WO2023090378A1 - Production method for dihydroxyphenylalanine - Google Patents

Production method for dihydroxyphenylalanine Download PDF

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WO2023090378A1
WO2023090378A1 PCT/JP2022/042624 JP2022042624W WO2023090378A1 WO 2023090378 A1 WO2023090378 A1 WO 2023090378A1 JP 2022042624 W JP2022042624 W JP 2022042624W WO 2023090378 A1 WO2023090378 A1 WO 2023090378A1
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amino acid
seq
acid sequence
protein
tyrosine phenol
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Japanese (ja)
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幸盛 辻
鏡士朗 野中
史員 高橋
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花王株式会社
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine

Definitions

  • the present invention relates to a method for producing dihydroxyphenylalanine.
  • L-DOPA 3,4-Dihydroxy-L-phenylalanine
  • L-tyrosine which is a non-essential amino acid
  • L-DOPA is hydroxylated by tyrosine hydroxylase and converted to L-DOPA, and further converted to dopamine, a neurotransmitter, by L-DOPA decarboxylase.
  • L-DOPA is a precursor of dopamine.
  • L-DOPA is also used as a therapeutic agent for Parkinson's disease because it can pass through the blood-brain barrier.
  • shikimate pathway is an important metabolic pathway for the biosynthesis of aromatic compounds by plants and microorganisms.
  • the formation of a 6-carbon ring is followed by the formation of a double bond, and protocatechuic acid derived from 3-dehydroshikimic acid produces aromatic compounds such as gallic acid and catechol. It is known that
  • Patent Document 1 Patent No. 3116102
  • the present invention relates to the following 1) to 3).
  • 1) A step of contact-reacting protocatechuic acid, pyruvic acid, and an ammonium salt with cultured cells of a microorganism producing protocatechuate decarboxylase and tyrosine phenol lyase, a crushed product thereof, or a cell extract of the microorganism.
  • 2) A vector or DNA fragment containing a polynucleotide encoding protocatechuate decarboxylase and tyrosine phenol lyase.
  • 3) A transformed cell containing the vector or DNA fragment of 2) above.
  • the present invention relates to providing a method for efficiently producing 3,4-dihydroxyphenylalanine from protocatechuic acid using cultured cells of microorganisms.
  • the present inventors have found a tyrosine phenol lyase that is less susceptible to inhibition by protocatechuic acid, and efficiently produce 3,4-dihydroxyphenylalanine from protocatechuic acid by using protocatechuic acid decarboxylase and a microorganism that produces the tyrosine phenol lyase. I found what I could do.
  • the present invention it becomes possible to efficiently produce 3,4-dihydroxyphenylalanine from protocatechuic acid. Since protocatechuic acid is produced via the shikimate pathway, the present invention enables the production of 3,4-dihydroxyphenylalanine using biomass sugar as a raw material.
  • the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, genetic information processing software GENETYX Ver. 12 homology analysis (Search homology) program is used, unit size to compare (ktup) is set to 2, and analysis is performed.
  • GENETYX Ver. 12 homology analysis (Search homology) program is used, unit size to compare (ktup) is set to 2, and analysis is performed.
  • amino acid sequences and nucleotide sequences means 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more. % or more, preferably 99% or more identity.
  • an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted means 1 or more and 20 or less, preferably 1 or more and 15 or less, more preferably 1 It refers to an amino acid sequence in which 10 or less, more preferably 1 or more and 5 or less, still more preferably 1 or more and 3 or less amino acids are deleted, substituted, added, or inserted.
  • nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted means 1 or more and 30 or less, preferably 1 or more and 24 or less, more preferably 1 or more and 15 It refers to a nucleotide sequence in which no more than 1, more preferably from 1 to 9 nucleotides have been deleted, substituted, added or inserted.
  • additional of amino acids or nucleotides includes addition of amino acids or nucleotides to one and both termini of a sequence. Mutations such as deletions, substitutions, insertions and additions can be introduced by introducing mutations into the base sequence of interest by, for example, site-directed mutagenesis.
  • upstream and downstream with respect to a gene refer to upstream and downstream in the transcription direction of the gene.
  • a gene located downstream of a promoter means that the gene is present on the 3' side of the promoter in the DNA sense strand
  • upstream of the gene means that the gene is located 5' of the gene in the DNA sense strand. means the area on the side.
  • control region such as a promoter and a gene
  • control region means that the gene and the control region are linked so that the gene can be expressed under the control of the control region.
  • Procedures for "operably linking" genes and regulatory regions are well known to those of skill in the art.
  • a "foreign gene” is an exogenous gene introduced into a cell from the outside.
  • a foreign gene may be derived from the same organism as the cell into which it is introduced, or from a different organism (ie, a heterologous gene).
  • 3,4-dihydroxyphenylalanine is a mixture of cultured cells of a microorganism that produces protocatechuate decarboxylase and tyrosine phenol lyase, a lysate thereof, or a cell extract of the microorganism, and protocatechuic acid ( PCA), pyruvic acid and an ammonium salt.
  • protocatechuic acid is converted to catechol by protocatechuic acid decarboxylase, and then the catechol, pyruvic acid and ammonium salt react with tyrosine phenol lyase (TPL) to form 3,4- It is a reaction that produces dihydroxyphenylalanine.
  • 3,4-dihydroxyphenylalanine includes 3,4-dihydroxy-L-phenylalanine (L-DOPA) and 3,4-dihydroxy-D-phenylalanine (D-DOPA), but 3,4-dihydroxy -L-phenylalanine (L-DOPA) is preferred.
  • DOPA may be in the form of zwitterions or salts
  • salts include sodium salts, potassium salts, calcium salts, hydrochlorides, and sulfates.
  • hydrochlorides and sulfates are more preferable.
  • the microorganism of the present invention has the ability to produce protocatechuate decarboxylase and tyrosine phenol lyase, and specifically includes a microorganism having genes encoding protocatechuate decarboxylase and tyrosine phenol lyase.
  • Protocatechuic acid decarboxylase refers to an enzyme that catalyzes the reaction of decarboxylating protocatechuic acid to produce catechol.
  • Enzymes having protocatechuate decarboxylase activity include protocatechuate decarboxylase (EC 4.1.1.63), gallic acid decarboxylase (EC 4.1.1.59), 4- Hydroxybenzoate decarboxylase (4-hydroxybenzoate decarboxylase, EC 4.1.1.61) and the like, and protocatechuate decarboxylase (EC 4.1.1.63) is preferred.
  • Protocatechuate decarboxylase includes, for example, the following proteins (A1) or (A2).
  • a protein consisting of the amino acid sequence shown in SEQ ID NO: 18 A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 18 and having protocatechuic acid decarboxylase activity
  • SEQ ID NO: 18 is protocatechuate decarboxylase (EcAroY) derived from Enterobacter cloacae.
  • the identity with the amino acid sequence of SEQ ID NO: 18 is preferably 95% or more, more preferably 97% or more, from the viewpoint of protocatechuate decarboxylase activity, and 98% or more is More preferably, 99% or more is even more preferable.
  • the amino acid sequence of the protein (A2) includes, for example, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence of SEQ ID NO:18.
  • whether the protein has protocatechuic acid decarboxylase activity can be confirmed, for example, by incubating the protein with a substrate (that is, protocatechuic acid) and measuring the protein- and substrate-dependent catechol production. .
  • the protocatechuate decarboxylase gene includes, for example, the gene encoding the protein (A1) or (A2). Specific examples include the following (a1) or (a2) polynucleotides.
  • (a1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17
  • (a2) comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 17 and encoding a polypeptide having protocatechuate decarboxylase activity
  • polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 represents a gene encoding protocatechuate decarboxylase (EcAroY) derived from Enterobacter cloacae.
  • the identity of the nucleotide of (a2) with the nucleotide sequence of SEQ ID NO: 17 is preferably 95% or more, more preferably 97% or more, in terms of protocatechuate decarboxylase activity, and 98% or more is More preferably, 99% or more is even more preferable.
  • the nucleotide sequence of the polynucleotide of (a2) includes, for example, a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted with respect to the nucleotide sequence shown in SEQ ID NO:17.
  • tyrosine phenol-lyase (EC 4.1.99.2) is an enzyme that catalyzes the reaction of degrading L-tyrosine into phenol, pyruvic acid and ammonia or the reverse reaction thereof.
  • Tyrosine phenol lyase can also catalyze the decomposition of 3,4-dihydroxyphenylalanine into catechol, pyruvate and ammonia, or the reverse reaction.
  • the activity of tyrosine phenol lyase is thought to be inhibited by catechol or its precursor, protocatechuic acid.
  • the tyrosine phenol lyase used in the present invention preferably exhibits a low rate of activity inhibition by protocatechuic acid.
  • the inhibition rate calculated by the formula is 91% or less, preferably 90% or less, preferably 75% or less, more preferably 50% or less, more preferably 40% or less, and still more preferably 36% or less. .
  • the tyrosine phenol lyase of the present invention preferably includes, for example, proteins selected from the group consisting of (B1), (B2), (C1), (C2), (D1) and (D2) below. .
  • B1 a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
  • B2 consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2
  • C1 sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6.
  • Protein consisting of an amino acid sequence (D2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having tyrosine phenol lyase activity
  • the protein consisting of the amino acid sequence of SEQ ID NO: 2 is a tyrosine phenol lyase (FnTPL) derived from Fusobacterium nucleatum.
  • the protein consisting of the amino acid sequence of SEQ ID NO: 4 is Citrobacter freundii-derived tyrosine phenol lyase (CfTPL).
  • the protein consisting of the amino acid sequence of SEQ ID NO: 6 is Erwinia herbicola-derived tyrosine phenol lyase (EwTPL).
  • the protein (B2), (C2) or (D2) has 95% or more identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, respectively, in terms of tyrosine phenol lyase activity. is preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more.
  • the amino acid sequence of the protein (B2), (C2) or (D2) includes, for example, deletion, substitution, insertion of one or several amino acids in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or added amino acid sequences are included.
  • the protein also has tyrosine phenol lyase activity, for example, by incubating the protein with a substrate (ie, phenol or catechol, pyruvic acid and ammonium salts), the protein and substrate-dependent tyrosine or 3,4-dihydroxyphenylalanine can be confirmed by measuring the generation of a substrate (ie, phenol or catechol, pyruvic acid and ammonium salts), the protein and substrate-dependent tyrosine or 3,4-dihydroxyphenylalanine can be confirmed by measuring the generation of a substrate (ie, phenol or catechol, pyruvic acid and ammonium salts), the protein and substrate-dependent tyrosine or 3,4-dihydroxyphenylalanine can be confirmed by measuring the generation of a substrate (ie, phenol or catechol, pyruvic acid and ammonium salts), the protein and substrate-dependent tyrosine or 3,4-dihydroxyphenylalanine can be confirmed by measuring the generation of a
  • the tyrosine phenol lyase gene of the present invention includes genes encoding the above proteins (B1), (B2), (C1), (C2), (D1) and (D2). Specific examples include polynucleotides selected from the group consisting of (b1), (b2), (c1), (c2), (d1) and (d2) below.
  • (b1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1; (b2) 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 tyrosine phenol lyase activity
  • Polynucleotide (c1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3
  • (c2) a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and having tyrosine phenol lyase activity
  • Polynucleotide encoding (d1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5
  • the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 represents a gene encoding tyrosine phenol lyase (FnTPL) derived from Fusobacterium nucleatum.
  • the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 represents a gene encoding tyrosine phenol lyase (CfTPL) derived from Citrobacter freundii.
  • the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 represents a gene encoding tyrosine phenol lyase (EwTPL) derived from Erwinia herbicola.
  • the nucleotides (b2), (c2), and (d2) have 95% or more identity with the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 in terms of tyrosine phenol lyase activity. is preferably 97% or more, more preferably 98% or more, even more preferably 99% or more.
  • the nucleotide sequence of the polynucleotide (b2), (c2) or (d2) for example, one or more nucleotides are deleted from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 , substituted, added, or inserted nucleotide sequences.
  • the Fusobacterium nucleatum-derived tyrosine phenol lyase (FnTPL) gene is particularly resistant to activity inhibition by protocatechuic acid and has excellent production efficiency of 3,4-dihydroxyphenylalanine.
  • FnTPL Fusobacterium nucleatum-derived tyrosine phenol lyase
  • a gene encoding the protein shown in (B1) or (B2) above, more specifically a polynucleotide consisting of the nucleotide sequence shown in (b1) or (b2) above is preferred.
  • the microorganism of the present invention preferably has the ability to produce flavin mononucleotide prenyltransferase, ie, a gene encoding flavin mononucleotide prenyltransferase.
  • Flavin mononucleotide prenyltransferase (EC 2.5.1.129) attaches a dimethylallyl structure from dimethylallyl monophosphate (DMAP) to the flavin backbone of flavin mononucleotide (FMN) to form prenylated-FMN.
  • Flavin mononucleotide prenyltransferases of the present invention include, for example, proteins (E1) or (E2) below.
  • E1 A protein consisting of the amino acid sequence shown in SEQ ID NO: 24
  • E2 A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24 and having flavin mononucleotide prenyltransferase activity
  • the protein consisting of a 24 amino acid sequence is Escherichia coli-derived flavin mononucleotide prenyltransferase (EcUbiX).
  • the identity with the amino acid sequence of SEQ ID NO: 24 is preferably 95% or more, more preferably 97% or more, and 98% or more in terms of flavin mononucleotide prenyltransferase activity. is more preferred, and 99% or more is even more preferred.
  • the amino acid sequence of the protein (E2) includes, for example, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence of SEQ ID NO:24.
  • the protein has flavin mononucleotide prenyltransferase activity, for example, see Nature, 2015, 522: 502-506, etc.
  • the protein is flavin mononucleotide (FMN) and dimethylallyl monophosphate (DMAP) It can be confirmed by incubating in the presence and tracking the absorption spectrum change due to the production of prenylated-FMN.
  • the flavin mononucleotide prenyltransferase gene of the present invention includes a gene encoding the protein (E1) or (E2). Specific examples include the following (e1) or (e2) polynucleotides.
  • (e1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:23; Encoding Polynucleotide
  • the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 23 represents a gene encoding Escherichia coli-derived flavin mononucleotide prenyltransferase (EcUbiX).
  • the identity of the nucleotide (e2) with the nucleotide sequence of SEQ ID NO: 23 is preferably 95% or more, more preferably 97% or more, and 98% or more in terms of flavin mononucleotide prenyltransferase activity. is more preferred, and 99% or more is even more preferred.
  • the nucleotide sequence of the polynucleotide (e2) includes, for example, a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted with respect to the nucleotide sequence shown in SEQ ID NO:23.
  • the microorganism of the present invention may have genes encoding protocatechuate decarboxylase and tyrosine phenol lyase, and optionally further flavin mononucleotide prenyltransferase (these are also referred to as "genes of the present invention"), but at least one It is preferably a gene exogenous to the host microorganism (foreign gene).
  • a foreign gene-introduced microorganism can be produced by preparing an expression vector or a gene expression cassette capable of expressing the gene in host cells, and then introducing it into host cells to transform the host cells.
  • Microorganisms as hosts for transformants may be either prokaryotes or eukaryotes, and prokaryotes such as microorganisms belonging to the genus Escherichia, microorganisms belonging to the genus Bacillus, actinomycetes, and coryneform bacteria.
  • Organisms or eukaryotic microorganisms such as yeast and filamentous fungi can be used.
  • Escherichia coli which is a microorganism belonging to the genus Escherichia
  • Erwinia herbicola which is a microorganism belonging to the genus Erwinia
  • Bacillus subtilis which is a microorganism belonging to the genus Bacillus
  • the genus Corynebacterium Corynebacterium glutamicum which is a microorganism belonging to the genus Pseudomonas, Pseudomonas putida, which is a microorganism belonging to the genus Pseudomonas
  • Rhodococcus jostii which is a microorganism belonging to the genus Rhodococcus.
  • Li Um glutamicum is more preferred.
  • any vector can be used as long as the gene of the present invention can be introduced into a host and the gene can be expressed in the host cell.
  • the vector comprises the gene of the invention and a control region operably linked thereto.
  • the vector may be a vector capable of autonomous replication and replication outside the chromosome, such as a plasmid, or a vector that is integrated into the chromosome.
  • Specific vectors include, for example, pBluescript II SK (-) (Agilent Technologies), pUC-based vectors such as pUC18/19 and pUC118/119 (Takara Bio Inc.), pET-based vectors (Merck), and pGEX-based vectors.
  • the gene of the present invention may be constructed as a DNA fragment containing it.
  • the DNA fragments include, for example, PCR-amplified DNA fragments and restriction enzyme-cleaved DNA fragments.
  • the DNA fragment may be an expression cassette comprising the gene of the invention and a control region operably linked thereto.
  • the control region contained in the above vector or DNA fragment is a sequence for expressing the gene of the present invention in a host cell into which the vector or DNA fragment has been introduced. points, etc.
  • the type of control region can be appropriately selected according to the type of host microorganism into which the vector or DNA fragment is introduced. If necessary, the vector or DNA fragment further has a selection marker such as an antibiotic resistance gene, an amino acid synthesis-related gene (e.g., drug resistance genes such as ampicillin, neomycin, kanamycin, chloramphenicol, etc.).
  • the gene of the present invention can be ligated with the regulatory region or marker gene sequence by a method such as a seamless cloning method. Procedures for introducing gene sequences into vectors are well known in the art.
  • the types of control regions such as promoter regions, terminators, and secretory signal regions are not particularly limited, and commonly used promoters and secretory signal sequences can be appropriately selected and used according to the host into which the gene is introduced.
  • control region include strong control regions capable of enhancing expression compared to the wild type, such as known high-expression promoters such as T7 promoter, lac promoter, tac promoter, trp promoter, gap promoter, tuf promoter, etc. are exemplified, but are not particularly limited to these.
  • Transformed cells can be obtained by introducing a vector containing the gene of the present invention into a host or by introducing a DNA fragment containing the gene of the present invention into the genome of the host.
  • Methods for introducing vectors or DNA fragments into hosts include, for example, the heat shock method, electroporation method, transformation method, transfection method, conjugation method, protoplast method, particle gun method, Agrobacterium method, and the like. be able to.
  • the method for introducing the gene of the present invention into the genome of the host is not particularly limited, but examples include a double crossover method using a DNA fragment containing the gene.
  • the DNA fragment may be introduced downstream of the promoter sequence of a gene highly expressed in the host cell described above, or a fragment previously prepared by operably linking the DNA fragment with the control region described above,
  • the ligated fragment may be introduced into the genome of the host.
