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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
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  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/15—Corynebacterium

Definitions

• the present invention relates to a modified microorganism capable of producing 4-acetylamino-3-hydroxybenzoic acid, and a method for producing 4-acetylamino-3-hydroxybenzoic acids or salts thereof using the microorganism.
• Polybenzoxazole is known as an engineering plastic with excellent heat resistance and mechanical strength, and is used for fiber materials and insulating films of semiconductor elements (Non-Patent Document 1).
• the benzoxazole skeleton is produced by condensation of an o-aminophenol skeleton with a carboxylic acid. Therefore, 4-amino-3-hydroxybenzoic acids (4,3-AHBAs) having these functional groups in the molecule are expected to be useful as PBO monomers.
• 4,3-AHBAs 4-amino-3-hydroxybenzoic acids having these functional groups in the molecule are expected to be useful as PBO monomers.
• the synthesis of polybenzoxazole using 4,3-AHBAs and evaluation of their physical properties have been investigated (Non-Patent Document 2). It has been reported that such 4,3-AHBAs can be efficiently produced using a microorganism capable of expressing a polypeptide having 4-aminobenzoic acid hydroxylation activity (Patent Document 1).
• 4,3-AHBA is oxidized and converted to 2-aminophenoxazin-3-one-7-carboxylic acid, which becomes colored (Patent Document 4), and it is useful to acetylate the amino group of the AHBA.
• Patent Document 4 the enzyme that acetylates 4,3-AHBA is not known.
• Patent Document 1 JP 2021-073914 A
• Patent Document 2 JP 5445453 A
• Patent Document 3 International Patent Publication No. 2013/179711
• Patent Document 4 JP 2021-101628 A
• Hirotaka Murase, SENI GAKKAISHI Textiles and Industry
• Vol. 66 No. 6 (2010)
• Non-patent Document 2 Lon J. Mathias et al., Macromolecules, Vol.18, No.4, pp.616-622 (1985)
• the present invention relates to the following 1) and 2).
• FIG. 2 is a schematic diagram showing the pathway for the production of 4-acetylamino-3-hydroxybenzoic acid in the modified microorganism of the present invention (host: coryneform bacterium).
• aroG and aroF are genes encoding 2-dehydro-3-deoxyarabinoheptonate aldolase
• aroB is a gene encoding 3-dehydroquinate synthase
• aroD is a gene encoding dehydroquinate dehydratase
• qsuD is a gene encoding quinate/shikimate dehydrogenase
• aroE3 is shikimate dehydrogenase
• aroA is a gene encoding 5-enolate pyruvylshikimate-3-phosphate synthase
• aroC is a gene encoding chorismate synthase
• aroK is a gene encoding shikimate kinase
• pabAB is a gene en
• phbh* is a gene encoding a polypeptide having hydroxylation activity at the 3-position of 4-aminobenzoic acid, which is a mutant of 4-hydroxybenzoic acid hydroxylase derived from Caulobacter vibrioides, and is a metabolic pathway that is initiated by exogenous gene introduction.
• PEP is phosphoenolpyruvic acid
• DAHP is 3-deoxy-D-arabino-heptulosonic acid 7-phosphate
• DHQ is dehydroquinic acid
• DHS is dehydroshikimic acid
• SHK is shikimic acid
• S3P is shikimic acid-3-phosphate
• EPSP is 5-enoylpyruvinylshikimic acid-3-phosphate
• 4ABA 4-aminobenzoic acid
• 43AHBA is 4-amino-3-hydroxybenzoic acid
• 43AcAHBA is 4-acetylamino-3-hydroxybenzoic acid.
• the present invention relates to a modified microorganism capable of producing 4-acetylamino-3-hydroxybenzoic acid, and a method for producing 4-acetylamino-3-hydroxybenzoic acids or salts thereof using the modified microorganism.
