WO2006016432A1 - Novel aromatic dehydrogenase gene and process for producing aromatic carboxylic acid - Google Patents

Novel aromatic dehydrogenase gene and process for producing aromatic carboxylic acid Download PDF

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WO2006016432A1
WO2006016432A1 PCT/JP2005/004163 JP2005004163W WO2006016432A1 WO 2006016432 A1 WO2006016432 A1 WO 2006016432A1 JP 2005004163 W JP2005004163 W JP 2005004163W WO 2006016432 A1 WO2006016432 A1 WO 2006016432A1
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acid
aromatic
peptide
seq
dna
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PCT/JP2005/004163
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French (fr)
Japanese (ja)
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Xue Peng
Hironori Taki
Kaneo Kanoh
Norihiko Misawa
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Marine Biotechnology Institute Co., Ltd.
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Priority to JP2006531252A priority Critical patent/JP4881160B2/en
Publication of WO2006016432A1 publication Critical patent/WO2006016432A1/en

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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0093—Oxidoreductases (1.) acting on CH or CH2 groups (1.17)

Definitions

  • the present invention relates to a novel enzyme that oxidizes an aromatic compound having a hydroxymethyl group or an aldehyde group, a gene encoding the same, and a microorganism into which the gene has been introduced.
  • a microorganism into which this gene has been introduced using a microorganism into which this gene has been introduced,
  • the present invention relates to a method for producing an aromatic carboxylic acid from an aromatic compound having a methyl group or an aldehyde group.
  • Alcohol dehydrogenase is an enzyme that reversibly converts benzilanolol to benzaldehyde
  • XylB enzyme in the toluene metabolism system encoded by the TOL plasmid pWWO of Pseudomonas putida is the best studied.
  • XylB depends on NAD (P) + and zinc ions, and not only benzyl alcohol but also-, ⁇ 1-, -methylbenzylanolol, m-, £ -methoxybenzyl alcohol as substrates. c but into a body, if the 2_ methylbenzyl alcohol and 2-main butoxy benzyl alcohol was used as a substrate, the conversion efficiency was poor (Shaw, JP Schwager, F., and Harayama, S.
  • NAD (P) + and Zinc A gene encoding 4-nitrobenzyl alcohol dehydrogenase (NtnD) that is not dependent on ON and has low homology with XylB of the TOL plasmid was isolated from the TW3 strain of the genus Pseudomonas (James, KD, Hughes, MA, and Will iams, PA, Cloning and expression of ntnD, encoding a novel NAD (P) +-independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp. Strain TW3. J. Bacteriol. 182 (11), 3136-3141, 2000 ).
  • NtnD consists of Benzenorea Noreconole, m-, £ —Nitrobenzinoreanoreconole, 0—, m-, £ —Metinorepenzino Renoreco Inore, m—Hydroxy Benzenore Arconore, £ — Each aldehyde was synthesized using ethinolevenoleanoreconole, 2, 4-, 3, 4-, 3, 5-dimethylbenzyl alcohol as a substrate. 0-, £ -Hydroxybenzinoleol alcohol and 2,5-dimethylenorebenzylanorecoinole could not be converted.
  • aromatic alcohol (benzyl alcohol) dehydrogenase AreB isolated from the ADP1 strain of the genus Acinetobacter genus Acinetobacter is highly homologous to XylB of the T0L plasmid pWWO. , £ -Methinolenginole alcohol, ⁇ -, ra-, £ -Hydroxybenzyl alcohol reported activity (Jones, RM., Collier, LS, Neidle, EL, and Williams, PA, areABC genes determine the catabol ism of aryl esters in Acinetobacter sp. ADPl, J. Bacteriol. 181, 4568-4575, 1999).
  • Aromatic aldehyde dehydrogenase catalyzes the reaction from benzaldehyde to benzoic acid.
  • the best known is the XylC enzyme encoded by the T0L plasmid pWWO, whose enzyme activity depends on NAD (P) + (Harayama et al., Supra).
  • xylB and xylC genes were isolated from Acinetobacter calcoaceticus NCIB 8250, and the amino acid sequences encoded by them were highly homologous to T0L plasmids XylB and XylC, and the substrate specificity was almost the same.
  • alcohol dehydrogenase and aldehyde dehydrogenase Genases are enzymes that convert aromatic alcohols and aromatic aldehydes into aromatic aldehydes and aromatic carboxylic acids, respectively.
  • a monocyclic aromatic compound having a hydroxymethyl group such as benzyl alcohol is converted to its carboxylic acid.
  • metabolic pathways at the cell (cell) level of polycyclic aromatic hydrocarbons have been speculated in many polycyclic aromatic hydrocarbon-utilizing bacteria.
  • the metabolic pathway of 2, 6-dimethylnaphthalene, one of the polycyclic aromatic hydrocarbons is Flavobacteria (Barns ⁇ ey, n.A., Metabolism ⁇ 2, o dimethy ⁇ naphthalene by flavobacteria. Appl. Environ. Microbiol. 54 (2), 428-433, 1988), Pseudomonas sp.
  • D-87 (Miyachi, N., Tanaka, T., Suzuki, T., Hotta, Y., and Omori, T., Microbial oxidation of dimethylnaphthalene isomers. Appl. Environ. Microbiol. 59 (5), 1504-1506, 1993), Psudomonas cepacia F297 (Grifoll, M., Selifonov, SA, Gatlln, CV, and Chapman, PJ, Actions of a versatile fluorene -degrading bacteria ⁇ isolate on polycyclic aromatic compounds. Appl. Environ. Microbiol.
  • the monooxygenase gene that catalyzes the first reaction was reported by Bramucci et al. In the Sphingomonas genus ASU 1 (Bramucci, M., Singh, M., and
  • An object of the present invention is to obtain a gene encoding an enzyme that oxidizes an aromatic compound having various hydroxymethyl groups or aldehyde groups and converts it into a carboxylic acid. Furthermore, the present invention provides a method for producing various aromatic carboxylic acids using a recombinant microorganism into which this gene has been introduced and expressed.
  • the present inventors have already encoded a novel aromatic compound dioxygenase that synthesizes 1,4-dihydroxymethylnaphthalene, a commercially available aromatic compound, using 1,4-dimethylnaphthalene as a substrate.
  • phnA! A2A3A4 was acquired from the genus Cycloc lasticus (Japanese Patent Application 2003-324117).
  • Escherichia coli having the present gene transferred 1,4-dimethylnaphthalene to produce 1,4-dihydroxymethylnaphthalene.
  • the present inventors isolated the 14DN61 strain of the genus Sphingomonas that can use 1,4-dimethylnaphthalene as the sole carbon source from the soil of the Niigata Pref.
  • the present inventors can convert 1,4-dihydroxymethylnaphthalene by using the above 1,4-dihydroxymethylnaphthalene as a substrate and mixed culture with Escherichia coli introduced with DNA derived from the genus Sphingomonas bacterium 14DN61.
  • the key oxidase (dehydrogenase) gene required to convert 1,4-dihydroxymethylnaphthalene to 4-hydroxymethyl-1-naphthoic acid was identified, and the present invention was completed.
  • the gene according to the present invention encoded an aromatic aldehyde dehydrogenase having an activity of converting an aromatic aldehyde into an aromatic carboxylic acid.
  • Microorganisms such as Escherichia coli introduced with this gene were able to convert aromatic alcohol into aromatic carboxylic acid. This was totally unexpected. Microorganisms such as Escherichia coli are inherently weak but convert aromatic alcohol to aldehyde. It has its own aromatic alcohol dehydrogenase activity, and the coexistence of the PhnN enzyme (phnN gene product) improves the rotational efficiency of its own aromatic alcohol dehydrogenase. This is because aromatic carboxylic acids must be produced.
  • PhnN enzyme phnN gene product
  • coli expressing the EiinU gene is not only an aromatic compound having a naphthalene skeleton having a hydroxymethyl group, but also a very wide range of aromatic compounds ranging from aromatic compounds having a benzene skeleton having a hydroxymethyl group. could be converted to an aromatic carboxylic acid. This also reversed the expectation of the inventors, that is, the conventional common sense.
  • the present inventors have reported that the aromatic alcohol dehydrogenase gene derived from the o-xylene-utilizing bacterium Rhodococcus opacus TKN14 strain has various aromatic compounds (aromatic compounds having a hydroxymethyl group in the molecule).
  • aromatic compounds aromatic compounds having a hydroxymethyl group in the molecule.
  • benzyl alcohol and various benzyl alcohol substitutes, and also a novel enzyme that converts hydroxymethylnaphthalene to the respective aldehydes.
  • the inventors of the present invention together with the aromatic alcohol dehydrogenase gene, E. coli expressing the aromatic aldehyde dehydrogenase gene described above, each of the above-mentioned various aromatic hydroxymethyl compounds have their respective strengths. It was clarified that it can be converted to sulfonic acid.
  • the present invention has been completed based on the above findings.
  • the present invention provides the following (1) to (6).
  • the transformant according to (3) to (4) is cultured in a medium containing an aromatic compound having a hydroxymethyl group or an aldehyde group, and aromatic rubonic acid is obtained from the culture or the cell.
  • a process for producing an aromatic carboxylic acid characterized in that
  • the resulting aromatic carboxylic acid is 4-hydroxymethyl-1-naphthoic acid, 6-methyl-2-naphthoic acid, 1-naphthoic acid, 2-naphthoic acid, 4-methylbenzoic acid, 3 _Methylbenzoic acid, 2-Methylbenzoic acid, Benzoic acid, 4-Hydroxymethylbenzoic acid, 4-Hydroxybenzoic acid, 3-Hydroxybenzoic acid, 2-Hydroxybenzoic acid, 4-Methoxybenzoic acid A small amount selected from peroxy acid, 3-methoxybenzoic acid, 2-methoxybenzoic acid, 4_ethyl benzoic acid, 4-isopropylpyrubenzoic acid, 3-dioxybenzoic acid and 3-phenylpropionic acid
  • the method for producing an aromatic carboxylic acid according to (5) characterized in that it is at least one kind of aromatic carboxylic acid.
  • a peptide comprising an amino acid sequence in which one or a plurality of amino acids are attached, deleted or substituted in the amino acid sequence described in self-sequence number 5, and having an aromatic alcohol dehydrogenase activity;
  • the transformant according to (9) or (10) is cultured in a medium containing an aromatic compound having a hydroxymethyl group to obtain an aromatic carboxylic acid from the culture or the cell.
  • a process for producing an aromatic carboxylic acid is produced.
  • the resulting aromatic carboxylic acid is 2-naphthoic acid, benzoic acid, 2-, 3-, 4-methylbenzoic acid, 4-hydroxymethylbenzoic acid, 2-, 3-, 4-hydroxy. It is characterized by being at least one aromatic carboxylic acid selected from the group consisting of benzoic acid, 2-, 3-, 4-methoxyoxybenzoic acid, 3-chlorobenzoic acid, cinnamic acid and vanillic acid.
  • Fig. 1a is a diagram showing an example of conversion of an aromatic alcohol compound by E. coli having a £ ⁇ gene.
  • Fig. 1b shows an example of conversion of an aromatic alcohol compound by E. coli with a gene.
  • FIG. 2 is a diagram showing an example of conversion of an aromatic aldehyde compound by E. coli having a phnN gene.
  • Figure 3 shows the results of analysis of GC-MS using TMS of the conversion product of 1,4-dihydroxymethylnaphthalene pMA9-54 by E. coli JM109 transformant.
  • the lower part b) is a TMS mass spectrum of 1-hydroxymethylnaphthoic acid as a conversion product.
  • Figure 4 shows the restriction map of the 7-kb Kpnl fragment and the location of each gene.
  • the 1,4-dihydroxymethylnaphthalene conversion activity of each subclone is shown on the right. + Indicates conversion activity and-indicates no conversion activity.
  • Figure 5 shows the alignment of the sequences used for primer design.
  • Acineto means the areB gene of Acinetobacter sp. ADP1 strain, “xylB—AcinetoJ tastes Acinetobacter calcoaceticus NCIB 8250 xylB ⁇ zs; child”, “xylB_T0L” means Pseudomonas putida T0L plasmid p0 It means dragon 0's xylB gene.
  • Fig. 6 is a photograph of plasmid pl8-34 digested with various restriction enzymes and subjected to agarose gel electrophoresis.
  • Figure 7 shows the phylogenetic tree of XylB and related enzymes, where the numbers are bootscrap values (1000 tests).