  • the DNA fragment may be ligated in advance with a marker (drug resistance gene, auxotrophic complementary gene, etc.) for selecting cells into which the gene of the present invention has been appropriately introduced.
  • Transformed cells into which the vector or DNA fragment of interest has been introduced can be selected using a selection marker.
  • a selection marker is an antibiotic resistance gene
  • transformed cells into which the desired vector or DNA fragment has been introduced can be selected by culturing in the antibiotic-supplemented medium.
  • the selectable marker is an amino acid synthesis-related gene
  • transformed cells into which the target vector or DNA fragment has been introduced using the presence or absence of the amino acid auxotrophy as an indicator, after gene introduction into the host microorganism having the amino acid auxotrophy.
  • introduction of the desired vector or DNA fragment can be confirmed by examining the DNA sequence of transformed cells by PCR or the like.
  • the cultured cells of the microorganism thus obtained, the crushed product thereof, or the cell extract of the microorganism, protocatechuic acid, pyruvic acid, and an ammonium salt are brought into contact reaction.
  • the cultured cells of microorganisms used here are microbial culture solutions during and after culturing, cells isolated from microbial cultures, and powders by freeze-drying or spray-drying the cultured cells. powdered fungus bodies obtained by immobilizing the cultured fungus bodies on a carrier.
  • the disrupted bacterial cells include a disrupted bacterial cell solution containing protocatechuate decarboxylase and tyrosine phenol lyase of the present invention, a microsomal fraction, and the like.
  • the cell extract of the microorganism the cell of the microorganism is lysed by a bacteriophage, an agent such as an organic solvent or a surfactant, an enzyme, a mechanical force, a temperature shock, an osmotic shock, or the like. extracts of protocatechuate decarboxylase and tyrosine phenol lyase.
  • Microorganisms can be cultured using media (natural media, synthetic media) containing carbon sources, nitrogen sources, inorganic salts, and other nutrients.
  • Carbon sources include monosaccharides such as glucose, fructose, mannose, arabinose, xylose and galactose; disaccharides such as cellobiose, sucrose (sucrose), lactose, maltose, trehalose, cellobiose and xylobiose; dextrin or soluble starch.
  • 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 paraffins and the like
  • Nitrogen sources include, for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, corn steep liquor, alkylamines such as methylamine, nitrogen-containing organic compounds such as amino acids, ammonia or salts thereof.
  • inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate
  • urea aqueous ammonia, sodium nitrate, potassium nitrate and the like
  • inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate.
  • Other nutritional substances include soybean protein hydrolysates, amino acids and the like. Tyrosine or tyrosine substitutes, vitamin B6s, catechol, 3,4-dihydroxyphenylalanine, protocatechuic acid, antifoaming agents and the like can be added to the medium.
  • a culture temperature of 15°C to 45°C is appropriate.
  • the culture pH and culture time are not particularly limited, but, for example, a method of continuing culture for 6 to 72 hours while controlling the pH to 6.0 to 8.0 can be employed.
  • antibiotics such as ampicillin and kanamycin may be added to the medium during the culture, if necessary.
  • the contact reaction between the cultured cells of the microorganism or the crushed product thereof or the cell extract of the microorganism and protocatechuic acid, pyruvic acid and ammonium salt is carried out by mixing these and usually at 20 ° C. to 50 ° C. for a predetermined time ( For example, 30 minutes to 60 hours, preferably 1 hour to 24 hours), with stirring or shaking as necessary.
  • the origin of protocatechuic acid used as a raw material is not particularly limited, and it may be produced by any of an organic synthesis method and a method using microorganisms or enzymes. is preferably produced from saccharides such as glucose.
  • the concentration of protocatechuic acid is preferably 0.5-100 g/L, more preferably 5-15 g/L.
  • the concentration of pyruvic acid is preferably 0.5-100 g/L, more preferably 5-15 g/L.
  • the concentration of the ammonium salt is preferably 5-150 g/L, more preferably 50-80 g/L.
  • the concentration of the cultured cells, the crushed product thereof, or the cell extract of the microorganism is suitably in the range of 2 to 400 g/L, preferably 50 to 200 g/L. 3,4-dihydroxyphenylalanine can be accumulated by intermittently adding protocatechuic acid, pyruvic acid, and an ammonium salt to the reaction solution.
  • reducing agents such as sodium sulfite, sodium ascorbate and cysteine, chelating agents such as EDTA and citric acid, vitamin B6 compounds such as pyridoxal phosphate, and buffers such as Tris/HCl are added to the reaction system.
  • concentration of the reducing agent is preferably 0.5-20 g/L, more preferably 2-5 g/L.
  • the concentration of the chelating agent is preferably 0.5-20 g/L, more preferably 2-5 g/L.
  • concentration of pyridoxal phosphate is preferably 0.05-5 mM, more preferably 0.5-2 mM.
  • the buffer concentration is preferably 5-200 mM, more preferably 50-100 mM.
  • the reaction temperature is preferably 5 to 60°C, more preferably 15 to 30°C.
  • the reaction pH is suitably in the range of 5.0-10.0, preferably 6.5-8.5.
  • the reaction time is appropriately determined depending on the amount of activity of protocatechuate decarboxylase and tyrosine phenol lyase used and the substrate concentration, and is usually 0.5 to 72 hours, preferably 6 to 24 hours.
  • the 3,4-dihydroxyphenylalanine produced in the reaction solution is separated and recovered by centrifugation, filtration, etc., and then combined with conventional methods such as ion-exchange resin treatment and crystallization as appropriate. can be further purified by
  • ⁇ 1> including a step of contact-reacting protocatechuic acid, pyruvic acid and ammonium salt with cultured cells of a microorganism that produces protocatechuate decarboxylase and tyrosine phenol lyase, a crushed product thereof, or a cell extract of the microorganism; A method for producing 4-dihydroxyphenylalanine.
  • ⁇ 2> The method of ⁇ 1>, wherein the microorganism has genes encoding protocatechuate decarboxylase and tyrosine phenol lyase.
  • ⁇ 3> The method of ⁇ 2>, wherein the microorganism further has a gene encoding flavin mononucleotide prenyltransferase.
  • ⁇ 4> The method of ⁇ 2> or ⁇ 3>, wherein at least one of the genes is a foreign gene.
  • the tyrosine phenol lyase has an activity inhibition rate by protocatechuic acid of 91% or less, preferably 75% or less, more preferably 50% or less, more preferably 40% or less, still more preferably 36% or less. , ⁇ 1> to ⁇ 4>.
  • the tyrosine phenol lyase is a protein selected from the group consisting of the following (B1), (B2), (C1), (C2), (D1) and (D2), ⁇ 1> to ⁇ 5 > either method.
  • B1 a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
  • B2 consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2
  • C1 sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6.
  • Protein consisting of an amino acid sequence (D2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having tyrosine phenol lyase activity ⁇ 7>
  • Tyrosine phenol lyase is the above (B1) Or the method of ⁇ 6>, which is the protein of (B2).
  • ⁇ 8> The method according to any one of ⁇ 1> to ⁇ 7>, wherein the protocatechuate decarboxylase is the following protein (A1) or (A2).
  • A1 A protein consisting of the amino acid sequence shown in SEQ ID NO: 18
  • A2 A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 18 and having protocatechuic acid decarboxylase activity
  • E1 A protein consisting of the amino acid sequence shown in SEQ ID NO: 24
  • E2 A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24 and having flavin mononucleotide prenyltransferase activity
  • ⁇ 10 > The method according to any one of ⁇ 1> to ⁇ 9>, wherein the protocatechuic acid is produced from a saccharide as a starting material.
  • ⁇ 11> The method according to any one of ⁇ 1> to ⁇ 10>, comprising the step of recovering 3,4-dihydroxyphenylalanine from the reaction solution.
  • ⁇ 12> A vector or DNA fragment containing a polynucleotide encoding protocatechuate decarboxylase and tyrosine phenol lyase.
  • Tyrosine phenol lyase is a protein selected from the group consisting of the following (B1), (B2), (C1), (C2), (D1) and (D2) ⁇ 12> or ⁇ 13 > vectors or DNA fragments.
  • (B1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, and having tyrosine phenol lyase activity (C1) sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4. Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6.
  • Protocatechuate decarboxylase has the following ( The vector or DNA fragment of any one of ⁇ 12> to ⁇ 14>, which is the protein of A1) or (A2).
  • A1 A protein consisting of the amino acid sequence shown in SEQ ID NO: 18
  • A2 A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 18 and having protocatechuic acid decarboxylase activity ⁇ 16>
  • E1 A protein consisting of the amino acid sequence shown in SEQ ID NO: 24
  • E2 A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24 and having flavin mononucleotide prenyltransferase activity
  • ⁇ 17 > A transformed cell containing the vector or DNA fragment of any one of ⁇ 12> to ⁇ 16>.
  • ⁇ 18> The method according to any one of ⁇ 1> to ⁇ 11>, wherein the microorganism is the transformed cell of ⁇ 17>.
  • Example 1 Preparation of Plasmids Containing Various Genes
  • PCR was performed using KOD One PCR Master Mix (Toyobo).
  • plasmids pET_FnTPL, pET_CfTPL, pET_EwTPL
  • FnTPL genes encoding tyrosine phenol lyase
  • FnTPL Fusobacterium nucleatum
  • SEQ ID NO: 3 A DNA fragment (SEQ ID NO: 3) of a gene encoding tyrosine phenol lyase (CfTPL) derived from Citrobacter freundii and a gene encoding tyrosine phenol lyase (EwTPL) derived from Erwinia herbicola
  • SEQ ID NO: 5 was prepared by artificial gene synthesis (Eurfin Genomics), and using this
  • Each plasmid (pET_FnTPL, pET_CfTPL, pET_EwTPL) was constructed by ligating the purified DNA fragments with an In-Fusion HD Cloning Kit (Takara Bio Inc.). Using the obtained plasmid solution, ECOS competent E. coli DH5 ⁇ (Nippon Gene), and spread the cell solution on LBAmp agar medium (Bacto Tryptone 1%, Yeast Extract 0.5%, NaCl 1%, ampicillin sodium 50 ⁇ g/mL, agar 1.5%).
  • a DNA fragment for insert was obtained by PCR using primers EcoroYins F (SEQ ID NO: 19, GAAGGAGATATACATATGCAAAACCCGATAAATGAC) and EcoroYins R (SEQ ID NO: 20, GTGGTGGTGGTGGTGTTATTTCTTATCGCTAAATAACTC). Subsequently, a DNA fragment for a vector was obtained by PCR using primers pET vec F (SEQ ID NO: 13) and pET vec R (SEQ ID NO: 14) and pET21a plasmid as a template.
  • each DNA fragment was purified by NucleoSpin Gel and PCR clean-up (Takara Bio Inc.).
  • a plasmid (pET_EcAroY) was constructed by ligating the purified DNA fragment using In-Fusion HD Cloning Kit (Takara Bio Inc.).
  • ECOS competent E. coli DH5 ⁇ (Nippon Gene)
  • the cell solution was spread on an LBAmp agar medium, allowed to stand overnight at 37° C., and the resulting colonies were treated with the primer pET CPCR2 F (SEQ ID NO: 21, AGATCTCGATCCCGCGAAAT).
  • pET CPCR2 R (SEQ ID NO: 22, TTTAGAGGCCCCAAGGGGTT) was performed to select a transformant in which introduction of the target DNA fragment was confirmed.
  • the resulting transformants were inoculated into 2 mL of LBAmp liquid medium and cultured overnight at 37°C.
  • a plasmid was purified from this culture medium using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
  • a vector DNA fragment was obtained by PCR using primers EcAroY vec R (SEQ ID NO: 27, GATTCAAACCTCCTTTATTTCTTATCGCTAAATAACTCTG) and EcAroY vec F (SEQ ID NO: 28, CACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC) for the plasmid (pET_EcAroY) prepared in (a). .
  • EcAroY vec R SEQ ID NO: 27, GATTCAAACCTCCTTTATTTCTTATCGCTAAATAACTCTG
  • EcAroY vec F SEQ ID NO: 28, CACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC
  • the target plasmid (pET_EcAroY-EcUbiX) was constructed by ligating the purified DNA fragments with In-Fusion HD Cloning Kit (Takara Bio Inc.). Using the obtained plasmid solution, ECOS competent E. E. coli DH5 ⁇ (Nippon Gene) was transformed, the cell solution was spread on LBAmp agar medium, allowed to stand overnight at 37° C., and the resulting colonies were treated with primers pET CPCR2 F (SEQ ID NO: 21) and pET. A PCR reaction was performed using CPCR2 R (SEQ ID NO: 22), and transformants in which introduction of the target DNA fragment was confirmed were selected. The resulting transformants were inoculated into 2 mL of LBAmp liquid medium and cultured overnight at 37°C. A plasmid was purified from this culture medium using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
  • PCR was performed using primers EcAroY-EcUbiX vec F (SEQ ID NO: 35, AGGAGGTTTGATTCATGCAAAACCCGATAAATGAC) and EcAroY-EcUbiX vec R (SEQ ID NO: 36, ATGTATATCTCCTTCTTAAAGTTAA).
  • a vector DNA fragment was obtained by After treating these PCR products with DpnI (Takara Bio Inc.), each DNA fragment was purified by NucleoSpin Gel and PCR clean-up (Takara Bio Inc.).
  • the target plasmids (pET_FnTPL-EcAroY-EcUbiX, pET_CfTPL-EcAroY-EcUbiX, pET_EwTPL-EcAroY-EcUbiX) were constructed by ligating the purified DNA fragments using In-Fusion HD Cloning Kit (Takara Bio Inc.). Using the obtained plasmid solution, ECOS competent E. E. coli DH5 ⁇ (Nippon Gene) was transformed, the cell solution was spread on LBAmp agar medium, allowed to stand at 37° C.
  • Example 2 Evaluation of inhibition rate of tyrosine phenol lyase by protocatechuic acid (1) Introduction of plasmid into host cell Each plasmid (pET_FnTPL, pET_CfTPL, pET_EwTPL) obtained in Example 1(1) was used to transform ECOS competent E. coli. coli BL21(DE3) (Nippon Gene) was transformed by the heat shock method. The resulting transformed cell suspension was plated on an LBAmp agar medium and allowed to stand at 37° C. for 16 hours, and the resulting colonies were used as transformants.
  • ECOS competent E. coli. coli BL21(DE3) Nippon Gene
  • Example 3 Production of 3,4-dihydroxyphenylalanine using protocatechuic acid as a substrate using a bacterial cell catalyst
  • plasmid pET_FnTPL-EcAroY-EcUbiX, pET_CfTPL-EcAroY-EcUbiX, pET_EwTPL-EcAroY-EcUbiX
  • ECOS competent E. coli. coli BL21(DE3) Nippon Gene
  • the resulting transformed cell suspension was plated on an LBAmp agar medium and allowed to stand at 37° C. for 16 hours, and the resulting colonies were used as transformants.
  • the concentration of 3,4-dihydroxyphenylalanine in the resulting reaction solution was determined according to the method of Reference Example 1, and the ability to produce 3,4-dihydroxyphenylalanine using protocatechuic acid as a substrate was calculated according to the following formula.
  • Gradient elution was performed under the conditions of 70% methanol as the eluent B, a flow rate of 1.0 mL/min, and a column temperature of 40°C.
  • a UV detector detection wavelength 280 nm was used to detect 3,4-dihydroxyphenylalanine.
  • a concentration calibration curve was prepared using a standard sample [3,4-dihydroxy-L-phenylalanine (seller code A11311, Fujifilm Wako Pure Chemical Industries, Ltd.)], and based on the concentration calibration curve, 3,4-dihydroxyphenylalanine Quantification was performed.

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Abstract

Provided is a method for efficiently producing 3,4-dihydroxyphenylalanine from protocatechuic acid using cultured cells of a microorganism.　According to the present invention, a production method for 3,4-dihydroxyphenylalanine includes a step for performing a contact reaction between protocatechuic acid, pyruvic acid, and an ammonium salt and cultured cells, crushed cultured cells, or a cultured cell extract of a microorganism that produces protocatechuic acid decarboxylase and tyrosine phenol-lyase.

Description

ジヒドロキシフェニルアラニンの製造方法Method for producing dihydroxyphenylalanine
　本発明は、ジヒドロキシフェニルアラニンの製造方法に関する。 The present invention relates to a method for producing dihydroxyphenylalanine.
　３，４－ジヒドロキシ－Ｌ－フェニルアラニン（Ｌ－ＤＯＰＡ）は、ある種の植物（例えばムクナマメ）や哺乳類の体内で生合成される化合物である。哺乳類では非必須アミノ酸であるＬ－チロシンがチロシン水酸化酵素により水酸化されてＬ－ＤＯＰＡに変換され、さらにＬ－ＤＯＰＡ脱炭酸酵素により神経伝達物質であるドーパミンに変換される。すなわちＬ－ＤＯＰＡはドーパミンの前駆体である。また、Ｌ－ＤＯＰＡは血液脳関門を通過できることから、パーキンソン病の治療薬としても用いられる。 　3,4-Dihydroxy-L-phenylalanine (L-DOPA) is a compound biosynthesized in the body of certain plants (eg Mucuna bean) and mammals. In mammals, L-tyrosine, which is a non-essential amino acid, is hydroxylated by tyrosine hydroxylase and converted to L-DOPA, and further converted to dopamine, a neurotransmitter, by L-DOPA decarboxylase. L-DOPA is a precursor of dopamine. L-DOPA is also used as a therapeutic agent for Parkinson's disease because it can pass through the blood-brain barrier.
　従来、Ｌ－ＤＯＰＡの製造法として、バニリンを原料とする化学合成法や、微生物の有する酵素系を用いた方法等が知られ、安価且つ効率的な方法として、β－チロシナーゼ（チロシンフェノールリアーゼ）活性を有するエルウィニア属微生物の培養物をカテコール、ピルビン酸及びアンモニウムイオン、又はカテコール及びＬ－セリンに接触反応させて製造する方法が報告されている（例えば、特許文献１）。 Conventionally, as a method for producing L-DOPA, a chemical synthesis method using vanillin as a raw material, a method using an enzyme system possessed by microorganisms, etc. are known. A method has been reported in which a culture of an active Erwinia microorganism is brought into contact with catechol, pyruvic acid and ammonium ions, or catechol and L-serine (eg, Patent Document 1).
