loss of monocarboxylic acid transporter expression.
The expression level of the monocarboxylic acid transporter is compared by measuring the expression level of the polypeptide by well-known immunological techniques such as Western blotting and immunohistochemical staining.

Transformed cells in which the expression of the monocarboxylic acid transporter is reduced or lost can be specifically obtained by suppressing the function of the gene encoding the monocarboxylic acid transporter on the chromosomal DNA of the host cell. can. Here, suppression of function may be either complete suppression (inhibition) or incomplete suppression of function.
A gene encoding a monocarboxylic acid transporter means a DNA comprising a transcriptional region containing an ORF and a transcriptional regulatory region such as the promoter of the gene. In the present invention, the gene encoding the monocarboxylic acid transporter preferably includes the following polynucleotide (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 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) encoding a monocarboxylic acid transporter derived from Corynebacterium glutamicum.

Examples of nucleotide sequences having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 include deletions, substitutions, additions, or An inserted nucleotide sequence is included. Methods for introducing mutations such as deletion, substitution, addition, or insertion of nucleotides into the nucleotide sequence are as described above. The polynucleotide may be in single- or double-stranded form, and may be DNA or RNA. The DNA may be cDNA, artificial DNA such as chemically synthesized DNA.

Suppression of the function of a gene encoding such a monocarboxylic acid transporter is a mutation that deletes or inactivates the coding region, non-coding region, transcription or translation initiation region of the gene encoding the monocarboxylic acid transporter. (deletion or inactivation of a gene encoding a monocarboxylic acid transporter), or a polynucleotide having the activity of degrading a transcript of a gene encoding a monocarboxylic acid transporter, a protein from the transcript It can be carried out by suppressing transcription or translation by introducing a polynucleotide that suppresses translation into .

In one aspect, deletion or inactivation of a gene encoding a monocarboxylic acid transporter involves removing part or all of the nucleotide sequence of the gene encoding the monocarboxylic acid transporter from the genome, or removing other nucleotides from the genome. sequence replacement, inserting other polynucleotide fragments into the sequence of the gene encoding the monocarboxylic acid transporter, mutating the transcription or translation initiation region of the gene encoding the monocarboxylic acid transporter, etc. It can be realized by Preferably, part or all of the nucleotide sequence of the gene encoding the monocarboxylic acid transporter is deleted or inactivated. More specific examples include a method for specifically deleting or inactivating a gene encoding a monocarboxylic acid transporter on the genome of cells, and random deletion or inactivation of the gene in cells. Examples include a method in which cells having desired mutations are selected by evaluating the expression level and activity of the monocarboxylic acid transporter, or conducting genetic analysis after giving mutation mutations.

Specific deletion or inactivation of genes encoding monocarboxylic acid transporters includes, for example, methods by homologous recombination. That is, a DNA fragment of a gene encoding a monocarboxylic acid transporter into which an inactivating mutation has been introduced by polynucleotide substitution, insertion, or the like, or a monocarboxylic acid transporter containing an outer region of a gene encoding a monocarboxylic acid transporter. By constructing a DNA fragment that does not contain a gene encoding a porter and incorporating it into the parent cell to cause homologous recombination in the region containing the gene encoding the monocarboxylic acid transporter in the genome of the parent cell, the genome Genes encoding the above monocarboxylic acid transporters can be deleted or inactivated.
Alternatively, a recombinant vector (such as a plasmid) having a DNA fragment containing a partial region of the gene encoding the monocarboxylic acid transporter is incorporated into the parent cell, and the gene encoding the monocarboxylic acid transporter in the genome of the parent cell. It is also possible to inactivate the gene encoding the monocarboxylic acid transporter by disrupting the partial region by homologous recombination.
As a method for randomly deleting or inactivating genes in cells, a DNA fragment obtained by randomly cloning a gene introduced with an inactivating mutation is introduced into a cell, and a gene on the genome of the cell Examples include a method of causing homologous recombination, a method of irradiating cells with ultraviolet rays, γ-rays, etc. to induce mutations, and the like. A gene inactivating mutation means a mutation in which the original function of the target gene is lost due to silence mutation, missense mutation, nonsense mutation, frameshift mutation, or the like. For example, genes introduced with inactivating mutations either do not express proteins or express proteins with impaired native activity.

