Method for producing aromatic compound

Abstract

Provided is a method for efficiently producing an aromatic compound using a transformed coryneform bacterium. A method for producing an aromatic compound or a salt thereof, said method comprising a step for culturing a modified coryneform bacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted.

Description

Technical Field

[0001] The present invention relates to a method for producing an aromatic compound using modified coryneform bacteria. Background Art

[0002] In recent years, it is hoped that microorganisms can be used to produce useful aromatic compounds represented by gallic acid from glucose of cheap raw materials. Microorganisms have a metabolic pathway (shikimic acid pathway) for biosynthesizing aromatic compounds. That is, phosphoenolpyruvic acid obtained by the glycolysis system is combined with erythrose 4-phosphate supplied by the pentose phosphate pathway to form 3-deoxy-D-arabinoheptulose acid 7-phosphate (DAHP), and shikimic acid is formed via 3-dehydroquinic acid (DHQ) and 3-dehydroshikimic acid (DHS). In addition, shikimic acid transfers a phosphate group from adenosine triphosphate to become 3-phosphoshikimic acid, and chorismic acid is formed via 3-phosphoenolpyruvylshikimic acid. In the shikimate pathway, after the formation of a six-membered carbon ring, a double bond is formed, and aromatic compounds such as gallic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), catechol, p-aminobenzoic acid (4-ABA), and 4-amino-3-hydroxybenzoic acid (4,3-AHBA) are produced from protocatechuic acid derived from DHS ( Figure 1 ).

[0003] Among them, gallic acid is used as a developer for photographs and a raw material for the manufacture of blue inks due to its strong reducing property. In addition, esters such as propyl gallate are used as antioxidants for oils or butter. In addition, pyrogallic acid synthesized by decarboxylation of gallic acid is used as electronic materials, organic synthesis reagents, developing solutions for photographs, mordants for wool fabrics, etc. Therefore, it is beneficial to produce gallic acid efficiently. In addition, polybenzoxazole (PBO), an engineering plastic with excellent heat resistance and mechanical strength, is used in fiber materials and insulating films of semiconductor elements, etc. (Non-patent Document 1), and 4-amino-3-hydroxybenzoic acid (4,3-AHBA), which can be a raw material for its manufacture, has also attracted much attention in recent years. In addition, para-aminobenzoic acid (4-ABA) is not only a raw material for ultraviolet absorbers or pharmaceuticals, but also a raw material for polymers. It was previously obtained by chemical synthesis, but this method requires a lot of energy, so it is hoped that a fermentation production technology as an environmentally friendly process will be developed.

[0004] On the other hand, coryneform bacteria are aerobic bacteria and use oxygen as an electron acceptor in respiration. It is known that there are two types of terminal oxidases in the respiratory chain of coryneform bacteria, cytochrome aa3 type oxidase and cytochrome bd type oxidase (Non-Patent Document 2).

[0005] In the past, regarding the relationship between the respiratory chain-related enzymes and substance production of coryneform bacteria, for example, it has been reported that the productivity of lysine, a derivative of the TCA cycle, has been improved in strains in which cytochrome bd-type oxidase has been destroyed (non-patent document 3); and the productivity of diacetyl has been improved in strains in which cytochrome bd-type oxidase is overexpressed (non-patent document 4). In addition, in Patent Document 1, a microorganism in which the respiratory pathway is inactivated by genetic modification is disclosed in order to increase the amount of NAD(P)H that can be used in oxidoreductases that require NAD(P)H.

[0006] However, it has not been known until now that the productivity of aromatic compounds is improved when the function of cytochrome bd-type oxidase is inhibited or deleted.

[0007] Patent Document 1: U.S. Patent Application Publication No. 2008 / 0293101

[0008] Non-patent document 1: Murase Hiroki, SENI GAKKAISHI (Textiles and Industry), Vol. 66, No. 6 (2010)

[0009] Non-patent document 2: Michael Bott et al., Journal of Biotechnology, Vol. 104, No. 1-3, 2003, 129-153

[0010] Non-patent document 3: Armin Kabus et al., American Society for Microbiology Applied and Environmental Microbiology, Vol. 73, 2006, No. 3, pp. 861-868

[0011] Non-patent document 4: Cong Chen et al., Journal of Biotechnology, Vol. 332, 2021, 20-28 Summary of the invention

[0012] The present invention relates to the following 1) to 2).

[0013] 1) A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a modified coryneform bacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted.

[0014] 2) A coryneform bacterium, which is a modified coryneform bacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted, wherein the expression of at least one polypeptide selected from the following (C), (D), (E) and (F) is enhanced:

[0015] (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity,

[0016] (D) a polypeptide having 3-dehydroshikimate dehydratase activity,

[0017] (E) a polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate,

[0018] (F) A polypeptide having p-aminobenzoate hydroxylase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the production route of various aromatic compounds when Corynebacterium glutamicum is used as a host. In the figure, aroG and aroF represent genes encoding 2-dehydro-3-deoxyarabinoheptulose acid aldolase, aroB is a gene encoding 3-dehydroquinate synthetase, aroD is a gene encoding dehydroquinate dehydratase, qsuD is a gene encoding quinate / shikimate dehydrogenase, aroE3 is a gene encoding shikimate dehydrogenase, qsuB is a gene encoding dehydroshikimate dehydratase, aroA is a gene encoding 5-enoylpyruvylshikimate-3-phosphate synthetase, aroC is a gene encoding chorismate synthetase, and aroK is a gene encoding shikimate kinase, pabAB is a gene encoding 4-amino-4-deoxychorismate synthetase, and pabC is a gene encoding 4-amino-4-deoxychorismate lyase, which is an endogenous gene group of Corynebacterium glutamicum. pobA* is a gene encoding a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity as a mutant of p-hydroxybenzoate hydroxylase (pobA) from Corynebacterium glutamicum, phbh* is a gene encoding a polypeptide having p-aminobenzoate hydroxylase activity as a mutant of p-hydroxybenzoate hydroxylase from Caulobacter vibrioides, and is a metabolic pathway by introducing genes from the outside. In the figure, E4P is erythrose 4-phosphate, PEP is phosphoenolpyruvate, DAHP is 3-deoxy-D-arabinoheptulose 7-phosphate, DHQ is dehydroquinic acid, DHS is dehydroshikimic acid, SHK is shikimic acid, S3P is shikimic acid-3-phosphate, and EPSP is 5-enolpyruvylshikimic acid-3-phosphate. DETAILED DESCRIPTION

[0020] The present invention provides a method for efficiently producing aromatic compounds using transformed coryneform bacteria.

[0021] The inventors of the present invention have found that the productivity of aromatic compounds represented by gallic acid, p-aminobenzoic acid and 4-amino-3-hydroxybenzoic acid is improved in transformants in which the function of bd-type oxidase, one of the terminal oxidases of the respiratory chain of coryneform bacteria, is suppressed or deleted.

[0022] According to the present invention, an aromatic compound such as gallic acid, p-aminobenzoic acid, or 4-amino-3-hydroxybenzoic acid or a salt thereof can be efficiently produced by a fermentation method with a small environmental load.

[0023] In the present invention, the identity of amino acid sequences or nucleotide sequences was calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver.12 was used to analyze and calculate the identity by setting the Unit size to compare (ktup) to 2.

[0024] In the present invention, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence preferably means an identity of 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more.

[0025] In the present invention, "an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted" refers to an amino acid sequence in which more than one and less than ten amino acids are deleted, substituted, added or inserted, preferably more than one and less than eight, more preferably more than one and less than five, and further preferably more than one and less than three amino acids are deleted, substituted, added or inserted. In addition, "a nucleotide sequence in which one or more nucleotides are deleted, substituted, added or inserted" refers to a nucleotide sequence in which more than one and less than thirty nucleotides are deleted, substituted, added or inserted, preferably more than one and less than twenty-four, more preferably more than one and less than fifteen, and further more preferably more than one and less than nine nucleotides are deleted, substituted, added or inserted. In the present invention, "addition" of amino acids or nucleotides includes addition of amino acids or nucleotides at one end and both ends of the sequence.

[0026] In the present invention, the aromatic compound refers to an organic aromatic compound biosynthesized in coryneform bacteria, specifically, an aromatic compound synthesized via the shikimic acid pathway, preferably an aromatic compound derived from 3-dehydroshikimic acid (DHS) or chorismic acid ( Figure 1 ).

[0027] Specific examples thereof include protocatechuic acid, catechol, gallic acid, phenylalanine, tyrosine, tryptophan, p-hydroxybenzoic acid, 4-aminobenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, and 4-amino-3-hydroxybenzoic acid.

[0028] Among them, preferred are protocatechuic acid derived from DHS; gallic acid, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), and catechol derived from protocatechuic acid; p-hydroxybenzoic acid, p-aminobenzoic acid (4-ABA), 4-amino-3-hydroxybenzoic acid (4,3-AHBA), 2,5-pyridinedicarboxylic acid (2,5-PDCA), tyrosine, tryptophan, etc. derived from branched acid, and more preferred are gallic acid, p-aminobenzoic acid, and 4-amino-3-hydroxybenzoic acid.

[0029] 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 inorganic acid salts such as hydrochlorides, sulfates, nitrates, and phosphates.

[0030] The method for producing an aromatic compound or a salt thereof of the present invention uses a coryneform bacterium in which the function of the cytochrome bd type oxidase complex is suppressed or deleted.