　一方、シキミ酸経路は、植物や微生物が芳香族化合物を生合成する重要な代謝経路である。シキミ酸経路では、炭素６員環の形成に続いて二重結合の形成が行われるが、３－デヒドロシキミ酸から誘導されるプロトカテク酸からは、没食子酸、カテコール等の芳香族化合物が生産されることが知られている。 On the other hand, the shikimate pathway is an important metabolic pathway for the biosynthesis of aromatic compounds by plants and microorganisms. In the shikimate pathway, the formation of a 6-carbon ring is followed by the formation of a double bond, and protocatechuic acid derived from 3-dehydroshikimic acid produces aromatic compounds such as gallic acid and catechol. It is known that
　　〔特許文献１〕特許第３１１６１０２号公報 [Patent Document 1] Patent No. 3116102
　本発明は、以下の１）～３）に係るものである。
　１）プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼを生産する微生物の培養菌体若しくはその破砕物又は当該微生物の菌体抽出物と、プロトカテク酸、ピルビン酸及びアンモニウム塩を接触反応させる工程を含む３，４－ジヒドロキシフェニルアラニンの製造方法。
　２）プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼをコードするポリヌクレオチドを含むベクター又はＤＮＡ断片。
　３）上記２）のベクター又はＤＮＡ断片を含有する形質転換細胞。 The present invention relates to the following 1) to 3).
1) A step of contact-reacting protocatechuic acid, pyruvic acid, and an ammonium salt with cultured cells of a microorganism producing protocatechuate decarboxylase and tyrosine phenol lyase, a crushed product thereof, or a cell extract of the microorganism. - A process for the preparation of dihydroxyphenylalanine.
2) A vector or DNA fragment containing a polynucleotide encoding protocatechuate decarboxylase and tyrosine phenol lyase.
3) A transformed cell containing the vector or DNA fragment of 2) above.
発明の詳細な説明Detailed description of the invention
　本発明は、微生物の培養菌体等を用いてプロトカテク酸から３，４－ジヒドロキシフェニルアラニンを効率よく製造する方法を提供することに関する。 The present invention relates to providing a method for efficiently producing 3,4-dihydroxyphenylalanine from protocatechuic acid using cultured cells of microorganisms.
　本発明者らは、プロトカテク酸に阻害を受けにくいチロシンフェノールリアーゼを見出し、プロトカテク酸デカルボキシラーゼ及び該チロシンフェノールリアーゼを生産する微生物を用いることにより、プロトカテク酸から３，４－ジヒドロキシフェニルアラニンを効率良く製造できることを見出した。 The present inventors have found a tyrosine phenol lyase that is less susceptible to inhibition by protocatechuic acid, and efficiently produce 3,4-dihydroxyphenylalanine from protocatechuic acid by using protocatechuic acid decarboxylase and a microorganism that produces the tyrosine phenol lyase. I found what I could do.
　本発明によれば、プロトカテク酸から３，４－ジヒドロキシフェニルアラニンを効率よく製造することが可能になる。プロトカテク酸はシキミ酸経路を介して生成されることから、本発明によれば、バイオマス糖を原料として３，４－ジヒドロキシフェニルアラニンの生産が可能となる。 According to the present invention, it becomes possible to efficiently produce 3,4-dihydroxyphenylalanine from protocatechuic acid. Since protocatechuic acid is produced via the shikimate pathway, the present invention enables the production of 3,4-dihydroxyphenylalanine using biomass sugar as a raw material.
　本発明において、アミノ酸配列又はヌクレオチド配列の同一性は、Ｌｉｐｍａｎ－Ｐｅａｒｓｏｎ法（Science,1985,227:1435-1441）によって計算される。具体的には、遺伝情報処理ソフトウェアＧＥＮＥＴＹＸ　Ｖｅｒ．１２のホモロジー解析（Ｓｅａｒｃｈ　ｈｏｍｏｌｏｇｙ）プログラムを用いて、Ｕｎｉｔ　ｓｉｚｅ　ｔｏ　ｃｏｍｐａｒｅ（ｋｔｕｐ）を２として解析を行うことにより算出される。 In the present invention, the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, genetic information processing software GENETYX Ver. 12 homology analysis (Search homology) program is used, unit size to compare (ktup) is set to 2, and analysis is performed.
　本発明において、アミノ酸配列及びヌクレオチド配列に関する「少なくとも９０％の同一性」とは、９０％以上、好ましくは９５％以上、より好ましくは９６％以上、さらに好ましくは９７％以上、さらにより好ましくは９８％以上、なお好ましくは９９％以上の同一性をいう。 In the present invention, "at least 90% identity" with respect to amino acid sequences and nucleotide sequences means 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more. % or more, preferably 99% or more identity.
　本発明において、「１又は数個のアミノ酸が欠失、置換、付加、又は挿入されたアミノ酸配列」とは、１個以上２０個以下、好ましくは１個以上１５個以下、より好ましくは１個以上１０個以下、より好ましくは１個以上５個以下、さらにより好ましくは１個以上３個以下のアミノ酸が欠失、置換、付加、又は挿入されたアミノ酸配列をいう。また、「１又は数個のヌクレオチドが欠失、置換、付加、又は挿入されたヌクレオチド配列」とは、１個以上３０個以下、好ましくは１個以上２４個以下、より好ましくは１個以上１５個以下、さらにより好ましくは１個以上９個以下のヌクレオチドが欠失、置換、付加、又は挿入されたヌクレオチド配列をいう。本発明において、アミノ酸又はヌクレオチドの「付加」には、配列の一末端及び両末端へのアミノ酸又はヌクレオチドの付加が含まれる。
　斯かる欠失、置換、挿入、付加等の変異の導入は、例えば部位特異的な変異導入法等により、対象の塩基配列に変異を導入することにより実施できる。 In the present invention, "an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted" means 1 or more and 20 or less, preferably 1 or more and 15 or less, more preferably 1 It refers to an amino acid sequence in which 10 or less, more preferably 1 or more and 5 or less, still more preferably 1 or more and 3 or less amino acids are deleted, substituted, added, or inserted. In addition, "a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted" means 1 or more and 30 or less, preferably 1 or more and 24 or less, more preferably 1 or more and 15 It refers to a nucleotide sequence in which no more than 1, more preferably from 1 to 9 nucleotides have been deleted, substituted, added or inserted. In the present invention, "addition" of amino acids or nucleotides includes addition of amino acids or nucleotides to one and both termini of a sequence.
Mutations such as deletions, substitutions, insertions and additions can be introduced by introducing mutations into the base sequence of interest by, for example, site-directed mutagenesis.
　本発明において、遺伝子に関する「上流」及び「下流」とは、該遺伝子の転写方向の上流及び下流をいう。例えば、「プロモーターの下流に配置された遺伝子」とは、ＤＮＡセンス鎖においてプロモーターの３’側に該遺伝子が存在することを意味し、遺伝子の上流とは、ＤＮＡセンス鎖における該遺伝子の５’側の領域を意味する。 In the present invention, "upstream" and "downstream" with respect to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter in the DNA sense strand, and "upstream of the gene" means that the gene is located 5' of the gene in the DNA sense strand. means the area on the side.
　本発明において、プロモーター等の制御領域と遺伝子の「作動可能な連結」とは、遺伝子と制御領域とが、該遺伝子が該制御領域の制御の下で発現し得るように連結されていることをいう。遺伝子と制御領域との「作動可能な連結」の手順は当業者に周知である。 In the present invention, "operably linked" between a control region such as a promoter and a gene means that the gene and the control region are linked so that the gene can be expressed under the control of the control region. say. Procedures for "operably linking" genes and regulatory regions are well known to those of skill in the art.
　本発明において、「外来遺伝子」とは、細胞に外部から導入された外因性の遺伝子である。外来遺伝子は、それが導入された細胞と同種の生物由来であっても、異種の生物由来（すなわち異種遺伝子）であってもよい。 In the present invention, a "foreign gene" is an exogenous gene introduced into a cell from the outside. A foreign gene may be derived from the same organism as the cell into which it is introduced, or from a different organism (ie, a heterologous gene).
　本発明の方法において、３，４－ジヒドロキシフェニルアラニン（ＤＯＰＡ）は、プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼを生産する微生物の培養菌体若しくはその破砕物又は当該微生物の菌体抽出物と、プロトカテク酸（ＰＣＡ）、ピルビン酸及びアンモニウム塩を接触反応させることにより製造される。
　当該反応は、下記式で示すように、プロトカテク酸がプロトカテク酸デカルボキシラーゼによってカテコールに変換された後、当該カテコールとピルビン酸及びアンモニウム塩がチロシンフェノールリアーゼ（ＴＰＬ）によって反応して、３，４－ジヒドロキシフェニルアラニンを生成する反応である。 In the method of the present invention, 3,4-dihydroxyphenylalanine (DOPA) is a mixture of cultured cells of a microorganism that produces protocatechuate decarboxylase and tyrosine phenol lyase, a lysate thereof, or a cell extract of the microorganism, and protocatechuic acid ( PCA), pyruvic acid and an ammonium salt.
As shown in the following formula, protocatechuic acid is converted to catechol by protocatechuic acid decarboxylase, and then the catechol, pyruvic acid and ammonium salt react with tyrosine phenol lyase (TPL) to form 3,4- It is a reaction that produces dihydroxyphenylalanine.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　本発明において、３，４－ジヒドロキシフェニルアラニンは、３，４－ジヒドロキシ－Ｌ－フェニルアラニン（Ｌ-ＤＯＰＡ）及び３，４－ジヒドロキシ－Ｄ－フェニルアラニン（Ｄ-ＤＯＰＡ）を含むが、３，４－ジヒドロキシ－Ｌ－フェニルアラニン（Ｌ-ＤＯＰＡ）が好ましい。 In the present invention, 3,4-dihydroxyphenylalanine includes 3,4-dihydroxy-L-phenylalanine (L-DOPA) and 3,4-dihydroxy-D-phenylalanine (D-DOPA), but 3,4-dihydroxy -L-phenylalanine (L-DOPA) is preferred.
　本発明においてＤＯＰＡは、両性イオン又は塩の形態であっても良く、塩としては、例えば、ナトリウム塩、カリウム塩、カルシウム塩、塩酸塩、硫酸塩などを挙げることができる。これらの中でも、塩酸塩、硫酸塩がより好ましい。 In the present invention, DOPA may be in the form of zwitterions or salts, and examples of salts include sodium salts, potassium salts, calcium salts, hydrochlorides, and sulfates. Among these, hydrochlorides and sulfates are more preferable.
　本発明の微生物は、プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼの生産能を有し、具体的にはプロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼをコードする遺伝子を有する微生物が挙げられる。
　プロトカテク酸デカルボキシラーゼは、プロトカテク酸を脱炭酸してカテコールを生成する反応を触媒する酵素をいう。プロトカテク酸デカルボキシラーゼ活性を有する酵素としては、プロトカテク酸デカルボキシラーゼ（Ｐｒｏｔｏｃａｔｅｃｈｕａｔｅ　ｄｅｃａｒｂｏｘｙｌａｓｅ、ＥＣ　４．１．１．６３）、没食子酸デカルボキシラーゼ（Ｇａｌｌｉｃ　ａｃｉｄ　ｄｅｃａｒｂｏｘｙｌａｓｅ、ＥＣ　４．１．１．５９）、４－ヒドロキシ安息香酸デカルボキシラーゼ（４－ｈｙｄｒｏｘｙｂｅｎｚｏａｔｅ　ｄｅｃａｒｂｏｘｙｌａｓｅ、ＥＣ　４．１．１．６１）等が挙げられるが、プロトカテク酸デカルボキシラーゼ（ＥＣ　４．１．１．６３）が好ましい。
　プロトカテク酸デカルボキシラーゼとしては、例えば、以下の（Ａ１）又は（Ａ２）のタンパク質が挙げられる。
（Ａ１）配列番号１８に示すアミノ酸配列からなるタンパク質
（Ａ２）配列番号１８に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつプロトカテク酸デカルボキシラーゼ活性を有するタンパク質
　配列番号１８のアミノ酸配列からなるタンパク質は、エンテロバクター・クロアカ（Ｅｎｔｅｒｏｂａｃｔｅｒ　ｃｌｏａｃａｅ）由来のプロトカテク酸デカルボキシラーゼ（ＥｃＡｒｏＹ）である。 The microorganism of the present invention has the ability to produce protocatechuate decarboxylase and tyrosine phenol lyase, and specifically includes a microorganism having genes encoding protocatechuate decarboxylase and tyrosine phenol lyase.
Protocatechuic acid decarboxylase refers to an enzyme that catalyzes the reaction of decarboxylating protocatechuic acid to produce catechol. Enzymes having protocatechuate decarboxylase activity include protocatechuate decarboxylase (EC 4.1.1.63), gallic acid decarboxylase (EC 4.1.1.59), 4- Hydroxybenzoate decarboxylase (4-hydroxybenzoate decarboxylase, EC 4.1.1.61) and the like, and protocatechuate decarboxylase (EC 4.1.1.63) is preferred.
Protocatechuate decarboxylase includes, for example, the following proteins (A1) or (A2).
(A1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 18 (A2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 18 and having protocatechuic acid decarboxylase activity SEQ ID NO: 18 is protocatechuate decarboxylase (EcAroY) derived from Enterobacter cloacae.
　（Ａ２）のタンパク質において、プロトカテク酸デカルボキシラーゼ活性の点から、配列番号１８のアミノ酸配列との同一性は、９５％以上であることが好ましく、より好ましくは９７％以上であり、９８％以上がさらに好ましく、９９％以上がよりさらに好ましい。
　当該（Ａ２）のタンパク質のアミノ酸配列には、例えば配列番号１８のアミノ酸配列において１又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列が包含される。
　また、当該タンパク質がプロトカテク酸デカルボキシラーゼ活性を有することは、例えば、タンパク質を基質（すなわちプロトカテク酸）とインキュベートし、当該タンパク質及び基質依存的なカテコールの生成を測定することにより、確認することができる。 In the protein (A2), the identity with the amino acid sequence of SEQ ID NO: 18 is preferably 95% or more, more preferably 97% or more, from the viewpoint of protocatechuate decarboxylase activity, and 98% or more is More preferably, 99% or more is even more preferable.
The amino acid sequence of the protein (A2) includes, for example, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence of SEQ ID NO:18.
In addition, whether the protein has protocatechuic acid decarboxylase activity can be confirmed, for example, by incubating the protein with a substrate (that is, protocatechuic acid) and measuring the protein- and substrate-dependent catechol production. .
　プロトカテク酸デカルボキシラーゼ遺伝子としては、例えば、前記（Ａ１）又は（Ａ２）のタンパク質をコードする遺伝子が挙げられる。
　具体的には、以下の（ａ１）又は（ａ２）のポリヌクレオチドが挙げられる。
　（ａ１）配列番号１７に示すヌクレオチド配列からなるポリヌクレオチド
　（ａ２）配列番号１７に示すヌクレオチド配列と少なくとも９０％以上の同一性を有するヌクレオチド配列からなり、プロトカテク酸デカルボキシラーゼ活性を有するポリペプチドをコードするポリヌクレオチド
　ここで、配列番号１７に示すヌクレオチド配列からなるポリヌクレオチドは、エンテロバクター・クロアカ（Ｅｎｔｅｒｏｂａｃｔｅｒ　ｃｌｏａｃａｅ）由来プロトカテク酸デカルボキシラーゼ（ＥｃＡｒｏＹ）をコードする遺伝子を示す。
　（ａ２）のヌクレオチドにおいて、プロトカテク酸デカルボキシラーゼ活性の点から、配列番号１７のヌクレオチド配列との同一性は、９５％以上であることが好ましく、より好ましくは９７％以上であり、９８％以上がさらに好ましく、９９％以上がよりさらに好ましい。
　当該（ａ２）のポリヌクレオチドのヌクレオチド配列には、例えば配列番号１７で示されるヌクレオチド配列に対して１又は数個のヌクレオチドが欠失、置換、付加、又は挿入されたヌクレオチド配列が包含される。 The protocatechuate decarboxylase gene includes, for example, the gene encoding the protein (A1) or (A2).
Specific examples include the following (a1) or (a2) polynucleotides.
(a1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 (a2) comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 17 and encoding a polypeptide having protocatechuate decarboxylase activity Polynucleotide Here, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 represents a gene encoding protocatechuate decarboxylase (EcAroY) derived from Enterobacter cloacae.
The identity of the nucleotide of (a2) with the nucleotide sequence of SEQ ID NO: 17 is preferably 95% or more, more preferably 97% or more, in terms of protocatechuate decarboxylase activity, and 98% or more is More preferably, 99% or more is even more preferable.
The nucleotide sequence of the polynucleotide of (a2) includes, for example, a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted with respect to the nucleotide sequence shown in SEQ ID NO:17.
　本発明において、チロシンフェノールリアーゼ（Ｔｙｒｏｓｉｎｅ　ｐｈｅｎｏｌ－ｌｙａｓｅ、ＥＣ　４．１．９９．２）は、Ｌ－チロシンをフェノール、ピルビン酸、アンモニアに分解する反応又はその逆反応を触媒する酵素である。また、チロシンフェノールリアーゼは、３，４－ジヒドロキシフェニルアラニンをカテコール、ピルビン酸、アンモニアに分解する反応又はその逆反応も触媒することが可能である。
　チロシンフェノールリアーゼによるカテコールから３，４－ジヒドロキシフェニルアラニンへの変換反応を含む反応系において、チロシンフェノールリアーゼはカテコール又はその前駆体であるプロトカテク酸により、その活性が阻害されることが考えられる。
　したがって、本発明において用いられるチロシンフェノールリアーゼとしては、プロトカテク酸による活性阻害率が低いものであるのが好ましく、例えば、後述する実施例２で示される条件２～６の何れかの条件において、下記式で算出される阻害率が９１％以下、好ましくは９０％以下、好ましくは７５％以下、より好ましくは５０％以下、より好ましくは４０％以下、さらに好ましくは３６％以下であるものが挙げられる。 In the present invention, tyrosine phenol-lyase (EC 4.1.99.2) is an enzyme that catalyzes the reaction of degrading L-tyrosine into phenol, pyruvic acid and ammonia or the reverse reaction thereof. Tyrosine phenol lyase can also catalyze the decomposition of 3,4-dihydroxyphenylalanine into catechol, pyruvate and ammonia, or the reverse reaction.
In a reaction system involving conversion of catechol to 3,4-dihydroxyphenylalanine by tyrosine phenol lyase, the activity of tyrosine phenol lyase is thought to be inhibited by catechol or its precursor, protocatechuic acid.