  • MyrB_Pseudo ⁇ as means Anolechol dehydrogenase MyrB of Pseudomonas sp.
  • XylB—TKN14J means XylB (protein according to the present invention) of Rhodococcus opacus TKN14 strain
  • TerpD_Pseudomonas Bacterial TerpD means “XylB—Ac inetoII J means Acyletobater cal coaceti cus NCIB 8250 ylB,“ AreB 1 AcinetoJ means Acinetobater sp.
  • XylB—pWW0 putida T0L plasmid means XylB (Accession number, D63341) coded by pWWO
  • XylB—T0L is XylB (Shaw, JP, Rekik, coded by T0L plus.mid of Pseudononas putida mt-2) M., Schwager, F., and Harayama, Kinetic studies on benzyl alcohol dehydrogenase encoded by T0L p ⁇ asmid pWWO, J. Biological Chemistry,
  • ADH1— Man is a human alcohol dehydrogenase 1 gene.
  • FEBS Lett. (Matsuo, Y., Yokoyama, S. Molecular structure of the human alcohol dehydrogenase 1 gene. , 243, 57-60, 1989).
  • Figure 8 shows the structure of the prepared plasmid, where Sp stands for l And B means BamHI, A means, Xo means, EV means EcoRV, E means EcoRI, N means Notl, and P means Pstl.
  • the peptide according to the present invention has an aromatic compound dehydrogenase activity, for example, from an aromatic family compound aldehyde dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 3 and an amino acid sequence shown in SEQ ID NO: 5.
  • An aromatic alcohol dehydrogenase can be mentioned.
  • the aromatic compound aldehyde dehydrogenase activity means the activity of oxidizing an aromatic aldehyde to an aromatic carboxylic acid.
  • the aromatic family compound dehydrogenase consisting of the amino acid sequence of SEQ ID NO: 3 is a sphingomonas (Sphingomonas) that can be used as the sole carbon source from 1,4-dimethylnaphthalene isolated from the soil of the Niigata Petroleum. ) It can be isolated from the genus bacterium 14DN61 strain.
  • the base sequence encoding the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 2.
  • the peptide according to the present invention is based on an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 3 as long as it has aromatic compound dehydrogenase activity. It may be.
  • the plurality of amino acids means, for example, 1 to 50 amino acids, preferably 1 to 35 amino acids, more preferably 1 to 20 amino acids, and most preferably 1 to 10 amino acids. Means amino acid.
  • the stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • stringent conditions for example, DNAs having high homology (for example, DNAs having homology of 60% or more) hybridize and lower homology.
  • a condition where NAs do not hyper-predicate can be mentioned.
  • stringent conditions include washing in normal Southern high pridization. 60 ° C, 1 X SSC is a condition, 0. 1 0/0 SDS, preferably, 0. 1 X SSC, High Priestess soybeans conditions and the like at a salt concentration corresponding to 0. 1% SDS.
  • the peptide according to the present invention has an aromatic compound dehydrogenase activity is determined by adding an aromatic aldehyde (substrate) such as 2-naphthaldehydride to a solution containing the target peptide. Detect whether the solution after the reaction contains aromatic rubonic acid (reaction product) such as 2-naphthoic acid by GC-MS. As a result, if it can be confirmed that the aromatic aldehyde has been converted to an aromatic carboxylic acid, the target peptide has an aromatic compound dehydrogenase activity.
  • aromatic aldehyde substrate
  • 2-naphthaldehydride aromatic rubonic acid
  • the gene according to the present invention is a gene that codes for the above-described peptide, and comprises, for example, the base sequence of SEQ ID NO: 2.
  • the gene according to the present invention has a nucleotide sequence having a homology of 60% or more, preferably 70% or more, more preferably 80% or more of the nucleotide sequence of SEQ ID NO: 2. It may comprise a sequence and may comprise a DNA encoding a peptide having the above-mentioned aromatic compound dehydrogenase activity.
  • the homology is a numerical value obtained, for example, by executing a command of the Maxim matching method using DNASIS (Hitachi Software Engineering) which is sequence analysis software. The parameters at that time shall be the default settings (initial settings).
  • the aromatic alcohol dehydrogenase activity means an activity of oxidizing an aromatic alcohol (aromatic hydroxymethyl compound) having a hydroxymethyl group in the molecule to an aromatic aldehyde.
  • the aromatic alcohol dehydrogenase comprising the amino acid sequence of SEQ ID NO: 5 can be isolated from the 2-xylene-utilizing bacterium Rhodococcus opacus TKN14.
  • the base sequence encoding the amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 4.
  • Rhodococcus opacus TKN14 strain can be obtained from Marine Biotechnology Co., Ltd. under the culture collection number “MBIC 05572”.
  • the gene encoding the above-mentioned aromatic alcohol dehydrogenase (base sequence of SEQ ID NO: 4) can be obtained from the obtained mouthdococcus opacus TKN14 strain according to a conventional method.
  • the gene encoding the above aromatic alcohol dehydrogenase is represented by the nucleotide sequence information of SEQ ID NO: 4. It can also be obtained by chemical synthesis based on this.
  • amino acids in the amino acid sequence shown in SEQ ID NO: 5 are added, deleted or substituted. It may consist of an array.
  • the plurality of amino acids means, for example, 1 to 50 amino acids, preferably 1 to 35 amino acids, more preferably 1 to 20 amino acids, most preferably 1 to Means 10 amino acids.
  • the peptide according to the present invention hybridizes under stringent conditions to DNA consisting of the base sequence of SEQ ID NO: 4 or DNA complementary thereto as long as it has aromatic compound dehydrogenase activity. It may be one encoded by DNA.
  • the stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • stringent conditions for example, DNAs with high homology (for example, DNAs having homology of 60% or more) hybridize and DNAs with lower homology do not hybridize with each other.
  • stringent conditions include washing conditions in normal Southern hybridization, 60 ° C, 1 X SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1 A condition for hybridizing at a salt concentration corresponding to% SDS is mentioned.
  • whether or not the peptide according to the present invention has an aromatic compound alcohol dehydrogenase activity is determined by, for example, determining whether the peptide containing the peptide of interest has an aromatic hydroxymethyl compound such as 2-hydroxymethylnaphthalene (Substrate) is added, and GC-MS detects whether the solution after the reaction contains aromatic aldehydes (reaction products) such as 2-naphthalaldehyde. As a result, if it can be confirmed that the aromatic alcohol has been converted to an aromatic aldehyde, the subject peptide will have an aromatic alcohol dehydrogenase activity.
  • aromatic aldehydes reaction products
  • the gene according to the present invention is a gene that codes for the above-mentioned peptide, and consists of the base sequence of SEQ ID NO: 4, for example.
  • the gene according to the present invention is SEQ ID NO:
  • the base sequence consists of a base sequence having 60% or more homology, preferably 70% or more homology, more preferably 80% or more homology with respect to the base sequence of 4. It may be composed of DNA that encodes a peptide having compound alcohol dehydrogenase activity.
  • the homology is a numerical value obtained by, for example, executing a command of the Maxim matching method using DNASIS (Hitachi Software Engineering), which is sequence analysis software. The parameters at that time are the default settings (initial settings).
  • the gene according to the present invention can be a recombinant vector obtained by ligation (insertion) into a commonly used vector.
  • the vector for inserting the gene according to the present invention is not particularly limited as long as it can be replicated in the host, and examples thereof include plasmid emergence and phage DNA.
  • the plasmid DNA includes plasmids for E. coli hosts such as pBR322, pBR325, pUC118, and pUC119, plasmids for Bacillus subtilis such as pUB110 and pTP5, plasmids for yeast hosts such as YEpl3, YEp24, and YCp50, ⁇ 221, ⁇ 21, etc.
  • E. coli hosts such as pBR322, pBR325, pUC118, and pUC119
  • Bacillus subtilis such as pUB110 and pTP5
  • plasmids for yeast hosts such as YEpl3, YEp24, and YCp50, ⁇ 221, ⁇ 21, etc.
  • yeast hosts such as YEpl3, YEp24, and YCp50, ⁇ 221, ⁇ 21, etc.
  • examples include plasmids for plant cell hosts, and examples of phage DNA include: phages.
  • the purified DNA having the gene according to the present invention is cleaved with an appropriate restriction enzyme, and a restriction enzyme site or a multicloning site of an appropriate vector DNA is cut.
  • a method of inserting into a vector and linking to a vector is employed.
  • the gene according to the present invention can be incorporated into a vector so that the function of the gene is exhibited. That is, the vector can be prepared so as to contain a promoter, a gene according to the present invention, a cis element such as an enhancer, a splicing signal, a polyaddition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like.
  • a selection marker for example, ampicillin resistance gene, neomycin resistance gene, dihydrofolate reductase gene and the like can be used.
  • a transformant can be obtained by introducing a recombinant vector containing the gene of the present invention into a host so that the gene can be expressed.
  • the host is not particularly limited as long as it can express the gene of the present invention.
  • Escherichia such as Escherichia coli
  • Bacillus such as Bacillus subtilis
  • Syudomonas such as Pseudomonas putida
  • Rhizobium me examples include bacteria belonging to the genus Rhizobium, such as Rhizobium mel iloti, and Saccharomyces cerevis iae, Schizosaccharomyces pombe, and Hihi no ichi ris.
  • yeast, etc. plant cells established from Arabidopsis thaliana, tobacco, corn, rice, carrots, etc., protoplasts prepared from these plants, animal cells such as COS cells, CH0 cells, etc. Or insect cells such as Sf9 and Sf21.
  • the recombinant vector of the present invention can autonomously replicate in the bacterium, and at the same time, is composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Preferably it is. Moreover, the gene which controls a promoter may be contained.
  • Any promoter can be used as long as it can be expressed in a host such as E. coli.
  • those derived from Escherichia coli such as trp promoter, lac promoter, PL promoter, PR promoter, and those derived from phage such as T7 promoter are used.
  • artificially designed and modified promoters such as the tac promoter may be used.
  • the method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria.
  • a method using calcium ions [Cohen, SN, et al .: Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972)], and an electroporation method.
  • yeast When yeast is used as a host, for example, Saccharomyces cereviche, Schizosaccharomyces bomb, Pichia nostris, etc. are used.
  • the promoter is not particularly limited as long as it can be expressed in yeast.
  • gal l promoter gal l O promoter
  • heat shock tannomi Quality promoter
  • MF CK I promoter MF CK I promoter
  • PH05 promoter PH05 promoter
  • PGK promoter GAP promoter
  • the method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast.
  • the electric mouth population method [Becker, DM, et al .: Methods. Enzymol., 194: 182-187 (1990)]
  • Spheroplast method [Hinnen, A. et al .: Proc. Natl. Acad. Sci., USA, 75: 1929-1933 (1978)]
  • lithium acetate method [Itoh, H. : J. Bacteriol., 153: 163-168 (1983)].
  • the promoter is not particularly limited as long as it can be expressed in a plant, and examples thereof include a power reframer mosaic virus 35S RNA promoter, an rd29A gene promoter, and an rbcS promoter.
  • monkey cells COS-7, Vero, Chinese hamster ovary cells (CH0 cells), mouse L cells, rat GH3, and human FL cells are used.
  • the promoter SRa promoter, SV40 promoter, LTR promoter, CMV promoter, etc. may be used, and a human megalovirus early gene promoter may also be used.
  • methods for introducing a recombinant vector into animal cells include the electoral position method, the calcium phosphate method, and the lipofection method.
  • Sf9 cells When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used.
  • a method for introducing a recombinant vector into an insect cell for example, a calcium phosphate method, a lipofection method, an electroporation method and the like are used.
  • An aromatic carboxylic acid is obtained from the culture and / or transformant by culturing the transformant obtained as described above in a medium containing an aromatic compound having a hydroxymethyl group or an aldehyde group.
  • the aromatic aldehyde dehydrogenase according to the present invention can increase the activity of an aromatic alcohol dehydrogenase that oxidizes an aromatic hydroxymethyl compound possessed by the host itself into an aromatic aldehyde. Therefore, transformation in a medium containing aromatic hydroxymethyl compounds By culturing the recombinant, the host's own aromatic alcohol dehydrogenase activity is increased, and as a result, an aromatic carboxylic acid can be obtained from the culture and / or the transformant.