• the inventors conducted extensive research to obtain 4-acetylamino-3-hydroxybenzoic acids using microorganisms capable of producing 4-amino-3-hydroxybenzoic acid (4,3-AHBA), and discovered that 4-acetylamino-3-hydroxybenzoic acids can be produced efficiently by using microorganisms modified to increase the activity of N-hydroxyarylamine O-acetyltransferase (NhoA).
• NhoA N-hydroxyarylamine O-acetyltransferase
• the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the Search homology program of the genetic information processing software GENETYX Ver. 12 with the Unit size to compare (ktup) set to 2.
• amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.
• an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted refers to an amino acid sequence in which 1 to 10, preferably 1 to 8, more preferably 1 to 5, and even more preferably 1 to 3 amino acids have been deleted, substituted, added, or inserted.
• a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted refers to a nucleotide sequence in which 1 to 30, preferably 1 to 24, more preferably 1 to 15, and even more preferably 1 to 9 nucleotides have been deleted, substituted, added, or inserted.
• “addition" of an amino acid or nucleotide includes addition of an amino acid or nucleotide to one end and both ends of a sequence.
• operably linked between a regulatory region and a gene means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region.
• the procedure for "operably linked" between a gene and a regulatory region is well known to those skilled in the art.
• the term "native" used in reference to a function, property, or trait of a cell is used to indicate that the function, property, or trait is present in the wild-type of the cell.
• the term “exogenous” is used to indicate a function, property, or trait that is not inherently present in the cell, but is introduced from outside.
• a "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from outside.
• the exogenous gene or polynucleotide may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).
• modified microorganism that has been modified to increase N-hydroxyarylamine O-acetyltransferase activity in a microorganism capable of producing 4-amino-3-hydroxybenzoic acid is used to produce 4-acetylamino-3-hydroxybenzoic acids or salts thereof.
• modified microorganism has the ability to produce 4-acetylamino-3-hydroxybenzoic acids or salts thereof (see Figure 1).
• N-hydroxyarylamine O-acetyltransferase activity refers to the enzyme activity of a polypeptide that catalyzes the reaction of transferring an acetyl group from acetyl-CoA to a terminal nitrogen group of a substrate (a polypeptide having N-hydroxyarylamine O-acetyltransferase activity (NhoA)).
• polypeptide having N-hydroxyarylamine O-acetyltransferase activity examples include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 and having N-hydroxyarylamine O-acetyltransferase activity.
• polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is NhoA derived from Escherichia coli.
• amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:2 is preferably an amino acid sequence having 95% or more identity, more preferably 96% or more identity, even more preferably 97% or more identity, even more preferably 98% or more identity, and even more preferably 99% or more identity, and includes, for example, an amino acid sequence in which one or several amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO:2.
• polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:2 and that has N-hydroxyarylamine O-acetyltransferase activity is a polypeptide derived from a bacterium of the genus Shigella that has N-hydroxyarylamine O-acetyltransferase activity.
• Examples of methods for introducing mutations such as deletion, substitution, addition, or insertion of amino acids into the amino acid sequence of the above-mentioned polypeptide include methods for introducing mutations such as deletion, substitution, addition, or insertion of nucleotides into the nucleotide sequence encoding the amino acid sequence.
• Examples of methods for introducing mutations into a nucleotide sequence include mutagenesis using chemical mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, and nitrous acid, or physical mutagens such as ultraviolet light, X-rays, gamma rays, and ion beams, site-directed mutagenesis, and the method described in Dieffenbach et al.
• site-specific mutagenesis technique examples include a method using splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), the ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and the Kunkel method (Kunkel, T. A., Proc. Natl. Acad. Sci. USA, 1985, 82, 488).
• SOE splicing overlap extension
• site-directed mutagenesis kits such as Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), Transformer TM Site-Directed Mutagenesis Kit (Clonetech), and KOD-Plus-Mutagenesis Kit (Toyobo) can also be used.