Site-directed mutagenesis is an example of a method for producing a DNA fragment containing a gene encoding a monocarboxylic acid transporter introduced with an inactivating mutation. Site-directed mutagenesis can be performed using mutagenic primers containing the nucleotide mutation to be introduced. For example, by PCR with a gene encoding a monocarboxylic acid transporter as a template using two sets of primers containing nucleotide mutations to be introduced, the upstream and downstream regions of the region containing the gene encoding the monocarboxylic acid transporter are detected. DNA fragments are amplified, respectively, and then these are ligated into one by SOE-PCR (splicing by overlap extension PCR) (Gene, 1989, 77(1): p61-68) to obtain the desired mutation. A DNA fragment can be constructed that contains: Alternatively, for site-directed mutagenesis, the inverse PCR method, annealing method, etc. (edited by Muramatsu et al., "Revised 4th Edition New Genetic Engineering Handbook", Yodosha, p82-88), or Stratagene's QuickChange II Site- Commercially available kits for site-directed mutagenesis such as Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit can also be used.

Mutation primers can be prepared by well-known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). A gene encoding a monocarboxylic acid transporter used as a template may be prepared from a host cell by a conventional method, or may be chemically synthesized.

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

Further, as a polynucleotide having an activity to degrade a transcription product of a gene encoding a monocarboxylic acid transporter or a polynucleotide that suppresses translation of the transcription product into a protein, a gene encoding a monocarboxylic acid transporter Polynucleotides comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of mRNA, or portions thereof, are included. Specifically, antisense RNA against the mRNA of the gene encoding the monocarboxylic acid transporter, siRNA against the mRNA of the gene encoding the monocarboxylic acid transporter, ribozyme against the mRNA of the gene encoding the monocarboxylic acid transporter, and the like. mentioned.

Cells in which the function of the gene encoding the monocarboxylic acid transporter is suppressed can be selected by confirming the genome sequence. Alternatively, cells in which the function of the gene encoding the monocarboxylic acid transporter is suppressed can be selected using the expression level or activity of the monocarboxylic acid transporter as an index.

In the present invention, the host cells may be cells suitable for producing aromatic compounds or salts thereof, and may be any of microbial cells, plant cells and animal cells, preferably microbial cells.
From the viewpoint of production efficiency of aromatic compounds or salts thereof, especially protocatechuic acid, gallic acid, shikimic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, L-DOPA, chorismic acid, 4-hydroxy In terms of production efficiency of aromatic compounds derived from 3-dehydroshikimic acid such as benzoic acid, 4-aminobenzoic acid, and 4-amino-3-hydroxybenzoic acid, or salts thereof, host cells are capable of producing 3-dehydroshikimic acid. It is more preferable to use microbial cells with improved production activity.

Examples of microbial cells include Escherichia coli, Bacillus subtilis, actinomycetes, Pseudomonas bacteria, Streptococcus bacteria, Lactobacillus bacteria, fungi (Neurospora, Aspergillus, Trichoderma, etc.), yeasts (Saccharomyces, Kluyveromyces, Schizosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Pichia, Candida, etc.) may be used. , actinomycetes are preferred.

As the actinomycete, a group of microorganisms defined as coryneform bacteria (Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974)) is preferable, specifically, the genus Corynebacterium and the genus Brevibacterium. bacteria, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, Micrococcus, and the like.
Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alcano Corynebacterium alkanolyticum, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium callunae and the like.
Brevibacterium ammoniagenes etc. are mentioned as a Brevibacterium genus microbe.
Arthrobacter globiformis includes Arthrobacter globiformis.
Mycobacterium genus includes Mycobacterium bovis and the like, and Micrococcus genus includes Micrococcus freudenreichii, Micrococcus leuteus, Micrococcus ureae, Micrococcus Coccus roseus (Micrococcus roseus) etc. are mentioned.
Among the coryneform bacteria, the genus Corynebacterium is preferred, and Corynebacterium glutamicum is more preferred.
The above microbial cells may be wild strains, mutant strains thereof, or artificial genetically modified strains thereof.