Therefore, the tyrosine phenol lyase used in the present invention preferably exhibits a low rate of activity inhibition by protocatechuic acid. The inhibition rate calculated by the formula is 91% or less, preferably 90% or less, preferably 75% or less, more preferably 50% or less, more preferably 40% or less, and still more preferably 36% or less. .
（数１）
　チロシンフェノールリアーゼのプロトカテク酸による阻害率（％）＝１００×［１－プロトカテク酸存在下におけるカテコールから３,４－ジヒドロキシフェニルアラニンへの変換率（％）〕／〔プロトカテク酸非存在下におけるカテコールから３,４－ジヒドロキシフェニルアラニンへの変換率（％）〕］
　ここで、カテコールから３,４－ジヒドロキシフェニルアラニンへの変換率（％）は、１００×〔反応終了時の３,４－ジヒドロキシフェニルアラニンの物質量（ｍｏｌ）〕／〔反応開始前のカテコールの物質量（ｍｏｌ）〕により算出される。 (Number 1)
Inhibition rate of tyrosine phenol lyase by protocatechuic acid (%) = 100 × [conversion rate (%) of catechol to 3,4-dihydroxyphenylalanine in the presence of 1-protocatechuic acid]/[3 from catechol in the absence of protocatechuic acid , Conversion rate to 4-dihydroxyphenylalanine (%)]]
Here, the conversion rate (%) of catechol to 3,4-dihydroxyphenylalanine is 100×[substance amount (mol) of 3,4-dihydroxyphenylalanine at the end of the reaction]/[substance amount of catechol before starting the reaction. (mol)].
　本発明のチロシンフェノールリアーゼとしては、好適には、例えば以下の（Ｂ１）、（Ｂ２）、（Ｃ１）、（Ｃ２）、（Ｄ１）及び（Ｄ２）からなる群より選択されるタンパク質が挙げられる。
（Ｂ１）配列番号２に示すアミノ酸配列からなるタンパク質
（Ｂ２）配列番号２に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
（Ｃ１）配列番号４に示すアミノ酸配列からなるタンパク質
（Ｃ２）配列番号４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
（Ｄ１）配列番号６に示すアミノ酸配列からなるタンパク質
（Ｄ２）配列番号６に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質 The tyrosine phenol lyase of the present invention preferably includes, for example, proteins selected from the group consisting of (B1), (B2), (C1), (C2), (D1) and (D2) below. .
(B1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, and having tyrosine phenol lyase activity (C1) sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4. Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6. Protein consisting of an amino acid sequence (D2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having tyrosine phenol lyase activity
　配列番号２のアミノ酸配列からなるタンパク質は、フソバクテリウム・ヌクレアタム(Ｆｕｓｏｂａｃｔｅｒｉｕｍ　ｎｕｃｌｅａｔｕｍ)由来のチロシンフェノールリアーゼ(ＦｎＴＰＬ)である。
　配列番号４のアミノ酸配列からなるタンパク質は、シトロバクタ―・フレウンディー(Ｃｉｔｒｏｂａｃｔｅｒ ｆｒｅｕｎｄｉｉ)由来のチロシンフェノールリアーゼ(ＣｆＴＰＬ)である。
　配列番号６のアミノ酸配列からなるタンパク質は、エルウィニア・ヘルビコラ(Ｅｒｗｉｎｉａ ｈｅｒｂｉｃｏｌａ)由来のチロシンフェノールリアーゼ(ＥｗＴＰＬ)である。　 The protein consisting of the amino acid sequence of SEQ ID NO: 2 is a tyrosine phenol lyase (FnTPL) derived from Fusobacterium nucleatum.
The protein consisting of the amino acid sequence of SEQ ID NO: 4 is Citrobacter freundii-derived tyrosine phenol lyase (CfTPL).
The protein consisting of the amino acid sequence of SEQ ID NO: 6 is Erwinia herbicola-derived tyrosine phenol lyase (EwTPL).
　（Ｂ２）、（Ｃ２）又は（Ｄ２）のタンパク質において、チロシンフェノールリアーゼ活性の点から、それぞれ配列番号２、配列番号４又は配列番号６のアミノ酸配列との同一性は、９５％以上であることが好ましく、より好ましくは９７％以上であり、９８％以上がさらに好ましく、９９％以上がよりさらに好ましい。
　当該（Ｂ２）、（Ｃ２）又は（Ｄ２）のタンパク質のアミノ酸配列には、例えば配列番号２、配列番号４又は配列番号６のアミノ酸配列において１又は数個のアミノ酸が欠失、置換、挿入、又は付加されたアミノ酸配列が包含される。
　また、当該タンパク質がチロシンフェノールリアーゼ活性を有することは、例えば、タンパク質を基質（すなわちフェノール又はカテコール、ピルビン酸及びアンモニウム塩）とインキュベートし、当該タンパク質及び基質依存的なチロシン又は３，４－ジヒドロキシフェニルアラニンの生成を測定することにより、確認することができる。 The protein (B2), (C2) or (D2) has 95% or more identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, respectively, in terms of tyrosine phenol lyase activity. is preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more.
The amino acid sequence of the protein (B2), (C2) or (D2) includes, for example, deletion, substitution, insertion of one or several amino acids in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or added amino acid sequences are included.
The protein also has tyrosine phenol lyase activity, for example, by incubating the protein with a substrate (ie, phenol or catechol, pyruvic acid and ammonium salts), the protein and substrate-dependent tyrosine or 3,4-dihydroxyphenylalanine can be confirmed by measuring the generation of
　本発明のチロシンフェノールリアーゼ遺伝子としては、前記（Ｂ１）、（Ｂ２）、（Ｃ１）、（Ｃ２）、（Ｄ１）及び（Ｄ２）の各タンパク質をコードする遺伝子が挙げられる。
　具体的には、以下の（ｂ１）、（ｂ２）、（ｃ１）、（ｃ２）、（ｄ１）及び（ｄ２）からなる群より選択されるポリヌクレオチドが挙げられる。
　（ｂ１）配列番号１に示すヌクレオチド配列からなるポリヌクレオチド
　（ｂ２）配列番号１に示すヌクレオチド配列と少なくとも９０％以上の同一性を有するヌクレオチド配列からなり、チロシンフェノールリアーゼ活性を有するポリペプチドをコードするポリヌクレオチド
　（ｃ１）配列番号３に示すヌクレオチド配列からなるポリヌクレオチド
　（ｃ２）配列番号３に示すヌクレオチド配列と少なくとも９０％以上の同一性を有するヌクレオチド配列からなり、チロシンフェノールリアーゼ活性を有するポリペプチドをコードするポリヌクレオチド
　（ｄ１）配列番号５に示すヌクレオチド配列からなるポリヌクレオチド
　（ｄ２）配列番号５に示すヌクレオチド配列と少なくとも９０％以上の同一性を有するヌクレオチド配列からなり、チロシンフェノールリアーゼ活性を有するポリペプチドをコードするポリヌクレオチド The tyrosine phenol lyase gene of the present invention includes genes encoding the above proteins (B1), (B2), (C1), (C2), (D1) and (D2).
Specific examples include polynucleotides selected from the group consisting of (b1), (b2), (c1), (c2), (d1) and (d2) below.
(b1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1; (b2) 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 tyrosine phenol lyase activity Polynucleotide (c1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 (c2) a polypeptide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and having tyrosine phenol lyase activity Polynucleotide encoding (d1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 (d2) a polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 5 and having tyrosine phenol lyase activity A polynucleotide encoding a peptide
　ここで、配列番号１に示すヌクレオチド配列からなるポリヌクレオチドは、フソバクテリウム・ヌクレアタム(Ｆｕｓｏｂａｃｔｅｒｉｕｍ　ｎｕｃｌｅａｔｕｍ)由来のチロシンフェノールリアーゼ(ＦｎＴＰＬ)をコードする遺伝子を示す。
　配列番号３に示すヌクレオチド配列からなるポリヌクレオチドは、シトロバクタ―・フレウンディー(Ｃｉｔｒｏｂａｃｔｅｒｆｒｅｕｎｄｉｉ)由来のチロシンフェノールリアーゼ(ＣｆＴＰＬ)をコードする遺伝子を示す。
　配列番号５に示すヌクレオチド配列からなるポリヌクレオチドは、エルウィニア・ヘルビコラ(Ｅｒｗｉｎｉａ ｈｅｒｂｉｃｏｌａ)由来のチロシンフェノールリアーゼ(ＥｗＴＰＬ)をコードする遺伝子を示す。 Here, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 represents a gene encoding tyrosine phenol lyase (FnTPL) derived from Fusobacterium nucleatum.
The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 represents a gene encoding tyrosine phenol lyase (CfTPL) derived from Citrobacter freundii.
The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 represents a gene encoding tyrosine phenol lyase (EwTPL) derived from Erwinia herbicola.
　（ｂ２）、（ｃ２）、（ｄ２）のヌクレオチドにおいて、チロシンフェノールリアーゼ活性の点から、配列番号１、配列番号３及び配列番号５のそれぞれのヌクレオチド配列との同一性は、９５％以上であることが好ましく、より好ましくは９７％以上であり、９８％以上がさらに好ましく、９９％以上がよりさらに好ましい。
　当該（ｂ２）、（ｃ２）又は（ｄ２）のポリヌクレオチドのヌクレオチド配列には、例えば配列番号１、配列番号３又は配列番号５で示されるヌクレオチド配列に対して１又は複数個のヌクレオチドが欠失、置換、付加、又は挿入されたヌクレオチド配列が包含される。 The nucleotides (b2), (c2), and (d2) have 95% or more identity with the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 in terms of tyrosine phenol lyase activity. is preferably 97% or more, more preferably 98% or more, even more preferably 99% or more.
In the nucleotide sequence of the polynucleotide (b2), (c2) or (d2), for example, one or more nucleotides are deleted from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 , substituted, added, or inserted nucleotide sequences.
　斯かるチロシンフェノールリアーゼをコードする遺伝子のうち、プロトカテク酸による活性阻害を受けにくく、３，４－ジヒドロキシフェニルアラニンの生産効率に優れる点で、フソバクテリウム・ヌクレアタム由来のチロシンフェノールリアーゼ（ＦｎＴＰＬ）の遺伝子、具体的には、上記（Ｂ１）又は（Ｂ２）で示されるタンパク質をコードする遺伝子、さらに具体的には上記（ｂ１）又は（ｂ２）で示されるヌクレオチド配列からなるポリヌクレオチドが好ましい。 Among the genes encoding such tyrosine phenol lyases, the Fusobacterium nucleatum-derived tyrosine phenol lyase (FnTPL) gene is particularly resistant to activity inhibition by protocatechuic acid and has excellent production efficiency of 3,4-dihydroxyphenylalanine. Specifically, a gene encoding the protein shown in (B1) or (B2) above, more specifically a polynucleotide consisting of the nucleotide sequence shown in (b1) or (b2) above is preferred.
　本発明の微生物は、プロトカテク酸によるチロシンフェノールリアーゼの活性阻害を抑制する点から、さらにフラビンモノヌクレオチドプレニルトランスフェラーゼ生産能を有するもの、すなわちフラビンモノヌクレオチドプレニルトランスフェラーゼをコードする遺伝子を有するのが好ましい。
　フラビンモノヌクレオチドプレニルトランスフェラーゼ（Ｆｌａｖｉｎ　ｐｒｅｎｙｌｔｒａｎｓｆｅｒａｓｅ、ＥＣ　２．５．１．１２９）は、ジメチルアリル一リン酸（ＤＭＡＰ）からジメチルアリル構造をフラビンモノヌクレオチド（ＦＭＮ）のフラビン骨格へと結合し、ｐｒｅｎｙｌａｔｅｄ－ＦＭＮを合成する反応を触媒する酵素である。
　本発明のフラビンモノヌクレオチドプレニルトランスフェラーゼとしては、例えば、以下の（Ｅ１）又は（Ｅ２）のタンパク質が挙げられる。
（Ｅ１）配列番号２４に示すアミノ酸配列からなるタンパク質
（Ｅ２）配列番号２４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつフラビンモノヌクレオチドプレニルトランスフェラーゼ活性を有するタンパク質
　配列番号２４のアミノ酸配列からなるタンパク質は、大腸菌由来フラビンモノヌクレオチドプレニルトランスフェラーゼ（ＥｃＵｂｉＸ）である。 From the viewpoint of suppressing inhibition of tyrosine phenol lyase activity by protocatechuic acid, the microorganism of the present invention preferably has the ability to produce flavin mononucleotide prenyltransferase, ie, a gene encoding flavin mononucleotide prenyltransferase.
Flavin mononucleotide prenyltransferase (EC 2.5.1.129) attaches a dimethylallyl structure from dimethylallyl monophosphate (DMAP) to the flavin backbone of flavin mononucleotide (FMN) to form prenylated-FMN. is an enzyme that catalyzes the synthesis of
Flavin mononucleotide prenyltransferases of the present invention include, for example, proteins (E1) or (E2) below.
(E1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 24 (E2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24 and having flavin mononucleotide prenyltransferase activity SEQ ID NO: The protein consisting of a 24 amino acid sequence is Escherichia coli-derived flavin mononucleotide prenyltransferase (EcUbiX).
　（Ｅ２）のタンパク質において、フラビンモノヌクレオチドプレニルトランスフェラーゼ活性の点から、配列番号２４のアミノ酸配列との同一性は、９５％以上であることが好ましく、より好ましくは９７％以上であり、９８％以上がさらに好ましく、９９％以上がよりさらに好ましい。
　当該（Ｅ２）のタンパク質のアミノ酸配列には、例えば配列番号２４のアミノ酸配列において１又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列が包含される。
　また、当該タンパク質がフラビンモノヌクレオチドプレニルトランスフェラーゼ活性を有することは、例えば、Ｎａｔｕｒｅ，２０１５，５２２：５０２－５０６等を参照でき、タンパク質をフラビンモノヌクレオチド（ＦＭＮ）及びジメチルアリル一リン酸（ＤＭＡＰ）の存在下でインキュベートし、ｐｒｅｎｙｌａｔｅｄ－ＦＭＮの生成による吸収スペクトル変化を追跡することにより、確認することができる。 In the protein (E2), the identity with the amino acid sequence of SEQ ID NO: 24 is preferably 95% or more, more preferably 97% or more, and 98% or more in terms of flavin mononucleotide prenyltransferase activity. is more preferred, and 99% or more is even more preferred.
The amino acid sequence of the protein (E2) includes, for example, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added to the amino acid sequence of SEQ ID NO:24.
In addition, the protein has flavin mononucleotide prenyltransferase activity, for example, see Nature, 2015, 522: 502-506, etc., the protein is flavin mononucleotide (FMN) and dimethylallyl monophosphate (DMAP) It can be confirmed by incubating in the presence and tracking the absorption spectrum change due to the production of prenylated-FMN.
　本発明のフラビンモノヌクレオチドプレニルトランスフェラーゼ遺伝子としては、前記（Ｅ１）又は（Ｅ２）のタンパク質をコードする遺伝子が挙げられる。
　具体的には、下記（ｅ１）又は（ｅ２）のポリヌクレオチドが挙げられる。
　（ｅ１）配列番号２３に示すヌクレオチド配列からなるポリヌクレオチド
　（ｅ２）配列番号２３に示すヌクレオチド配列と少なくとも９０％以上の同一性を有するヌクレオチド配列からなり、フラビンモノヌクレオチドプレニルトランスフェラーゼ活性を有するポリペプチドをコードするポリヌクレオチド
　ここで、配列番号２３に示すヌクレオチド配列からなるポリヌクレオチドは、大腸菌由来フラビンモノヌクレオチドプレニルトランスフェラーゼ（ＥｃＵｂｉＸ）をコードする遺伝子を示す。
　（ｅ２）のヌクレオチドにおいて、フラビンモノヌクレオチドプレニルトランスフェラーゼ活性の点から、配列番号２３のヌクレオチド配列との同一性は、９５％以上であることが好ましく、より好ましくは９７％以上であり、９８％以上がさらに好ましく、９９％以上がよりさらに好ましい。
　当該（ｅ２）のポリヌクレオチドのヌクレオチド配列には、例えば配列番号２３で示されるヌクレオチド配列に対して１又は数個のヌクレオチドが欠失、置換、付加、又は挿入されたヌクレオチド配列が包含される。 The flavin mononucleotide prenyltransferase gene of the present invention includes a gene encoding the protein (E1) or (E2).
Specific examples include the following (e1) or (e2) polynucleotides.
(e1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:23; Encoding Polynucleotide Here, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 23 represents a gene encoding Escherichia coli-derived flavin mononucleotide prenyltransferase (EcUbiX).
The identity of the nucleotide (e2) with the nucleotide sequence of SEQ ID NO: 23 is preferably 95% or more, more preferably 97% or more, and 98% or more in terms of flavin mononucleotide prenyltransferase activity. is more preferred, and 99% or more is even more preferred.
The nucleotide sequence of the polynucleotide (e2) includes, for example, a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted with respect to the nucleotide sequence shown in SEQ ID NO:23.
　本発明の微生物は、前記プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼ、所望によりさらにフラビンモノヌクレオチドプレニルトランスフェラーゼをコードする遺伝子（これらを「本発明の遺伝子」とも称する）を有すればよいが、少なくとも１つが宿主微生物に対して外因性の遺伝子（外来遺伝子）であるのが好ましい。
　外来遺伝子が導入された微生物は、当該遺伝子を宿主細胞中で発現させることのできる発現ベクターや遺伝子発現カセットを調製し、これを宿主細胞に導入して宿主細胞を形質転換させることにより作製できる。 The microorganism of the present invention may have genes encoding protocatechuate decarboxylase and tyrosine phenol lyase, and optionally further flavin mononucleotide prenyltransferase (these are also referred to as "genes of the present invention"), but at least one It is preferably a gene exogenous to the host microorganism (foreign gene).
A foreign gene-introduced microorganism can be produced by preparing an expression vector or a gene expression cassette capable of expressing the gene in host cells, and then introducing it into host cells to transform the host cells.