  • the aromatic alcohol dehydrogenase according to the present invention can produce an aromatic aldehyde that is possessed by the host itself or an aromatic aldehyde that becomes a substrate for the aromatic aldehyde dehydrogenase introduced into the host. Therefore, aromatic carboxylic acid can be obtained from the culture and / or transformant by culturing the transformant in a medium containing an aromatic hydroxymethyl compound.
  • the aromatic hydroxymethyl compound contained in the medium by the action of each enzyme Can be efficiently converted into aromatic rubonic acid.
  • FIGS. 1a and 1b The correspondence between the aromatic compound having a hydroxymethyl group as a substrate and the aromatic carboxylic acid as a reaction product is shown in FIGS. 1a and 1b.
  • Figure 4 shows the correspondence between an aromatic compound having an aldehyde group as a substrate and an aromatic carboxylic acid as a reaction product.
  • 1A to 19A represent substrates
  • 1B to 19B represent reaction products corresponding to 1A to 19A, respectively.
  • the compound names of 1A-19A and 1B-19B are as follows:
  • 1A 1,4-dihydroxymethylnaphthalene; 1B, 4-hydroxymethyl-1-naphthoic acid; 2A, 2-hydroxymethyl-6-methylenonaphthalene; 2B, 6-methyl-2-naphthoic acid; 3A, 1-hydroxymethylnaphthalene; 3B, 1-naphthoic acid; 4A, 2-hydroxymethylnaphthalene; 4B, 2-naphthoic acid; 5A, 4-methylbenzyl alcohol; 5B, 4-methylbenzoic acid; 6A 3B-Methylbenzyl alcohol; 6B, 3-Methylbenzoic acid; 7A, 2-Methylbenzyl alcohol; 7B, 2-Methylbenzoic acid; 8A, Benzyl alcohol; 8B, Benzoic acid; 9A, Xylene- ⁇ -Hin ' -Diol; 9 ⁇ , 4-hydroxymethylbenzoic acid;
  • 15B .2-Methoxybenzyl alcohol; 15B, 2-Methoxybenzoic acid; 16A, 4-Ethylbenzyl alcohol; 16B, 4-Ethylbenzoic acid; 17A, 14B, 3-Methoxybenzoic acid;
  • 17-B 4-Isopropyl benzoic acid; 18A, 3-chlorobenzil alcohol; 18B, 3-mouth benzoic acid; 19A, 3_phenylpropyl alcohol; 19B, 3 -Phenylpropionic acid.
  • 1A to 6A represent substrates
  • .1B to 6B represent reaction products corresponding to 1A to 6A, respectively.
  • the compound names 1A-6A and 1B-6B are as follows:
  • the concentration of the aromatic compound in the medium is not particularly limited.
  • the concentration is 10 mM, preferably 2 mM, more preferably 0.5 mM.
  • a medium suitable for the host of the transformant described above can be used.
  • LB medium can be used.
  • any medium that contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host and that can efficiently culture transformants can be used. May be used.
  • the carbon source carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salt of organic acid, and other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, etc. Is used.
  • Inorganic substances include potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, Sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
  • Culturing is usually performed at 30 to 37 ° C for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and agitation culture, depending on the host of the transformant.
  • the pH is maintained at 7.0 to 7.5.
  • the pH is adjusted using inorganic or organic acid, alkaline solution, etc.
  • an indeuser When culturing a microorganism transformed with a recombinant vector containing an inducible promoter as a promoter, an indeuser may be added to the medium as necessary.
  • an indeuser may be added to the medium as necessary.
  • an expression vector using the Lac promoter when cultivating a microorganism transformed with an expression vector using the Lac promoter, when culturing a microorganism transformed with isopropyl-jS-D-thiogalactopyranoside (IPTG) or the like with an expression vector using the trp promoter.
  • IPTG isopropyl-jS-D-thiogalactopyranoside
  • IAA Indoleacrylic acid
  • the peptide according to the present invention When the peptide according to the present invention is produced in cells or cells after culturing, the peptide can also be extracted by destroying the cells or cells. In addition, when the peptide is produced outside the cells or cells, the cells or cells are removed by centrifuging using the culture solution as it is. Then, general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, genomic chromatography, ion exchange chromatography, affinity mouth matography, etc., alone or in combination as appropriate. By using it, the peptide of the present invention can be isolated and purified from the culture.
  • Examples 1 to 10 describe the aromatic aldehyde dehydrogenase according to the present invention
  • Examples 11 to 18 describe the aromatic alcohol dehydrogenase according to the present invention.
  • W medium inorganic medium
  • 0.1% of a sample collected from an oil ejection source and 0.2% of 1,4-dimernaphthalene were added and cultured at 30 ° C with shaking.
  • the composition of W medium is shown below.
  • K P0 4 (0. 85 g / 1)
  • (NH 4 ) 2 S0 4 (0.
  • Routine genetic engineering experiments such as plasmid preparation, restriction enzyme treatment, ligation reaction, transformation, etc. are performed by Sambrook et al. In Molecular Cloning (Sambrook, J., Fritsch,
  • Example 3 Preparation of chromosomal DNA from Sphingomonas sp. Strain 14DN61
  • Sphingomonas sp. Strain 14DN61 obtained in Example 1 was cultured in 300 ml of Tryptic Soy Broth (TSB) medium (Difco) at 30 ° C. for 2 days.
  • TLB Tryptic Soy Broth
  • the cells were washed twice with STE buffer (100 mM NaCl, 10 mM Tris ⁇ HC1, 1 mM EDTA, pH 8.0), heat-treated at 68 ° C for 15 minutes, then 5 mg It was suspended in solution I (50 mM glucose, 25 mM Tris ⁇ HC1, 10 mM EDTA, pH 8.0) containing / ml lysozyme (Sigma) and 100 ig / ml RNAase (Sigma). After incubating at 37 ° C for 1 hour, Protenase K (Sigma) was added to 10 mg / ml, and incubated at 37 ° C for 10 minutes.
  • STE buffer 100 mM NaCl, 10 mM Tris ⁇ HC1, 1 mM EDTA, pH 8.0
  • N-Lauroylsarcosin ⁇ Na was added so that the final concentration would be 1%, and the mixture was gently mixed thoroughly by inversion and then incubated at 37 ° C for 3 hours. After several phenol / chloroform extractions, slowly add 2 volumes of ethanol, wind up the precipitated chromosomal DNA with a glass rod, rinse with 70% ethanol, and then add 2 ml of TE. It was dissolved in a buffer solution (10 mM Tris ⁇ HC1, 1 mM EDTA, pH 8.0) to obtain a chromosomal DNA solution. '
  • the chromosomal DNA of Sphingomonas genus 14DN61 was partially cleaved with 3AI so that a DNA fragment of about 30-42 kb in length could be collected and treated with phosphatase (CIAP, manufactured by TAKARA).
  • the cleavage pattern of DNA by Sau3AI was confirmed by electrophoresis using a TAE agarose gel.
  • SuperCos 1 (Stratagene) was used as a smid vector, digested with ⁇ I, and then treated with phosphatase.
  • this vector DNA was digested with ⁇ HI, and then subjected to a ligation reaction (Ligation High T0Y0B0) with the Sau3AI partial degradation product of the above chromosomal DNA.
  • Sphingomonas sp. Strain 14DN61 was expected to cleave 1,4-dimethylnaphthalene to catechol and then cleave it with catechol dioxygenase.
  • the target gene may be in the vicinity of the force lever gene.
  • the catechol degraded by catechol dioxygenase turns yellow, Because of the ease of screening, we first attempted to isolate this gene.
  • a few hundred out of about 1,000 E. coli clones on the LB plate were used to spray an aqueous solution of 50 mM catechol and left at 37 ° C for 30 minutes. As a result, one clone (pMA13-10) turned yellow.
  • pMA13-10 did not have the ability to convert 1,4-dihydroxymethylnaphthalene, a clone overlapping pMA13-10 (pMA9-54) was newly isolated from the library by colony hybridization.
  • the bacterial solution further 1, 4 dihydric mud xylene was added 100 RAM DMS0 solution of methyl naphthalene to a final concentration of 0. 1 mM, was-2 days co-culture with 30 P C.
  • 100 l of this culture solution 150 ⁇ l of ethyl acetate and 10 ⁇ l of 3% hydrochloric acid were added, vortexed and centrifuged for 5 minutes at 15,000 rpm, and the supernatant ethyl acetate phase was recovered and dried.
  • it was trimethylsilylated with a trimethylsilylating agent (DG Science Co., Ltd.) to prepare a sample for gas chromatography / mass spectrum (GC-MS).
  • DG Science Co., Ltd. trimethylsilylating agent
  • the GC-MS used was a Shimadzu model QP5050A, and the column used was a DB-5 column (30 x 0.25 mm) manufactured by J & W Scientific.
  • the column temperature was initially maintained at 50 ° C for 5 minutes, and then increased to 300 ° C at a rate of 10 ° C nomin, and maintained at 300 ° C for 4 minutes. ).
  • the injection temperature and detection temperature were 300 ° C.
  • Figure 3 shows the analysis results. As a conversion product of 1,4-dihydroxymethylnaphthalene, a peak with a retention time of 25.1 minutes was detected, and its molecular weight was 346. Calculated from this molecular weight, it was estimated to be 4-hydroxymethyl ⁇ -1-naphthoic acid.
  • PMA9-54 was cleaved with Kpnl, and the 7-kb] ⁇ 1 fragment was inserted into the E. coli vector pBluescript II KS + to construct plasmid pMA7K5 and plasmid pMAl in which the inserted fragment was reversed.
  • pMA7K5 and pMA7Kl were cleaved with various restriction enzymes, and various subclones were constructed as shown in FIG.
  • pNB2.18N is a plasmid prepared by inserting the 2.18-kb Not BamHI fragment in the 7-kb Kpnl fragment into the Notl-BamHI site of pBluescript II SK +.
  • PGR Thermal Cycler MP (Takara Shuzo), BigDye Terminator Cycle Sequencing Ready Reaction Kit Ver. 2.0 (Applied Biosystems), 96 ° C with 5% dimethylsulfoxide (DMS0) 5 minutes, 92 ° C 10 seconds, 50. C 5 seconds, 60 ° C
  • the reaction product is Applied Biosystems 3700 DNA Analyzer
  • PhnN the encoded enzyme
  • the nucleotide sequence of the 14DN61 strain (base numbers 3222 to 4721 in SEQ ID NO: 1) and the encoded amino acid sequence are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the enzyme which has a certain degree of homology with the deduced amino acid sequence of PhnN in the database and whose function has been clarified, is the chloroaceta nodehyde hydrogenase (AldB) (Bergeron, H., Xanthobacter autotrophicus GJ10). Labbe, D., Turmel, C.
  • KUC-1 enzymologi cal characteri stics and functional properti es. Biochera. Biophys. Res. Commun. 298 (5), 632-637, 2002), Al cal igenes eutropha, ) (Pri ef ert, H., Kruger, N., j endrossek, D., Scnmidt, B., and Steinbuchel, A., Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcal igenes eutrophus. J. Bacteriol. 174 (3), 899-907, Aldehyde dehydrogenase such as 1992).
  • PhnN's amino acid sequences 216, 218 and 222 were considered to be NAD + binding sites.
  • Glu, No. 294 Cys was preserved.
  • acetaldehyde dehydrogenase gene (hnl), 4-hydroxy-2-oxovalerate aldorase gene (phnj), and 4-oxalosrotonate decarboxylase gene (E_hnK) (Hwang, S., Kim, SJ, Kim, CK) of SphinRomonas chungbukensis DJ77 , Kim, Y., Kim, SJ and Kim, YC
  • the phnl.T genes encoding acetaldehyde dehydrogenase (acylating) and 4-hydroxy-2 ⁇ oxovalerate aldolase in Pseudomonas sp. DJ77 and their evolutionary impl ications. Biochem.
  • the structure of 4-hydroxymethyl-1-naphthoic acid was determined as follows. pNB2. After 12 hours incubation at 30 ° C in LB medium containing ampicillin 100 g / ml of E. coli JM109 1 L containing 18N, after recovered by centrifugation of the bacteria, ampicillin 100 g / m l, The cells were dissolved in 1 L of M9 medium containing 0.2% (w / v) glucose, 0.01% (w / v) thiamine and 1 mM IPTG.
  • E. coli JM109 transformant containing ⁇ 2 ⁇ 18N was cultured at 30 ° C for 12 hours in LB medium containing 3 ml of ampicillin 100 ⁇ g / ral, and then LB containing 200 ml of ampicillin 100 ⁇ g / ml.