• modified to increase N-hydroxyarylamine O-acetyltransferase activity includes that the N-hydroxyarylamine O-acetyltransferase activity is higher than the specific activity of the microorganism before modification, that the number of molecules of polypeptide having N-hydroxyarylamine O-acetyltransferase activity per cell is increased, that the N-hydroxyarylamine O-acetyltransferase activity per molecule is increased, etc.
• modifications for increasing N-hydroxyarylamine O-acetyltransferase activity include modifications that express or enhance the expression of a polypeptide having N-hydroxyarylamine O-acetyltransferase activity, and preferably modifications that enhance the expression of a polynucleotide encoding a polypeptide having N-hydroxyarylamine O-acetyltransferase activity.
• Means for enhancing the expression of a polynucleotide encoding the polypeptide include introducing a polynucleotide encoding the polypeptide into a host so that the polynucleotide can be expressed, and modifying the control region of the polynucleotide encoding the polypeptide on the host genome to improve the transcription amount of the polynucleotide, and the polypeptide may be foreign or may be inherent to the microorganism.
• Introducing the polynucleotide so that the polynucleotide can be expressed includes introducing the polynucleotide so that the degree of expression is enhanced. Specifically, examples of such methods include introducing a vector or DNA fragment containing the polynucleotide and a control region operably linked thereto, and replacing the control region of the polynucleotide with a strong control region.
• examples of polynucleotides encoding a polypeptide having N-hydroxyarylamine O-acetyltransferase activity include a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1, or a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:1 and encoding a polypeptide having N-hydroxyarylamine O-acetyltransferase activity.
• nucleotide sequences having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:1 include nucleotide sequences having preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and still more preferably 99% or more identity, and include, for example, nucleotide sequences in which one or more nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequence shown in SEQ ID NO:1.
• the method of introducing a mutation such as a deletion, substitution, addition, or insertion of a nucleotide into a nucleotide sequence is as described above.
• the polynucleotide may be in the form of a single strand or a double strand, and may be DNA or RNA.
• the DNA may be artificial DNA such as cDNA or chemically synthesized DNA.
• the polynucleotide may be incorporated into a vector.
• the vector containing the polynucleotide is an expression vector.
• the vector is an expression vector capable of introducing the polynucleotide into a host microorganism and expressing the polynucleotide in the host microorganism.
• the vector contains the polynucleotide and a control region operably linked thereto.
• the vector may be a vector capable of autonomously replicating and replicating outside a chromosome, such as a plasmid, or may be a vector that is incorporated into a chromosome.
• vectors include pBluescript II SK(-) (Stratagene), pUC18/19, pUC118/119 and other pUC vectors (Takara Bio), pET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T.
• the polynucleotide may be constructed as a DNA fragment containing the polynucleotide.
• the DNA fragment include a PCR-amplified DNA fragment and a restriction enzyme-cleaved DNA fragment.
• the DNA fragment may be an expression cassette containing the polynucleotide and a control region operably linked thereto.
• control region contained in the vector or DNA fragment is a sequence for expressing the polynucleotide in a host cell into which the vector or DNA fragment has been introduced, and examples of such control regions include expression regulatory regions such as promoters and terminators, and replication origins.
• the type of control region can be appropriately selected depending on the type of host microorganism into which the vector or DNA fragment is introduced. If necessary, the vector or DNA fragment may further have a selection marker such as an antibiotic resistance gene or an amino acid synthesis-related gene.
• transformation methods such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, and Agrobacterium can be used.
• a modified microorganism into which a vector or DNA fragment of interest has been introduced can be selected using a selection marker.
• the selection marker is an antibiotic resistance gene
• cells into which a vector or DNA fragment of interest has been introduced can be selected by culturing in a medium containing the antibiotic.
• the selection marker is an amino acid synthesis-related gene
• after gene introduction into a host cell that requires that amino acid cells into which a vector or DNA fragment of interest has been introduced can be selected using the presence or absence of that amino acid requirement as an indicator.
• introduction of the vector or DNA fragment of interest can be confirmed by examining the DNA sequence of transformed cells using PCR or the like.