　形質転換体の宿主としての微生物は原核生物、真核生物のいずれであってもよく、エシェリキア（Ｅｓｃｈｅｒｉｃｈｉａ）属に属する微生物やバシラス（Ｂａｃｉｌｌｕｓ）属に属する微生物、放線菌、コリネ型細菌等の原核生物、又は酵母や糸状菌等の真核微生物を用いることができる。なかでも、エシェリキア属に属する微生物である大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）、エルウィニア属に属する微生物であるエルウィニア・ヘルビコラ(Ｅｒｗｉｎｉａ ｈｅｒｂｉｃｏｌａ)、バシラス属に属する微生物である枯草菌（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ）、コリネバクテリウム属に属する微生物であるコリネバクテリウム・グルタミカム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ）、シュードモナス属に属する微生物であるシュードモナス・プチダ（Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ）、ロドコッカス属に属する微生物であるロドコッカス・ジョスティ（Ｒｈｏｄｏｃｏｃｃｕｓ　ｊｏｓｔｉｉ）が好ましく、大腸菌又はコリネバクテリウム・グルタミカムがより好ましい。 Microorganisms as hosts for transformants may be either prokaryotes or eukaryotes, and prokaryotes such as microorganisms belonging to the genus Escherichia, microorganisms belonging to the genus Bacillus, actinomycetes, and coryneform bacteria. Organisms or eukaryotic microorganisms such as yeast and filamentous fungi can be used. Among them, Escherichia coli which is a microorganism belonging to the genus Escherichia, Erwinia herbicola which is a microorganism belonging to the genus Erwinia, Bacillus subtilis which is a microorganism belonging to the genus Bacillus, and the genus Corynebacterium Corynebacterium glutamicum, which is a microorganism belonging to the genus Pseudomonas, Pseudomonas putida, which is a microorganism belonging to the genus Pseudomonas, and Rhodococcus jostii, which is a microorganism belonging to the genus Rhodococcus. Li Um glutamicum is more preferred.
　発現ベクターの母体となるベクターとしては、本発明の遺伝子を宿主に導入することができ、宿主細胞内で当該遺伝子を発現可能なベクターであればよい。好ましくは、該ベクターは、本発明の遺伝子、及びこれと作動可能に連結された制御領域を含む。該ベクターは、プラスミド等の染色体外で自立増殖及び複製可能なベクターであってもよく、又は染色体内に組み込まれるベクターであってもよい。
　具体的なベクターとしては、例えばｐＢｌｕｅｓｃｒｉｐｔ　ＩＩ　ＳＫ（－）（アジレント・テクノロジー社）、ｐＵＣ１８／１９、ｐＵＣ１１８／１１９等のｐＵＣ系ベクター（タカラバイオ社）、ｐＥＴ系ベクター（メルク社）、ｐＧＥＸ系ベクター（メルク社）、ｐＣｏｌｄ系ベクター（タカラバイオ社）、ｐＨＹ３００ＰＬＫ（タカラバイオ社）、ｐＵＢ１１０（Ｍｃｋｅｎｚｉｅ，Ｔ．ｅｔ　ａｌ．，１９８６，Ｐｌａｓｍｉｄ　１５（２）：９３－１０３）、ｐＢＲ３２２（タカラバイオ社）、ｐＭＷ２１８／２１９（ニッポンジーン社）、ｐＲＩ９０９／９１０等のｐＲＩ系ベクター（タカラバイオ社）、ｐＢＩ系ベクター（クロンテック社）、ＩＮ３系ベクター（インプランタイノベーションズ社）、ｐＰＴＲ１／２（タカラバイオ社）、ｐＤＪＢ２（Ｄ．Ｊ．Ｂａｌｌａｎｃｅｅｔ ａｌ．，Ｇｅｎｅ，３６，３２１－３３１，１９８５）、ｐＡＢ４－１（ｖａｎ Ｈａｒｔｉｎｇｓｖｅｌｄｔ Ｗ ｅｔ ａｌ．，Ｍｏｌ Ｇｅｎ Ｇｅｎｅｔ，２０６，７１－７５，１９８７）、ｐＬｅｕ４（Ｍ．Ｉ．Ｇ．Ｒｏｎｃｅｒｏ ｅｔ ａｌ．，Ｇｅｎｅ，８４，３３５－３４３，１９８９）、ｐＰｙｒ２２５（Ｃ．Ｄ．Ｓｋｏｒｙ ｅｔ ａｌ．，Ｍｏｌ Ｇｅｎｅｔ Ｇｅｎｏｍｉｃｓ，２６８，３９７－４０６，２００２）、ｐＦＧ１（Ｇｒｕｂｅｒ，Ｆ．ｅｔ ａｌ．，Ｃｕｒｒ Ｇｅｎｅｔ，１８，４４７－４５１，１９９０）等が挙げられる。特に、宿主が大腸菌の場合は、ｐＥＴ系ベクターが好ましく用いられる。 As a parent vector of an expression vector, any vector can be used as long as the gene of the present invention can be introduced into a host and the gene can be expressed in the host cell. Preferably, the vector comprises the gene of the invention and a control region operably linked thereto. The vector may be a vector capable of autonomous replication and replication outside the chromosome, such as a plasmid, or a vector that is integrated into the chromosome.
Specific vectors include, for example, pBluescript II SK (-) (Agilent Technologies), pUC-based vectors such as pUC18/19 and pUC118/119 (Takara Bio Inc.), pET-based vectors (Merck), and pGEX-based vectors. (Merck), pCold vector (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2):93-103), pBR322 (Takara Bio) , pMW218/219 (Nippon Gene), pRI vectors such as pRI909/910 (Takara Bio), pBI vectors (Clontech), IN3 vectors (Inplanta Innovations), pPTR1/2 (Takara Bio), pDJB2 (DJ Ballance et al., Gene, 36, 321-331, 1985), pAB4-1 (van Hartingsveldt W et al., Mol Gen Genet, 206, 71-75, 1987), pLeu4 (MI G. Roncero et al., Gene, 84, 335-343, 1989), pPyr225 (CD Skory et al., Mol Genet Genomics, 268, 397-406, 2002), pFG1 (Gruber, F. et al. al., Curr Genet, 18, 447-451, 1990). In particular, when the host is Escherichia coli, pET-based vectors are preferably used.
　また、本発明の遺伝子はこれを含むＤＮＡ断片として構築されていてもよい。該ＤＮＡ断片としては、例えば、ＰＣＲ増幅ＤＮＡ断片及び制限酵素切断ＤＮＡ断片が挙げられる。好ましくは、該ＤＮＡ断片は、本発明の遺伝子、及びこれと作動可能に連結された制御領域を含む発現カセットであり得る。 In addition, the gene of the present invention may be constructed as a DNA fragment containing it. The DNA fragments include, for example, PCR-amplified DNA fragments and restriction enzyme-cleaved DNA fragments. Preferably, the DNA fragment may be an expression cassette comprising the gene of the invention and a control region operably linked thereto.
　上記ベクター又はＤＮＡ断片に含まれる制御領域は、該ベクター又はＤＮＡ断片が導入された宿主細胞内で本発明の遺伝子を発現させるための配列であり、例えばプロモーターやターミネーター等の発現調節領域、複製開始点等が挙げられる。該制御領域の種類は、ベクター又はＤＮＡ断片を導入する宿主微生物の種類に応じて適宜選択することができる。必要に応じて、該ベクター又はＤＮＡ断片はさらに、抗生物質耐性遺伝子、アミノ酸合成関連遺伝子等の選択マーカー（例えば、アンピシリン、ネオマイシン、カナマイシン、クロラムフェニコールなどの薬剤の耐性遺伝子）を有していてもよい。
　本発明の遺伝子と上記制御領域や、マーカー遺伝子配列との連結は、シームレスクローニング法などの方法によって行うことができる。ベクターへの遺伝子配列の導入手順は、当該分野で周知である。プロモーター領域、ターミネーター、分泌シグナル領域等の制御領域の種類は、特に限定されず、導入する宿主に応じて、通常使用されるプロモーターや分泌シグナル配列を適宜選択して用いることができる。 The control region contained in the above vector or DNA fragment is a sequence for expressing the gene of the present invention in a host cell into which the vector or DNA fragment has been introduced. points, etc. The type of control region can be appropriately selected according to the type of host microorganism into which the vector or DNA fragment is introduced. If necessary, the vector or DNA fragment further has a selection marker such as an antibiotic resistance gene, an amino acid synthesis-related gene (e.g., drug resistance genes such as ampicillin, neomycin, kanamycin, chloramphenicol, etc.). may
The gene of the present invention can be ligated with the regulatory region or marker gene sequence by a method such as a seamless cloning method. Procedures for introducing gene sequences into vectors are well known in the art. The types of control regions such as promoter regions, terminators, and secretory signal regions are not particularly limited, and commonly used promoters and secretory signal sequences can be appropriately selected and used according to the host into which the gene is introduced.
　該制御領域の好適な例としては、野生型に比較して発現を強化できる強制御領域、例えば公知の高発現プロモーターであるＴ７プロモーター、ｌａｃプロモーター、ｔａｃプロモーター、ｔｒｐプロモーター、ｇａｐプロモーター、ｔｕｆプロモーター等が例示されるが、これらに特に限定されない。 Preferable examples of the control region include strong control regions capable of enhancing expression compared to the wild type, such as known high-expression promoters such as T7 promoter, lac promoter, tac promoter, trp promoter, gap promoter, tuf promoter, etc. are exemplified, but are not particularly limited to these.
　本発明の遺伝子を含むベクターを宿主へ導入するか、又は本発明の遺伝子を含むＤＮＡ断片を宿主のゲノムに導入することにより、形質転換細胞を得ることができる。
　宿主へのベクター又はＤＮＡ断片の導入の方法としては、例えばヒートショック法、エレクトロポレーション法、トランスフォーメーション法、トランスフェクション法、接合法、プロトプラスト法、パーティクル・ガン法、アグロバクテリウム法等を用いることができる。 Transformed cells can be obtained by introducing a vector containing the gene of the present invention into a host or by introducing a DNA fragment containing the gene of the present invention into the genome of the host.
Methods for introducing vectors or DNA fragments into hosts include, for example, the heat shock method, electroporation method, transformation method, transfection method, conjugation method, protoplast method, particle gun method, Agrobacterium method, and the like. be able to.
　また、本発明の遺伝子を宿主のゲノムに導入する方法としては、特に限定されないが、例えば、該遺伝子を含むＤＮＡ断片を用いた２重交差法が挙げられる。該ＤＮＡ断片は、上述する宿主細胞において発現量の多い遺伝子のプロモーター配列の下流に導入されてもよく、あるいは、予め該ＤＮＡ断片と上述した制御領域とを作動可能に連結した断片を作製し、当該連結断片を宿主のゲノムに導入してもよい。さらに、該ＤＮＡ断片は、本発明の遺伝子が適切に導入された細胞を選択するためのマーカー（薬剤耐性遺伝子や栄養要求性相補遺伝子など）と予め連結されていてもよい。 The method for introducing the gene of the present invention into the genome of the host is not particularly limited, but examples include a double crossover method using a DNA fragment containing the gene. The DNA fragment may be introduced downstream of the promoter sequence of a gene highly expressed in the host cell described above, or a fragment previously prepared by operably linking the DNA fragment with the control region described above, The ligated fragment may be introduced into the genome of the host. Furthermore, the DNA fragment may be ligated in advance with a marker (drug resistance gene, auxotrophic complementary gene, etc.) for selecting cells into which the gene of the present invention has been appropriately introduced.
　目的のベクター又はＤＮＡ断片が導入された形質転換細胞は、選択マーカーを利用して選択することができる。例えば、選択マーカーが抗生物質耐性遺伝子である場合、該抗生物質添加培地で培養することで、目的のベクター又はＤＮＡ断片が導入された形質転換細胞を選択することができる。また例えば、選択マーカーがアミノ酸合成関連遺伝子である場合、該アミノ酸要求性の宿主微生物に遺伝子導入した後、該アミノ酸要求性の有無を指標に、目的のベクター又はＤＮＡ断片が導入された形質転換細胞を選択することができる。あるいは、ＰＣＲ等によって形質転換細胞のＤＮＡ配列を調べることで目的のベクター又はＤＮＡ断片の導入を確認することもできる。 Transformed cells into which the vector or DNA fragment of interest has been introduced can be selected using a selection marker. For example, when the selection marker is an antibiotic resistance gene, transformed cells into which the desired vector or DNA fragment has been introduced can be selected by culturing in the antibiotic-supplemented medium. Alternatively, for example, when the selectable marker is an amino acid synthesis-related gene, transformed cells into which the target vector or DNA fragment has been introduced, using the presence or absence of the amino acid auxotrophy as an indicator, after gene introduction into the host microorganism having the amino acid auxotrophy. can be selected. Alternatively, introduction of the desired vector or DNA fragment can be confirmed by examining the DNA sequence of transformed cells by PCR or the like.
　本発明の３，４－ジヒドロキシフェニルアラニンの製造においては、斯くして得られた微生物の培養菌体若しくはその破砕物又は当該微生物の菌体抽出物と、プロトカテク酸、ピルビン酸及びアンモニウム塩が接触反応に供されるが、ここで用いられる微生物の培養菌体は、培養中及び後の微生物培養液、微生物の培養物より分離された菌体、該培養菌体を凍結乾燥やスプレードライによって粉末にした粉末菌体、該培養菌体を担体に固定化した固定化菌体を挙げることができる。菌体破砕物としては、例えば本発明のプロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼを含む菌体破砕液やミクロソーム画分等が挙げられる。また、当該微生物の菌体抽出物としては、当該微生物菌体をバクテリオファージ、有機溶媒や界面活性剤等の薬剤、酵素、機械的な力、温度ショック及び浸透圧ショック等によって溶菌させ、本発明のプロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼを抽出した抽出物が挙げられる。 In the production of 3,4-dihydroxyphenylalanine of the present invention, the cultured cells of the microorganism thus obtained, the crushed product thereof, or the cell extract of the microorganism, protocatechuic acid, pyruvic acid, and an ammonium salt are brought into contact reaction. However, the cultured cells of microorganisms used here are microbial culture solutions during and after culturing, cells isolated from microbial cultures, and powders by freeze-drying or spray-drying the cultured cells. powdered fungus bodies obtained by immobilizing the cultured fungus bodies on a carrier. Examples of the disrupted bacterial cells include a disrupted bacterial cell solution containing protocatechuate decarboxylase and tyrosine phenol lyase of the present invention, a microsomal fraction, and the like. In addition, as the cell extract of the microorganism, the cell of the microorganism is lysed by a bacteriophage, an agent such as an organic solvent or a surfactant, an enzyme, a mechanical force, a temperature shock, an osmotic shock, or the like. extracts of protocatechuate decarboxylase and tyrosine phenol lyase.
　微生物の培養は、炭素源、窒素源、無機塩類、その他の栄養物質を含有する培地（天然培地、合成培地）を用いて行うことができる。
　ここで、炭素源としては、グルコース、フルクトース、マンノース、アラビノース、キシロース、ガラクトース等の単糖類；セロビオース、スクロース(ショ糖)、ラクトース、マルトース、トレハロース、セロビオース、キシロビオース等の二糖類；デキストリン又は可溶性澱粉等の多糖類等が挙げられる。また、糖類の他に、マンニトール、ソルビトール、キシリトール、グリセリンのような糖アルコール；酢酸、クエン酸、乳酸、フマル酸、マレイン酸、グルコン酸のような有機酸；エタノール、プロパノールのようなアルコール；ノルマルパラフィンのような炭化水素等も用いることができる。
　窒素源としては、例えば、ペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ抽出物、コーンスティープリカー、メチルアミン等のアルキルアミン類、アミノ酸等の含窒素有機化合物、アンモニアもしくはその塩（塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウム、酢酸アンモニウムのような無機又は有機アンモニウム化合物）、尿素、アンモニア水、硝酸ナトリウム、硝酸カリウム等を使用することができる。
　無機塩類としては、リン酸第一カリウム、リン酸第二カリウム、硫酸マグネシウム、塩化ナトリウム、硝酸第一鉄、硫酸マンガン、硫酸亜鉛、硫酸コバルト、炭酸カルシウム等が挙げられる。
　その他の栄養物質としては、大豆蛋白加水分解物、アミノ酸類等が挙げられる。
　なお、培地には、チロシン又はチロシン代替物質、ビタミンＢ６類、カテコール、３，４－ジヒドロキシフェニルアラニン、プロトカテク酸、消泡剤等を添加することができる。 Microorganisms can be cultured using media (natural media, synthetic media) containing carbon sources, nitrogen sources, inorganic salts, and other nutrients.
Carbon sources include monosaccharides such as glucose, fructose, mannose, arabinose, xylose and galactose; disaccharides such as cellobiose, sucrose (sucrose), lactose, maltose, trehalose, cellobiose and xylobiose; dextrin or soluble starch. and polysaccharides such as In addition to 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 paraffins and the like can also be used.
Nitrogen sources include, for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, corn steep liquor, alkylamines such as methylamine, nitrogen-containing organic compounds such as amino acids, ammonia or salts thereof. (Inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate), urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like can be used.
Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate.
Other nutritional substances include soybean protein hydrolysates, amino acids and the like.
Tyrosine or tyrosine substitutes, vitamin B6s, catechol, 3,4-dihydroxyphenylalanine, protocatechuic acid, antifoaming agents and the like can be added to the medium.
　培養条件としては、培養温度は１５℃～４５℃が適当である。培養ｐＨ及び培養時間については特に限定されないが、例えば、ｐＨを６．０～８．０に制御しつつ６～７２時間培養を続ける方法が採用できる。また、培養中は必要に応じてアンピシリンやカナマイシン等の抗生物質を培地に添加してもよい。 As for the culture conditions, a culture temperature of 15°C to 45°C is appropriate. The culture pH and culture time are not particularly limited, but, for example, a method of continuing culture for 6 to 72 hours while controlling the pH to 6.0 to 8.0 can be employed. In addition, antibiotics such as ampicillin and kanamycin may be added to the medium during the culture, if necessary.
　上記微生物の培養菌体若しくはその破砕物又は当該微生物の菌体抽出物と、プロトカテク酸、ピルビン酸及びアンモニウム塩との接触反応は、これらを混合し、通常２０℃～５０℃で、所定時間（例えば、３０分～６０時間、好ましくは１時間～２４時間）、必要に応じ撹拌又は振とうしながら行うことができる。 The contact reaction between the cultured cells of the microorganism or the crushed product thereof or the cell extract of the microorganism and protocatechuic acid, pyruvic acid and ammonium salt is carried out by mixing these and usually at 20 ° C. to 50 ° C. for a predetermined time ( For example, 30 minutes to 60 hours, preferably 1 hour to 24 hours), with stirring or shaking as necessary.
　ここで、原料として用いるプロトカテク酸の由来は特に限定されず、有機合成法及び微生物や酵素を用いる方法の何れにより生産されたものでも良いが、プロトカテク酸生産能を有する微生物を用いて、炭素源であるグルコース等の糖類から生産されたものが好ましい。
　アンモニウム塩としては、酢酸アンモニウム、塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウム、リン酸アンモニウム、有機酸アンモニウム塩等を用いることができるが、硫酸アンモニウムを用いるのが好ましい。 Here, the origin of protocatechuic acid used as a raw material is not particularly limited, and it may be produced by any of an organic synthesis method and a method using microorganisms or enzymes. is preferably produced from saccharides such as glucose.