[0031] The "cytochrome bd-type oxidase complex" is an enzyme involved in aerobic respiration of coryneform bacteria, and specifically, is one of the terminal oxidases of the respiratory chain.

[0032] It is known that rod-shaped bacteria have bd-type oxidase and aa3-type oxidase as the terminal oxidase of the respiratory chain. It is shown that the bd-type oxidase complex is composed of subunit I and subunit II, and according to the analysis of the N-terminal sequence, it is shown that they are equivalent to CydA and CydB, respectively (Kusumoto et al., 2000, "Arch Microbiol" 173: 390-397).

[0033] It is known that aa3 oxidase is composed of four subunits (CtaD, CtaC, CtaE, and CtaF), and forms a complex with the cytochrome bc1 complex (QcrB, QcrA, and QcrC) (Non-Patent Document 2 mentioned above).

[0034] The proton removal efficiency of BD oxidase and BC1-AA3 complex is known to be +When the O / O ratio is 2 and 6, it is reported that: since the proton discharge efficiency that can be pumped out per molecule of oxygen by the bd-type oxidase is low, the efficiency of ATP synthesis obtained in the end is also low (Matsushita K. (2013) Respiratory Chain and Energy Metabolism of Corynebacterium glutamicum. In: Yukawa H., Inui M. (eds) "Corynebacterium glutamicum. Microbiology Monographs", Vol. 23. Springer-Verlag, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 978-3-642-29857-8_11).

[0035] In the present invention, the cytochrome bd-type oxidase complex preferably includes a polypeptide complex having cytochrome bd-type oxidase activity as shown in the following (A) or (B).

[0036] (A) A complex comprising:

[0037] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and

[0038] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4

[0039] constituted.

[0040] (B) a complex consisting of:

[0041] 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 functioning as a cytochrome bd-type oxidase subunit I, and

[0042] A polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as a cytochrome bd-type oxidase subunit II

[0043] constituted.

[0044] Among them, in (A), the polypeptide composed of the amino acid sequence shown in sequence number 2 refers to cytochrome bd-type oxidase subunit I from Corynebacterium glutamicum, and the polypeptide composed of the amino acid sequence shown in sequence number 4 refers to cytochrome bd-type oxidase subunit II from Corynebacterium glutamicum.

[0045] In the polypeptide of (A), the identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is preferably 95% or more, more preferably 96% or more, further preferably 97% or more, further more preferably 98% or more, and even more preferably 99% or more.

[0046] Examples of amino acid sequences that are at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 include amino acid sequences in which one or more amino acids are deleted, substituted, added, or inserted into the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0047] The activity of the cytochrome bd type oxidase complex can be determined, for example, by referring to the non-patent literature described above (Kusumoto et al., 2000, Arch Microbiol 173: 390-397). Specifically, the cytochrome bd type oxidase, which is a membrane protein, is extracted from the bacteria, purified using a chromatographic column, etc., and the obtained enzyme is used to carry out a reaction of oxidizing hydroquinone to quinone, and the reaction can be confirmed by measuring the reaction.

[0048] In (B), as 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 functioning as a cytochrome bd-type oxidase subunit I, and a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as a cytochrome bd-type oxidase subunit II, for example, cytochrome bd-type oxidase subunit I and cytochrome bd-type oxidase subunit II from Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium SP.Sa1YVA5, Corynebacterium hadale, Corynebacterium gottingense, Corynebacterium godavarianum, Corynebacterium senegalense, etc. can be listed.

[0049] As a method for introducing mutations such as deletion, substitution, addition, or insertion of amino acids into the amino acid sequence of the above-mentioned polypeptide, for example, there can be mentioned a method for introducing mutations such as deletion, substitution, addition, or insertion of nucleotides into a nucleotide sequence encoding the amino acid sequence. As a method for introducing mutations into a nucleotide sequence, for example, there can be mentioned mutagenesis caused by chemical mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, nitrous acid, or physical mutagens such as ultraviolet rays, X-rays, gamma rays, and ion beams, site-directed mutagenesis, and the method described by Dieffenbach et al. (Cold Spring Harbar Laboratory Press, New York, 581-621, 1995). As methods for site-directed mutagenesis, there can be mentioned 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), etc. Alternatively, the site-directed mutagenesis system Mutan-SuperExpress Km kit (Takara Bio Co., Ltd.), Transformer TM Site-directed mutagenesis kit (Transformer TM Commercially available site-directed mutagenesis kits include Site-Directed Mutagenesis Kit (Clonetech), KOD-Plus-Mutagenesis Kit (Toyobo Co., Ltd.), and the like.

[0050] In the present invention, the modified rod-shaped bacteria (also referred to as "transformants") whose function as a cytochrome bd-type oxidase complex is suppressed or lost include: bacterial strains in which the expression of the cytochrome bd-type oxidase complex in the host bacteria is reduced or lost, and the function of the protein as a cytochrome bd-type oxidase complex is suppressed or lost; preferably, bacterial strains in which the expression of the cytochrome bd-type oxidase complex is reduced or lost compared to the parent bacteria by introducing a mutation, and the function of the protein as a cytochrome bd-type oxidase complex is reduced or lost (mutation-introduced strain).

[0051] In the present invention, "expression" of a protein means that a translation product is produced from a gene encoding the protein and localized to its site of action in a state where it can exert its function. Reduction or loss of expression of a cytochrome bd type oxidase complex by introducing a mutation means a state in which the amount of cytochrome bd type oxidase complex protein present in the transformant is reduced or lost as a result of changes at the gene level, transcription level, post-transcriptional regulation level, translation level, or post-translational modification level, preferably a state in which the amount is significantly reduced or lost compared to the amount in the parent bacterium.

[0052] "The expression of cytochrome bd type oxidase complex is reduced compared with the parent bacterium" means that the expression level of cytochrome bd type oxidase complex present in the transformant is reduced compared with the parent bacterium, and more specifically, it means that the expression level of the protein is reduced to generally 50% or less, preferably 20% or less, and more preferably 10% or less compared with the parent bacterium, and thus, its activity is also reduced. Most preferably, the expression level of cytochrome bd type oxidase complex is 0%, that is, the expression of cytochrome bd type oxidase complex is lost.

[0053] In addition, the comparison of the expression level of the cytochrome bd-type oxidase complex is carried out by measuring the expression level of the polypeptide using a known immunological method such as Western blotting or immunohistostaining.

[0054] The modified coryneform bacteria in which the expression of the cytochrome bd type oxidase complex is reduced or lost can be obtained by inhibiting the function of the gene encoding the cytochrome bd type oxidase complex on the chromosomal DNA of the host bacteria. The "inhibition of function" can be either complete inhibition (hindering) or incomplete inhibition of function.

[0055] The gene encoding the cytochrome bd type oxidase complex refers to a DNA consisting of a transcription region including an ORF and a transcriptional regulatory region such as a promoter of the gene. In the present invention, the gene encoding the cytochrome bd type oxidase complex preferably includes the following polynucleotides (a) or (b).

[0056] (a) a polynucleotide encoding:

[0057] A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, and

[0058] A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3

[0059] The complex formed.

[0060] (b) a polynucleotide encoding:

[0061] A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and functioning as a cytochrome bd-type oxidase subunit I, and

[0062] A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and functioning as a cytochrome bd-type oxidase subunit II

[0063] The complex formed.

[0064] Among them, in (a), the polynucleotide composed of the nucleotide sequence shown in sequence number 1 refers to the gene cg1301 (cydA) encoding the cytochrome bd-type oxidase subunit I from Corynebacterium glutamicum, and the polynucleotide composed of the nucleotide sequence shown in sequence number 3 refers to the gene cg1300 (cydB) encoding the cytochrome bd-type oxidase subunit II from Corynebacterium glutamicum.

[0065] As an example of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3, there can be cited: a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3. The method for introducing a mutation such as deletion, substitution, addition, or insertion of nucleotides into a nucleotide sequence is as described above. The above-mentioned polynucleotide can be in the form of a single strand or a double strand, or can be DNA or RNA. The DNA can be an artificial DNA such as cDNA or chemically synthesized DNA.

[0066] Regarding inhibiting the function of the gene encoding the cytochrome bd oxidase complex, it can be done by the following operations: introducing a deletion or inactivation mutation (deletion or inactivation of the gene encoding the cytochrome bd oxidase complex) into the coding region, non-coding region, transcription or translation start region of the gene encoding the cytochrome bd oxidase complex; or, inhibiting transcription or translation by introducing a polynucleotide having the activity of decomposing the transcription product of the gene encoding the cytochrome bd oxidase complex or a polynucleotide that inhibits the translation of the transcription product into protein.

[0067] As one embodiment, the deletion or inactivation of the gene encoding the cytochrome bd oxidase complex can be achieved by: removing part or all of the nucleotide sequence of the gene encoding the cytochrome bd oxidase complex from the genome or replacing it with another nucleotide sequence; inserting another polynucleotide fragment into the sequence of the gene encoding the cytochrome bd oxidase complex; imparting a mutation to the transcription or translation start region of the gene encoding the cytochrome bd oxidase complex; etc. Preferably, a part or all of the nucleotide sequence of the gene encoding the cytochrome bd oxidase complex is deleted or inactivated. As a more specific example, there can be cited: a method of specifically deleting or inactivating the gene encoding the cytochrome bd oxidase complex on the genome of the cell; and a method of imparting a random deletion or inactivation mutation to the gene in the bacterial cell, and then evaluating the expression level or activity of the cytochrome bd oxidase complex, or gene analysis, to select cells with the desired mutation.