• strong regulatory regions include known high expression promoters such as T7 promoter, lac promoter, tac promoter, trp promoter, tu promoter, and gap promoter, but are not particularly limited to these.
• an inducible promoter derived from a prokaryote can be used, and examples thereof include, but are not limited to, the vanA promoter which is induced by the addition of ferulic acid, vanillic acid, or vanillin, the rhcH promoter which is induced by the addition of resorcinol or 2,4-dihydroxybenzoic acid, the pcaI promoter which is induced by the addition of 4-hydroxybenzoic acid, the promoter of the nagI (cg3351) gene which is induced by the addition of 3-hydroxybenzoic acid, the promoter of the benA (cg2637) gene which is induced by the addition of benzoic acid (hereinafter, abbreviated as Pben), or the promoter
• Methods for replacing the regulatory region of the polynucleotide present on the genome of a host cell with a strong regulatory region include a method in which a DNA fragment containing the strong regulatory region and a polynucleotide sequence of a selection marker is introduced into a host cell, and cells transformed by homologous recombination or non-homologous recombination are selected.
• the N-hydroxyarylamine O-acetyltransferase activity and the degree of increase in activity can be confirmed by measuring the N-hydroxyarylamine O-acetyltransferase activity in a cell extract of the microorganism and comparing it with the activity of the microorganism before modification.
• the N-hydroxyarylamine O-acetyltransferase activity can be measured, for example, by the method described in Biochim. Biophys. Acta. 1475 (2000), 10-16.
• the modified microorganism thus produced can then be cultured, the productivity of 4-acetylamino-3-hydroxybenzoic acids or salts thereof evaluated, and an appropriate microbial strain selected, thereby making it possible to obtain a useful strain producing 4-acetylamino-3-hydroxybenzoic acids or salts thereof.
• the host microorganism to be modified as described above is a microorganism capable of producing 4-amino-3-hydroxybenzoic acid.
• microbial species include Escherichia coli, Bacillus subtilis, actinomycetes, Pseudomonas bacteria, Streptococcus bacteria, Lactobacillus bacteria, fungi (such as Neurospora, Aspergillus, and Trichoderma), yeasts (such as Saccharomyces, Kluyveromyces, Schizosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Pichia, and Candida), and the like. Actinomycetes are preferred.
• coryneform bacteria As actinomycetes, a group of microorganisms defined as coryneform bacteria (Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974)) is preferred, and specific examples thereof include bacteria of the genus Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Rhodococcus, Streptomyces, Micrococcus, etc. Examples of bacteria of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum.
• Examples of the Brevibacterium genus include Brevibacterium ammoniagenes.
• Examples of the Arthrobacter genus include Arthrobacter globiformis.
• Examples of bacteria belonging to the genus Mycobacterium include Mycobacterium bovis, and examples of bacteria belonging to the genus Micrococcus include Micrococcus freudenreichii, Micrococcus leuteus, Micrococcus ureae, Micrococcus roseus, and the like.
• the coryneform bacteria the genus Corynebacterium is preferred, and Corynebacterium glutamicum is more preferred.
• coryneform bacteria may be mutant strains or artificially genetically modified strains.
• disruption strains of genes such as lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, and malate dehydrogenase may be included.
• LDH lactate dehydrogenase
• phosphoenolpyruvate carboxylase phosphoenolpyruvate carboxylase
• malate dehydrogenase may be included.
• a microorganism capable of producing 4-amino-3-hydroxybenzoic acid refers to a microorganism that inherently has the ability to produce 4-amino-3-hydroxybenzoic acid and a microorganism to which the ability to produce 4-amino-3-hydroxybenzoic acid has been imparted.
• Preferred examples of the microorganism include (A) a microorganism in which expression of a polypeptide having activity of hydroxylating 3-position of 4-aminobenzoic acid has been enhanced, and further, in addition to (A), (B) a microorganism in which expression of a polypeptide necessary for biosynthesis of 4-aminobenzoic acid from chorismic acid has been enhanced (see FIG. 1).