As the ammonium salt, ammonium acetate, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate, organic acid ammonium salt, etc. can be used, but ammonium sulfate is preferably used.
　プロトカテク酸の濃度は、０．５～１００ｇ／Ｌが好ましく、より好ましくは５～１５ｇ／Ｌである。
　ピルビン酸の濃度は、０．５～１００ｇ／Ｌが好ましく、より好ましくは５～１５ｇ／Ｌである。
　アンモニウム塩の濃度は、５～１５０ｇ／Ｌが好ましく、より好ましくは５０～８０ｇ／Ｌである。
　培養菌体しくはその破砕物又は当該微生物の菌体抽出物の濃度は、２～４００ｇ／Ｌの範囲が適当であり、好ましくは５０～２００ｇ／Ｌである。
　プロトカテク酸、ピルビン酸、及びアンモニウム塩を、反応溶液中に断続的に添加することによって、３，４－ジヒドロキシフェニルアラニンを蓄積させることができる。 The concentration of protocatechuic acid is preferably 0.5-100 g/L, more preferably 5-15 g/L.
The concentration of pyruvic acid is preferably 0.5-100 g/L, more preferably 5-15 g/L.
The concentration of the ammonium salt is preferably 5-150 g/L, more preferably 50-80 g/L.
The concentration of the cultured cells, the crushed product thereof, or the cell extract of the microorganism is suitably in the range of 2 to 400 g/L, preferably 50 to 200 g/L.
3,4-dihydroxyphenylalanine can be accumulated by intermittently adding protocatechuic acid, pyruvic acid, and an ammonium salt to the reaction solution.
　また、反応系には必要に応じ亜硫酸ナトリウム、アスコルビン酸ナトリウムやシステイン等の還元剤、ＥＤＴＡやクエン酸等のキレート剤、ピリドキサールリン酸等のビタミンＢ６類、Ｔｒｉｓ／ＨＣｌ等の緩衝液を添加してもよい。
　還元剤の濃度は、０．５～２０ｇ／Ｌが好ましく、より好ましくは２～５ｇ／Ｌである。
　キレート剤の濃度は、０．５～２０ｇ／Ｌが好ましく、より好ましくは２～５ｇ／Ｌである。
　ピリドキサールリン酸の濃度は、０．０５～５ｍＭが好ましく、より好ましくは０．５～２ｍＭである。
　緩衝液の濃度は、５～２００ｍＭが好ましく、より好ましくは５０～１００ｍＭである。 If necessary, reducing agents such as sodium sulfite, sodium ascorbate and cysteine, chelating agents such as EDTA and citric acid, vitamin B6 compounds such as pyridoxal phosphate, and buffers such as Tris/HCl are added to the reaction system. may
The concentration of the reducing agent is preferably 0.5-20 g/L, more preferably 2-5 g/L.
The concentration of the chelating agent is preferably 0.5-20 g/L, more preferably 2-5 g/L.
The concentration of pyridoxal phosphate is preferably 0.05-5 mM, more preferably 0.5-2 mM.
The buffer concentration is preferably 5-200 mM, more preferably 50-100 mM.
　反応温度は、５～６０℃が好ましく、より好ましくは１５～３０℃である。
　反応ｐＨは５．０～１０．０の範囲が適当であり、好ましくは６．５～８．５である。
　反応時間は、用いるプロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼの活性量や基質濃度に応じ適宜決定されるが、通常０．５～７２時間であり、好ましくは６時間～２４時間である。 The reaction temperature is preferably 5 to 60°C, more preferably 15 to 30°C.
The reaction pH is suitably in the range of 5.0-10.0, preferably 6.5-8.5.
The reaction time is appropriately determined depending on the amount of activity of protocatechuate decarboxylase and tyrosine phenol lyase used and the substrate concentration, and is usually 0.5 to 72 hours, preferably 6 to 24 hours.
　反応終了後、反応液中に生成した３，４－ジヒドロキシフェニルアラニンは菌体を遠心分離やろ過等によって分離・回収した後、適宜イオン交換樹脂処理法、晶析法等の通常の方法を組み合わせることによりさらに精製することができる。 After the completion of the reaction, the 3,4-dihydroxyphenylalanine produced in the reaction solution is separated and recovered by centrifugation, filtration, etc., and then combined with conventional methods such as ion-exchange resin treatment and crystallization as appropriate. can be further purified by
　上述した実施形態に関し、本発明においてはさらに以下の態様が開示される。
　＜１＞プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼを生産する微生物の培養菌体若しくはその破砕物又は当該微生物の菌体抽出物と、プロトカテク酸、ピルビン酸及びアンモニウム塩を接触反応させる工程を含む３，４－ジヒドロキシフェニルアラニンの製造方法。
　＜２＞微生物がプロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼをコードする遺伝子を有する、＜１＞の方法。
　＜３＞微生物が、さらにフラビンモノヌクレオチドプレニルトランスフェラーゼをコードする遺伝子を有する、＜２＞の方法。
　＜４＞遺伝子のうち少なくとも１つが外来遺伝子である、＜２＞又は＜３＞の方法。
　＜５＞チロシンフェノールリアーゼがプロトカテク酸による活性阻害率が９１％以下、好ましくは７５％以下、より好ましくは５０％以下、より好ましくは４０％以下、さらに好ましくは３６％以下のチロシンフェノールリアーゼである、＜１＞～＜４＞のいずれかの方法。
　＜６＞チロシンフェノールリアーゼが、以下の（Ｂ１）、（Ｂ２）、（Ｃ１）、（Ｃ２）、（Ｄ１）及び（Ｄ２）からなる群より選択されるタンパク質である、＜１＞～＜５＞のいずれかの方法。
（Ｂ１）配列番号２に示すアミノ酸配列からなるタンパク質
（Ｂ２）配列番号２に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
（Ｃ１）配列番号４に示すアミノ酸配列からなるタンパク質
（Ｃ２）配列番号４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
（Ｄ１）配列番号６に示すアミノ酸配列からなるタンパク質
（Ｄ２）配列番号６に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
　＜７＞チロシンフェノールリアーゼが、前記（Ｂ１）又は（Ｂ２）のタンパク質である、＜６＞の方法。
　＜８＞プロトカテク酸デカルボキシラーゼが、以下の（Ａ１）又は（Ａ２）のタンパク質である、＜１＞～＜７＞のいずれかの方法。
　（Ａ１）配列番号１８に示すアミノ酸配列からなるタンパク質
　（Ａ２）配列番号１８に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつプロトカテク酸デカルボキシラーゼ活性を有するタンパク質
　＜９＞フラビンモノヌクレオチドプレニルトランスフェラーゼが、以下の（Ｅ１）又は（Ｅ２）のタンパク質である、＜３＞～＜８＞のいずれかの方法。
　（Ｅ１）配列番号２４に示すアミノ酸配列からなるタンパク質
　（Ｅ２）配列番号２４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつフラビンモノヌクレオチドプレニルトランスフェラーゼ活性を有するタンパク質
　＜１０＞プロトカテク酸が糖類を原料として製造されたものである、＜１＞～＜９＞のいずれかの方法。
　＜１１＞反応溶液中より３，４－ジヒドロキシフェニルアラニンを回収する工程を含む、＜１＞～＜１０＞のいずれかの方法。
　＜１２＞プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼをコードするポリヌクレオチドを含むベクター又はＤＮＡ断片。
　＜１３＞さらにフラビンモノヌクレオチドプレニルトランスフェラーゼをコードするポリヌクレオチドを含む、＜１２＞のベクター又はＤＮＡ断片。
　＜１４＞チロシンフェノールリアーゼが、以下の（Ｂ１）、（Ｂ２）、（Ｃ１）、（Ｃ２）、（Ｄ１）及び（Ｄ２）からなる群より選択されるタンパク質である、＜１２＞又は＜１３＞のベクター又はＤＮＡ断片。
（Ｂ１）配列番号２に示すアミノ酸配列からなるタンパク質
（Ｂ２）配列番号２に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
（Ｃ１）配列番号４に示すアミノ酸配列からなるタンパク質
（Ｃ２）配列番号４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
（Ｄ１）配列番号６に示すアミノ酸配列からなるタンパク質
（Ｄ２）配列番号６に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
　＜１５＞プロトカテク酸デカルボキシラーゼが、以下の（Ａ１）又は（Ａ２）のタンパク質である、＜１２＞～＜１４＞のいずれかのベクター又はＤＮＡ断片。
　（Ａ１）配列番号１８に示すアミノ酸配列からなるタンパク質
　（Ａ２）配列番号１８に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつプロトカテク酸デカルボキシラーゼ活性を有するタンパク質
　＜１６＞フラビンモノヌクレオチドプレニルトランスフェラーゼが、以下の（Ｅ１）又は（Ｅ２）のタンパク質である、＜１３＞～＜１５＞のいずれかのベクター又はＤＮＡ断片。
　（Ｅ１）配列番号２４に示すアミノ酸配列からなるタンパク質
　（Ｅ２）配列番号２４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつフラビンモノヌクレオチドプレニルトランスフェラーゼ活性を有するタンパク質
　＜１７＞＜１２＞～＜１６＞のいずれかのベクター又はＤＮＡ断片を含有する形質転換細胞。
　＜１８＞微生物が、＜１７＞の形質転換細胞である、＜１＞～＜１１＞のいずれかの方法。 The following aspects are further disclosed in this invention regarding embodiment mentioned above.
<1> including a step of contact-reacting protocatechuic acid, pyruvic acid and ammonium salt with cultured cells of a microorganism that produces protocatechuate decarboxylase and tyrosine phenol lyase, a crushed product thereof, or a cell extract of the microorganism; A method for producing 4-dihydroxyphenylalanine.
<2> The method of <1>, wherein the microorganism has genes encoding protocatechuate decarboxylase and tyrosine phenol lyase.
<3> The method of <2>, wherein the microorganism further has a gene encoding flavin mononucleotide prenyltransferase.
<4> The method of <2> or <3>, wherein at least one of the genes is a foreign gene.
<5> The tyrosine phenol lyase has an activity inhibition rate by protocatechuic acid of 91% or less, preferably 75% or less, more preferably 50% or less, more preferably 40% or less, still more preferably 36% or less. , <1> to <4>.
<6> The tyrosine phenol lyase is a protein selected from the group consisting of the following (B1), (B2), (C1), (C2), (D1) and (D2), <1> to <5 > either method.
(B1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, and having tyrosine phenol lyase activity (C1) sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4. Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6. Protein consisting of an amino acid sequence (D2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having tyrosine phenol lyase activity <7> Tyrosine phenol lyase is the above (B1) Or the method of <6>, which is the protein of (B2).
<8> The method according to any one of <1> to <7>, wherein the protocatechuate decarboxylase is the following protein (A1) or (A2).
(A1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 18 (A2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 18 and having protocatechuic acid decarboxylase activity <9> The method according to any one of <3> to <8>, wherein the flavin mononucleotide prenyltransferase is the protein (E1) or (E2) below.
(E1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 24 (E2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24 and having flavin mononucleotide prenyltransferase activity <10 > The method according to any one of <1> to <9>, wherein the protocatechuic acid is produced from a saccharide as a starting material.
<11> The method according to any one of <1> to <10>, comprising the step of recovering 3,4-dihydroxyphenylalanine from the reaction solution.
<12> A vector or DNA fragment containing a polynucleotide encoding protocatechuate decarboxylase and tyrosine phenol lyase.
<13> The vector or DNA fragment of <12>, further comprising a polynucleotide encoding flavin mononucleotide prenyltransferase.
<14> Tyrosine phenol lyase is a protein selected from the group consisting of the following (B1), (B2), (C1), (C2), (D1) and (D2) <12> or <13 > vectors or DNA fragments.
(B1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, and having tyrosine phenol lyase activity (C1) sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4. Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6. Protein consisting of an amino acid sequence (D2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having tyrosine phenol lyase activity <15> Protocatechuate decarboxylase has the following ( The vector or DNA fragment of any one of <12> to <14>, which is the protein of A1) or (A2).
(A1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 18 (A2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 18 and having protocatechuic acid decarboxylase activity <16> The vector or DNA fragment according to any one of <13> to <15>, wherein the flavin mononucleotide prenyltransferase is the protein (E1) or (E2) below.
(E1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 24 (E2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24 and having flavin mononucleotide prenyltransferase activity <17 > A transformed cell containing the vector or DNA fragment of any one of <12> to <16>.
<18> The method according to any one of <1> to <11>, wherein the microorganism is the transformed cell of <17>.
実施例１　各種遺伝子を含むプラスミドの作製
　以下の実施例において、ＰＣＲはＫＯＤ　Ｏｎｅ ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（東洋紡社）を用いて行った。
(１)チロシンフェノールリアーゼをコードする遺伝子を含むプラスミド（ｐＥＴ＿ＦｎＴＰＬ、ｐＥＴ＿ＣｆＴＰＬ、ｐＥＴ＿ＥｗＴＰＬ）の作製
　フソバクテリウム・ヌクレアタム(Ｆｕｓｏｂａｃｔｅｒｉｕｍ　ｎｕｃｌｅａｔｕｍ)由来のチロシンフェノールリアーゼ(ＦｎＴＰＬ)をコードする遺伝子のＤＮＡ断片(配列番号１)、シトロバクタ―・フレウンディー(Ｃｉｔｒｏｂａｃｔｅｒ ｆｒｅｕｎｄｉｉ)由来のチロシンフェノールリアーゼ(ＣｆＴＰＬ)をコードする遺伝子のＤＮＡ断片(配列番号３)、及びエルウィニア・ヘルビコラ(Ｅｒｗｉｎｉａ ｈｅｒｂｉｃｏｌａ)由来のチロシンフェノールリアーゼ(ＥｗＴＰＬ)をコードする遺伝子のＤＮＡ断片(配列番号５)を人工遺伝子合成(ユーロフィンゲノミクス社)により作製し、これを鋳型として表１に示す各プライマーを用いたＰＣＲにてインサート用ＤＮＡ断片を得た。続いてプライマーｐＥＴ ｖｅｃ　Ｆ (配列番号１３、CACCACCACCACCACCACTGAG)及びｐＥＴ ｖｅｃ　Ｒ(配列番号１４、ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGG)を用いてｐＥＴ２１ａプラスミドを鋳型としたＰＣＲにてベクター用ＤＮＡ断片を得た。これらのＰＣＲ産物に対してＤｐｎＩ（タカラバイオ社）による処理を行った後、各ＤＮＡ断片をＮｕｃｌｅｏＳｐｉｎ Ｇｅｌ　ａｎｄ　ＰＣＲ　ｃｌｅａｎ－ｕｐ（タカラバイオ社）により精製した。精製後のＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　(タカラバイオ社)により連結することで各プラスミド（ｐＥＴ＿ＦｎＴＰＬ、ｐＥＴ＿ＣｆＴＰＬ、ｐＥＴ＿ＥｗＴＰＬ）を構築した。得られたプラスミド溶液を用いてＥＣＯＳ　ｃｏｍｐｅｔｅｎｔ　Ｅ．　ｃｏｌｉ　ＤＨ５α(ニッポン・ジーン社)に形質転換し、細胞液をＬＢＡｍｐ寒天培地（Ｂａｃｔｏ　Ｔｒｙｐｔｏｎｅ　１％、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　０．５％、ＮａＣｌ　１％、アンピシリンナトリウム　５０μｇ／ｍＬ、寒天　１．５％）に塗布した後３７℃にて一晩静置し、得られたコロニーに対しプライマーｐＥＴ ＣＰＣＲ１　Ｆ (配列番号１５、CGAAATTAATACGACTCACTATAGGGGAATTGTG)及びｐＥＴ ＣＰＣＲ１　Ｒ(配列番号１６、CCAAGGGGTTATGCTAGTTATTGCTCAG)を用いたＰＣＲ反応を行い、目的ＤＮＡ断片の導入が確認された形質転換株を選択した。得られた形質転換株をＬＢＡｍｐ液体培地（Ｂａｃｔｏ　Ｔｒｙｐｔｏｎｅ　１％、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　０．５％、ＮａＣｌ　１％、アンピシリンナトリウム　５０μｇ／ｍＬ）２ｍＬに接種した後３７℃で終夜培養した。この培養液よりＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ　(タカラバイオ社)を用いてプラスミドを精製した。 Example 1 Preparation of Plasmids Containing Various Genes In the following examples, PCR was performed using KOD One PCR Master Mix (Toyobo).