[0068] Regarding the specific deletion or inactivation of the gene encoding the cytochrome bd type oxidase complex, for example, a method by homologous recombination can be cited. That is, a DNA fragment of the gene encoding the cytochrome bd type oxidase complex into which an inactivation mutation is introduced by substitution or insertion of a polynucleotide, or a DNA fragment containing the outer region of the gene encoding the cytochrome bd type oxidase complex but not containing the gene encoding the cytochrome bd type oxidase complex is constructed, and it is integrated into the parent bacterium, so that the region containing the gene encoding the cytochrome bd type oxidase complex of the genome of the parent bacterium undergoes homologous recombination, thereby making it possible to delete or inactivate the gene encoding the cytochrome bd type oxidase complex on the genome.

[0069] Alternatively, by integrating a recombinant vector (plasmid, etc.) having a DNA fragment containing a portion of a gene encoding a cytochrome bd type oxidase complex into a parent bacterium, a portion of the gene encoding a cytochrome bd type oxidase complex in the genome of the parent bacterium is separated by homologous recombination, thereby inactivating the gene encoding a cytochrome bd type oxidase complex.

[0070] As methods for randomly deleting or inactivating genes in bacteria, there can be listed: a method of introducing a DNA fragment of a gene randomly cloned and introduced with an inactivating mutation into a cell so that it undergoes homologous recombination with a gene on the genome of the cell; a method of inducing mutations by irradiating cells with ultraviolet rays, gamma rays, etc. Inactivating mutations of genes refer to mutations that cause the original function of the target gene to be lost through silent mutations, missense mutations, nonsense mutations, frameshift mutations, etc. For example, a gene introduced with an inactivating mutation does not express a protein, or expresses a protein whose original activity is impaired.

[0071] As a method for preparing a DNA fragment containing a gene encoding a cytochrome bd type oxidase complex into which an inactivating mutation is introduced, site-directed mutagenesis can be cited. Site-directed mutagenesis can be performed by using a mutation primer containing the nucleotide mutation to be introduced. For example, by using two sets of primers containing the nucleotide mutation to be introduced and using the gene encoding the cytochrome bd type oxidase complex as a template for PCR, DNA fragments are prepared that amplify the upstream and downstream sides of the region containing the gene encoding the cytochrome bd type oxidase complex, respectively, and then they are connected into one body by SOE-PCR (splicing by overlap extension PCR) (Gene, 1989, 77 (1): p61-68), thereby constructing a DNA fragment containing the desired mutation. Alternatively, site-directed mutagenesis can be introduced using the inverse PCR method or the annealing method (Muramatsu et al., ed., "New Gene Engineering Handbook, Revised Fourth Edition", Yangtu Co., Ltd., p82-88), or commercially available site-directed mutagenesis kits such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit.

[0072] Primers for mutation can be prepared by known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids Research, 1989, 17: 7059-7071). The gene encoding the cytochrome bd oxidase complex as a template can be prepared from host bacteria by conventional methods or chemically synthesized.

[0073] The introduction of DNA fragments or vectors into host bacteria can be carried out by applying known techniques such as calcium phosphate method, electroporation method, liposome transfection method, gene gun method, PEG method, etc. For example, as methods applicable to coryneform bacteria, there can be mentioned: 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), etc.

[0074] In addition, examples of polynucleotides having an activity of decomposing a transcription product of a gene encoding a cytochrome bd type oxidase complex, or a polynucleotide that inhibits translation of the transcription product into a protein include a nucleotide sequence that is complementary or substantially complementary to a nucleotide sequence of mRNA of a gene encoding a cytochrome bd type oxidase complex, or a polynucleotide comprising a portion thereof. Specifically, examples include antisense RNA corresponding to mRNA of a gene encoding a cytochrome bd type oxidase complex, siRNA corresponding to mRNA of a gene encoding a cytochrome bd type oxidase complex, and ribozymes corresponding to mRNA of a gene encoding a cytochrome bd type oxidase complex.

[0075] Bacteria in which the function of the gene encoding the cytochrome bd type oxidase complex is suppressed can be selected by confirming the genome sequence thereof. Alternatively, bacteria in which the function of the gene encoding the cytochrome bd type oxidase complex is suppressed can be selected by using the expression level or activity of the cytochrome bd type oxidase complex as an indicator.

[0076] In the present invention, the host bacteria are coryneform bacteria. Coryneform bacteria include a group of microorganisms defined as coryneform bacteria in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), specifically, coryneform bacteria, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, Micrococcus, etc.

[0077] Examples of the Corynebacterium bacteria include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium crenatum, Corynebacterium crudilactis, and Corynebacterium callunae.

[0078] Examples of the bacteria belonging to the genus Brevibacterium include Brevibacterium ammoniagenes and the like.

[0079] Examples of the bacteria belonging to the genus Arthrobacter include Arthrobacter globiformis and the like.

[0080] Examples of the bacteria belonging to the genus Mycobacterium include Mycobacterium bovis, and examples of the bacteria belonging to the genus Micrococcus include Micrococcus freudenreichii, Micrococcus leuteus, Micrococcus ureae, and Micrococcus roseus.

[0081] Among the coryneform bacteria, bacteria of the genus Corynebacterium are preferred, and Corynebacterium glutamicum is more preferred.

[0082] The above-mentioned microbial cells may be wild strains, but may also be mutant strains or artificial gene recombinants thereof.

[0083] In the present invention, the coryneform bacteria used as the host can be suitable for the production of aromatic compounds or their salts. From the viewpoint of the production efficiency of aromatic compounds or their salts, especially from the viewpoint of the production efficiency of aromatic compounds or their salts derived from 3-dehydroshikimic acid or chorismic acid such as protocatechuic acid, gallic acid, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, catechol, p-hydroxybenzoic acid, p-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, it is preferred to use coryneform bacteria with improved 3-dehydroshikimic acid production activity and further chorismic acid production activity. Specifically, from the viewpoint of improving the production efficiency of compounds derived from 3-dehydroshikimic acid and chorismic acid, it is preferred to use coryneform bacteria with enhanced expression of one or more polypeptides selected from the following (C), (D), (E) and (F) as the host:

[0084] (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity;

[0085] (D) a polypeptide having 3-dehydroshikimate dehydratase activity;

[0086] (E) a polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate;

[0087] (F) A polypeptide having p-aminobenzoate hydroxylase activity.

[0088] Therefore, the modified coryneform bacterium of the present invention is preferably a modified coryneform bacterium in which the function of the cytochrome bd type oxidase complex is suppressed or deleted, wherein the expression of at least one or more polypeptides selected from the following (C), (D), (E) and (F) is enhanced:

[0089] (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity;

[0090] (D) a polypeptide having 3-dehydroshikimate dehydratase activity;

[0091] (E) a polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate;

[0092] (F) A polypeptide having p-aminobenzoate hydroxylase activity.

[0093] Among them, the polypeptide having 3,4-dihydroxybenzoate hydroxylase activity of (C) is a polypeptide that catalyzes the reaction of producing gallic acid from protocatechuic acid, and examples thereof include: (C1) a polypeptide consisting of the amino acid sequence shown in sequence number 5; or, (C2) a polypeptide consisting of an amino acid sequence that has at least 90% identity with the amino acid sequence shown in sequence number 5 and has 3,4-dihydroxybenzoate hydroxylase activity.

[0094] Among them, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 is a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity as a mutant of p-hydroxybenzoate hydroxylase (pobA) from Corynebacterium glutamicum ( Figure 1 in the literature, denoted as “pobA*”).

[0095] In addition, as a polypeptide (C2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 5 and having 3,4-dihydroxybenzoate hydroxylase activity, for example, a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity derived from a bacterium of the genus Corynebacterium can be listed, specifically, 4-hydroxybenzoate-3-monooxygenase from Corynebacterium glutamicum, 4-hydroxybenzoate-3-monooxygenase from Corynebacterium suranareeae, 4-hydroxybenzoate-3-monooxygenase from Corynebacterium crudilactis, etc.

[0096] The polypeptide having 3-dehydroshikimate dehydratase activity of (D) is a polypeptide that catalyzes the reaction of generating protocatechuic acid from 3-dehydroshikimate, and examples thereof include: (D1) a polypeptide consisting of the amino acid sequence shown in sequence number 6; or, (D2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 6 and having 3-dehydroshikimate dehydratase activity.

[0097] Among them, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 is 3-dehydroshikimate dehydratase from Corynebacterium glutamicum, and is known as "qsuB".

[0098] In addition, as a polypeptide (D2) composed of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 6 and having 3-dehydroshikimate dehydratase activity, for example, 3-dehydroshikimate dehydratase from bacteria of the genus Corynebacterium can be listed, specifically, 3-dehydroshikimate dehydratase from Corynebacterium glutamicum, 3-dehydroshikimate dehydratase from Corynebacterium suranum, etc.

[0099] As the polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate (E), (EI) 4-amino-4-deoxychorismate synthase and (EII) 4-amino-4-deoxychorismate lyase can be cited. Therefore, regarding "enhanced expression of the polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate", the mode of enhancing the expression of any one or more of these polypeptides can be cited.