Examples of microbial cells with improved 3-dehydroshikimic acid-producing activity include microbial cells in which genes necessary for producing 3-dehydroshikimic acid are enhanced. Specifically, the following (i), ( ii), (iii) and (iv) genetically engineered microbial cells, preferably any one of (i), (ii), (iii) and (iv) Two or more, more preferably three or more, and more preferably all of (i), (ii), (iii), and (iv) are genetically engineered microbial cells.
Here, gene enhancement includes introduction of a predetermined gene in an expressible state, introduction of a mutation into a predetermined gene or the control region of the gene, and the like.
(i) enhancement of one or more genes selected from dehydroshikimate dehydratase gene, dehydroquinate dehydratase gene, quinate dehydrogenase gene and shikimate dehydrogenase gene;
(ii) enhancement of one or more genes selected from a group of genes related to the shikimate synthesis pathway consisting of 2-dehydro-3-deoxyarabinoheptonate aldolase gene, 3-dehydroquinate synthase gene, and shikimate dehydrogenase gene;
(iii) glucose-6-phosphate dehydrogenase gene, 6-phosphogluconolactonase gene, phosphogluconate dehydrogenase gene, ribose-5-phosphate isomerase gene, ribulose-5-phosphate-3-epimerase gene, transke Enhancement of one or more genes selected from the gene group involved in the pentose phosphate pathway consisting of the tolase gene and the transaldolase gene.
(iv) enhancement of genes encoding polypeptides with 3,4-dihydroxybenzoate hydroxylase activity;

By culturing the thus produced transformed cells, evaluating the productivity of aromatic compounds or salts thereof, and selecting suitable transformed cells, useful aromatic compound or salt-producing cells can be obtained. Obtainable. The product can be measured according to the method described in Reference Examples below.

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 sugars, and recovering the desired aromatic compound or salt thereof.
Glucose is preferred as the sugar, but monosaccharides such as fructose, mannose, arabinose, xylose and galactose, as well as sugars capable of producing glucose by metabolism can also be used. Such sugars include oligosaccharides or polysaccharides having glucose units, disaccharides such as cellobiose, sucrose, lactose, maltose, trehalose, cellobiose, xylobiose; polysaccharides such as dextrin or soluble starch; is mentioned.
Molasses can also be used, for example, as a raw material containing these raw material compounds. Inedible agricultural waste such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw, etc.), bagasse and corn stover, energy crops such as switchgrass, napier grass and miscanthus, and wood waste Alternatively, a saccharified solution containing a plurality of sugars such as glucose, which is obtained by saccharifying waste paper or the like with a saccharifying enzyme or the like, can also be used.

The medium for culturing the transformed cells contains a carbon source, a nitrogen source, inorganic salts, etc., and can be either a natural medium or a synthetic medium as long as it is a medium capable of efficiently culturing the transformed cells of the present invention. may be used.
As the carbon source, the above sugars or molasses or saccharified solutions containing them are used. In addition to the above sugars, sugar alcohols such as mannitol, sorbitol, xylitol and glycerin; , maleic acid, and gluconic acid; alcohols, such as ethanol and propanol; and hydrocarbons, such as normal paraffin. A carbon source can be used individually by 1 type or in mixture of 2 or more types.
The concentration of the saccharide, which is the raw material compound, 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%.

Nitrogen sources include, for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, alkylamines such as methylamine, nitrogen-containing organic compounds such as amino acids, ammonia or its salts (ammonium chloride, Inorganic or organic ammonium compounds such as ammonium sulfate, ammonium nitrate and ammonium acetate), urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like can be used.

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

As culture media for coryneform bacteria, A medium [J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)], BT medium [J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)], CGXII medium [Patent No. 6322576] and the like can be mentioned, and these media can be used with the saccharide concentration within the above range.

Prior to the reaction or culture containing sugars, it is preferable to grow the transformants by culturing them in the same medium under aerobic conditions at a temperature of about 25-38°C for about 12-48 hours.