• the polypeptide (A) having 4-aminobenzoic acid 3-position hydroxylation activity is a polypeptide that catalyzes a reaction to produce 4-amino-3-hydroxybenzoic acid from 4-aminobenzoic acid, and examples thereof include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, or a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 3 and having 4-aminobenzoic acid 3-position hydroxylation activity.
• polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 is a polypeptide having 4-aminobenzoic acid 3-position hydroxylation activity, which is a mutant of 4-hydroxybenzoic acid hydroxylase derived from Caulobacter vibrioides (V47L, M106A double mutant: Japanese Patent Application No. 2022-117492) (referred to as "phbh *" in FIG. 1).
• examples of the polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:3 and having the activity of hydroxylating 3-position of 4-aminobenzoic acid include mutants of 4-hydroxybenzoate hydroxylase derived from bacteria of the genus Caulobacter, specifically mutants of 4-hydroxybenzoate hydroxylase derived from Caulobacter rhizosphaerae and 4-hydroxybenzoate hydroxylase derived from Caulobacter sp.
• mutants include one or more mutants selected from the mutants described in JP 2021-073914 A, the mutants described in JP 2021-101626 A, the mutants described in JP 2021-101627 A, and the mutants described in JP 2022-047939 A, and preferably one or more mutants selected from a mutant in which the 47th position or a position equivalent thereto is leucine, a mutant in which the 106th position or a position equivalent thereto is alanine, a mutant in which the 201st position or a position equivalent thereto is phenylalanine, a mutant in which the 222nd position or a position equivalent thereto is phenylalanine, and a mutant in which the 294th position or a position equivalent thereto is serine.
• polypeptides (B) required for the biosynthesis of 4-aminobenzoic acid from chorismate examples include (B1) 4-amino-4-deoxychorismate synthase and (B2) 4-amino-4-deoxychorismate lyase.
• the enhancement of expression of the polypeptides required for the biosynthesis of 4-aminobenzoic acid from chorismate includes an embodiment in which the expression of any one or more of these polypeptides is enhanced.
• 4-Aminobenzoic acid is produced from chorismate via 4-amino-4-deoxychorismic acid.
• chorismate to 4-amino-4-deoxychorismic acid involves para-aminobenzoate synthetase component II (PabA) and para-aminobenzoate synthetase component I (PabB), and 4-amino-4-deoxychorismic acid is converted to para-aminobenzoic acid by 4-amino-4-deoxychorismic acid lyase (PabC).
• the (B1) 4-amino-4-deoxychorismate synthase is a polypeptide that catalyzes a reaction to produce 4-amino-4-deoxychorismate from chorismate, and examples thereof include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4 and having 4-amino-4-deoxychorismate synthase activity.
• the (B2) 4-amino-4-deoxychorismate lyase is a polypeptide that catalyzes a reaction to produce 4-aminobenzoic acid from 4-amino-4-deoxychorismate, and examples thereof include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5, or a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:5 and having 4-amino-4-deoxychorismate lyase activity.
• polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is 4-amino-4-deoxychorismate synthase derived from Corynebacterium glutamicum and is known as "pabAB”
• polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 is 4-amino-4-deoxychorismate lyase derived from Corynebacterium glutamicum and is known as "pabC”.
• the expression of the above polypeptide can be enhanced by introducing a polynucleotide necessary for the expression of the polypeptide so that it can be expressed, and the method for this can be the same as that for the expression and introduction of the polypeptide having hydroxyarylamine O-acetyltransferase activity described above.
• the production of 4-acetylamino-3-hydroxybenzoic acids or salts thereof comprises a step of culturing the above-mentioned modified microorganism, preferably in the presence of sugars, and is carried out by recovering 4-acetylamino-3-hydroxybenzoic acids from the medium.
• a microorganism modified to have increased N-hydroxyarylamine O-acetyltransferase activity has the ability to produce 4-acetylamino-3-hydroxybenzoic acids, and the modified microorganism is characterized in that it also has improved productivity of its precursor, 4-amino-3-hydroxybenzoic acids.