[0100] Para-aminobenzoic acid is produced from chorismate via 4-amino-4-deoxychorismate. Para-aminobenzoate synthetase component II (PabA) and para-aminobenzoate synthetase component I (PabB) are involved in the conversion of chorismate to 4-amino-4-deoxychorismate, and 4-amino-4-deoxychorismate is converted into para-aminobenzoic acid by 4-amino-4-deoxychorismate lyase (PabC).

[0101] The above-mentioned (EI) 4-amino-4-deoxychorismate synthase is a polypeptide that catalyzes the reaction of generating 4-amino-4-deoxychorismate from chorismate, and examples thereof include: (EI-1) a polypeptide consisting of the amino acid sequence shown in sequence number 7; or, (EI-2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 7 and having 4-amino-4-deoxychorismate synthase activity.

[0102] The above-mentioned (EII) 4-amino-4-deoxychorismate lyase is a polypeptide that catalyzes the reaction of producing para-aminobenzoic acid from 4-amino-4-deoxychorismate, and examples thereof include: (EII-1) a polypeptide consisting of the amino acid sequence shown in sequence number 8; or, (EII-2) a polypeptide consisting of an amino acid sequence that has at least 90% identity with the amino acid sequence shown in sequence number 8 and has 4-amino-4-deoxychorismate lyase activity.

[0103] Among them, the polypeptide composed of the amino acid sequence shown in sequence number 7 is the 4-amino-4-deoxychorismate synthase from Corynebacterium glutamicum, known as "pabAB"; the polypeptide composed of the amino acid sequence shown in sequence number 8 is the 4-amino-4-deoxychorismate lyase from Corynebacterium glutamicum, known as "pabC".

[0104] The polypeptide having p-aminobenzoate hydroxylase activity of (F) is a polypeptide that catalyzes the reaction of producing 4-amino-3-hydroxybenzoic acid from p-aminobenzoic acid, and examples thereof include: (F1) a polypeptide consisting of the amino acid sequence shown in sequence number 9; or, (F2) a polypeptide consisting of an amino acid sequence that has at least 90% identity with the amino acid sequence shown in sequence number 9 and having p-aminobenzoate hydroxylase activity.

[0105] Among them, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 is a polypeptide having p-aminobenzoate hydroxylase activity as a mutant of p-hydroxybenzoate hydroxylase from Caulobacter vibrioides (in Figure 1 in Chinese, written as “phbh*”).

[0106] In addition, as a polypeptide (F2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 9 and having p-aminobenzoate hydroxylase activity, for example, p-hydroxybenzoate hydroxylase from bacteria of the genus Caulobacter can be listed, specifically, p-hydroxybenzoate hydroxylase from Caulobacter rhizosphaerae, p-hydroxybenzoate hydroxylase from Caulobacter sp., etc.

[0107] Among them, in the production of gallic acid, it is preferred to use a host in which expression of a polypeptide having (C) 3,4-dihydroxybenzoate hydroxylase activity is enhanced, and it is more preferred to use a host in which expression of a polypeptide having (D) dehydroshikimate dehydratase activity is enhanced.

[0108] In addition, in the production of para-aminobenzoic acid, it is preferred to use a host (E) in which the expression of a polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate is enhanced; in the production of 4-amino-3-hydroxybenzoic acid, it is preferred to use a host (F) in which the expression of a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is enhanced, and it is further preferred to use a host (E) in which the expression of a polypeptide required for the biosynthesis of para-aminobenzoic acid from chorismate is enhanced.

[0109] In addition, as the coryneform bacteria used as the host in the present invention, from the viewpoint of improving the 3-dehydroshikimic acid production or chorismic acid production activity, it is preferable to use coryneform bacteria in which the expression of the polypeptides represented by (G) to (O) below is also enhanced:

[0110] (G) a polypeptide having 3-deoxy-D-arabinoheptulose-7-phosphate synthase activity;

[0111] (H) a polypeptide having 3-dehydroquinate synthetase activity;

[0112] (I) a polypeptide having 3-dehydroquinate dehydratase activity;

[0113] (J) a polypeptide having 5-shikimate dehydrogenase activity;

[0114] (K) a polypeptide having shikimate kinase activity;

[0115] (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyl transferase activity;

[0116] (M) a polypeptide having chorismate synthase activity;

[0117] (N) a polypeptide having pyruvate phosphate dikinase activity;

[0118] (O) An enzyme group involved in the pentose phosphate pathway: at least one selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase and transaldolase.

[0119] The host coryneform bacteria in which the expression of the above-mentioned polypeptide is enhanced are, specifically, coryneform bacteria into which a polynucleotide required for the expression of the polypeptide is introduced in a manner that enables expression, and the polypeptide may be foreign or originally possessed by the bacterium. For example, coryneform bacteria into which the polynucleotide is introduced in a manner that enables expression, and coryneform bacteria in which the degree of expression of the polynucleotide is enhanced can be cited. Specifically, coryneform bacteria into which a vector or DNA fragment containing the polynucleotide and a control region operably linked thereto is introduced, and coryneform bacteria in which the control region of the polynucleotide is replaced with a strong control region can be cited.

[0120] Examples of the strong control region include, but are not particularly limited to, T7 promoter, lac promoter, tac promoter, trp promoter, tu promoter, gap promoter, and the like, which are well-known high expression promoters.

[0121] In addition, as a strong control region, an inducible promoter from a prokaryotic organism can be used, and examples thereof include: a vanA promoter induced by adding ferulic acid, vanillic acid or vanillin; an rhcH promoter induced by adding resorcinol or 2,4-dihydroxybenzoic acid; a pcaI promoter induced by adding 4-hydroxybenzoic acid; a promoter of the nagI (cg3351) gene induced by adding 3-hydroxybenzoic acid; a promoter of the benA (cg2637) gene (hereinafter referred to as Pben) induced by adding benzoic acid; or a promoter of the cg2118 gene or a promoter of the ptsS (cg2925) gene induced by adding either fructose or sucrose, etc., but are not particularly limited to these.

[0122] Methods for replacing the control region of the above-mentioned polynucleotide present in the genome of the host cell with a strong control region include: introducing a DNA fragment containing a polynucleotide sequence of a strong control region and a selection marker into the host cell, and selecting cells transformed by homologous recombination or non-homologous recombination, etc.

[0123] By culturing the modified coryneform bacteria thus prepared, evaluating the productivity of the aromatic compound or its salt, and selecting appropriate transformants, a useful aromatic compound or its salt-producing bacterium can be obtained. The method for measuring the product can be carried out according to the method described in the Examples below.

[0124] The method for producing an aromatic compound or a salt thereof of the present invention is carried out by culturing the modified coryneform bacteria described above, preferably in the presence of a sugar, and recovering the target aromatic compound or a salt thereof.

[0125] As the sugar, glucose is preferred, but in addition to monosaccharides such as fructose, mannose, arabinose, xylose, and galactose, sugars that can generate glucose by metabolism can also be used. Such sugars include oligosaccharides or polysaccharides having glucose units, and examples thereof include disaccharides such as cellobiose, sucrose, lactose, maltose, trehalose, cellobiose, and xylobiose; polysaccharides such as dextrin or soluble starch, and the like.

[0126] In addition, as a raw material containing these raw material compounds, molasses can also be used. In addition, inedible agricultural wastes such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw, etc.), bagasse, corn straw, etc.; energy crops such as switchgrass, elephant grass, and miscanthus; saccharified liquid containing various sugars such as glucose obtained by saccharifying sawdust, waste paper, etc. using saccharifying enzymes, etc. can also be used.

[0127] The medium for culturing the transformant contains a carbon source, a nitrogen source, inorganic salts, etc., and any natural medium or synthetic medium can be used as long as it can effectively culture the modified coryneform bacteria of the present invention.

[0128] As the carbon source, the above-mentioned sugars or molasses or saccharified liquid containing the above-mentioned sugars can be used. In addition to the above-mentioned sugars, sugar alcohols such as mannitol, sorbitol, xylitol, and glycerol; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; alcohols such as ethanol and propanol; hydrocarbons such as normal paraffins, etc. can be used. As for the carbon source, one kind can be used alone, or two or more kinds can be mixed and used.

[0129] The concentration of the sugar as a raw material compound in the culture solution is preferably 1 to 20 w / v%, more preferably 2 to 10 w / v%, and further preferably 2 to 5 w / v%.

[0130] As nitrogen sources, for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract, alkylamines such as methylamine, nitrogen-containing organic compounds such as amino acids, ammonia or its salts (inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate), urea, ammonia water, sodium nitrate, potassium nitrate, etc. can be used.

[0131] Examples of the inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate.

[0132] Furthermore, vitamins, defoaming agents, etc. may be added as necessary. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and niacin.

[0133] Examples of culture 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)], CGXII medium [Japanese Patent No. 6322576], etc. In these culture media, the sugar concentration can be set to the above-mentioned range for use.

[0134] Furthermore, prior to the reaction or culture involving sugars, the transformant is preferably cultured in the same medium under aerobic conditions at a temperature of about 25 to 38° C. for about 12 to 48 hours to proliferate.

[0135] The culture temperature or reaction temperature is preferably 15 to 45°C, more preferably 25 to 37°C.

[0136] In addition, the culture or reaction time is 24 hours to 168 hours, preferably 24 hours to 96 hours, and more preferably 24 hours to 72 hours, and the culture or reaction can be carried out while stirring or shaking as needed. In addition, antibiotics such as ampicillin and / or kanamycin can be added to the culture medium as needed during the culture.