The culture temperature or reaction temperature is preferably 15 to 45°C, more preferably 25 to 37°C.
The culture or reaction time is 24 hours to 168 hours, preferably 24 hours to 96 hours, more preferably 24 hours to 72 hours, and can be performed with stirring or shaking as necessary. In addition, antibiotics such as ampicillin and kanamycin may be added to the medium during the culture, if necessary.
Cultivation may be of batch type, fed-batch type, or continuous type. Among them, a batch system is preferable.
Cultivation or reaction may be carried out under aerobic conditions or reducing conditions, but is preferably carried out under aerobic conditions.
When the reaction or culture is carried out under aerobic conditions, it is preferable to carry out under conditions that suppress excessive growth of the transformant from the viewpoint of the production efficiency of the aromatic compound or its salt.
If the aromatic compound is susceptible to oxidation, the culture is preferably carried out under conditions of low dissolved oxygen concentration. For example, in the production of gallic acid, specifically, the dissolved oxygen concentration is preferably 0.1 to 3 ppm, more preferably 0.1 to 1 ppm.

The method of collecting and purifying the aromatic compound or its salt from the culture is not particularly limited. That is, it can be carried out by combining well-known ion exchange resin method, precipitation method, crystallization method, recrystallization method, concentration method and other methods. For example, after the cells are removed by centrifugation or the like, ionic substances are removed with a cation and anion exchange resin, and the mixture is concentrated to obtain an aromatic compound or a salt thereof. Aromatic compounds or salts thereof accumulated in the culture may be used as they are without isolation.

The following aspects are further disclosed in this invention regarding embodiment mentioned above.
<1> A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a transformed cell in which the function of the 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 having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 and having monocarboxylic acid transporter activity <2> A transformed cell in which the function of the monocarboxylic acid transporter is suppressed or deleted is obtained by suppressing the function of the gene encoding the monocarboxylic acid transporter on the chromosomal DNA of the host cell. The method according to <1>, wherein the gene encoding the monocarboxylic acid transporter is the 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.
<3> The method according to <1> or <2>, wherein a microbial cell with improved 3-dehydroshikimic acid-producing activity is used as the host.
<4> The microbial cell with improved 3-dehydroshikimic acid-producing activity is a microbial cell genetically engineered by one or more of the following (i), (ii), (iii) and (iv) , <3>.
(i) enhancement of one or more genes selected from dehydroshikimate dehydratase gene, dehydroquinate dehydratase gene, quinate dehydrogenase gene and shikimate dehydrogenase gene;
(ii) enhancement of one or more genes selected from a group of genes related to the shikimate synthesis pathway consisting of 2-dehydro-3-deoxyarabinoheptonate aldolase gene, 3-dehydroquinate synthase gene, and shikimate dehydrogenase gene;
(iii) glucose-6-phosphate dehydrogenase gene, 6-phosphogluconolactonase gene, phosphogluconate dehydrogenase gene, ribose-5-phosphate isomerase gene, ribulose-5-phosphate-3-epimerase gene, transke Enhancement of one or more genes selected from the gene group involved in the pentose phosphate pathway consisting of the tolase gene and the transaldolase gene.
(iv) enhancement of genes encoding polypeptides with 3,4-dihydroxybenzoate hydroxylase activity;
<5> The microbial cell with improved 3-dehydroshikimic acid-producing activity is a microbial cell genetically engineered with any two or more of (i), (ii), (iii), and (iv). , the method described in <4>.
<6> The microbial cell with improved 3-dehydroshikimic acid-producing activity is a microbial cell genetically engineered with any three or more of (i), (ii), (iii), and (iv). , the method described in <4>.
<7> The microbial cell with improved 3-dehydroshikimic acid-producing activity is the microbial cell genetically engineered in (i), (ii), (iii), and (iv), according to <4>. the method of.
<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 bacterium belongs to the genus Corynebacterium.
<10> The Corynebacterium bacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium carnae, Corynebacterium clenatum or Corynebacterium The method according to <9>, which is Umm cruzilactis.
<11> The method according to <9>, wherein the Corynebacterium bacterium is Corynebacterium glutamicum.
<12> The method according to any one of <1> to <11>, wherein the aromatic compound or its salt is derived from 3-dehydroshikimic acid or its salt.
<13> 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-aminobenzoic acid, The method according to any one of <1> to <11>, which is 4-amino-3-hydroxybenzoic acid or a salt thereof.
<14> 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. Any method described.
<15> The method according to <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 its salt is gallic acid or its salt.
<17> The method according to any one of <1> to <16>, wherein the transformed cell is cultured in the presence of sugar.

Although the present invention will be described in more detail below using examples, the technical scope of the present invention is not limited to the following examples.