(1) Preparation of plasmids (pET_FnTPL, pET_CfTPL, pET_EwTPL) containing genes encoding tyrosine phenol lyase DNA fragments of genes encoding tyrosine phenol lyase (FnTPL) derived from Fusobacterium nucleatum (SEQ ID NO: 1), A DNA fragment (SEQ ID NO: 3) of a gene encoding tyrosine phenol lyase (CfTPL) derived from Citrobacter freundii and a gene encoding tyrosine phenol lyase (EwTPL) derived from Erwinia herbicola A DNA fragment (SEQ ID NO: 5) was prepared by artificial gene synthesis (Eurfin Genomics), and using this as a template, PCR was performed using each primer shown in Table 1 to obtain a DNA fragment for insert. Subsequently, using primers pET vec F (SEQ ID NO: 13, CACCACCACCACCACCACTGAG) and pET vec R (SEQ ID NO: 14, ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGG), a vector DNA fragment was obtained by PCR using the pET21a plasmid as a template. After treating these PCR products with DpnI (Takara Bio Inc.), each DNA fragment was purified by NucleoSpin Gel and PCR clean-up (Takara Bio Inc.). Each plasmid (pET_FnTPL, pET_CfTPL, pET_EwTPL) was constructed by ligating the purified DNA fragments with an In-Fusion HD Cloning Kit (Takara Bio Inc.). Using the obtained plasmid solution, ECOS competent E. coli DH5α (Nippon Gene), and spread the cell solution on LBAmp agar medium (Bacto Tryptone 1%, Yeast Extract 0.5%, NaCl 1%, ampicillin sodium 50 μg/mL, agar 1.5%). After that, it was allowed to stand overnight at 37° C., and the obtained colonies were subjected to PCR reaction using primers pET CPCR1 F (SEQ ID NO: 15, CGAAATTAATACGACTCACTATAGGGGAATTGTG) and pET CPCR1 R (SEQ ID NO: 16, CCAAGGGGTTATGCTAGTTATTGCTCAG) to obtain the target DNA. A transformant in which introduction of the fragment was confirmed was selected. The resulting transformants were inoculated into 2 mL of LBAmp liquid medium (Bacto Tryptone 1%, Yeast Extract 0.5%, NaCl 1%, ampicillin sodium 50 µg/mL) and cultured overnight at 37°C. A plasmid was purified from this culture medium using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
(２)チロシンフェノールリアーゼ、プロトカテク酸デカルボキシラーゼ及びフラビンモノヌクレオチドプレニルトランスフェラーゼをコードする遺伝子を含むプラスミドの作製　（ａ）プロトカテク酸デカルボキシラーゼをコードする遺伝子を含むプラスミド（ｐＥＴ＿ＥｃＡｒｏＹ）の作製
　エンテロバクター・クロアカ（Ｅｎｔｅｒｏｂａｃｔｅｒ　ｃｌｏａｃａｅ）由来プロトカテク酸デカルボキシラーゼ（ＥｃＡｒｏＹ）をコードする遺伝子のＤＮＡ断片（配列番号１７）を人工遺伝子合成(ユーロフィンゲノミクス社)により作製した。これを鋳型としてプライマーＥｃａｒｏＹ　ｉｎｓ　Ｆ（配列番号１９、GAAGGAGATATACATATGCAAAACCCGATAAATGAC）及びＥｃａｒｏＹ　ｉｎｓ　Ｒ（配列番号２０、GTGGTGGTGGTGGTGTTATTTCTTATCGCTAAATAACTC）を用いたＰＣＲにてインサート用ＤＮＡ断片を得た。続いてプライマーｐＥＴ　ｖｅｃ　Ｆ（配列番号１３）及びｐＥＴ　ｖｅｃ　Ｒ（配列番号１４）を用いてｐＥＴ２１ａプラスミドを鋳型としたＰＣＲにてベクター用ＤＮＡ断片を得た。これらのＰＣＲ産物に対してＤｐｎＩ（タカラバイオ社）による処理を行った後、各ＤＮＡ断片をＮｕｃｌｅｏＳｐｉｎ Ｇｅｌ ａｎｄ ＰＣＲ ｃｌｅａｎ－ｕｐ(タカラバイオ社)により精製した。精製後のＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　(タカラバイオ社)により連結することでプラスミド（ｐＥＴ＿ＥｃＡｒｏＹ）を構築した。得られたプラスミド溶液を用いてＥＣＯＳ　ｃｏｍｐｅｔｅｎｔ　Ｅ．　ｃｏｌｉ　ＤＨ５α(ニッポン・ジーン社)に形質転換し、細胞液をＬＢＡｍｐ寒天培地に塗布した後３７℃にて一晩静置し、得られたコロニーに対しプライマーｐＥＴ ＣＰＣＲ２ Ｆ（配列番号２１、AGATCTCGATCCCGCGAAAT）及びｐＥＴ ＣＰＣＲ２ Ｒ（配列番号２２、TTTAGAGGCCCCAAGGGGTT）を用いたＰＣＲ反応を行い、目的ＤＮＡ断片の導入が確認された形質転換株を選択した。得られた形質転換株をＬＢＡｍｐ液体培地２ｍＬに接種した後３７℃で終夜培養した。この培養液よりＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ　(タカラバイオ社)を用いてプラスミドを精製した。 (2) Preparation of plasmid containing gene encoding tyrosine phenol lyase, protocatechuate decarboxylase and flavin mononucleotide prenyltransferase (a) Preparation of plasmid (pET_EcAroY) containing gene encoding protocatechuate decarboxylase Enterobacter cloacae ( A DNA fragment (SEQ ID NO: 17) of the gene encoding protocatechuate decarboxylase (EcAroY) derived from Enterobacter cloacae was prepared by artificial gene synthesis (Eurfin Genomics). Using this as a template, a DNA fragment for insert was obtained by PCR using primers EcoroYins F (SEQ ID NO: 19, GAAGGAGATATACATATGCAAAACCCGATAAATGAC) and EcoroYins R (SEQ ID NO: 20, GTGGTGGTGGTGGTGTTATTTCTTATCGCTAAATAACTC). Subsequently, a DNA fragment for a vector was obtained by PCR using primers pET vec F (SEQ ID NO: 13) and pET vec R (SEQ ID NO: 14) and pET21a plasmid as a template. After treating these PCR products with DpnI (Takara Bio Inc.), each DNA fragment was purified by NucleoSpin Gel and PCR clean-up (Takara Bio Inc.). A plasmid (pET_EcAroY) was constructed by ligating the purified DNA fragment using In-Fusion HD Cloning Kit (Takara Bio Inc.). Using the obtained plasmid solution, ECOS competent E. coli DH5α (Nippon Gene), the cell solution was spread on an LBAmp agar medium, allowed to stand overnight at 37° C., and the resulting colonies were treated with the primer pET CPCR2 F (SEQ ID NO: 21, AGATCTCGATCCCGCGAAAT). and pET CPCR2 R (SEQ ID NO: 22, TTTAGAGGCCCCAAGGGGTT) was performed to select a transformant in which introduction of the target DNA fragment was confirmed. The resulting transformants were inoculated into 2 mL of LBAmp liquid medium and cultured overnight at 37°C. A plasmid was purified from this culture medium using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
　（ｂ）プロトカテク酸デカルボキシラーゼ及びフラビンモノヌクレオチドプレニルトランスフェラーゼをコードする遺伝子を含むプラスミド（ｐＥＴ＿ＥｃＡｒｏＹ－ＥｃＵｂｉＸ）の作製
　大腸菌由来フラビンモノヌクレオチドプレニルトランスフェラーゼ（ＥｃＵｂｉＸ）をコードする遺伝子のＤＮＡ断片（配列番号２３）を人工遺伝子合成(ユーロフィンゲノミクス社)により作製した。これを鋳型としてプライマーＥｃＵｂｉＸ　ｉｎｓ　Ｆ（配列番号２５、AAGGAGGTTTGATTCATGAAACGGCTTATTGTGGGCATTT）及びＥｃＵｂｉＸ　ｉｎｓ　Ｒ（配列番号２６、GTGGTGGTGGTGGTGGTGTTAAGCACCTTGCCACCGGGCA）を用いたＰＣＲにてインサート用ＤＮＡ断片を得た。（ａ）にて作製したプラスミド（ｐＥＴ＿ＥｃＡｒｏＹ）に対して、プライマーＥｃＡｒｏＹ　ｖｅｃ　Ｒ　(配列番号２７、GAATCAAACCTCCTTTATTTCTTATCGCTAAATAACTCTG)及びＥｃＡｒｏＹ　ｖｅｃ　Ｆ　(配列番号２８、CACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC)を用いたＰＣＲにてベクター用ＤＮＡ断片を得た。これらのＰＣＲ産物に対してＤｐｎＩ（タカラバイオ社）による処理を行った後、各ＤＮＡ断片をＮｕｃｌｅｏＳｐｉｎ Ｇｅｌ ａｎｄ ＰＣＲ ｃｌｅａｎ－ｕｐ(タカラバイオ社)により精製した。精製後のＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　 (タカラバイオ社)により連結することで目的とするプラスミド（ｐＥＴ＿ＥｃＡｒｏＹ－ＥｃＵｂｉＸ）を構築した。得られたプラスミド溶液を用いてＥＣＯＳ　ｃｏｍｐｅｔｅｎｔ　Ｅ．　ｃｏｌｉ　ＤＨ５α(ニッポン・ジーン社)に形質転換し、細胞液をＬＢＡｍｐ寒天培地に塗布した後３７℃にて一晩静置し、得られたコロニーに対しプライマーｐＥＴ ＣＰＣＲ２ Ｆ（配列番号２１）及びｐＥＴ ＣＰＣＲ２ Ｒ（配列番号２２）を用いたＰＣＲ反応を行い、目的ＤＮＡ断片の導入が確認された形質転換株を選択した。得られた形質転換株をＬＢＡｍｐ液体培地２ｍＬに接種した後３７℃で終夜培養した。この培養液よりＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ　(タカラバイオ社)を用いてプラスミドを精製した。 (b) Preparation of plasmid (pET_EcAroY-EcUbiX) containing genes encoding protocatechuate decarboxylase and flavin mononucleotide prenyltransferase It was prepared by artificial gene synthesis (Eurofin Genomics). Using this as a template, a DNA fragment for insert was obtained by PCR using primers EcUbiXins F (SEQ ID NO: 25, AAGGAGGTTTGATTCATGAAACGGCTTATTGTGGGCATTT) and EcUbiXins R (SEQ ID NO: 26, GTGGTGGTGGTGGTGGTGTTAAGCACCTTGCCACCGGGCA). A vector DNA fragment was obtained by PCR using primers EcAroY vec R (SEQ ID NO: 27, GATTCAAACCTCCTTTATTTCTTATCGCTAAATAACTCTG) and EcAroY vec F (SEQ ID NO: 28, CACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC) for the plasmid (pET_EcAroY) prepared in (a). . After treating these PCR products with DpnI (Takara Bio Inc.), each DNA fragment was purified by NucleoSpin Gel and PCR clean-up (Takara Bio Inc.). The target plasmid (pET_EcAroY-EcUbiX) was constructed by ligating the purified DNA fragments with In-Fusion HD Cloning Kit (Takara Bio Inc.). Using the obtained plasmid solution, ECOS competent E. E. coli DH5α (Nippon Gene) was transformed, the cell solution was spread on LBAmp agar medium, allowed to stand overnight at 37° C., and the resulting colonies were treated with primers pET CPCR2 F (SEQ ID NO: 21) and pET. A PCR reaction was performed using CPCR2 R (SEQ ID NO: 22), and transformants in which introduction of the target DNA fragment was confirmed were selected. The resulting transformants were inoculated into 2 mL of LBAmp liquid medium and cultured overnight at 37°C. A plasmid was purified from this culture medium using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
　（ｃ）チロシンフェノールリアーゼ、プロトカテク酸デカルボキシラーゼ及びフラビンモノヌクレオチドプレニルトランスフェラーゼをコードする遺伝子を含むプラスミドの作製
　（１）にて作製した各プラスミド（ｐＥＴ＿ＦｎＴＰＬ、ｐＥＴ＿ＣｆＴＰＬ、ｐＥＴ＿ＥｗＴＰＬ）を鋳型として表２に示す各プライマーを用いたＰＣＲにてインサート用ＤＮＡ断片を得た。続いて、（ｂ）にて作製したプラスミド（ｐＥＴ＿ＥｃＡｒｏＹ－ＥｃＵｂｉＸ）を鋳型としてプライマーＥｃＡｒｏＹ－ＥｃＵｂｉＸ　ｖｅｃ　Ｆ（配列番号３５、AGGAGGTTTGATTCATGCAAAACCCGATAAATGAC）及びＥｃＡｒｏＹ－ＥｃＵｂｉＸ　ｖｅｃ　Ｒ（配列番号３６、ATGTATATCTCCTTCTTAAAGTTAA）を用いてＰＣＲにてベクター用ＤＮＡ断片を得た。これらのＰＣＲ産物に対してＤｐｎＩ（タカラバイオ社）による処理を行った後、各ＤＮＡ断片をＮｕｃｌｅｏＳｐｉｎ Ｇｅｌ ａｎｄ ＰＣＲ ｃｌｅａｎ－ｕｐ(タカラバイオ社)により精製した。精製後のＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　(タカラバイオ社)により連結することで目的とするプラスミド（ｐＥＴ＿ＦｎＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ、ｐＥＴ＿ＣｆＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ、ｐＥＴ＿ＥｗＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ）を構築した。得られたプラスミド溶液を用いてＥＣＯＳ　ｃｏｍｐｅｔｅｎｔ　Ｅ．　ｃｏｌｉ　ＤＨ５α(ニッポン・ジーン社)に形質転換し、細胞液をＬＢＡｍｐ寒天培地に塗布した後３７℃にて一晩静置し、得られたコロニーに対しプライマーｐＥＴ ＣＰＣＲ２ Ｆ（配列番号２０）及びｐＥＴ ＣＰＣＲ２ Ｒ（配列番号２１）を用いたＰＣＲ反応を行い、目的ＤＮＡ断片の導入が確認された形質転換株を選択した。得られた形質転換株をＬＢＡｍｐ液体培地２ｍＬに接種した後３７℃で終夜培養した。この培養液よりＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ　(タカラバイオ社)を用いてプラスミドを精製した。 (c) Preparation of plasmids containing genes encoding tyrosine phenol lyase, protocatechuate decarboxylase and flavin mononucleotide prenyltransferase Each plasmid (pET_FnTPL, pET_CfTPL, pET_EwTPL) prepared in (1) shown in Table 2 as a template A DNA fragment for insert was obtained by PCR using primers. Subsequently, using the plasmid (pET_EcAroY-EcUbiX) prepared in (b) as a template, PCR was performed using primers EcAroY-EcUbiX vec F (SEQ ID NO: 35, AGGAGGTTTGATTCATGCAAAACCCGATAAATGAC) and EcAroY-EcUbiX vec R (SEQ ID NO: 36, ATGTATATCTCCTTCTTAAAGTTAA). A vector DNA fragment was obtained by After treating these PCR products with DpnI (Takara Bio Inc.), each DNA fragment was purified by NucleoSpin Gel and PCR clean-up (Takara Bio Inc.). The target plasmids (pET_FnTPL-EcAroY-EcUbiX, pET_CfTPL-EcAroY-EcUbiX, pET_EwTPL-EcAroY-EcUbiX) were constructed by ligating the purified DNA fragments using In-Fusion HD Cloning Kit (Takara Bio Inc.). Using the obtained plasmid solution, ECOS competent E. E. coli DH5α (Nippon Gene) was transformed, the cell solution was spread on LBAmp agar medium, allowed to stand at 37° C. overnight, and the resulting colonies were treated with primers pET CPCR2 F (SEQ ID NO: 20) and pET. A PCR reaction was performed using CPCR2 R (SEQ ID NO: 21) to select transformants in which introduction of the target DNA fragment was confirmed. The resulting transformants were inoculated into 2 mL of LBAmp liquid medium and cultured overnight at 37°C. A plasmid was purified from this culture medium using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
実施例２　プロトカテク酸によるチロシンフェノールリアーゼの阻害率評価
(１)プラスミドの宿主細胞への導入
　実施例１（１）で得られた各プラスミド（ｐＥＴ＿ＦｎＴＰＬ、ｐＥＴ＿ＣｆＴＰＬ、ｐＥＴ＿ＥｗＴＰＬ）を用いて、ＥＣＯＳ　ｃｏｍｐｅｔｅｎｔ　Ｅ．　ｃｏｌｉ　ＢＬ２１（ＤＥ３） (ニッポン・ジーン社)にヒートショック法により形質転換した。得られた形質転換細胞液をＬＢＡｍｐ寒天培地に塗布後３７℃にて１６時間静置し、得られたコロニーを形質転換体とした。 Example 2 Evaluation of inhibition rate of tyrosine phenol lyase by protocatechuic acid
(1) Introduction of plasmid into host cell Each plasmid (pET_FnTPL, pET_CfTPL, pET_EwTPL) obtained in Example 1(1) was used to transform ECOS competent E. coli. coli BL21(DE3) (Nippon Gene) was transformed by the heat shock method. The resulting transformed cell suspension was plated on an LBAmp agar medium and allowed to stand at 37° C. for 16 hours, and the resulting colonies were used as transformants.
(２)形質転換株の培養
　（１）で得られた形質転換体を表３に示す培地（アンピシリンナトリウム　５０μｇ／ｍＬを含む）１０ｍＬを含む大型試験管に接種し、３７℃にて一晩培養し目的タンパク質を発現させた。培養液を遠心分離（３，０００ｒｐｍ，１０分）することにより培養上清を除去し、目的タンパク質を含有する菌体を得た。各プラスミド（ｐＥＴ＿ＦｎＴＰＬ、ｐＥＴ＿ＣｆＴＰＬ、ｐＥＴ＿ＥｗＴＰＬ)を、形質転換後培養して得られた菌体をそれぞれ菌体１、菌体２、及び菌体３と称する。菌体１～３の宿主及び発現される酵素を表４に記載した。 (2) Culture of transformed strain (1) The transformant obtained in (1) was inoculated into a large test tube containing 10 mL of the medium shown in Table 3 (containing ampicillin sodium 50 µg/mL) and cultured overnight at 37°C. to express the target protein. The culture medium was centrifuged (3,000 rpm, 10 minutes) to remove the culture supernatant to obtain cells containing the target protein. Cells obtained by culturing after transformation with each plasmid (pET_FnTPL, pET_CfTPL, pET_EwTPL) are referred to as cell 1, cell 2, and cell 3, respectively. Table 4 lists the hosts and expressed enzymes of cells 1 to 3.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（３）菌体1～３を用いたプロトカテク酸によるチロシンフェノールリアーゼの阻害率の測定
　（２）で得られた菌体１、菌体２、及び菌体３を用いて、プロトカテク酸の存在下におけるカテコールを基質とした３,４－ジヒドロキシフェニアラニンへの変換率から、プロトカテク酸による各種チロシンフェノールリアーゼの阻害率を求めた。各種濃度のプロトカテク酸を含む表５の条件１～６に記載の反応溶液(総量２００μＬ)にて、カテコールから３,４－ジヒドロキシフェニアラニンへの変換反応を終濃度１００ｇ／Ｌの菌体を用いて３０℃、４時間行った。得られた反応溶液中の３,４－ジヒドロキシフェニルアラニン濃度を参考例１の方法に従って定量し、下記式に従いカテコールから３,４－ジヒドロキシフェニルアラニンへの変換率を算出した。 (3) Measurement of inhibition rate of tyrosine phenol lyase by protocatechuic acid using cells 1 to 3 Using cells 1, 2, and 3 obtained in (2), The inhibition rate of various tyrosine phenol lyases by protocatechuic acid was obtained from the conversion rate of catechol to 3,4-dihydroxyphenialanine using catechol as a substrate. The conversion reaction from catechol to 3,4-dihydroxyphenylalanine was carried out using the reaction solution (total amount: 200 μL) described in conditions 1 to 6 in Table 5 containing various concentrations of protocatechuic acid, using cells with a final concentration of 100 g/L. and 30° C. for 4 hours. The concentration of 3,4-dihydroxyphenylalanine in the resulting reaction solution was determined according to the method of Reference Example 1, and the conversion rate of catechol to 3,4-dihydroxyphenylalanine was calculated according to the following formula.