[0137] The culture may be carried out in any of the following ways: batch culture, fed-batch culture or continuous culture, among which the batch culture is preferred.

[0138] The culture or reaction may be carried out under aerobic conditions or under reducing conditions, but is preferably carried out under aerobic conditions.

[0139] When the reaction or culture is performed under aerobic conditions, it is preferably performed under conditions that suppress excessive growth of transformants from the viewpoint of the production efficiency of the aromatic compound or its salt.

[0140] There is no particular limitation on the method for recovering and purifying aromatic compounds or their salts from the culture. That is, it can be implemented by combining known ion exchange resin methods, precipitation methods, crystallization methods, recrystallization methods, concentration methods, and other methods. For example, after the bacterial cells are removed by centrifugation, ionic substances are removed by cation and anion exchange resins, and concentrated, aromatic compounds or their salts can be obtained. The aromatic compounds or their salts accumulated in the culture can be used directly without separation.

[0141] Regarding the above-mentioned embodiment, the present invention further discloses the following aspects.

[0142] <1> A method for producing an aromatic compound or a salt thereof, comprising the step of culturing a modified coryneform bacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted.

[0143] <2> The method according to <1>, wherein the modified coryneform bacterium is a bacterial strain in which expression of a cytochrome bd-type oxidase complex is reduced or lost compared to a parent bacterium.

[0144] <3> The method according to <1> or <2>, wherein the cytochrome bd-type oxidase complex is a polypeptide complex having cytochrome bd-type oxidase activity as shown in (A) or (B) below,

[0145] (A) A complex comprising:

[0146] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and

[0147] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4

[0148] constituted by

[0149] (B) a complex consisting of:

[0150] 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 functioning as a cytochrome bd-type oxidase subunit I, and

[0151] A polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as a cytochrome bd-type oxidase subunit II

[0152] constituted.

[0153] <4> The method according to <2> or <3>, wherein the modified coryneform bacterium is a bacterial strain in which a part or all of the nucleotide sequence of the polynucleotide (a) or (b) described below is deleted or inactivated,

[0154] (a) a polynucleotide encoding:

[0155] A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, and

[0156] A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3

[0157] The complex formed

[0158] (b) a polynucleotide encoding:

[0159] A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and functioning as a cytochrome bd-type oxidase subunit I, and

[0160] A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and functioning as a cytochrome bd-type oxidase subunit II

[0161] The complex formed.

[0162] <5> The method according to any one of <1> to <4>, wherein a coryneform bacterium in which expression of at least one polypeptide selected from the following (C), (D), (E), and (F) is enhanced is used as a host:

[0163] (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity;

[0164] (D) a polypeptide having 3-dehydroshikimate dehydratase activity;

[0165] (E) a polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate;

[0166] (F) A polypeptide having p-aminobenzoate hydroxylase activity.

[0167] <6> The method as described in <5>, wherein (C) the polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is (C1) a polypeptide consisting of the amino acid sequence shown in Sequence Number 5, or (C2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence Number 5 and having 3,4-dihydroxybenzoate hydroxylase activity.

[0168] <7> The method as described in <5>, wherein (D) the polypeptide having 3-dehydroshikimate dehydratase activity is (D1) a polypeptide consisting of the amino acid sequence shown in Sequence Number 6, or (D2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence Number 6 and having 3-dehydroshikimate dehydratase activity.

[0169] <8> The method as described in <5>, wherein (E) the polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate is one or more selected from (EI) 4-amino-4-deoxychorismate synthase and (EII) 4-amino-4-deoxychorismate lyase.

[0170] <9> The method as described in <8>, wherein (EI) 4-amino-4-deoxychorismate synthetase is (EI-1) a polypeptide consisting of the amino acid sequence shown in sequence number 7, or (EI-2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 7 and having 4-amino-4-deoxychorismate synthetase activity, and (EII) 4-amino-4-deoxychorismate lyase is (EII-1) a polypeptide consisting of the amino acid sequence shown in sequence number 8, or (EII-2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 8 and having 4-amino-4-deoxychorismate lyase activity.

[0171] <10> The method as described in <5>, wherein (F) the polypeptide having p-aminobenzoate hydroxylase activity is (F1) a polypeptide consisting of the amino acid sequence shown in Sequence Number 9, or (F2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence Number 9 and having p-aminobenzoate hydroxylase activity.

[0172] <11> The method according to any one of <5> to <10>, wherein a coryneform bacterium in which expression of one or more polypeptides selected from the following (G) to (O) is enhanced is used as a host:

[0173] (G) a polypeptide having 3-deoxy-D-arabinoheptulose-7-phosphate synthase activity;

[0174] (H) a polypeptide having 3-dehydroquinate synthetase activity;

[0175] (I) a polypeptide having 3-dehydroquinate dehydratase activity;

[0176] (J) a polypeptide having 5-shikimate dehydrogenase activity;

[0177] (K) a polypeptide having shikimate kinase activity;

[0178] (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyl transferase activity;

[0179] (M) a polypeptide having chorismate synthase activity;

[0180] (N) a polypeptide having pyruvate phosphate dikinase activity;

[0181] (O) An enzyme group involved in the pentose phosphate pathway: at least one selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase and transaldolase.

[0182] <12> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is gallic acid, protocatechuic acid, catechol, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, p-hydroxybenzoic acid, p-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

[0183] <13> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is gallic acid, protocatechuic acid, p-hydroxybenzoic acid, p-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

[0184] <14> The method according to any one of <1> to <11>, wherein the aromatic compound or a salt thereof is gallic acid, p-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

[0185] <15> The method according to any one of <1> to <14>, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.

[0186] <16> The method according to <15>, wherein the Corynebacterium bacterium is Corynebacterium glutamicum, Corynebacterium efferogenes, Corynebacterium ammoniagenes, Corynebacterium halodurans, Corynebacterium alkanolyticum, Corynebacterium sphaeroides, Corynebacterium serratum, or Corynebacterium kluyveri.

[0187] <17> The method according to <16>, wherein the Corynebacterium bacterium is Corynebacterium glutamicum.

[0188] <18> A coryneform bacterium, which is a modified coryneform bacterium in which the function of a cytochrome bd-type oxidase complex is suppressed or deleted, wherein:

[0189] The expression of at least one polypeptide selected from the following (C), (D), (E) and (F) is enhanced:

[0190] (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity;

[0191] (D) a polypeptide having 3-dehydroshikimate dehydratase activity;

[0192] (E) a polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate;

[0193] (F) A polypeptide having p-aminobenzoate hydroxylase activity.

[0194] <19> The coryneform bacterium according to <18>, wherein the cytochrome bd-type oxidase complex is a polypeptide complex having cytochrome bd-type oxidase activity as shown in the following (A) or (B),

[0195] (A) A complex comprising:

[0196] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and

[0197] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4

[0198] constituted by

[0199] (B) a complex consisting of:

[0200] 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 functioning as a cytochrome bd-type oxidase subunit I, and

[0201] A polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as a cytochrome bd-type oxidase subunit II

[0202] constituted.

[0203] <20> The coryneform bacterium according to <18> or <19>, wherein a part or all of the nucleotide sequence of the polynucleotide of (a) or (b) below is deleted or inactivated,

[0204] (a) a polynucleotide encoding:

[0205] A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, and

[0206] A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3

[0207] The complex formed

[0208] (b) a polynucleotide encoding:

[0209] A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and functioning as a cytochrome bd-type oxidase subunit I, and

[0210] A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and functioning as a cytochrome bd-type oxidase subunit II

[0211] The complex formed.

[0212] <21> The coryneform bacterium according to any one of <18> to <20>, wherein (C) the polypeptide having 3,4-dihydroxybenzoate hydroxylase activity is (C1) a polypeptide consisting of the amino acid sequence shown in Sequence Number 5, or (C2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence Number 5 and having 3,4-dihydroxybenzoate hydroxylase activity.

[0213] <22> The rod-shaped bacterium as described in any one of <18> to <20>, wherein (D) the polypeptide having 3-dehydroshikimate dehydratase activity is (D1) a polypeptide consisting of the amino acid sequence shown in Sequence Number 6, or (D2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence Number 6 and having 3-dehydroshikimate dehydratase activity.

[0214] <23> The coryneform bacterium according to any one of <18> to <20>, wherein the polypeptide (E) required for the biosynthesis of p-aminobenzoic acid from chorismate is one or more selected from (EI) 4-amino-4-deoxychorismate synthase and (EII) 4-amino-4-deoxychorismate lyase.

[0215] <24> The rod-shaped bacterium as described in <23>, wherein: (EI) 4-amino-4-deoxychorismate synthase is (EI-1) a polypeptide consisting of the amino acid sequence shown in sequence number 7, or (EI-2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 7 and having 4-amino-4-deoxychorismate synthase activity; (EII) 4-amino-4-deoxychorismate lyase is (EII-1) a polypeptide consisting of the amino acid sequence shown in sequence number 8, or (EII-2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in sequence number 8 and having 4-amino-4-deoxychorismate lyase activity.

[0216] <25> The coryneform bacterium as described in any one of <18> to <20>, wherein (F) the polypeptide having p-aminobenzoate hydroxylase activity is (F1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, or (F2) a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9 and having p-aminobenzoate hydroxylase activity.