(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-dihydroxybenzoic acid hydroxylase activity The genome sequence information was obtained from the GB database of NCBI as accession number NC_006958.
PrimeSTAR Max DNA Polymerase (TaKaRa) was used as an enzyme for PCR.

The genomic DNA of the ATCC 13032 strain (= NBRC 12168 strain) was used as a template and amplified with primers OT20 and OT21 to obtain a DNA fragment on the 5' side of the cg0620 gene region. In addition, genomic DNA was used as a template and amplified with primers OT23 and OT24 to obtain a DNA fragment on the 3' side of the cg0620 gene region. In addition, a DNA fragment (OT25) containing the promoter (hereinafter referred to as tu promoter) of the tuf gene (cg0587) of Corynebacterium glutamicum ATCC13032 strain was produced by artificial gene synthesis. Using this as a template, it was amplified with primers OT26 and OT27 to obtain a DNA fragment of the promoter region. In addition, two types of DNA fragments (SEQ ID NOS: 3 and 4) containing a polypeptide gene (hereinafter abbreviated as hfm145VF) having 3,4-dihydroxybenzoate hydroxylase activity were prepared by artificial gene synthesis. Each DNA fragment was used as a template and amplified with two kinds of DNA primers (OT30 and OT31 and OT32 and OT33) to obtain two kinds of DNA fragments. Using pHKPsacB1 as a template, a vector fragment was amplified with primers OT34 and OT35. The resulting PCR product was treated with DpnI (Takara Bio). For the 6 types of PCR products obtained, each DNA fragment was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated with the In-Fusion HD Cloning Kit (Takara Bio) to create the plasmid pHKPsacB_cg0620- Ptu-hfm145VF-hfm145VFopt was produced. Using the obtained plasmid solution, ECOS Competent E.　Coli DH5α strain (Nippon Gene) was transformed, and the cell solution was spread on an LB agar medium containing kanamycin and allowed to stand overnight at 37°C. A transformant having the plasmid was inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. A plasmid was purified from this culture using NucleoSpin Plasmid EasyPure (TaKaRa) to obtain pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt.

2) Preparation of a polypeptide gene-introduced strain having 3,4-dihydroxybenzoate hydroxylase activity Using a transformation method by electroporation (Bio-rad), the above plasmid pHKPsacB_cg0620-Ptu-hfm145VF-hfm145VFopt was transformed into the CY44 strain. (The CY44 strain is a tkt strain described in Reference Example 14 of Japanese Patent No. 6322576, and its expression is enhanced by controlling transcription of the transketolase (sometimes referred to as tkt) gene by the tu promoter. Transcription of the dehydroshikimate dehydratase gene (sometimes called qsuB) and vanR (cg2615) gene can be induced by addition of benzoic acid, and the shikimate dehydrogenase (sometimes called aroE3) gene is induced by the VanR repressor. The KC148sr strain was obtained by introducing the strain into the spp. and selecting for kanamycin resistance. When the KC148sr strain was analyzed by the PCR method (Sapphire Amp (Takara Bio)) using primers OT20 and OT36, the expected results were obtained. It was confirmed to be a single-crossover homologous recombinant introduced into the gene region.
The KC148sr strain was cultured in 1 mL of LB liquid medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) for 24 hours, and a portion of the culture was spread on 20% sucrose-containing LB agar medium. KC148 strain was obtained by culturing. By the PCR method (Sapphire Amp (Takara Bio)) using primers OT36 and OT37, the KC148 strain is a double-crossover homologous recombinant in which the Ptu-hfm145VF-hfm145VFop gene has been introduced into the cg0620 gene region as expected. It was confirmed.