  • the medium was inoculated and cultured at 37 ° C so that 0D (600 nm) was 0.5.
  • I mM IPTG was added and induced for 4 hours.
  • the cells were collected by centrifugation and then dissolved in 10 ml of 50 mM Tris-HC1 (pH 7.5).
  • the cells were disrupted twice with a Otake Seisakusho punch press (l, 500 MPa), centrifuged at 15000 rpm, and the supernatant was used as the cell extract. Protein concentrations in cell extracts are measured by Bradford (Bradford, MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the priciple of protein-dye binding. Anal. Biochem. 72, 248-254, 1976) It went according to the method of.
  • Aldehyde dehydrogenase activity was measured using 1 ml of 50 raM Tris-HCl (pH 7.5) in a final concentration of 50 ⁇ M NAD +, 1 mg of pNB2.18N E. coli JM109 transformant as a cell extract and substrate. 50 ⁇ 2-naphthalaldehyde was added. After reacting at 30 ° C for 1 hour, extraction was performed with ethyl acetate according to the method of [Example 5], and the reaction product was analyzed by GC-MS. As a result, 2-naphthoic acid and 2-hydroxymethylnaphthalene were detected as reaction products from 2-naphthalaldehyde.
  • Alcohol dehydrogenase activity was measured using 1 ml glycine / NaOH buffer (pH 9.4) in a final concentration of 50 ⁇ M NAD +, 1 mg ⁇ 2 ⁇ 18N E. coli JM109 transformant cell extract, as substrate 50 2-hydroxymethylnaphthalene was added. After reacting at 30 ° C for 1 hour, extraction with ethyl acetate was performed according to the method of [Example 5], and the reaction product was analyzed by GC-MS. As a result, 2-naphthoic acid was detected as a reaction product from 2-hydroxymethylnaphthalene. Based on the above experimental results, PhnN It was proved that Escherichia coli expressing the enzyme activity of both alcohol dehydrogenase and aldehyde dehydrogenase.
  • a 1.8 pkb Bgin-Hindlll fragment containing the Eimil gene was inserted into the Bglll-HindlH site of pTrcHisA to produce plasmid pTrcNl.
  • a cell extract of E. coli JM109 transformant containing pTrcNl was prepared according to [Example 8]. The cell extract was purified on Amersharm Biosciences HisTrap TM HP column. The adsorption buffer was 20 mM phosphate, 30 mM imidazole, 0.5 M NaCl, and the elution buffer was 20 mM phosphate, 500 mM imidazole, 0.5 M NaCl.
  • the eluted sample was dialyzed against 20 mM Tris-HCl (pH 8) and further purified with Mono Q 5/5 HR manufactured by Amersharm Biosciences.
  • the adsorption buffer was 20 mM Tris-HCl (pH 8), and the elution buffer was 1 M NaCl added to the adsorption buffer.
  • SDS-PAGE confirming the eluted sample, it was almost a single panda.
  • the alcohol dehydrogenase activity was measured using the purified enzyme according to the method of [Example 8] using 2-hydroxymethylnaphthalene as a substrate. As a result, no enzyme activity was detected.
  • aldehyde dehydrogenase activity using a purified enzyme was measured according to the method of [Example 8] using 2-naphthalaldehyde as a substrate. As a result, enzyme activity was detected.
  • PhnN is an aldehyde dehydrogenase, and the conversion of aromatic alcohols to carboxylic acids by E. coli transformants expressing the gene indicates that the alcohol dehydrogenase of Ph. It was revealed that this was done by the joint action of drogenase.
  • E. coli JM109 containing 18N was cultured for 12 hours at 30 ° C in LB medium containing 3 ml of ampicillin 100 g / ml, and the cells were collected by centrifugation, and then ampicillin 100 ⁇ g / The cells were dissolved in 3 ml of M9 medium containing ml, 0.2% (w / v) glucose, 0.01% (w / v) thiamine and 1 mM IPTG. This bacterial cell solution was prepared as many as the number of substrates.
  • Each substrate 100 mM DMS0 solution was added to the bacterial cell solution so that the final concentration of the substrate was 0.5 mM, transferred to a test tube with a stopper, and the lid was sealed and cultured at 30 ° C for 2 days.
  • 100 ⁇ l of each culture was extracted with ethyl acetate by the method described in Example 5, and the ethyl acetate phase was analyzed by GC / MS under the conditions described in Example 5.
  • 2-hydroxymethyl-6-methylnaphthalene, 6-methyl-2-naphthoic acid, and 1-2-hydroxymethylnaphthalene 1- 2-, 3-naphthoic acid, 2-, 3-, 4-methylbenzyl alcohol, 2-, 3-, 4-methylbenzoic acid, benzyl alcohol, benzoic acid, xylene- ⁇ - ⁇ '-diol, 4-hydroxymethyl Benzoic acid, 2-, 3-, 4-hydroxybenzyl alcohol 2-, 3-, 4-hydroxybenzoic acid, 2-, 3-, 4-methoxybenzyl 2-, respectively.
  • E. coli JM109 containing pNB2.18N is 1-, 2-naphthoaldehyde from 1-, 2-naphthoic acid, 2-3-, 4-methinolevene aldehyde from 2-, 3-, 4-, respectively.
  • a conversion reaction from methylbenzoic acid and benzaldehyde to benzoic acid was also shown. These products were hardly detected in the conversion experiment using E. coli JM109 containing only vector pBluescript II SK.
  • the PhnN enzyme synthesized in E. coli was able to convert toxic aromatic aldehyde compounds to carboxylic acids at a high rate, and the alcohol dehydrogenase activity originally possessed by E. coli was thought to be enhanced.
  • the PhnN enzyme isolated this time has aldehyde dehydrogenase activity with a wide substrate specificity.
  • the PhnN enzyme is compatible with microorganisms such as Escherichia coli, and the transformant of E. coli expressing the gene is a polycyclic compound such as benzyl alcohol from a monocyclic compound such as benzyl alcohol. Since a very wide range of aromatic compounds leading to the compound could be converted to carboxylic acid, this enzyme was considered to be a novel and industrially useful enzyme.
  • Routine genetic manipulation experiments such as plasmid preparation, restriction enzyme treatment, ligation reaction, transformation, etc., described below, are described in Sambrook et al., Molecular Cloning (Sambrook, J., ritsch, E., and Mamatis, T., 1989). , Molecular cloning -a laboratory manual, 2nd edition, or Sambrook, J., Russel, DW, 2001, "Molecular cloning -a laboratory manual, Third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) Or the method shown in the protocol attached to the reagent used in the nuclear test system.
  • Rhodococcus opacus TKN14 is a 2-xylene-utilizing bacterium that is dominated by £ -xylene from soil in Kamaishi City, Iwate Prefecture. It was isolated from Kamaishi soil by the Marine Biotechnology Research Institute, Inc. to which they belong (Publication No. 2002-238551). This TKN14 strain was classified as a Rhodococcus bacterium by analysis of the 16S rDNA and gyrB gene sequences.
  • 2-xylene is known to be extremely difficult to degrade in soil compared to nr-xylene and £ -xylene, and there was little knowledge of 2-xylene-degrading bacteria and degradation genes. .
  • the present inventor has confirmed that the TKN14 bacteria are metabolized not by direct oxidation of the benzene ring but by oxidation of the methyl group of the side chain in the degradation of 2-xylene.
  • R. opacus TKN14 strain has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Accession number: FERM P-18195, accession date: February 2, 2001).
  • Rhodococcus opacus TKN14 strain was deposited under the name [“Rhodococcus sp. KN-14” at the Deposit Center. Rhodococcus opacus TKN14 strain can be obtained from Marine Biotechnology Co., Ltd. under the culture collection number “MBIC 05572”. The R. opacus TKN14 strain was cultured in 300 ml of Tryptic Soy Broth (TSB) medium (Difco) at 30 ° C. for 2 days.
  • TTB Tryptic Soy Broth
  • N-Lauroyl sarcosin.Na was added to a final concentration of 1%, and the mixture was gently mixed thoroughly by inversion and then incubated at 37 ° C for 3 hours. After several phenol / chloroform extractions, slowly add 2 volumes of ethanol, wind up the precipitated chromosomal DNA with a glass rod and rinse with 70% ethanol. It was dissolved in ml of TE buffer (10 mM Tris ⁇ HCl, 1 mM EDTA, pH 8.0) to obtain a chromosomal DNA solution.
  • TE buffer 10 mM Tris ⁇ HCl, 1 mM EDTA, pH 8.0
  • Chromosomal DNA of TKN14 strain can be recovered with a DNA fragment of about 30-42 kb in length
  • the chromosomal DNA of strain TKN14 was made into a saddle shape, and PCR was performed with the designed primers. As a result, a 214 bp fragment was amplified and cloned into a vector by TA cloning. The base sequence of the cloned 214 bp fragment was determined using the T7 promoter of the vector. The determined base sequence is shown in SEQ ID NO: 6.
  • the probe sequence was converted to RNA from the T7 promoter sequence of the vector containing the 214 bp amplified DNA fragment cloned in Example 14 by T7 RNA polymerase.
  • the probe was prepared as follows. 6 ⁇ of Sol. 1 (Transcription buffer, 2.0 1; DIG RNA labeling mixture, 2.0 ⁇ ; T7 RNA polymerase, 1.0 1) was added to 14 ⁇ 1 of the vertical DNA solution. Heated at 37 ° C for 90 minutes. The solution 21 was electrophoresed to confirm the transcript.
  • DNase l 1 ⁇ 1 and RNase Inhibitor 1 tl were added to the remaining 18 ⁇ 1 of the above solution and heated at 37 ° C. for 30 minutes.
  • DEPC treated water 80 ⁇ 1, 3 ⁇ sodium acetate ( ⁇ 5.2) 10 ⁇ 1, 100% ethanol 250 ⁇ 1 was added, and ethanol precipitation was performed at ⁇ 80 ° C. for 1-2 hours. After centrifugation at 15000 rpm for 30 minutes, the supernatant was discarded and the precipitate was washed with cold 100% ethanol. The precipitate was dried and dissolved in DEPC-treated water 201. Probe labeling was performed using a PCR DIG probe synthesis kit (Roche).
  • Example 16 Subcloning of colony hybridization After transferring the colonies to membrane, the membrane was dried at 80 ° C for 5 minutes. The dried membrane was wrapped in aluminum and fixed at 80 ° C for 2.5 hours. Menpure down 100 cm 2 diary 20 ml of pre-hybridization solution was sealed bar Tsu grayed After placing the membrane and back, KoTsuta 2 hours Purehaipuri at 37 ° C. The probe solution containing the probe was added to the hybrid bag where the probe was diluted with the hybrid solution and the pre-high solution was discarded, and the sample was subjected to high-pressure at 37 ° C for 3 hours.
  • the membrane was washed twice with 0.2 X SSC (SSC composition; 0.3 sodium trisodium citrate, 3 M sodium chloride) to remove the probe that had not been amplified.
  • the membrane was washed with 0.1 X SSC, equilibrated with the buffer used for washing for 1 minute, and shaken with the prokking solution for 60 minutes.
  • the antibody diluted solution was added and permeated at room temperature for 30 minutes, and then washed twice with 100 ml of washing buffer for 15 minutes.
  • the detection buffer was added and the membrane was equilibrated for 2 minutes, and CSPD diluted 100-fold with the detection buffer was applied to the membrane.
  • Membrene was sealed in a high-ply bag so that air bubbles would not enter, and heated at 37 ° C for 15-30 minutes. After the addition, the membrane was placed on an X-ray film in a dark room, and the cover with the underline for exposure was tightened for 10 to 15 seconds. The exposed film was developed and the target band was detected. One clone showing the signal was isolated as described above (named 18-34).
  • Terminator Cycle Sequencing Ready Reaction Kit ver. 2.0 (Applied Biosystems) and adding 5% dimethyl sulfoxide (DMS0), 96 ° C for 5 minutes.
  • the test was performed at 92 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes, and 25 cycles.
  • the reaction product was analyzed with Applied Biosystems 3700 DNA Analyzer (Applied Biosystems).
  • SEQ ID NO: 7 shows the 3,874 bp nucleotide sequence obtained as a result of the analysis.
  • the homology analysis of the gene sequence and the search for 0RF were carried out from the NCBI homepage using BLAST 2.0 (gapped BLAST) and ORF binder, respectively.