• 4-aminobenzoic acid (4-ABA), a precursor of 4-amino-3-hydroxybenzoic acid, is also acetylated by N-hydroxyarylamine O-acetyltransferase (Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1475, Issue 1, 1 June 2000, Pages 10-16).
• the modified microorganism of the present invention is characterized in that 4-acetylaminobenzoic acid, which is the acetylated form of 4-aminobenzoic acid, is hardly produced, and thus 4-acetylamino-3-hydroxybenzoic acids can be efficiently produced.
• the 4-acetylamino-3-hydroxybenzoic acid is specifically represented by the following general formula (1):
• R 1 represents a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH 3 ), an amino group (-NH 2 ), a fluorine atom (-F), a chlorine atom (-Cl), a bromine atom (-Br), an iodine atom (-I), a carboxy group (-COOH), a methyl group (-CH 3 ), or an ethyl group (-CH 2 CH 3 );
• R 2 represents a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH 3 ), an amino group (-NH 2 ), a fluorine atom (-F), a chlorine atom (-Cl), a bromine atom (-Br), an iodine atom (-I), a carboxy group (-COOH), a methyl group (-CH 3 ), or an ethyl group (-CH 2 CH 3 ); and X 1 and X 2
• the functional group represented by R 1 is preferably a hydrogen atom, a hydroxy group (--OH), a methoxy group (--OCH 3 ), a fluorine atom (--F) or a methyl group (--CH 3 ).
• the functional group represented by R 2 is preferably a hydrogen atom, a hydroxy group (--OH), a methoxy group (--OCH 3 ), a fluorine atom (--F) or a methyl group (--CH 3 ). It is more preferable that R 1 and R 2 are both hydrogen atoms.
• both X 1 and X 2 may be hydroxy groups, but it is preferable that either one of X 1 or X 2 is a hydroxy group.
• Salts of 4-acetylamino-3-hydroxybenzoic acids include, for example, salts with alkali metals such as sodium and potassium, and salts with alkaline earth metals such as calcium and magnesium.
• glucose is preferable, but in addition to monosaccharides such as fructose, mannose, arabinose, xylose, galactose, etc., saccharides that can generate glucose by metabolism can also be used.
• saccharides include oligosaccharides or polysaccharides having a glucose unit, such as disaccharides such as cellobiose, sucrose (cane sugar), lactose, maltose, trehalose, cellobiose, xylobiose, etc.; polysaccharides such as dextrin or soluble starch, etc. Molasses can also be used as a raw material containing these raw material compounds.
• inedible agricultural waste such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw, etc.), bagasse, corn stover, etc.
• energy crops such as switchgrass, napier grass, and miscanthus, wood chips, waste paper, etc.
• saccharification enzyme or the like to produce a saccharified liquid containing multiple sugars such as glucose.
• the medium for culturing the modified microorganism contains a carbon source, a nitrogen source, inorganic salts, etc., and may be either a natural medium or a synthetic medium, as long as it is a medium in which the modified coryneform bacterium of the present invention can be efficiently cultured.
• the above-mentioned sugars or molasses or saccharified solution containing them can be used, but in addition to the above-mentioned sugars, sugar alcohols such as mannitol, sorbitol, xylitol, and glycerin, organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid, alcohols such as ethanol and propanol, hydrocarbons such as normal paraffin, etc.
• the carbon source can be used alone or in combination of two or more.
• the concentration of the saccharides as raw material compounds in the culture medium is preferably 1 to 20 w/v %, more preferably 2 to 10 w/v %, and even more preferably 2 to 5 w/v %.
• Nitrogen sources that can be used include, for example, peptone, meat extract, yeast extract, casein hydrolysate, alkaline extract of soybean meal, alkylamines such as methylamine, nitrogen-containing organic compounds such as amino acids, ammonia or its salts (inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate), urea, aqueous ammonia, sodium nitrate, and potassium nitrate.