(2) Preparation of lactate dehydrogenase (sometimes referred to as ldh) disrupted strain 1) Preparation of plasmid for disrupting ldh (cg3219) gene PrimeSTAR Max DNA Polymerase (TaKaRa) was used as a PCR enzyme. Using pHKPsacB1 (described in Japanese Patent No. 6322576) as a template, the vector fragment was amplified with primers pHKPsacB-F2 and pHKPsacB-R2. A DNA fragment on the 5' side of the cg3219 gene amplified with primers 3219-up-F and 3219-up-R using the genomic DNA of the ATCC 13032 strain (= NBRC 12168 strain) as a template, and the genomic DNA as the template with the primer 3219- A DNA fragment on the 3' side of the cg3219 gene amplified by down-F and 3219-down-R was obtained.
The resulting PCR product was treated with DpnI (Takara Bio). For the three types of PCR products obtained, each DNA fragment was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and then ligated with In-Fusion HD cloning kit (Clontech) to obtain pHKBsacB-Δldh. was made. Using the obtained plasmid solution, ECOS Competent E. coli strain DH5α (Nippon Gene) was transformed, and the cell solution was spread on an LB agar medium containing kanamycin and allowed to stand overnight at 37°C. Using the obtained colony as a template, colony PCR was performed using Sapphire Amp (TaKaRa) as an enzyme. Using primers 3219-up-F and 3219-down-R, introduction of the target DNA fragment was confirmed. A transformant having a plasmid in which gene transfer was confirmed was inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. A plasmid was purified from this culture using NucleoSpin Plasmid EasyPure (TaKaRa) to obtain pHKBsacB-Δldh.

2) Acquisition of ldh gene (cg3219) disrupted strain Using the plasmid pHKBsacB-Δldh obtained above, KC148 was transformed by electroporation (Bio-rad). KC148Δldh-sr was obtained by selecting for kanamycin resistance. Using the resulting colony as a template, PCR (Sapphire Amp) was performed using primers sacB-1 and 3219-up1500, and the expected results were obtained. It was confirmed that it was introduced into the cg3219 gene region by recombination. KC148Δldh-sr was cultured in 1 mL of LB liquid medium for 24 hours, and a portion of the culture was smear cultured on 20% sucrose-containing LB agar medium to obtain KC148Δldh strain. Colony PCR (Sapphire Amp) using primers 3219-coloP-F and 3219-coloP-R confirmed that the ldh gene (cg3219) was deleted by double-crossover homologous recombination. In addition, deletion of kanamycin resistance gene and sacB gene was confirmed.

(3) Preparation of mctC-disrupted strain 1) Preparation of plasmid for disrupting mctC (cg0953) gene PrimeSTAR Max DNA Polymerase (TaKaRa) was used as an enzyme for PCR. Using pHKPsacB1 (described in Japanese Patent No. 6322576) as a template, the vector fragment was amplified with primers pHKPsacB-F2 and pHKPsacB-R2. A DNA fragment on the 5' side of the cg0953 gene amplified with primers ocJK197 and ocJK198 using the genomic DNA of ATCC 13032 strain (= NBRC 12168 strain) as a template, and a cg0953 gene amplified with primers ocJK199 and ocJK200 using genomic DNA as a template. A 3' DNA fragment was obtained. The resulting PCR product was treated with DpnI (Takara Bio). For the three types of PCR products obtained, each DNA fragment was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), ligated with In-Fusion HD cloning kit (Clontech), and pHKBsacB-ΔmctC. was made. Using the obtained plasmid solution, ECOS Competent E. coli strain DH5α (Nippon Gene) was transformed, and the cell solution was spread on an LB agar medium containing kanamycin and allowed to stand overnight at 37°C. Using the obtained colony as a template, colony PCR was performed using Sapphire Amp (TaKaRa) as an enzyme. Using ocJK216 and ocJK217 as primers, introduction of the target DNA fragment was confirmed. A transformant having a plasmid in which gene transfer was confirmed was inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. A plasmid was purified from this culture using NucleoSpin Plasmid EasyPure (TaKaRa) to obtain pHKBsacB-ΔmctC.

2) Acquisition of mctC-disrupted strain Using the plasmid pHKBsacB-ΔmctC obtained above, KC148Δldh strain was transformed by electroporation (Bio-rad). KC148ΔldhΔmctC-sr was obtained by selecting for kanamycin resistance. Using the resulting colony as a template, PCR (Sapphire Amp) was performed using primers ocJK217 and ocJK224, and the expected results were obtained. It was confirmed that it was introduced into the gene region. KC148ΔldhΔmctC-sr was cultured in 1 mL of LB liquid medium for 24 hours, and a part of the culture was smear cultured on 20% sucrose-containing LB agar medium to obtain the KC148ΔldhΔmctC strain. Colony PCR (Sapphire Amp) using primers ocJK491 and ocJK492 confirmed that the mctC gene (cg0953) was deleted by double-crossover homologous recombination. In addition, deletion of kanamycin resistance gene and sacB gene was confirmed.