  • the amino acid sequence (SEQ ID NO: 5) encoded by one 0RF (SEQ ID NO: 4) is the known XylB [XylB encoded by the T0L. Plasmid of P. putida and XylB derived from the genus Acinetobacter (AreB )] Has 33-44% homology (identity) with the amino acid K sequence of)], so it was named xylB gene.
  • XylB of TKN14 strain is an alcoholic monoaldehydro savvyase (MyrB) (Iurescia, S., Marconi, AM, Tofani, D. , Gambacorta, A., Peterno, A., Devirgi l ns, C, van der Werf, M., and Zennaro., E., Identif icat ion and sequencing of ⁇ -myrcene catabo ⁇ ism genes from Pseudomonas sp. Strain Ml, Appl.
  • the aromatic alcohol dehydration ⁇ “Ize” (SEQ ID NO: 5) encoded by the xylB gene derived from opacus TKN14 has no more than 49% homology (similarity) with the existing one.
  • the phylogenetic tree of XylB of this TKN14 strain and the aromatic alcohol dehydrogenase reported so far are shown in Fig. 7.
  • XylB of TKN14 is encoded on the T0L plasmid involved in aromatic metabolism that has been well studied so far.
  • Alcohol dehydro which is separate from the XylB group, is involved in isoprenoid metabolism It was shown that it is classified into the same group as MyrB and TerpD which are geneases. Therefore, it can be said that the liL gene (SEQ ID NO: 4) derived from the TKN14 strain isolated this time is a completely new aromatic alcohol dehydrogenase gene.
  • amino acid sequence (SEQ ID NO: 7) encoded by another 0RF (SEQ ID NO: 8) is Pseudomonas genus Cychrome P ⁇ 450terp (Peterson, JA, Lu, J.-Y., Geisselsoder, J. , Graham-] Lorence, S,. Carmona, C., Witney, F., and Lorence, MC, Cytochrome P ⁇ 450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J. Biol. Chem. 267 , 14193-14203, 1992) and 46% homology (identity).
  • Example 18 Aromatic alcohol conversion activity of Escherichia coli with ill ⁇ gene A 1,844 bp Xhol-Pstl fragment containing the xylB gene was excised from the 3.9-kb S ⁇ hl DNA fragment and pBluescript KS II It was inserted into the Xhol-Pstl site to prepare pXylB8 (Fig. 8). In addition, a 1.7 kb Xhol-Sphl fragment containing the xylB gene was inserted into the Xhol-Sphl site of plasmid ⁇ 2 ⁇ 18N prepared in Example 6 to prepare pPhnN-XylB (FIG. 8).
  • the plasmid pNB2.18N is a plasmid in which a 2 ⁇ 18-kb Notl-BamHI fragment containing the hnN gene is inserted into the Notl-BamHI site of pBluescript II SK +.
  • Escherichia coli with the plasmid P PhnN_XylB prepared in this example has a smooth conversion from aromatic aldehyde to carboxylic acid, and the xylB gene product activity can be evaluated by quantifying the amount of carboxylic acid produced. Conceivable.
  • the xylB gene was obtained as described above, but the xylB gene can also be obtained by chemical synthesis based on the nucleotide sequence information of SEQ ID NO: 4.
  • E. coli JM101 containing either plasmid pPhnN or pPhnN-XylB C was cultured in LB medium containing 10 ml of ampicillin 100 Aig / ml at 30 ° C for 12 hours. After recovery by centrifugation, ampicillin 100 g / ml, 0.2% (w / v) glucose, 0.01% (w / v) thiamine, M9 medium containing 1 mM IPTG (Na 2 HP0 4 , 6 g / L;
  • KH 2 P0 4 3 g / L; NaCl, 0.5 g / L; NH 4 C1, 1 g / L; MgS04, 1 raM; CaCl 2 , 0.1 mM; FeS0 4 ,
  • the flow rate was 1 ml / min 0 after the injection of sampnore, followed by flowing for 2 minutes with solvent A (0.1% phosphate in water) and then applying a gradient of up to 70% with solvent B (0.1% phosphate in acetonitrile). It was flushed with 70% solvent B for 5 minutes. For detection, a maximum of 230 to 280 nm was measured.
  • E. coli JM101 with the plasmid pPhnN-XylB is 2-hydroxymethylnaphthalene to 2-naphthoic acid, benzyl alcohol to benzoic acid, 2-, 3 2-, 3-, 4-methyl benzoic acid from-, 4-methyl benzyl alcohol, xylene- ⁇ -, 4-diol to 4-hydroxymethyl benzoic acid, 2-, 3-, 4-hydride, respectively 2-, 3--4-hydroxybenzoic acid from roxybenzyl alcohol, 2--, 3--4-hydroxybenzoinoleol, 2--, 3-, 4-methoxybenzoic acid, 3-chloro mouth respectively Conversion reactions from benzyl alcohol to 3-chlorobenzoic acid, cinnamyl alcohol to cinnamic acid, and vanyl alcohol to vanillic acid were confirmed. Since the amount of carboxylic acid produced was much higher than that of E. coli JM101 with the plasm
  • a novel peptide having an aromatic dehydrogenase activity capable of converting a very wide range of aromatic compounds from monocyclic compounds to polycyclic compounds into carboxylic acids, and a gene encoding the peptide are provided.
  • an aromatic compound having a hydroxymethyl group or an aldehyde group is converted into an aromatic group.
  • Useful transformants that can be converted into carboxylic acids can be provided.
  • the present invention can provide a method for producing an aromatic carboxylic acid from an aromatic compound having a hydroxymethyl group or an aldehyde group, using the transformant.

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Abstract

A novel gene coding for an enzyme capable of oxidizing any of various hydroxymethylated or aldehyde-group-having aromatic compounds to thereby convert the same to a carboxylic acid; and a process for producing various aromatic carboxylic acids with the use of a recombinant microbe wherein the above gene is introduced and expressed. There are provided peptides including: (a) peptide consisting of an amino acid sequence of SEQ ID NO: 3, (b) peptide consisting of the same amino acid sequence as that of SEQ ID NO: 3 except for deletion or addition of one or two or more amino acid residues or substitution thereof with other amino acid residues, the peptide having an aromatic compound dehydrogenase activity, and (c) microbe-derived peptide coded for by DNA consisting of the base sequence of SEQ ID NO: 2 or DNA capable of hybridizing under stringent conditions with DNA complementary thereto, the peptide having an aromatic compound dehydrogenase activity.

Description

新規な芳香族デヒ ドロゲナーゼ遺伝子及び芳香族カルボン酸の製造法 技術分野  Novel aromatic dehydrogenase gene and method for producing aromatic carboxylic acid
本発明は、 ヒ ドロキシメチル基またはアルデヒ ド基を持った芳香族化合物を酸 化する新規な酵素、 それをコードする遺伝子、 この遺伝子を導入した微生物に関 するものである。 また、 この遺伝子が導入された微生物を利用した、 ヒ ドロキシ 明  The present invention relates to a novel enzyme that oxidizes an aromatic compound having a hydroxymethyl group or an aldehyde group, a gene encoding the same, and a microorganism into which the gene has been introduced. In addition, using a microorganism into which this gene has been introduced,
メチル基またはアルデヒ ド基を持った芳香族化合物から芳香族カルボン酸への製 造法に関するものである。 The present invention relates to a method for producing an aromatic carboxylic acid from an aromatic compound having a methyl group or an aldehyde group.
書 背景技術  Background art
ベンジルアルコールから安息香酸への変換はアルコールデヒ ドロゲナーゼ ( alcohol dehydrogenase ) およぴァノレァヒ ドデヒ ドロゲナーゼ ( aldehyde dehydrogenase) という二つの酵素で行われている。 アルコールデヒ ドロゲナーゼ はべンジルァノレコールをベンズアルデヒドに可逆的に変換する酵素で、 シユード モナス 'プチダ (Pseudomonas putida) の TOLプラスミ ド pWWOにコードされてい るトルエン代謝系の XylB酵素が最もよく研究されている （Harayama, S. Rekik, M Wubbolts, M. , Rose, K. , Leppik, R. A. and Timmis, K. , Characterization of five genes in the upper-pathway operon of POL plasmid pWWO from Pseudomonas Dut i da and identification of the gene products. J. Bacteriol. 171 (9)， 5048-5055, 1989)。 XylBはその酵素活性が NAD (P) +および亜鉛イオンに依存し、 ベンジルアルコールだけではなく -， Π1-, -メチルベンジルァノレコール、 m-, £ -メ トキシベンジルアルコールも基質としてそれぞれのアルデヒド体に変換する c しかし、 2_メチルベンジルアルコール及び 2—メ トキシベンジルアルコールを基質 とした場合、 変換効率は悪かった （Shaw, J. P. Schwager, F. , and Harayama, S.Conversion of benzyl alcohol to benzoic acid is carried out by two enzymes, alcohol dehydrogenase and aldehyde dehydrogenase. Alcohol dehydrogenase is an enzyme that reversibly converts benzilanolol to benzaldehyde, and the XylB enzyme in the toluene metabolism system encoded by the TOL plasmid pWWO of Pseudomonas putida is the best studied. (Harayama, S. Rekik, M Wubbolts, M., Rose, K., Leppik, RA and Timmis, K., Characterization of five genes in the upper-pathway operon of POL plasmid pWWO from Pseudomonas Dut i da and identification of the gene products. J. Bacteriol. 171 (9), 5048-5055, 1989). XylB depends on NAD (P) + and zinc ions, and not only benzyl alcohol but also-, Π1-, -methylbenzylanolol, m-, £ -methoxybenzyl alcohol as substrates. c but into a body, if the 2_ methylbenzyl alcohol and 2-main butoxy benzyl alcohol was used as a substrate, the conversion efficiency was poor (Shaw, JP Schwager, F., and Harayama, S.
Substrate-spec ificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by TOL plasmid pWWO. Metabol ic and mechanistic impl ications. Biochem. J. 283 789-794, 1992)。 最近、 NAD (P) +および亜鉛ィ オンに依存しなく、 TOLプラスミ ドの XylBとも相同性の低い 4-二トロベンジルァ ルコールデヒ ドロゲナーゼ ( NtnD ) をコードする遺伝子がシユー ドモナス (Pseudomonas) 属 TW3株から単離された （James, K. D. , Hughes, M. A. , and Will iams, P. A. , Cloning and expression of ntnD, encoding a novel NAD (P) +- independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp. strain TW3. J. Bacteriol. 182 (11) , 3136-3141, 2000)。 NtnD は、 ベンジノレア ノレコーノレ、 m-, £—二トロべンジノレアノレコーノレ、 0—， m-, £—メチノレペンジノレアノレコ 一ノレ、 m—ヒ ドロキシベンジノレアルコーノレ、 £—ェチノレべンジノレアノレコーノレ、 2， 4 -、 3, 4-, 3, 5-ジメチルベンジルアルコールを基質として利用しそれぞれのアルデヒ ド体を合成したが、 2—二 ト口べンジルアルコール、 0-, £ -ヒ ドロキシベンジノレア ルコール、 2， 5-ジメチノレベンジルァノレコ一ノレは変換できなかつた。 また、 アジ一 ノレエーテル资化菌ァシネトパクター (Acinetobacter) 属 ADPl株から単離された 芳香族アルコール (ベンジルアルコール)デヒ ドロゲナーゼ AreBは T0Lプラスミ ド pWWOの XylBと高い相同性を持ち、 0-, ra-, £ -メチノレ ンジノレアルコール、 ο-, ra-, £ -ヒ ドロキシベンジルアルコールに対する活性が報告された (Jones, R. M. .， Coll ier, L. S. , Neidle, E. L. , and Wi ll iams, P. A. , areABC genes determine the catabol ism of aryl esters in Acinetobacter sp. ADPl, J. Bacteriol. 181， 4568-4575, 1999)。 Substrate-specificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by TOL plasmid pWWO. Metabolic and mechanistic impl ications. Biochem. J. 283 789-794, 1992). Recently, NAD (P) + and Zinc A gene encoding 4-nitrobenzyl alcohol dehydrogenase (NtnD) that is not dependent on ON and has low homology with XylB of the TOL plasmid was isolated from the TW3 strain of the genus Pseudomonas (James, KD, Hughes, MA, and Will iams, PA, Cloning and expression of ntnD, encoding a novel NAD (P) +-independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp. Strain TW3. J. Bacteriol. 182 (11), 3136-3141, 2000 ). NtnD consists of Benzenorea Noreconole, m-, £ —Nitrobenzinoreanoreconole, 0—, m-, £ —Metinorepenzino Renoreco Inore, m—Hydroxy Benzenore Arconore, £ — Each aldehyde was synthesized using ethinolevenoleanoreconole, 2, 4-, 3, 4-, 3, 5-dimethylbenzyl alcohol as a substrate. 0-, £ -Hydroxybenzinoleol alcohol and 2,5-dimethylenorebenzylanorecoinole could not be converted. In addition, the aromatic alcohol (benzyl alcohol) dehydrogenase AreB isolated from the ADP1 strain of the genus Acinetobacter genus Acinetobacter is highly homologous to XylB of the T0L plasmid pWWO. , £ -Methinolenginole alcohol, ο-, ra-, £ -Hydroxybenzyl alcohol reported activity (Jones, RM., Collier, LS, Neidle, EL, and Williams, PA, areABC genes determine the catabol ism of aryl esters in Acinetobacter sp. ADPl, J. Bacteriol. 181, 4568-4575, 1999).