[0217] <26> The coryneform bacterium according to any one of <18> to <25>, wherein expression of one or more polypeptides selected from the following (G) to (O) is also enhanced:

[0218] (G) a polypeptide having 3-deoxy-D-arabinoheptulose-7-phosphate synthase activity;

[0219] (H) a polypeptide having 3-dehydroquinate synthetase activity;

[0220] (I) a polypeptide having 3-dehydroquinate dehydratase activity;

[0221] (J) a polypeptide having 5-shikimate dehydrogenase activity;

[0222] (K) a polypeptide having shikimate kinase activity;

[0223] (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyl transferase activity;

[0224] (M) a polypeptide having chorismate synthase activity;

[0225] (N) a polypeptide having pyruvate phosphate dikinase activity;

[0226] (O) An enzyme group involved in the pentose phosphate pathway: at least one selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase and transaldolase.

[0227] <27> The coryneform bacterium according to any one of <18> to <26>, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.

[0228] <28> The coryneform bacterium according to <27>, wherein the bacterium of the genus Corynebacterium is Corynebacterium glutamicum, Corynebacterium efferogenes, Corynebacterium ammoniagenes, Corynebacterium halodurans, Corynebacterium alkanolyticum, Corynebacterium sphaeroides, Corynebacterium serratum, or Corynebacterium kluyveri.

[0229] <29> The coryneform bacterium according to <28>, wherein the bacterium of the genus Corynebacterium is Corynebacterium glutamicum.

[0230] Example

[0231] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to the following examples.

[0232] <Preparation of strains with enhanced chorismic acid production activity>

[0233] 1) Construction of a plasmid with the tu promoter introduced into the cg1829 (aroC) promoter region

[0234] The 5' upstream region (sequence number 10) of the cg1829 gene region was amplified by PCR using two DNA primers (sequence number 11 and sequence number 12), and the 5' region (sequence number 13) in the cg1829 gene ORF was amplified by PCR using two DNA primers (sequence number 14 and sequence number 15), thereby obtaining a DNA fragment. In addition, a DNA fragment (sequence number 16) containing the tu promoter was amplified by PCR using two DNA primers (sequence number 17 and sequence number 18) using the genome of the ATCC13032 strain as a template, thereby obtaining a DNA fragment. In addition, pHKPsacB1 (see Japanese Patent Application No. 2014-523757) was used as a template, and amplified by PCR using two DNA primers (sequence number 19 and sequence number 20), and the obtained PCR product was treated with DpnI (Takara Bio). The four PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and ligated using In-FusionHD Cloning Kit (Takara Bio) to prepare plasmid pHKPsacB_Ptu-aroC.

[0235] 2) Preparation of strains with tu promoter introduced into the cg1829 (aroC) promoter region

[0236] The plasmid pHKPsacB_Ptu-aroC was introduced into the KC315 strain (Special Application No. 2021-201877) by electroporation, and the strain KC367sr was obtained by selection for kanamycin resistance. The KC367sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence numbers 11 and 21, and the expected results were obtained, thereby confirming that the KC367sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_Ptu-aroC was introduced.

[0237] The KC367sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC367 strain. By using the PCR method (Sapphire Amp (TakaraBio)) using primers of sequence number 21 and sequence number 22, it was confirmed that the KC367 strain was a double crossover homologous recombinant with the tu promoter introduced into the cg1829 (aroC) promoter region as expected.

[0238] 3) Construction of a plasmid with the tu promoter introduced into the cg1774 (tkt) promoter region

[0239] The 5' upstream region (sequence number 23) of the cg1774 gene region was amplified by PCR using two DNA primers (sequence number 24 and sequence number 25), and the 5' region (base number 26) in the cg1774 gene ORF was amplified by PCR using two DNA primers (sequence number 27 and sequence number 28), thereby obtaining a DNA fragment. In addition, a DNA fragment (sequence number 16) containing the tu promoter was amplified by PCR using two DNA primers (sequence number 17 and sequence number 18) using the genome of the ATCC13032 strain as a template, thereby obtaining a DNA fragment. In addition, pHKPsacB1 was used as a template, and PCR was amplified by using two DNA primers (sequence number 19 and sequence number 20), and the resulting PCR product was treated with DpnI (Takara Bio). The four PCR products obtained were purified using NucleoSpin Gel and PCRClean-up (Takara Bio), and ligated using In-Fusion HD Cloning Kit (Takara Bio) to construct plasmid pHKPsacB_Ptu-tkt.

[0240] 4) Construction of strains with tu promoter introduced into the cg1774 (tkt) promoter region

[0241] The plasmid pHKPsacB_Ptu-tkt was introduced into the KC367 strain by electroporation, and the strain KC376sr was obtained by selection for kanamycin resistance. The KC376sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 24 and sequence number 29, and the expected results were obtained, thereby confirming that the KC376sr strain was a single crossover homologous recombinant in which the plasmid pHKPsacB_Ptu-tkt was introduced into the promoter region of cg1774.

[0242] The KC376sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC376 strain. By using the PCR method (Sapphire Amp (TakaraBio)) using primers of sequence number 29 and sequence number 30, it was confirmed that the KC376 strain was a double crossover homologous recombinant with the tu promoter introduced into the cg1774 (tkt) promoter region as expected.

[0243] 5) Construction of a plasmid with the tu promoter introduced into the cg0644 (ppsA) promoter region

[0244] The 5' upstream region (sequence number 31) of the cg0644 gene region was amplified by PCR using two DNA primers (sequence number 32 and sequence number 33), and the 5' region (base number 34) in the cg0644 gene ORF was amplified by PCR using two DNA primers (sequence number 35 and sequence number 36), thereby obtaining a DNA fragment. In addition, a DNA fragment (sequence number 16) containing the tu promoter was amplified by PCR using two DNA primers (sequence number 17 and sequence number 18) using the genome of the ATCC13032 strain as a template, thereby obtaining a DNA fragment. In addition, pHKPsacB1 was used as a template, and PCR was amplified using two DNA primers (sequence number 19 and 20), and the resulting PCR product was treated with DpnI (Takara Bio). The four PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and ligated using In-Fusion HD Cloning Kit (Takara Bio) to prepare plasmid pHKPsacB_Ptu-ppsA.

[0245] 6) Construction of a strain with tu promoter introduced into the cg0644 (ppsA) promoter region

[0246] The plasmid pHKPsacB_Ptu-ppsA was introduced into the KC376 strain by electroporation, and the strain KC408sr was obtained by selection for kanamycin resistance. The KC408sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 32 and sequence number 37, and the expected results were obtained, thereby confirming that the KC408sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_Ptu-tkt was introduced.

[0247] The KC408sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC408 strain. By using the PCR method (Sapphire Amp (TakaraBio)) using primers of sequence number 37 and sequence number 38, it was confirmed that the KC408 strain was a double crossover homologous recombinant with the tu promoter introduced into the cg0644 (tkt) promoter region as expected.

[0248] <Evaluation of productivity of 4-aminobenzoic acid (4-ABA)>

[0249] (1) Preparation of 4-ABA producing strain

[0250] 1) Construction of a plasmid for introducing the promoter of the cg2875 gene into the cg1134 (pabAB) promoter region

[0251] The 5' upstream region (sequence number 39) of the cg1134 gene region was amplified by PCR using two DNA primers (sequence numbers 40 and 41), and the 5' region (sequence number 42) in the cg1134 gene ORF was amplified by PCR using two DNA primers (sequence numbers 43 and 44), thereby obtaining a DNA fragment. In addition, a DNA fragment (sequence number 45) containing the promoter of the cg2875 gene was amplified by PCR using two DNA primers (sequence numbers 46 and 47) using the genome of the ATCC13032 strain as a template, thereby obtaining a DNA fragment. In addition, pHKPsacB1 was used as a template, and PCR was amplified by using two DNA primers (sequence numbers 19 and 20), and the resulting PCR product was treated with DpnI (TakaraBio). The four PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) and ligated using the In-Fusion HD Cloning Kit (Takara Bio) to create plasmid pHKPsacB_Pcg2875-pabAB.

[0252] 2) Preparation of a strain in which the promoter of the cg2875 gene was introduced into the cg1134 (pabAB) promoter region

[0253] The plasmid pHKPsacB_Pcg2875-pabAB was introduced into the KC408 strain by electroporation, and the strain KC412sr was obtained by selection for kanamycin resistance. The KC412sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 40 and sequence number 48, and the expected results were obtained, thereby confirming that the KC412sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_Pcg2875-pabAB was introduced.

[0254] The KC412sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC412 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 48 and sequence number 49, it was confirmed that the KC412 strain was a double crossover homologous recombinant in which the promoter of the cg2875 gene was introduced into the cg1134 (pabAB) promoter region as expected.

[0255] (2) Preparation of 4-ABA-producing cydAB-disrupted strain

[0256] 1) Construction of a plasmid for deleting the cg1300-1301 (cydAB) gene region

[0257] The 5' upstream region (sequence number 50) of the cg1300 gene region was amplified by PCR using two DNA primers (sequence number 51 and sequence number 52), and the 3' downstream region (base number 53) of the cg1301 gene region was amplified by PCR using two DNA primers (sequence numbers 54 and 55), thereby obtaining a DNA fragment. In addition, pHKPsacB1 was used as a template and amplified by PCR using two DNA primers (sequence numbers 19 and 20), and the obtained PCR products were treated with DpnI (Takara Bio). The three PCR products obtained were purified using NucleoSpin Geland PCR Clean-up (Takara Bio), and connected using the In-Fusion HD Cloning Kit (TakaraBio), thereby producing the plasmid pHKPsacB_ΔcydAB.