• alkylamines such as methylamine
• nitrogen-containing organic compounds such as amino acids
• ammonia or its salts inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate
• urea aqueous ammonia
• sodium nitrate sodium nitrate
• potassium nitrate potassium nitrate
• inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate.
• vitamins, antifoaming agents, etc. can be added.
• vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.
• 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)], and CGXII medium [Patent No. 6322576]. These media can be used with the sugar concentration in the above range.
• the culture temperature or reaction temperature is preferably 15 to 45°C, more preferably 25 to 37°C.
• the culture or reaction time is 24 to 168 hours, preferably 24 to 96 hours, more preferably 24 to 72 hours, and can be performed with stirring or shaking as necessary.
• antibiotics such as ampicillin and kanamycin may be added to the medium as necessary.
• the culture may be any of a batch system, a fed-batch system, and a continuous system, of which the batch system is preferred.
• the culture or reaction may be carried out under aerobic conditions or under reducing conditions, but is preferably carried out under aerobic conditions. When the reaction or culture is carried out under aerobic conditions, it is preferable to carry out the reaction or culture under conditions that suppress excessive growth of microorganisms, from the viewpoint of the production efficiency of the aromatic compound or a salt thereof.
• the method for recovering and purifying 4-acetylamino-3-hydroxybenzoic acids from the culture is not particularly limited. In other words, it can be carried out by combining well-known ion exchange resin methods, precipitation methods, crystallization methods, recrystallization methods, concentration methods, and other methods.
• 4-acetylamino-3-hydroxybenzoic acids can be obtained by removing the bacterial cells by centrifugation or the like, removing ionic substances with cation and anion exchange resins, and concentrating the mixture.
• the 4-acetylamino-3-hydroxybenzoic acids accumulated in the culture may be used as is without isolation.
• the present invention further discloses the following aspects.
• a method for producing 4-acetylamino-3-hydroxybenzoic acids or a salt thereof comprising a step of culturing a modified microorganism that has been modified to have increased N-hydroxyarylamine O-acetyltransferase activity in a microorganism capable of producing 4-amino-3-hydroxybenzoic acid.
• the modification that increases the N-hydroxyarylamine O-acetyltransferase activity is a modification that causes expression or enhances expression of a polypeptide having N-hydroxyarylamine O-acetyltransferase activity.
• polypeptide having N-hydroxyarylamine O-acetyltransferase activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, and still more preferably 99% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having N-hydroxyarylamine O-acetyltransferase activity.
• ⁇ 4> The method according to ⁇ 2> or ⁇ 3>, wherein the expression of the polypeptide having N-hydroxyarylamine O-acetyltransferase activity is achieved by enhancing the expression of a polynucleotide encoding a polypeptide having N-hydroxyarylamine O-acetyltransferase activity.
• ⁇ 5> The method according to any one of ⁇ 1> to ⁇ 4>, wherein the microorganism is a coryneform bacterium.
• ⁇ 6> The method according to ⁇ 5>, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.
• ⁇ 7> The method according to ⁇ 6>, wherein the Corynebacterium bacterium is Corynebacterium glutamicum.
• 4-acetylamino-3-hydroxybenzoic acids are represented by the following general formula (1):
• R 1 represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group
• R 2 represents a hydrogen atom, a hydroxy group, a methoxy group, an amino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a carboxy group, a methyl group, or an ethyl group
• X 1 and X 2 represent a hydrogen atom or a hydroxy group, with at least one of them representing a hydroxy group.
• modified microorganism according to ⁇ 9> wherein the modification for increasing the N-hydroxyarylamine O-acetyltransferase activity is a modification for expressing or enhancing the expression of a polypeptide having N-hydroxyarylamine O-acetyltransferase activity.
• polypeptide having N-hydroxyarylamine O-acetyltransferase activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, and still more preferably 99% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having N-hydroxyarylamine O-acetyltransferase activity.