芳香族アルデヒ ドデヒ ドロゲナーゼはべンズアルデヒ ドから安息香酸までの反 応を触媒する。 最もよく知られているのは T0Lプラスミ ド pWWO にコードされる XylC酵素であり、その酵素活性は NAD (P) +に依存する（上掲の Harayamaらの論文）。 また、ァシネトパクタ一.カルコァセチカス (Acinetobacter calcoaceticus) NCIB 8250からも xylBと xylC遺伝子が単離され、 それらがコードするアミノ酸配列は T0Lプラスミ ドの XylB、 XylCと高い相同性を持ち、 基質特異性もほぼ同じであつ た ( Gillooly, D. J. , Robertson, A. G. S. , and Fewson, C. A.， Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus. Biochem. J. 330， 1375-1381， 1998) o  Aromatic aldehyde dehydrogenase catalyzes the reaction from benzaldehyde to benzoic acid. The best known is the XylC enzyme encoded by the T0L plasmid pWWO, whose enzyme activity depends on NAD (P) + (Harayama et al., Supra). Also, xylB and xylC genes were isolated from Acinetobacter calcoaceticus NCIB 8250, and the amino acid sequences encoded by them were highly homologous to T0L plasmids XylB and XylC, and the substrate specificity was almost the same. (Gillooly, DJ, Robertson, AGS, and Fewson, CA, Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus. Biochem. J. 330, 1375-1381, 1998) o
以上述べてきたように、 アルコールデヒ ドロゲナーゼ及びアルデヒ ドデヒ ドロ ゲナーゼは、 芳香族アルコール及び芳香族アルデヒドをそれぞれ、 芳香族アルデ ヒ ド及び芳香族カルボン酸に変換する酵素である。 これら 2つの酵素の働きによ り、 ベンジルアルコールのようなヒ ドロキシメチル基を有する単環式芳香族化合 物がそのカルボン酸に変換されるのである。 一方、 ヒ ドロキシメチルナフタレン .のような多環式芳香族化合物がそのカルボン酸へ変換される酵素やそれをコード する遺伝子の例は報告されていなかつた。 As described above, alcohol dehydrogenase and aldehyde dehydrogenase Genases are enzymes that convert aromatic alcohols and aromatic aldehydes into aromatic aldehydes and aromatic carboxylic acids, respectively. By the action of these two enzymes, a monocyclic aromatic compound having a hydroxymethyl group such as benzyl alcohol is converted to its carboxylic acid. On the other hand, there has been no report of an enzyme that converts a polycyclic aromatic compound such as hydroxymethylnaphthalene into its carboxylic acid and the gene that encodes it.
一方、 多環式芳香族炭化水素の菌体 （細胞） レベルでの代謝経路は、 多くの多 環式芳香族炭化水素資化細菌において推察されている。 たとえば、 多環式芳香族 炭化水素の 1つである 2, 6-ジメチルナフタレンの代謝経路は、 Flavobacteria (Barns丄 ey, n. A. , Metabolism οι 2， o一 dimethy丄 naphthalene by flavobacteria. Appl. Environ. Microbiol. 54(2), 428-433, 1988) 、 Pseudomonas sp. D- 87 (Miyachi, N. , Tanaka, T.， Suzuki, T.， Hotta, Y. , and Omori, T.， Microbial oxidation of dimethylnaphthalene isomers. Appl. Environ. Microbiol. 59(5)， 1504-1506, 1993)、 Psudomonas cepacia F297 (Grifoll, M.， Selifonov, S. A.， Gatlln, C. V. , and Chapman, P. J. , Actions of a versatile fluorene - degrading bacteria丄 isolate on polycyclic aromatic compounds. Appl. Environ. Microbiol. 61 (10), 3711-3723, 1995)、 及ぴ、 Shingomonas paucimobilis 2322 (Dutta, T. K. , Selifonov, S. A. , and Gunsalus, I. C.， Oxidation of methyl-substituted naphthalenes： pathways in a versatile Spningomonas paucimobilis strain. Appl. Environ. Microbiol. 64(5), 1884-1889， 1998) 等の文献において報告されてい る。 2， 6 -ジメチルナフタレンはまず、 モノォキシゲナーゼによって 2 -ヒ ドロキシ メチル- 6 -メチルナフタレンに酸化される。 そして、アルコールデヒ ドロゲナーゼ ' によって 6-メチル -2 -ナフタアルデヒ ドに変換された後、 アルデヒ ドデヒドロゲ ナーゼの触媒作用で 6-メチル -2-ナフトェ酸 （6- methyl- 2- naphthoic acid) に変 換される。最初の反応を触媒するモノォキシゲナーゼ遺伝子は Bramucci らによつ てスフインゴモナス (Sphingomonas) 属 ASU 1株 (Bramucci, M. , Singh, M. , andOn the other hand, metabolic pathways at the cell (cell) level of polycyclic aromatic hydrocarbons have been speculated in many polycyclic aromatic hydrocarbon-utilizing bacteria. For example, the metabolic pathway of 2, 6-dimethylnaphthalene, one of the polycyclic aromatic hydrocarbons, is Flavobacteria (Barns 丄 ey, n.A., Metabolism οι2, o dimethy 丄 naphthalene by flavobacteria. Appl. Environ. Microbiol. 54 (2), 428-433, 1988), Pseudomonas sp. D-87 (Miyachi, N., Tanaka, T., Suzuki, T., Hotta, Y., and Omori, T., Microbial oxidation of dimethylnaphthalene isomers. Appl. Environ. Microbiol. 59 (5), 1504-1506, 1993), Psudomonas cepacia F297 (Grifoll, M., Selifonov, SA, Gatlln, CV, and Chapman, PJ, Actions of a versatile fluorene -degrading bacteria 丄 isolate on polycyclic aromatic compounds. Appl. Environ. Microbiol. 61 (10), 3711-3723, 1995), Shingomonas paucimobilis 2322 (Dutta, TK, Selifonov, SA, and Gunsalus, IC, Oxidation of methyl-substituted naphthalenes: pathways in a versatile Spningomonas paucimobilis strain. Appl. Environ. Microbiol. 64 (5), 1884-1889, 1998) There Ru. 2,6-Dimethylnaphthalene is first oxidized by monooxygenase to 2-hydroxymethyl-6-methylnaphthalene. Then, 6-methyl-2 by alcohol dehydrogenase '- after being converted into Nafutaarudehi de, 6-methyl catalyzed the aldehyde Dodehidoroge kinase - 2 - Nafute acid converted to (6 - - methyl- 2 naphthoic acid ) Is done. The monooxygenase gene that catalyzes the first reaction was reported by Bramucci et al. In the Sphingomonas genus ASU 1 (Bramucci, M., Singh, M., and
Nagara j an, V.， Biotransformation of £— xylene and 2， 6— dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate. Appl. Microbiol.Nagara j an, V., Biotransformation of £ — xylene and 2, 6— dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate. Appl. Microbiol.
Biotechnol. 59(6)， 679-684， 2002) から単離されたが、 これ以降の代謝経路を 担うアルコールデヒ ドロゲナーゼ遺伝子およびアルデヒ ドデヒ ドロゲナーゼ遺伝 子を単離したと言う報告は存在しない。 また、 細菌菌体を利用した、 2， 6 -ジメチ ルナフタレンから 2, 6-ナフタレンジカルボン酸への生変換に関する特許がレ、く つか出願されている （特許第 2715260 号公報、 特開平 5 - 15365 号公報、 特開平3-80091号公報、 特開平 8-280380号公報）。 発明の開示 Biotechnol. 59 (6), 679-684, 2002). There are no reports that the responsible alcohol dehydrogenase gene and aldehyde dehydrogenase gene have been isolated. In addition, several patents relating to the bioconversion of 2,6-dimethylnaphthalene to 2,6-naphthalenedicarboxylic acid using bacterial cells have been filed (Patent No. 2715260, Japanese Patent Laid-open No. Hei 5- No. 15365, JP-A-3-80091, JP-A-8-280380). Disclosure of the invention
本発明の課題は、 種々のヒ ドロキシメチル基またはアルデヒ ド基を有する芳香 族化合物を酸化しカルボン酸に変換する酵素をコードする遺伝子を取得すること である。 そして更に、 この遺伝子を導入 '発現させた組換え微生物を利用した、 種々の芳香族カルボン酸の製造法を提供することである。  An object of the present invention is to obtain a gene encoding an enzyme that oxidizes an aromatic compound having various hydroxymethyl groups or aldehyde groups and converts it into a carboxylic acid. Furthermore, the present invention provides a method for producing various aromatic carboxylic acids using a recombinant microorganism into which this gene has been introduced and expressed.
本発明者らはすでに、 1， 4 -ジメチルナフタレンを基質として、 市販されていな い芳香族化合物である 1， 4-ジヒ ドロキシメチルナフタレンを合成する新規の芳 香族化合物ジォキシゲナーゼをコードする遺伝子 (phnA!A2A3A4) を海洋細菌サイ クロクラスティカス (Cycloc lasticus)属ょり取得していた（特願 2003-324117)。 すなわち、 本遗伝子を有する大腸菌は、 1， 4-ジメチルナフタレンを変換し 1, 4-ジ ヒ ドロキシメチルナフタレンを製造した。 一方で本発明者らは、 新潟県石油噴出 所の土壌より、 1，4 -ジメチルナフタレンを唯一の炭素源として利用できるスフィ ンゴモナス (Sphingomonas) 属細菌 14DN61株を単離した。 本発明者らは、 上記の 1, 4 -ジヒ ドロキシメチルナフタレンを基質として、 このスフインゴモナス属細菌 14DN61株由来の DNAを導入した大腸菌と混合培養して 1， 4 -ジヒ ドロキシメチルナ フタレンを変換できるものをスクリ一ユングした結果ついに、 1，4-ジヒドロキシ メチルナフタレンを 4-ヒ ドロキシメチル- 1-ナフトェ酸に変換するのに必要な鍵 となる酸化酵素 （デヒドロゲナーゼ） 遺伝子を突き止め、 本発明を完成するに至 つた。 なお、 本発明による遺伝子 (phnNと命名) は、 芳香族アルデヒドを芳香族 カルボン酸に変換する活性を有する芳香族アルデヒ ドデヒ ドロゲナーゼをコード していた。 この 遺伝子を導入した大腸菌等の微生物は、芳香族アルコールを 芳香族カルボン酸に変換することができた。 これは全く予想外のことであった。 大腸菌等の微生物は、 本来的に弱いながらも芳香族アルコールをアルデヒ ドに変 換する自前の芳香族アルコールデヒ ドロゲナーゼ活性を有しており、 PhnN 酵素 (phnN遺伝子産物) が共存することにより、 自前の芳香族アルコールデヒドロゲ ナーゼの回転効率がよくなり、 一気に芳香族アルコールから芳香族カルボン酸が 生産されたと考えざるを得ないからである。 また、 EiinU遺伝子を発現した大腸菌 は、ヒ ドロキシメチル基を有するナフタレン骨格を持つ芳香族化合物だけでなく、 ヒ ドロキシメチル基を有するベンゼン骨格を持つ芳香族化合物に至るまでの非常 に広い範囲の芳香族化合物を芳香族カルボン酸に変換することができた。 このこ とも発明者らの予想、 '即ち従来の常識を覆すものであった。 The present inventors have already encoded a novel aromatic compound dioxygenase that synthesizes 1,4-dihydroxymethylnaphthalene, a commercially available aromatic compound, using 1,4-dimethylnaphthalene as a substrate. (phnA! A2A3A4) was acquired from the genus Cycloc lasticus (Japanese Patent Application 2003-324117). In other words, Escherichia coli having the present gene transferred 1,4-dimethylnaphthalene to produce 1,4-dihydroxymethylnaphthalene. On the other hand, the present inventors isolated the 14DN61 strain of the genus Sphingomonas that can use 1,4-dimethylnaphthalene as the sole carbon source from the soil of the Niigata Pref. The present inventors can convert 1,4-dihydroxymethylnaphthalene by using the above 1,4-dihydroxymethylnaphthalene as a substrate and mixed culture with Escherichia coli introduced with DNA derived from the genus Sphingomonas bacterium 14DN61. Finally, the key oxidase (dehydrogenase) gene required to convert 1,4-dihydroxymethylnaphthalene to 4-hydroxymethyl-1-naphthoic acid was identified, and the present invention was completed. It was The gene according to the present invention (named phnN) encoded an aromatic aldehyde dehydrogenase having an activity of converting an aromatic aldehyde into an aromatic carboxylic acid. Microorganisms such as Escherichia coli introduced with this gene were able to convert aromatic alcohol into aromatic carboxylic acid. This was totally unexpected. Microorganisms such as Escherichia coli are inherently weak but convert aromatic alcohol to aldehyde. It has its own aromatic alcohol dehydrogenase activity, and the coexistence of the PhnN enzyme (phnN gene product) improves the rotational efficiency of its own aromatic alcohol dehydrogenase. This is because aromatic carboxylic acids must be produced. E. coli expressing the EiinU gene is not only an aromatic compound having a naphthalene skeleton having a hydroxymethyl group, but also a very wide range of aromatic compounds ranging from aromatic compounds having a benzene skeleton having a hydroxymethyl group. Could be converted to an aromatic carboxylic acid. This also reversed the expectation of the inventors, that is, the conventional common sense.