(4) Evaluation of aromatic compound productivity The KC148Δldh strain and the KC148ΔldhΔmctC strain were streaked on an LB plate and cultured at 30°C for 3 days. The cells grown on the plate were inoculated into a round-bottom spitz (Eiken Chemical Co., Ltd.) filled with 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 Bio Jr. 8 (ABLE Co., Ltd.) was charged with 100 mL. 1 mL of the preculture solution was inoculated, and cultured with shaking for 18 hours under conditions of 32° C., 700 rpm, and 100 mL/min aeration rate to evaluate the productivity of aromatic compounds. The culture solution was appropriately diluted with dilute sulfuric acid, centrifuged to remove the cells, and then the supernatant was recovered. Gallic acid and protocatechuic acid concentrations in the supernatant were quantified. Table 3 shows the results. Compared to the KC148Δldh strain, the KC148ΔldhΔmctC strain had a gallic acid concentration of 2.3 times, confirming the productivity-enhancing effect of aromatic compounds.

Reference Example 1 Quantification of Gallic Acid Insoluble matter was removed from the collected supernatant using an Acroprep 96 filter plate (0.2 μm GHP membrane, Nippon Pole), and the reaction solution was subjected to HPLC.
The HPLC apparatus used was Chromaster (Hitachi High-Tech Science). Using an L-column ODS (4.6 mm ID × 150 mm, Chemical Substances Evaluation and Research Organization), eluent A was a 0.1% phosphoric acid solution of 0.1 M potassium dihydrogen phosphate, and eluent B was 70%. % methanol, a flow rate of 1.0 mL/min, and a column temperature of 40° C., gradient elution was performed. A UV detector (detection wavelength 210) was used for detection.

Claims (13)

1. 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.
   (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; (B) a polypeptide 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
2. The method according to claim 1, wherein a microbial cell with improved 3-dehydroshikimic acid-producing activity is used as a host.
3. The claim that the microbial cell with improved 3-dehydroshikimic acid-producing activity is a microbial cell genetically engineered by any one or more of the following (i), (ii), (iii) and (iv): 2. The method described in 2.
   (i) enhancement of one or more genes selected from dehydroshikimate dehydratase gene, dehydroquinate dehydratase gene, quinate dehydrogenase gene and shikimate dehydrogenase gene;
   (ii) enhancement of one or more genes selected from a group of genes related to the shikimate synthesis pathway consisting of 2-dehydro-3-deoxyarabinoheptonate aldolase gene, 3-dehydroquinate synthase gene, and shikimate dehydrogenase gene;
   (iii) glucose-6-phosphate dehydrogenase gene, 6-phosphogluconolactonase gene, phosphogluconate dehydrogenase gene, ribose-5-phosphate isomerase gene, ribulose-5-phosphate-3-epimerase gene, transke Enhancement of one or more genes selected from the gene group involved in the pentose phosphate pathway consisting of the tolase gene and the transaldolase gene.
   (iv) enhancement of genes encoding polypeptides with 3,4-dihydroxybenzoate hydroxylase activity;
4. The method according to claim 2 or 3, wherein the microbial cells are coryneform bacteria.
5. The method according to claim 4, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.
6. The Corynebacterium bacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium carnae, Corynebacterium clenatum or Corynebacterium 6. The method of claim 5, which is Cruzilactis.
7. The method according to claim 5, wherein the Corynebacterium bacterium is Corynebacterium glutamicum.
8. The method according to any one of claims 1 to 7, wherein the aromatic compound or its salt is an aromatic compound or its salt derived from 3-dehydroshikimic acid.
9. Aromatic compounds or their salts are 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, the method according to any one of claims 1 to 7.
10. Claims 1 to 7, wherein the aromatic compound or a salt thereof is gallic acid, protocatechuic acid, L-DOPA, 4-hydroxybenzoic acid, 4-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof. A method according to any one of
11. The method according to claims 1 to 7, wherein the aromatic compound or its salt is gallic acid, protocatechuic acid, or a salt thereof.
12. The method according to any one of claims 1 to 7, wherein the aromatic compound or its salt is gallic acid or its salt.
13. The method according to any one of claims 1 to 12, wherein the transformed cells are cultured in the presence of sugar.

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