[0258] 2) Preparation of cg1300-1301 (cydAB) gene region defect strain

[0259] The plasmid pHKPsacB_ΔcydAB was introduced into the KC412 strain by electroporation, and the strain KC418sr was obtained by selection for kanamycin resistance. The KC418sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 51 and sequence number 56, and the expected results were obtained, thereby confirming that the KC418sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_ΔcydAB was introduced.

[0260] The KC418sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC418 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 56 and sequence number 57, it was confirmed that the KC418 strain was a double crossover homologous recombinant with a defect in the cg1300-1301 (cydAB) gene region, as expected.

[0261] (3) Preparation of 4-ABA production ctaCF disrupted strain

[0262] 1) Construction of a plasmid for deleting the cg2408-2409 (ctaCF) gene region

[0263] The 5' upstream region (sequence number 58) of the cg2408 gene region was amplified by PCR using two DNA primers (sequence number 59 and sequence number 60), and the 3' region (base number 61) of the cg2409 gene region was amplified by PCR using two DNA primers (sequence number 62 and sequence number 63), thereby obtaining a DNA fragment. In addition, pHKPsacB1 was used as a template and amplified by PCR using two DNA primers (sequence number 19 and sequence number 20), and the obtained PCR products were treated with DpnI (Takara Bio). The three PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and connected using the In-Fusion HD Cloning Kit (Takara Bio), thereby producing the plasmid pHKPsacB_ΔctaCF.

[0264] 2) Construction of cg2408-2409 (ctaCF) gene region defect strain

[0265] The plasmid pHKPsacB_ΔctaCF was introduced into the KC412 strain by electroporation, and the KC469sr strain was obtained by selection for kanamycin resistance. The KC469sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 59 and sequence number 64, and the expected results were obtained, thereby confirming that the KC469sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_ΔctaCF was introduced.

[0266] The KC469sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC469 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 64 and sequence number 65, it was confirmed that the KC469 strain was a double crossover homologous recombinant with a defect in the cg2408-2409 (ctaCF) gene region, as expected.

[0267] (4) Cultivation of transformants

[0268] 1) Streak the transformant obtained above on an LB culture plate and culture at 30°C for 3 days. Inoculate the bacteria grown on the culture plate into 6 mL of CGTG15 medium (Table 4) and perform shaking culture at 30°C and 250 rpm for 24 hours (pre-culture). Add 60 mL of CGTG15 medium (urea removed from the composition) to the culture tank of Bio Jr.8 (Able Co., Ltd.). Inoculate 1.2 mL of the above pre-culture solution, perform shaking culture for 30 hours at 32°C, pH 6.9, 700 rpm, and aeration of 60 mL / min, and evaluate the productivity of aromatic compounds. Dilute the culture solution appropriately with dilute sulfuric acid, remove the bacteria by centrifugation, and recover the supernatant. Quantify the concentration in the supernatant. The results are shown in the table.

[0269] 2) Results

[0270] The results of the comparison showed that the 4-ABA concentration in KC418 increased, indicating that the disruption of the cydAB gene had a positive effect on production. In addition, the 4-ABA concentration in KC469 decreased, indicating that the disruption of the ctaCF gene had an adverse effect on production (Table 1-1, Table 1-2).

[0271] [Table 1-1]

[0272] Concentration (g / L) KC412 3.1 KC418 4.0

[0273] [Table 1-2]

[0274] Concentration (g / L) KC412 3.4 KC469 0.088

[0275] <Evaluation of productivity of 4-amino-3-hydroxybenzoic acid (4,3-AHBA)>

[0276] (1) Preparation of 4,3-AHBA-producing bacteria

[0277] 1) Construction of a plasmid for introducing the HFM122 mutant gene bound to the promoter of the cg2875 gene into the cg2484 gene region

[0278] Using pECsf_gapS_pabABC_tuD_HFM122 (see Japanese Patent Application No. 2019-203523) as a template, PCR was amplified using two DNA primers (SEQ ID NO. 63 and SEQ ID NO. 64), and the resulting PCR product was treated with DpnI (Takara Bio). Two fragments of the HFM122 mutant gene (SEQ ID NO. 68 and SEQ ID NO. 69) were gene synthesized (Eurofin Genomics and Genscript), and amplified by PCR using two DNA primers (SEQ ID NO. 70 and SEQ ID NO. 71, and SEQ ID NO. 72 and SEQ ID NO. 73), respectively, to obtain DNA fragments. The three PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and ligated using In-Fusion HD Cloning Kit (Takara Bio) to construct plasmids pECsf_gapS_pabABC_tuD_HFM122LA_OPT1-HFM122LA_OPT2.

[0280] The 5' upstream region of the cg2484 gene region (sequence number 74) was amplified by PCR using two kinds of DNA primers (sequence number 75 and sequence number 76), and the 3' region of the cg2484 gene region (sequence number 77) was amplified by PCR using two kinds of DNA primers (sequence number 78 and sequence number 79), thereby obtaining a DNA fragment. In addition, a DNA fragment (sequence number 45) containing the promoter of the cg2875 gene was amplified by PCR using two kinds of DNA primers (sequence number 46 and sequence number 47) using the genome of the ATCC13032 strain as a template, thereby obtaining a DNA fragment. In addition, the above-mentioned plasmid pECsf_gapS_pabABC_tuD_HFM122LA_OPT1-HFM122LA_OPT2 was amplified by PCR using two kinds of DNA primers (sequence number 69 and sequence number 80), and the obtained PCR product was treated with DpnI (Takara Bio). In addition, pHKPsacB1 was used as a template and amplified by PCR using two DNA primers (SEQ ID NO: 19 and SEQ ID NO: 20), and the obtained PCR products were treated with DpnI (Takara Bio). The five PCR products obtained were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and ligated using the In-Fusion HD Cloning Kit (Takara Bio), thereby producing the plasmid pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2.

[0281] 2) Preparation of a strain in which the HFM122 mutant gene bound to the promoter of the cg2875 gene was introduced into the cg2484 gene region

[0282] The plasmid pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2 was introduced into the KC412 strain by electroporation, and the strain KC417sr was obtained by selection for kanamycin resistance. The KC417sr strain was analyzed by PCR (Sapphire Amp (TakaraBio)) using primers of sequence number 67 and sequence number 81, and the expected results were obtained, thereby confirming that the KC417sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_Δcg2484::Pcg2875-HFM122LA_OPT1-HFM122LA_OPT2 was introduced.

[0283] The KC417sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC417 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 81 and sequence number 82, it was confirmed that the KC417 strain was a double crossover homologous recombinant in which the HFM122 mutant gene bound to the promoter of the cg2875 gene was introduced into the cg2484 gene region as expected.

[0284] (2) Preparation of 4,3-AHBA-producing cydAB-disrupted strain

[0285] The plasmid pHKPsacB_ΔcydAB was introduced into the KC417 strain by electroporation, and the KC427sr strain was obtained by selection for kanamycin resistance. The KC427sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 51 and sequence number 56, and the expected results were obtained, thereby confirming that the KC427sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_ΔcydAB was introduced.

[0286] The KC427sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC427 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 56 and sequence number 57, it was confirmed that the KC427 strain was a double crossover homologous recombinant with a defect in the cg1300-1301 (cydAB) gene region, as expected.

[0287] (3) Preparation of 4,3-AHBA-producing ctaCF-disrupted strain

[0288] The plasmid pHKPsacB_ΔctaCF was introduced into the KC417 strain by electroporation, and the strain KC470sr was obtained by selection for kanamycin resistance. The KC470sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 59 and sequence number 64, and the expected results were obtained, thereby confirming that the KC470sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_ΔctaCF was introduced.

[0289] The KC470sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC470 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 64 and sequence number 65, it was confirmed that the KC470 strain was a double crossover homologous recombinant with a defect in the cg2408-2409 (ctaCF) gene region, as expected.

[0290] (4) Cultivation of transformants

[0291] 1) Streak the transformant obtained above on an LB culture plate and culture at 30°C for 3 days. Inoculate the bacteria grown on the culture plate into 6 mL of CGTG15 medium and perform shaking culture at 30°C and 250 rpm for 24 hours (pre-culture). Add 60 mL of CGTG15 medium (urea removed from the constituent components) to the culture tank of Bio Jr.8 (Able Co., Ltd.). Inoculate 1.2 mL of the above pre-culture solution, perform shaking culture for 30 hours at 32°C, pH 6.9, 700 rpm, and an aeration volume of 60 mL / min, and evaluate the productivity of aromatic compounds. Dilute the culture solution appropriately with dilute sulfuric acid, remove the bacteria by centrifugation, and recover the supernatant. Quantify the concentration in the supernatant. The results are shown in the table.

[0292] 2) Results

[0293] The results of the comparison showed that the 4,3-AHBA concentration in KC427 increased, indicating that the disruption of the cydAB gene had a positive effect on production. In addition, the 4,3-AHBA concentration in KC470 decreased, indicating that the disruption of the ctaCF gene had an adverse effect on production.