一方、本発明者らは、o-キシレン資化細菌口ドコッカス 'ォパカス (Rhodococcus opacus) TKN14株由来の芳香族アルコールデヒ ドロゲナーゼ遺伝子が、 ヒドロキ シメチル基を分子内に有する様々な芳香族化合物 （芳香族ヒ ドロキシメチル化合 物）、 たとえば、 ベンジルアルコールや種々のべンジルアルコールの置換体、 さら には、 ヒ ドロキシメチルナフタレンをそれぞれのアルデヒ ドに変換する新規の酵 素であることを発見した。 さらに本発明者らは、 この芳香族アルコールデヒ ドロ ゲ^ "一ゼ遺伝子とともに、 上記の芳香族アルデヒ ドデヒドロゲナーゼ遺伝子を発 現した大腸菌が、 上記の様々な芳香族ヒドロキシメチル化合物をそれぞれの力ル ボン酸に変換できることを明らかにした。  On the other hand, the present inventors have reported that the aromatic alcohol dehydrogenase gene derived from the o-xylene-utilizing bacterium Rhodococcus opacus TKN14 strain has various aromatic compounds (aromatic compounds having a hydroxymethyl group in the molecule). For example, benzyl alcohol and various benzyl alcohol substitutes, and also a novel enzyme that converts hydroxymethylnaphthalene to the respective aldehydes. Furthermore, the inventors of the present invention, together with the aromatic alcohol dehydrogenase gene, E. coli expressing the aromatic aldehyde dehydrogenase gene described above, each of the above-mentioned various aromatic hydroxymethyl compounds have their respective strengths. It was clarified that it can be converted to sulfonic acid.
本発明は以上のような知見を基に完成されたものである。  The present invention has been completed based on the above findings.
即ち、 本発明は以下の （1 ) 〜 （6 ) を提供するものである。  That is, the present invention provides the following (1) to (6).
( 1 ) 以下の （a )、 （b )、 又は （c ) に示すペプチド：  (1) Peptides shown in the following (a), (b), or (c):
( a ) 配列番号 3記載のアミノ酸配列からなるぺプチド、  (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3,
( b ) 配列番号 3記載のアミノ酸配列において 1もしくは複数個のアミノ酸が付 カ卩、 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物アルデヒ ドデヒ ドロゲナーゼ活性を有するペプチド、  (b) a peptide comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 3, and having an aromatic compound aldehyde dehydrogenase activity,
( c ) 配列番号 2記載の塩基配列からなる DNA又はそれと相補的な DNA とストリ ンジェントな条件下でハイブリダィズする DNAがコードする細菌由来のぺプチド であって、 芳香族化合物アルデヒ ドデヒドロゲナーゼ活性を有するペプチド。  (c) a peptide derived from a bacterium encoded by DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or DNA complementary thereto, and has aromatic aldehyde dehydrogenase activity peptide.
( 2 ) 以下の （a )、 （b )、 又は （c ) に示すペプチドをコードする遺伝子： (2) The gene encoding the peptide shown in (a), (b), or (c) below:
( a ) 配列番号 3記載のアミノ酸配列からなるぺプチド、 (b) 配列番号 3記載のアミノ酸配列において 1もしくは複数個のアミノ酸が付 カロ、 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物アルデヒ ドデヒ ドロゲナーゼ活性を有するペプチド、 (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3, (b) a peptide comprising an amino acid sequence in which one or a plurality of amino acids are appended, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 3 and having an aromatic compound aldehyde dehydrogenase activity,
( c ) 配列番号 2記載の塩基配列からなる DNA又はそれと相捕的な DNAとストリ ンジヱントな条件下でハイプリダイズする DNAがコードする細菌由来のペプチド であって、 芳香族化合物アルデヒドデヒド口ゲナーゼ活性を有するぺプチド。  (c) a peptide derived from a bacterium encoded by a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a DNA complementary thereto and a DNA that is hybridized under a stringent condition, and having an aromatic compound aldehyde dehydrogenase activity Peptides having
(3) (2) に記載の遺伝子を導入して得られる形質転換体であって、 ヒ ドロキシ メチル基またはアルデヒ ド基を有する芳香族化合物を芳香族カルボン酸に変換で きる形質転換体。  (3) A transformant obtained by introducing the gene according to (2), which can convert an aromatic compound having a hydroxymethyl group or an aldehyde group into an aromatic carboxylic acid.
(4) 形質転換体が大腸菌であることを特徴とする （3) に記載の形質転換体。 (4) The transformant according to (3), wherein the transformant is Escherichia coli.
(5) (3) 乃至 （4) に記載の形質転換体を、 ヒ ドロキシメチル基またはアルデ ヒド基を有する芳香族化合物を含む培地で培養して培養物又は菌体から芳香族力 ルボン酸を得ることを特徴とする、 芳香族カルボン酸の製造法。 (5) The transformant according to (3) to (4) is cultured in a medium containing an aromatic compound having a hydroxymethyl group or an aldehyde group, and aromatic rubonic acid is obtained from the culture or the cell. A process for producing an aromatic carboxylic acid, characterized in that
(6) 得られた芳香族カルボン酸が、 4 -ヒ ドロキシメチル -1-ナフ トェ酸、 6 -メチ ル- 2 -ナフトェ酸、 1-ナフトェ酸、 2-ナフトェ酸、 4-メチル安息香酸、 3_メチル安 息香酸、 2 -メチル安息香酸、 安息香酸、 4-ヒ ドロキシメチル安息香酸、 4-ヒ ドロ キシ安息香酸、 3 -ヒ ドロキシ安息香酸、 2 -ヒ ドロキシ安息香酸、 4 -メ トキシ安息 香酸、 3-メ トキシ安息香酸、 2-メ トキシ安息香酸、 4_ェチル安息香酸、 4 -イソプ 口ピル安息香酸、 3 -ク口口安息香酸及び 3-フヱニルプロピオン酸から選ばれる少 なくとも 1種の芳香族カルボン酸であることを特徴とする （5) に記載の芳香族 カルボン酸の製造法。  (6) The resulting aromatic carboxylic acid is 4-hydroxymethyl-1-naphthoic acid, 6-methyl-2-naphthoic acid, 1-naphthoic acid, 2-naphthoic acid, 4-methylbenzoic acid, 3 _Methylbenzoic acid, 2-Methylbenzoic acid, Benzoic acid, 4-Hydroxymethylbenzoic acid, 4-Hydroxybenzoic acid, 3-Hydroxybenzoic acid, 2-Hydroxybenzoic acid, 4-Methoxybenzoic acid A small amount selected from peroxy acid, 3-methoxybenzoic acid, 2-methoxybenzoic acid, 4_ethyl benzoic acid, 4-isopropylpyrubenzoic acid, 3-dioxybenzoic acid and 3-phenylpropionic acid The method for producing an aromatic carboxylic acid according to (5), characterized in that it is at least one kind of aromatic carboxylic acid.
(7) 以下の （a)、 （b)、 又は （c) に示すペプチド：  (7) Peptides shown in the following (a), (b), or (c):
( a ) 配列番号 5記載のァミノ酸配列からなるぺプチド、  (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 5,
(b) 配列番号 5記載のアミノ酸配列において 1もしくは複数個のアミノ酸が付 カロ、 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物アルコー ルデヒ ドロゲナーゼ活性を有するペプチド、  (b) a peptide comprising an amino acid sequence in which one or a plurality of amino acids are appended, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 5 and having an aromatic compound alcoholase activity,
( c ) 配列番号 4記載の塩基配列からなる DNA又はそれと相補的な DNAとストリ ンジェンドな条件下でハイブリダィズする DNAがコードする細菌由来のペプチド であって、 芳香族アルコールデヒ ドロゲナーゼ活性を有するペプチド。 (8) 以下の （a)、 （b)、 又は （c) に示すペプチドをコードする遺伝子： ( a ) 配列番号 5記載のァミノ酸配列からなるぺプチド、 (c) a peptide derived from a bacterium encoded by DNA that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 4 or DNA complementary thereto, and having aromatic alcohol dehydrogenase activity . (8) A gene encoding a peptide shown in the following (a), (b), or (c): (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 5,
(b) 己列番号 5記載のアミノ酸配列において 1もしくは複数個のアミノ酸が付 カ卩、 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物アルコー ルデヒ ドロゲナーゼ活性を有するぺプチド、  (b) a peptide comprising an amino acid sequence in which one or a plurality of amino acids are attached, deleted or substituted in the amino acid sequence described in self-sequence number 5, and having an aromatic alcohol dehydrogenase activity;
( c ) 配列番号 4記載の塩基配列からなる DNA又はそれと相補的な DNAとストリ ンジェントな条件下でハイプリダイズする DNAがコードする細菌由来のぺプチド であって、 芳香族アルコールデヒドロゲナーゼ活性を有するペプチド。  (c) a peptide derived from a bacterium encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4 or DNA complementary thereto and DNA that is hybridized under stringent conditions, and having an aromatic alcohol dehydrogenase activity .
(9) (8) に記載の遺伝子を導入して得られる微生物であって、 ヒドロキシメチ ル基を有する芳香族化合物を芳香族カルボン酸に変換できる形質転換体。  (9) A microorganism obtained by introducing the gene according to (8), which is capable of converting an aromatic compound having a hydroxymethyl group into an aromatic carboxylic acid.
(1 0) 微生物が大腸菌であることを特徴とする （9) に記載の形質転換体。 (1 0) The transformant according to (9), wherein the microorganism is Escherichia coli.
(1 1) (9) 又は （1 0) に記載の形質転換体を、 ヒ ドロキシメチル基を有する 芳香族化合物を含む培地で培養して培養物又は菌体から芳香族カルボン酸を得る ことを特徴とする、 芳香族カルボン酸の製造法。 (1) The transformant according to (9) or (10) is cultured in a medium containing an aromatic compound having a hydroxymethyl group to obtain an aromatic carboxylic acid from the culture or the cell. A process for producing an aromatic carboxylic acid.