（数２）
　カテコールから３,４－ジヒドロキシフェニルアラニンへの変換率（％）＝１００×〔反応終了時の３,４－ジヒドロキシフェニルアラニンの物質量（ｍｏｌ）〕／〔反応開始前のカテコールの物質量（ｍｏｌ）〕 (Number 2)
Conversion rate (%) of catechol to 3,4-dihydroxyphenylalanine=100×[substance amount (mol) of 3,4-dihydroxyphenylalanine at the end of the reaction]/[substance amount (mol) of catechol before starting the reaction]
　また、チロシンフェノールリアーゼのプロトカテク酸による阻害率は下記式に従って算出した。
（数３）
　チロシンフェノールリアーゼのプロトカテク酸による阻害率（％）＝１００×［１―〔プロトカテク酸存在下におけるカテコールから３,４－ジヒドロキシフェニルアラニンへの変換率（％）〕／〔プロトカテク酸非存在下におけるカテコールから３,４－ジヒドロキシフェニルアラニンへの変換率（％）〕］ In addition, the inhibition rate of tyrosine phenol lyase by protocatechuic acid was calculated according to the following formula.
(Number 3)
Inhibition rate of tyrosine phenol lyase by protocatechuic acid (%) = 100 × [1 - [conversion rate (%) of catechol to 3,4-dihydroxyphenylalanine in the presence of protocatechuic acid]/[from catechol in the absence of protocatechuic acid Conversion rate to 3,4-dihydroxyphenylalanine (%)]]
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　ＦｎＴＰＬ、ＣｆＴＰＬ及びＥｗＴＰＬのプロトカテク酸による阻害率を比較した（表６）。
　表６に示すように、いずれのＴＰＬにおいても阻害率の値はプロトカテク酸の濃度に依存して上昇した。条件２～６のいずれにおいてもＦｎＴＰＬのプロトカテク酸による阻害率が最も低い値となった。 The inhibition rates of FnTPL, CfTPL and EwTPL by protocatechuic acid were compared (Table 6).
As shown in Table 6, the inhibition rate increased depending on the concentration of protocatechuic acid in all TPLs. In all conditions 2 to 6, the inhibition rate of FnTPL by protocatechuic acid was the lowest value.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
実施例３　菌体触媒を用いたプロトカテク酸を基質とする３,４－ジヒドロキシフェニルアラニンの生産
(１)プラスミドの宿主細胞への導入
　実施例１（２）で得られた各プラスミド（ｐＥＴ＿ＦｎＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ、ｐＥＴ＿ＣｆＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ、ｐＥＴ＿ＥｗＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ）を用いて、ＥＣＯＳ　ｃｏｍｐｅｔｅｎｔ　Ｅ．　ｃｏｌｉ　ＢＬ２１（ＤＥ３） (ニッポン・ジーン社)にヒートショック法により形質転換した。得られた形質転換細胞液をＬＢＡｍｐ寒天培地に塗布後３７℃にて１６時間静置し、得られたコロニーを形質転換体とした。 Example 3 Production of 3,4-dihydroxyphenylalanine using protocatechuic acid as a substrate using a bacterial cell catalyst
(1) Introduction of plasmid into host cell Each plasmid (pET_FnTPL-EcAroY-EcUbiX, pET_CfTPL-EcAroY-EcUbiX, pET_EwTPL-EcAroY-EcUbiX) obtained in Example 1(2) was used to transform ECOS competent E. coli. coli BL21(DE3) (Nippon Gene) was transformed by the heat shock method. The resulting transformed cell suspension was plated on an LBAmp agar medium and allowed to stand at 37° C. for 16 hours, and the resulting colonies were used as transformants.
(２)形質転換株の培養
　（１）で得られた形質転換体を表３に示す培地（アンピシリンナトリウム　５０μｇ／ｍＬを含む）１０ｍＬを含む大型試験管に接種し、３７℃にて一晩培養し目的タンパク質を発現させた。培養液を遠心分離（３，０００ｒｐｍ, １０分)することにより培養上清を除去し、目的タンパク質を含有する菌体を得た。各プラスミド（ｐＥＴ＿ＦｎＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ、ｐＥＴ＿ＣｆＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ、及びｐＥＴ＿ＥｗＴＰＬ－ＥｃＡｒｏＹ－ＥｃＵｂｉＸ)を、形質転換後培養して得られた菌体をそれぞれ菌体４、菌体５、及び菌体６と称する。菌体４～６の宿主及び発現される酵素を表７に記載した。 (2) Culture of transformed strain (1) The transformant obtained in (1) was inoculated into a large test tube containing 10 mL of the medium shown in Table 3 (containing ampicillin sodium 50 µg/mL) and cultured overnight at 37°C. to express the target protein. The culture medium was centrifuged (3,000 rpm, 10 minutes) to remove the culture supernatant to obtain cells containing the target protein. Each plasmid (pET_FnTPL-EcAroY-EcUbiX, pET_CfTPL-EcAroY-EcUbiX, and pET_EwTPL-EcAroY-EcUbiX) was transformed and cultured. called. Hosts and expressed enzymes of cells 4-6 are listed in Table 7.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
（３）菌体４～６を用いたプロトカテク酸を基質とする３,４－ジヒドロキシフェニルアラニンの生産能の測定
　（２）で得られた菌体４、菌体５、及び菌体６を用いてプロトカテク酸を基質とする３,４－ジヒドロキシフェニルアラニンの生産能を評価した。各種濃度のプロトカテク酸及びピルビン酸を含む表８の条件７～１１に記載の反応溶液(総量２００μＬ)にて、プロトカテク酸から３,４－ジヒドロキシフェニアラニンへの変換反応を終濃度１００ｇ／Ｌの菌体を用いて３０℃、４時間行った。得られた反応溶液中の３,４－ジヒドロキシフェニルアラニン濃度を参考例１の方法に従って定量し、下記式に従いプロトカテク酸を基質とする３,４－ジヒドロキシフェニルアラニンの生産能を算出した。 (3) Measurement of the ability to produce 3,4-dihydroxyphenylalanine using protocatechuic acid as a substrate using cells 4 to 6 Using cells 4, 5 and 6 obtained in (2) The ability to produce 3,4-dihydroxyphenylalanine using protocatechuic acid as a substrate was evaluated. The conversion reaction from protocatechuic acid to 3,4-dihydroxyphenialanine was carried out at a final concentration of 100 g/L in reaction solutions (total amount: 200 μL) described in conditions 7 to 11 in Table 8 containing various concentrations of protocatechuic acid and pyruvic acid. It was performed at 30° C. for 4 hours using the cells. The concentration of 3,4-dihydroxyphenylalanine in the resulting reaction solution was determined according to the method of Reference Example 1, and the ability to produce 3,4-dihydroxyphenylalanine using protocatechuic acid as a substrate was calculated according to the following formula.
（数４）
　プロトカテク酸を基質とする３,４－ジヒドロキシフェニルアラニンの生産能（％）＝１００×〔反応終了時の３,４－ジヒドロキシフェニルアラニンの物質量（ｍｏｌ）〕／〔反応開始前のプロトカテク酸の物質量（ｍｏｌ）〕 (Number 4)
Production capacity (%) of 3,4-dihydroxyphenylalanine using protocatechuic acid as a substrate=100×[substance amount (mol) of 3,4-dihydroxyphenylalanine at the end of the reaction]/[substance amount of protocatechuic acid before the start of the reaction (mol)]
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　菌体４、５、６を用いたプロトカテク酸からの３，４－ジヒドロキシフェニルアラニンの生産能を評価した（表９）。
　表９の示すように条件７～１１において、３，４－ジヒドロキシフェニルアラニンの生産能は、ＦｎＴＰＬを発現させた菌体４を用いた場合には６０～８０％、ＣｆＴＰＬを発現させた菌体５を用いた場合には４４～５７％、ＥｗＴＰＬを発現させた菌体６を用いた場合には２４～３８％であった。プロトカテク酸からの３，４－ジヒドロキシフェニルアラニンの生産能はいずれの条件においても、プロトカテク酸による活性阻害を受けにくいＦｎＴＰＬを発現させた菌体４を使用した場合に最も高い値を示した。 The ability to produce 3,4-dihydroxyphenylalanine from protocatechuic acid using cells 4, 5 and 6 was evaluated (Table 9).
As shown in Table 9, under conditions 7 to 11, the ability to produce 3,4-dihydroxyphenylalanine was 60 to 80% when using fungus 4 expressing FnTPL, and 60 to 80% when using fungus 5 expressing CfTPL. was 44 to 57% when EwTPL was used, and 24 to 38% when EwTPL-expressing bacterial cell 6 was used. The ability to produce 3,4-dihydroxyphenylalanine from protocatechuic acid showed the highest value under all conditions when using cell 4 expressing FnTPL, which is less susceptible to activity inhibition by protocatechuic acid.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
参考例１ ３,４－ジヒドロキシフェニルアラニンの定量
　３,４－ジヒドロキシフェニルアラニンの定量はＨＰＬＣにより行った。ＨＰＬＣ分析に供する反応液を０．１％リン酸にて適宜希釈した後、アクロプレップ９６フィルタープレート（０．２μｍＧＨＰ膜、日本ポール社）を用いて不溶物の除去を行った。ＨＰＬＣの装置は、Ｃｈｒｏｍａｓｔｅｒ（日立ハイテクサイエンス社）を用いた。分析カラムには、Ｌ－カラム ＯＤＳ（４．６ｍｍ Ｉ．Ｄ．×１５０ｍｍ、化学物質評価研究機構）を用い、溶離液Ａを０．１Ｍ リン酸二水素カリウムの０．１％リン酸溶液、溶離液Ｂを７０％メタノールとし、流速１．０ｍＬ／分、カラム温度４０℃の条件にてグラジエント溶出を行った。３,４－ジヒドロキシフェニルアラニンの検出にはＵＶ検出器（検出波長２８０ｎｍ）を用いた。標準試料〔３,４－ジヒドロキシ－Ｌ－フェニルアラニン（販売元コードＡ１１３１１、富士フイルム和光純薬株式会社）〕を用いて濃度検量線を作成し、濃度検量線に基づいて３,４－ジヒドロキシフェニルアラニンの定量を行った。 Reference Example 1 Determination of 3,4-dihydroxyphenylalanine Determination of 3,4-dihydroxyphenylalanine was performed by HPLC. After appropriately diluting the reaction solution to be subjected to HPLC analysis with 0.1% phosphoric acid, insoluble matter was removed using an Acroprep 96 filter plate (0.2 μm GHP membrane, Nippon Pall Co., Ltd.). The HPLC apparatus used was Chromaster (Hitachi High-Tech Science). L-Column ODS (4.6 mm ID × 150 mm, Chemical Substances Evaluation and Research Organization) was used as the analytical column, and eluent A was a 0.1% phosphoric acid solution of 0.1 M potassium dihydrogen phosphate. Gradient elution was performed under the conditions of 70% methanol as the eluent B, a flow rate of 1.0 mL/min, and a column temperature of 40°C. A UV detector (detection wavelength 280 nm) was used to detect 3,4-dihydroxyphenylalanine. A concentration calibration curve was prepared using a standard sample [3,4-dihydroxy-L-phenylalanine (seller code A11311, Fujifilm Wako Pure Chemical Industries, Ltd.)], and based on the concentration calibration curve, 3,4-dihydroxyphenylalanine Quantification was performed.

Claims (14)

  1. 　プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼを生産する微生物の培養菌体若しくはその破砕物又は当該微生物の菌体抽出物と、プロトカテク酸、ピルビン酸及びアンモニウム塩を接触反応させる工程を含む３，４－ジヒドロキシフェニルアラニンの製造方法。 3,4-dihydroxy, comprising a step of contact-reacting protocatechuic acid, pyruvic acid, and ammonium salt with cultured cells of a microorganism that produces protocatechuate decarboxylase and tyrosine phenol-lyase, or a crushed product thereof, or a cell extract of the microorganism; A method for producing phenylalanine.
  2. 　微生物がプロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼをコードする遺伝子を有する、請求項１記載の方法。 The method according to claim 1, wherein the microorganism has genes encoding protocatechuate decarboxylase and tyrosine phenol lyase.
  3. 　微生物が、さらにフラビンモノヌクレオチドプレニルトランスフェラーゼをコードする遺伝子を有する、請求項２記載の方法。 The method according to claim 2, wherein the microorganism further has a gene encoding flavin mononucleotide prenyltransferase.
  4. 　遺伝子のうち少なくとも１つが外来遺伝子である、請求項２又は３記載の方法。 The method according to claim 2 or 3, wherein at least one of the genes is a foreign gene.
  5. 　チロシンフェノールリアーゼがプロトカテク酸による活性阻害率が９１％以下のチロシンフェノールリアーゼである、請求項１～４のいずれか１項記載の方法。 The method according to any one of claims 1 to 4, wherein the tyrosine phenol lyase is a tyrosine phenol lyase whose activity inhibition rate by protocatechuic acid is 91% or less.
  6. 　チロシンフェノールリアーゼが、以下の（Ｂ１）、（Ｂ２）、（Ｃ１）、（Ｃ２）、（Ｄ１）及び（Ｄ２）からなる群より選択されるタンパク質である、請求項１～５のいずれか１項記載の方法。
    （Ｂ１）配列番号２に示すアミノ酸配列からなるタンパク質
    （Ｂ２）配列番号２に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
    （Ｃ１）配列番号４に示すアミノ酸配列からなるタンパク質
    （Ｃ２）配列番号４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
    （Ｄ１）配列番号６に示すアミノ酸配列からなるタンパク質
    （Ｄ２）配列番号６に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質     Any one of claims 1 to 5, wherein the tyrosine phenol lyase is a protein selected from the group consisting of the following (B1), (B2), (C1), (C2), (D1) and (D2) The method described in the section.
    (B1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, and having tyrosine phenol lyase activity (C1) sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4. Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6. Protein consisting of an amino acid sequence (D2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having tyrosine phenol lyase activity
  7. 　チロシンフェノールリアーゼが、前記（Ｂ１）又は（Ｂ２）のタンパク質である、請求項６記載の方法。 The method according to claim 6, wherein the tyrosine phenol lyase is the protein (B1) or (B2).
  8. 　プロトカテク酸が糖類を原料として製造されたものである、請求項１～７のいずれか１項記載の方法。 The method according to any one of claims 1 to 7, wherein protocatechuic acid is produced using sugar as a raw material.
  9. 　反応溶液中より３，４－ジヒドロキシフェニルアラニンを回収する工程を含む、請求項１～８のいずれか１項記載の方法。 The method according to any one of claims 1 to 8, comprising a step of recovering 3,4-dihydroxyphenylalanine from the reaction solution.
  10. 　プロトカテク酸デカルボキシラーゼ及びチロシンフェノールリアーゼをコードするポリヌクレオチドを含むベクター又はＤＮＡ断片。 A vector or DNA fragment containing a polynucleotide encoding protocatechuate decarboxylase and tyrosine phenol lyase.
  11. 　さらにフラビンモノヌクレオチドプレニルトランスフェラーゼをコードするポリヌクレオチドを含む、請求項１０記載のベクター又はＤＮＡ断片。 The vector or DNA fragment according to claim 10, further comprising a polynucleotide encoding flavin mononucleotide prenyltransferase.
  12. 　チロシンフェノールリアーゼが、以下の（Ｂ１）、（Ｂ２）、（Ｃ１）、（Ｃ２）、（Ｄ１）及び（Ｄ２）からなる群より選択されるタンパク質である、請求項１０又は１１記載のベクター又はＤＮＡ断片。
    （Ｂ１）配列番号２に示すアミノ酸配列からなるタンパク質
    （Ｂ２）配列番号２に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
    （Ｃ１）配列番号４に示すアミノ酸配列からなるタンパク質
    （Ｃ２）配列番号４に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質
    （Ｄ１）配列番号６に示すアミノ酸配列からなるタンパク質
    （Ｄ２）配列番号６に示すアミノ酸配列と少なくとも９０％以上の同一性を有するアミノ酸配列からなり、かつチロシンフェノールリアーゼ活性を有するタンパク質     The vector or according to claim 10 or 11, wherein the tyrosine phenol lyase is a protein selected from the group consisting of the following (B1), (B2), (C1), (C2), (D1) and (D2) DNA fragment.
    (B1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B2) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, and having tyrosine phenol lyase activity (C1) sequence Protein (C2) consisting of the amino acid sequence shown in SEQ ID NO: 4. Protein (D1) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having tyrosine phenol lyase activity (D1) shown in SEQ ID NO: 6. Protein consisting of an amino acid sequence (D2) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having tyrosine phenol lyase activity
  13. 　請求項９～１２のいずれか１項記載のベクター又はＤＮＡ断片を含有する形質転換細胞。 A transformed cell containing the vector or DNA fragment according to any one of claims 9-12.
  14. 　微生物が、請求項１３記載の形質転換細胞である、請求項１～９のいずれか１項記載の方法。 The method according to any one of claims 1 to 9, wherein the microorganism is the transformed cell according to claim 13.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05123177A (en) * 1991-10-30 1993-05-21 Ajinomoto Co Inc Production of l-3,4-dihydroxyphenylalanine
JPH0739385A (en) * 1993-07-30 1995-02-10 Ajinomoto Co Inc Production of l-3,4-dihydroxyphenylalanine
WO2011085311A1 (en) * 2010-01-08 2011-07-14 Draths Corporation Methods for producing isomers of muconic acid and muconate salts
WO2018199112A1 (en) * 2017-04-25 2018-11-01 国立大学法人長岡技術科学大学 Transgenic microorganism and use thereof
WO2019018302A1 (en) * 2017-07-18 2019-01-24 Arizona Board Of Regents On Behalf Of Arizona State University Synthetic metabolic funneling for biochemical production

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05123177A (en) * 1991-10-30 1993-05-21 Ajinomoto Co Inc Production of l-3,4-dihydroxyphenylalanine
JPH0739385A (en) * 1993-07-30 1995-02-10 Ajinomoto Co Inc Production of l-3,4-dihydroxyphenylalanine
WO2011085311A1 (en) * 2010-01-08 2011-07-14 Draths Corporation Methods for producing isomers of muconic acid and muconate salts
WO2018199112A1 (en) * 2017-04-25 2018-11-01 国立大学法人長岡技術科学大学 Transgenic microorganism and use thereof
WO2019018302A1 (en) * 2017-07-18 2019-01-24 Arizona Board Of Regents On Behalf Of Arizona State University Synthetic metabolic funneling for biochemical production

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