• ⁇ 12> The modified microorganism according to ⁇ 10> or ⁇ 11>, wherein the expression of the polypeptide having N-hydroxyarylamine O-acetyltransferase activity is achieved by enhancing the expression of a polynucleotide encoding the polypeptide having N-hydroxyarylamine O-acetyltransferase activity.
• ⁇ 13> The modified microorganism according to any one of ⁇ 9> to ⁇ 12>, wherein the microorganism is a coryneform bacterium.
• ⁇ 14> The modified microorganism according to ⁇ 13>, wherein the coryneform bacterium is a bacterium of the genus Corynebacterium.
• ⁇ 15> The modified microorganism according to ⁇ 14>, wherein the Corynebacterium bacterium is Corynebacterium glutamicum.
• ⁇ 16> The method according to ⁇ 4> or the modified microorganism according to ⁇ 12>, wherein the expression of a polynucleotide encoding a polypeptide having N-hydroxyarylamine O-acetyltransferase activity is enhanced by introducing the polynucleotide into a host so that the polynucleotide can be expressed, or by modifying a control region of the polynucleotide on the host genome.
• ⁇ 17> The method of ⁇ 4>, the modified microorganism of ⁇ 12>, or the method or improved microorganism of ⁇ 16>, wherein the polynucleotide encoding a polypeptide having N-hydroxyarylamine O-acetyltransferase activity is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide consisting of a nucleotide sequence having 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, and still more preferably 99% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide having N-hydroxyarylamine O-acetyltransferase activity.
• microorganism capable of producing 4-amino-3-hydroxybenzoic acid is (A) a microorganism in which expression of a polypeptide having activity of hydroxylating 3-position of 4-aminobenzoic acid is enhanced, or (B) a microorganism in which expression of a polypeptide necessary for biosynthesis of 4-aminobenzoic acid from chorismic acid is enhanced in addition to (A).
• polypeptide (A) having 4-aminobenzoic acid 3-hydroxylating activity is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, and still more preferably 99% or more to the amino acid sequence shown in SEQ ID NO: 3, and having 4-aminobenzoic acid 3-hydroxylating activity.
• Example 1 Production of 4-acetylamino-3-hydroxybenzoic acid
• Construction of plasmid pECsf_gapS_pabABC_tuD_HFM122 (see Patent Application No. 2019-203523) was used as a template to amplify by PCR using two types of DNA primers (SEQ ID NOs: 6 and 7), and the resulting PCR product was treated with DpnI (Takara Bio).
• a DNA fragment containing the benA promoter (SEQ ID NO: 8) was amplified using two types of DNA primers (SEQ ID NOs: 9 and 10) using the genome of the ATCC13032 strain as a template to obtain a DNA fragment.
• a fragment of the nhoA gene (SEQ ID NO: 1) codon-optimized for Corynebacterium glutamicum was gene-synthesized (Eurofin Genomics) and amplified by PCR using two types of DNA primers (SEQ ID NOs: 11 and 12) to obtain a DNA fragment.
• the three types of PCR products obtained were each purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) to obtain DNA fragments, which were then ligated using In-Fusion HD Cloning Kit (Takara Bio) to produce the plasmid pECsf_PbenA_nhoA_OPT.
• a UV detector (detection wavelength 280 nm) was used for the detection of 4,3-AHBA, 4-ABA, 4,3-AcAHBA, and 4-AcABA.
• the quantification of 4,3-AHBA, 4-ABA, 4,3-AcAHBA, and 4-AcABA was performed using a calibration curve prepared using a standard substance.
• Glucose was analyzed using ICsep ION-300 (7.8 mm x 300 mm, Tokyo Chemical Industry Co., Ltd.), and detection was performed using a 37 mM sulfuric acid solution as the eluent at a flow rate of 0.5 mL/min and a column temperature of 50° C.
• Glucose was detected using RI, and quantification was performed using a calibration curve prepared using a standard substance.
• Each standard material is available from Fujifilm Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., etc.

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