[0294] [Table 2-1]

[0295] Concentration (g / L) KC417 2.2 KC427 2.7

[0296] [Table 2-2]

[0297] Concentration (g / L) KC417 2.2 KC470 0.45

[0298] <Evaluation of the productivity of gallic acid (GAL)>

[0299] (1) Preparation of GAL-producing cydAB-disrupted strain

[0300] The plasmid pHKPsacB_ΔcydAB was introduced into the KC148 strain (Japanese Patent Application No. 2020-94649) by electroporation, and selected for kanamycin resistance to obtain the KC201sr strain. The KC201sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 51 and sequence number 56, and the expected results were obtained, thereby confirming that the KC201sr strain is a single crossover homologous recombinant into which the plasmid pHKPsacB_ΔcydAB was introduced.

[0301] The KC201sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC201 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 56 and sequence number 57, it was confirmed that the KC201 strain was a double crossover homologous recombinant with a defect in the cg1300-1301 (cydAB) gene region, as expected.

[0302] (2) Preparation of GAL-producing ctaCF-disrupted strain

[0303] The plasmid pHKPsacB_ΔctaCF was introduced into the KC148 strain by electroporation, and the KC468sr strain was obtained by selection for kanamycin resistance. The KC468sr strain was analyzed by PCR (Sapphire Amp (Takara Bio)) using primers of sequence number 59 and sequence number 64, and the expected results were obtained, thereby confirming that the KC468sr strain was a single crossover homologous recombinant into which the plasmid pHKPsacB_ΔctaCF was introduced.

[0304] The KC468sr strain was cultured in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for 24 hours, and a portion of the culture solution was spread on LB agar medium containing 20% ​​sucrose to obtain the KC468 strain. By using the PCR method (Sapphire Amp (TakaraBio)) with primers of sequence number 64 and sequence number 65, it was confirmed that the KC468 strain was a double crossover homologous recombinant with a defect in the cg2408-2409 (ctaCF) gene region, as expected.

[0305] (3) Cultivation of transformants

[0306] 1) Streak the transformant obtained above on an LB culture plate and culture at 30°C for 3 days. Inoculate the bacteria grown on the culture plate into 6 mL of CGTG15 medium and perform shaking culture at 30°C and 250 rpm for 24 hours (pre-culture). Add sodium benzoate to CGTG15 medium (urea is removed from the constituent components) to a final concentration of 1 mM, and add 60 mL to a culture tank of Bio Jr.8 (Able Co., Ltd.). Inoculate 1.2 mL of the above pre-culture solution, perform shaking culture for 30 hours at 32°C, pH 6.9, 600 rpm, and an aeration volume of 60 mL / min, and evaluate the productivity of GAL. Dilute the culture solution appropriately with dilute sulfuric acid, remove the bacteria by centrifugation, and recover the supernatant. Quantify the concentration in the supernatant. The results are shown in the table.

[0307] 2) Results

[0308] The results of the comparison showed that the GAL concentration in KC201 strain was increased, indicating that the disruption of the cydAB gene had a positive effect on production, while the GAL concentration in KC468 strain was decreased, indicating that the disruption of the ctaCF gene had an adverse effect on production.

[0309] [Table 3-1]

[0310] Concentration (g / L) KC148 1.1 KC201 4.8

[0311] [Table 3-2]

[0312] Concentration (g / L) KC148 2.4 KC468 0.96

[0313] [Table 4]

[0314]

[0315] (4) Analysis conditions

[0316] The collected supernatant was subjected to removal of insoluble matter using an AcroPrep 96-well filter plate (0.2 μm GHP membrane, Nihon Pall Ltd.), and the reaction solution was subjected to HPLC.

[0317] Chromaster (Hitachi High-Technologies) was used as the HPLC device. L-column ODS (4.6 mm ID×150 mm, Chemical Evaluation Research Institute) was used for the analysis of gallic acid. The eluent A was set to 0.1 M potassium dihydrogen phosphate in 0.1% phosphoric acid solution, and the eluent B was set to 70% methanol. Gradient elution was performed at a flow rate of 1.0 mL / min and a column temperature of 40°C. A UV detector (detection wavelength 280 nm) was used for the detection of gallic acid.

[0318] Glucose was analyzed using ICsep ION-300 (7.8 mm×300 mm, Tokyo Chemical Industry), with 37 mM sulfuric acid solution as the eluent, and detection was performed at a flow rate of 0.5 mL / min and a column temperature of 50° C. Glucose was detected using RI.

Claims

1. A method for producing an aromatic compound or a salt thereof, wherein: include: A process for culturing a modified coryneform bacterium in which the function of a cytochrome bd type oxidase complex is inhibited or deleted.

2. The method of claim 1, wherein: The cytochrome bd-type oxidase complex is a polypeptide complex having cytochrome bd-type oxidase activity as shown in the following (A) or (B), (A) A complex comprising: A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 constituted by (B) a complex consisting of: 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 functioning as a cytochrome bd-type oxidase subunit I, and A polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as a cytochrome bd-type oxidase subunit II constituted.

3. The method according to claim 1 or 2, wherein: The modified coryneform bacterium is a bacterial strain in which a part or all of the nucleotide sequence of the polynucleotide (a) or (b) described below is deleted or inactivated. (a) a polynucleotide encoding: A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, and A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 The complex formed (b) a polynucleotide encoding: A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and functioning as a cytochrome bd-type oxidase subunit I, and A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and functioning as a cytochrome bd-type oxidase subunit II The complex formed.

4. The method according to any one of claims 1 to 3, wherein: A coryneform bacterium in which expression of at least one polypeptide selected from the following (C), (D), (E) and (F) is enhanced is used as a host: (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity; (D) a polypeptide having 3-dehydroshikimate dehydratase activity; (E) a polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate; (F) A polypeptide having p-aminobenzoate hydroxylase activity.

5. The method of claim 4, wherein: A coryneform bacterium in which expression of one or more polypeptides selected from the following (G) to (O) is also enhanced is used as a host: (G) a polypeptide having 3-deoxy-D-arabinoheptulose-7-phosphate synthase activity; (H) a polypeptide having 3-dehydroquinate synthetase activity; (I) a polypeptide having 3-dehydroquinate dehydratase activity; (J) a polypeptide having 5-shikimate dehydrogenase activity; (K) a polypeptide having shikimate kinase activity; (L) a polypeptide having 3-phosphoshikimate-1-carboxyvinyl transferase activity; (M) a polypeptide having chorismate synthase activity; (N) a polypeptide having pyruvate phosphate dikinase activity; (O) An enzyme group involved in the pentose phosphate pathway: at least one selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase and transaldolase.

6. The method according to any one of claims 1 to 5, wherein: The aromatic compound or its salt is gallic acid, protocatechuic acid, catechol, 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, p-hydroxybenzoic acid, p-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

7. The method of claim 6, wherein: The aromatic compound or its salt is gallic acid, protocatechuic acid, p-hydroxybenzoic acid, p-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

8. The method of claim 7, wherein: The aromatic compound or its salt is gallic acid, 4-aminobenzoic acid, 4-amino-3-hydroxybenzoic acid, or a salt thereof.

9. The method according to any one of claims 1 to 8, wherein: The rod-shaped bacteria are bacteria of the genus Corynebacterium.

10. The method of claim 9, wherein: The bacterium of the genus Corynebacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halodurans, Corynebacterium alkanolyticum, Corynebacterium sphaeroides, Corynebacterium serratum, or Corynebacterium kluyveri.

11. The method of claim 10, wherein: The bacterium of the genus Corynebacterium is Corynebacterium glutamicum.

12. A coryneform bacterium, which is a modified coryneform bacterium in which the function of a cytochrome bd type oxidase complex is suppressed or deleted, wherein: The expression of at least one polypeptide selected from the following (C), (D), (E) and (F) is enhanced: (C) a polypeptide having 3,4-dihydroxybenzoate hydroxylase activity; (D) a polypeptide having 3-dehydroshikimate dehydratase activity; (E) a polypeptide required for the biosynthesis of p-aminobenzoic acid from chorismate; (F) A polypeptide having p-aminobenzoate hydroxylase activity.

13. The coryneform bacterium according to claim 12, wherein The cytochrome bd-type oxidase complex is a polypeptide complex having cytochrome bd-type oxidase activity as shown in the following (A) or (B), (A) A complex comprising: A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 constituted by (B) a complex consisting of: 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 functioning as a cytochrome bd-type oxidase subunit I, and A polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and functioning as a cytochrome bd-type oxidase subunit II constituted.

14. The coryneform bacterium according to claim 12 or 13, wherein A part or all of the nucleotide sequence of the polynucleotide of (a) or (b) below is deleted or inactivated, (a) a polynucleotide encoding: A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, and A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 The complex formed (b) a polynucleotide encoding: A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and functioning as a cytochrome bd-type oxidase subunit I, and A polynucleotide encoding a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and functioning as a cytochrome bd-type oxidase subunit II The complex formed.

15. The coryneform bacterium according to any one of claims 12 to 14, wherein The rod-shaped bacteria are bacteria of the genus Corynebacterium.

16. The coryneform bacterium according to claim 15, wherein The bacterium of the genus Corynebacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halodurans, Corynebacterium alkanolyticum, Corynebacterium sphaeroides, Corynebacterium serratum, or Corynebacterium kluyveri.

17. The coryneform bacterium according to claim 16, wherein The bacterium of the genus Corynebacterium is Corynebacterium glutamicum.

Images