(1 2) 得られた芳香族カルボン酸が、 2-ナフトェ酸、 安息香酸、 2 -， 3 -， 4-メ チル安息香酸、 4-ヒ ドロキシメチル安息香酸、 2 -， 3-, 4-ヒドロキシ安息香酸、 2 -， 3-, 4 -メ トキシ安息香酸、 3-クロ口安息香酸、 桂皮酸及びバニリン酸からな る群から選ばれる少なく とも 1種の芳香族カルボン酸であることを特徴とする (1 1) に記載の芳香族カルボン酸の製造法。  (1 2) The resulting aromatic carboxylic acid is 2-naphthoic acid, benzoic acid, 2-, 3-, 4-methylbenzoic acid, 4-hydroxymethylbenzoic acid, 2-, 3-, 4-hydroxy. It is characterized by being at least one aromatic carboxylic acid selected from the group consisting of benzoic acid, 2-, 3-, 4-methoxyoxybenzoic acid, 3-chlorobenzoic acid, cinnamic acid and vanillic acid. (1) The method for producing an aromatic carboxylic acid according to (1).
本明細書は本願の優先権の基礎である日本国特許出願 2004-235414号の明細書 および/または図面に記載される内容を包含する。 図面の簡単な説明  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2004-235414, which is the basis of the priority of the present application. Brief Description of Drawings
図 1 aは、 £ϋ遺伝子を持つ大腸菌による芳香族アルコール化合物の変換例を 示す図である。  Fig. 1a is a diagram showing an example of conversion of an aromatic alcohol compound by E. coli having a £ ϋ gene.
図 1 bは、 遺伝子を持つ大腸菌による芳香族アルコール化合物の変換例を 示す図である。  Fig. 1b shows an example of conversion of an aromatic alcohol compound by E. coli with a gene.
図 2は、 phnN遺伝子を持つ大腸菌による芳香族アルデヒ ド化合物の変換例を示 す図である。 図 3は、 上段 a)は 1, 4-ジヒ ドロキシメチルナフタレンの pMA9 - 54 の大腸菌 JM109形質転換体による変換産物を TMS化し、 GC - MSで分析した結果を示す図であ る。下段 b)は変換産物の 1 -ヒ ドロキシメチルナフトェ酸の TMS化マススぺク トル である。 FIG. 2 is a diagram showing an example of conversion of an aromatic aldehyde compound by E. coli having a phnN gene. Figure 3 shows the results of analysis of GC-MS using TMS of the conversion product of 1,4-dihydroxymethylnaphthalene pMA9-54 by E. coli JM109 transformant. The lower part b) is a TMS mass spectrum of 1-hydroxymethylnaphthoic acid as a conversion product.
図 4は、 7- kb Kpnl 断片の制限酵素マップおょぴ各遺伝子位置。 各サブクロ一 ンの 1，4 -ジヒ ドロキシメチルナフタレン変換活性は右に示している。 +は変換活 性あり、 -は変換活性なしである。  Figure 4 shows the restriction map of the 7-kb Kpnl fragment and the location of each gene. The 1,4-dihydroxymethylnaphthalene conversion activity of each subclone is shown on the right. + Indicates conversion activity and-indicates no conversion activity.
図 5は、 プライマー設計に用いた配列のァライメントを示しており、  Figure 5 shows the alignment of the sequences used for primer design.
「areB—Acineto」 は Acinetobacter sp. ADP1株の areB遺伝子を意味し、 「xylB— AcinetoJ は Acinetobacter calcoaceticus NCIB 8250の xylB逾ィ zs;子 ' 、' 味し、 「xylB_T0L」 は Pseudomonas putida T0L プラスミ ド p龍 0の xylB遺伝子を 意味する。  “AreB—Acineto” means the areB gene of Acinetobacter sp. ADP1 strain, “xylB—AcinetoJ tastes Acinetobacter calcoaceticus NCIB 8250 xylB 逾 zs; child”, “xylB_T0L” means Pseudomonas putida T0L plasmid p0 It means dragon 0's xylB gene.
図 6は、 プラスミ ド pl8 - 34を各種制限酵素で切断し ァガロースゲル電気泳動 した写真である。  Fig. 6 is a photograph of plasmid pl8-34 digested with various restriction enzymes and subjected to agarose gel electrophoresis.
図 7は、 XylBおよび関連酵素の系統樹を示し、 図中、 数字は bootscrap値であ る （1000回テス ト）。 図中 「MyrB_Pseudo匪 as」 は Pseudomonas sp. Ml株のァノレ コールデヒ ドロゲナーゼ MyrB を意味し、 ； 「XylB— TKN14J は Rhodococcus opacus TKN14株の XylB (本発明によるタンパク質） を意味し、 「TerpD_Pseudomonas」 は Pseudomonas 属細菌の TerpD を意味し、 「XylB— Ac inetoII J は Ac inetobater cal coaceti cus NCIB 8250の ylBを意味し、 「AreB一 AcinetoJ は Acinetobater sp. ADP1株の AreB を意味し、 「XylB— pWW0」 は Pseudomonas putida T0L プラスミ ド pWWOにコードされる XylB (Accession number, D63341) を意味し、 「XylB— T0L」 は Pseudononas putida mt- 2株の T0L プラス.ミ ドにコードされる XylB (Shaw, J. P. , Rekik, M. , Schwager, F. , and Harayama, ， Kinet ic studies on benzyl alcohol dehydrogenase encoded by T0L p丄 asmid pWWO, J. Biological Chemistry, Figure 7 shows the phylogenetic tree of XylB and related enzymes, where the numbers are bootscrap values (1000 tests). In the figure, “MyrB_Pseudo 匪 as” means Anolechol dehydrogenase MyrB of Pseudomonas sp. Ml strain; “XylB—TKN14J means XylB (protein according to the present invention) of Rhodococcus opacus TKN14 strain, and“ TerpD_Pseudomonas ” Bacterial TerpD means “XylB—Ac inetoII J means Acyletobater cal coaceti cus NCIB 8250 ylB,“ AreB 1 AcinetoJ means Acinetobater sp. ADP1 strain AreB, “XylB—pWW0” putida T0L plasmid means XylB (Accession number, D63341) coded by pWWO, and “XylB—T0L” is XylB (Shaw, JP, Rekik, coded by T0L plus.mid of Pseudononas putida mt-2) M., Schwager, F., and Harayama,, Kinetic studies on benzyl alcohol dehydrogenase encoded by T0L p 丄 asmid pWWO, J. Biological Chemistry,
268， 10842 - 10850， 1993) を意味し、 「ADH1— Man」 はヒ トのアルコールデヒ.ドロゲ ナーゼ (Matsuo, Y. , Yokoyama, S. Mol ecular structure of the human alcohol dehydrogenase 1 gene. FEBS Lett. , 243, 57—60， 1989) を意味する。 268, 10842-10850, 1993), and “ADH1— Man” is a human alcohol dehydrogenase 1 gene. FEBS Lett. (Matsuo, Y., Yokoyama, S. Molecular structure of the human alcohol dehydrogenase 1 gene. , 243, 57-60, 1989).
図 8は、 作製したプラスミ ドの構造を示す図であり、 図中、 Spは l を意味 し、 Bは BamHIを意味し、 Aは を意味し、 Xoは を意味し、 EVは EcoRV を意味し、 Eは EcoRIを意味し、 Nは Notlを意味し、 Pは Pstlを意味する。 発明を実施するための最良の形態 Figure 8 shows the structure of the prepared plasmid, where Sp stands for l And B means BamHI, A means, Xo means, EV means EcoRV, E means EcoRI, N means Notl, and P means Pstl. BEST MODE FOR CARRYING OUT THE INVENTION
本発明に係るペプチドは、 芳香族化合物デヒ ドロゲナーゼ活性を有しており、 例えば、 配列番号 3に示すアミノ酸配列からなる芳香族家化合物アルデヒドデヒ ド口ゲナーゼ及ぴ配列番号 5に示すァミノ酸配列からなる芳香族アルコールデヒ ドロゲナーゼを挙げることができる。  The peptide according to the present invention has an aromatic compound dehydrogenase activity, for example, from an aromatic family compound aldehyde dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 3 and an amino acid sequence shown in SEQ ID NO: 5. An aromatic alcohol dehydrogenase can be mentioned.
ここで、 芳香族化合物アルデヒ ドデヒドロゲナーゼ活性とは、 芳香族アルデヒ ドを芳香族カルボン酸に酸化する活性を意味する。 配列番号 3のアミノ酸配列か らなる芳香族家化合物デヒ ドロゲナーゼは、 新潟県石油噴出所の土壌より単離さ れた、 1, 4 -ジメチルナフタレンを唯一の炭素源として利用できるスフィンゴモナ ス (Sphingomonas) 属細菌 14DN61株から単離することができる。 なお、 配列番号 3のアミノ酸配列をコードする塩基配列を配列番号 2に示す。  Here, the aromatic compound aldehyde dehydrogenase activity means the activity of oxidizing an aromatic aldehyde to an aromatic carboxylic acid. The aromatic family compound dehydrogenase consisting of the amino acid sequence of SEQ ID NO: 3 is a sphingomonas (Sphingomonas) that can be used as the sole carbon source from 1,4-dimethylnaphthalene isolated from the soil of the Niigata Petroleum. ) It can be isolated from the genus bacterium 14DN61 strain. The base sequence encoding the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 2.
また、 本発明に係るぺプチドは、 芳香族化合物デヒ ドロゲナーゼ活性を有して いる限り、 配列番号 3に示すアミノ酸配列において 1若しくは複数個のアミノ酸 が付加、 欠失若しくは置換されたアミノ酸配列からなるものであっても良い。 こ こで、 複数個のアミノ酸とは、 例えば 1 〜 5 0個のアミノ酸、 好ましくは 1 〜 3 5個のアミノ酸、 より好ましくは 1 〜 2 0個のアミノ酸、 最も好ましくは 1 〜 1 0個のアミノ酸を意味する。  In addition, the peptide according to the present invention is based on an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 3 as long as it has aromatic compound dehydrogenase activity. It may be. Here, the plurality of amino acids means, for example, 1 to 50 amino acids, preferably 1 to 35 amino acids, more preferably 1 to 20 amino acids, and most preferably 1 to 10 amino acids. Means amino acid.
さらに、 本発明に係るペプチドは、 芳香族化合物デヒドロゲナーゼ活性を有し ている限り、 配列番号 2の塩基配列からなる DNA又はそれと相補的な DNAに対し て、 ストリンジユントな条件でハイプリダイズする DNAによりコードされるもの であっても良い。 ここで、 ストリンジェントな条件とは、 いわゆる特異的なハイ プリッドが形成され、 非特異的なハイブリッドが形成されない条件をいう。 ス ト リンジェントな条件下としては、例えば、相同性が高い D N A同士（例えば 6 0 % 以上の相同性を有する D N A同士）がハイブリダィズし、それより相同性が低い D Furthermore, as long as the peptide according to the present invention has an aromatic compound dehydrogenase activity, DNA that is hybridized under stringent conditions to DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or DNA complementary thereto. It may be coded by. Here, the stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. As stringent conditions, for example, DNAs having high homology (for example, DNAs having homology of 60% or more) hybridize and lower homology.
N A同士がハイプリダイズしない条件を挙げることができる。 具体的に、 ストリ ンジェントな条件としては、 通常のサザンハイプリダイゼーションにおける洗い の条件である 60°C、 1 X SSC、 0. 10/0SDS、 好ましくは、 0. 1 X SSC、 0. 1 %SDSに相当 する塩濃度でハイプリダイズする条件が挙げられる。 A condition where NAs do not hyper-predicate can be mentioned. Specifically, stringent conditions include washing in normal Southern high pridization. 60 ° C, 1 X SSC is a condition, 0. 1 0/0 SDS, preferably, 0. 1 X SSC, High Priestess soybeans conditions and the like at a salt concentration corresponding to 0. 1% SDS.
さらにまた、 本発明に係るぺプチドが芳香族化合物デヒ ドロゲナーゼ活性を有 しているか否かは、 例えば、 対象のペプチドを含む溶液に 2-ナフタルデヒド等の 芳香族アルデヒ ド （基質） を添加し、 反応後の溶液に 2-ナフトェ酸等の芳香族力 ルボン酸 （反応産物） が含まれるか否かを GC- MSによって検出する。 その結果、 芳香族アルデヒ ドが芳香族カルボン酸へ変換されていることが確認できれば、 対 象のぺプチドは芳香族化合物デヒ ドロゲナーゼ活性を有することとなる。  Furthermore, whether or not the peptide according to the present invention has an aromatic compound dehydrogenase activity is determined by adding an aromatic aldehyde (substrate) such as 2-naphthaldehydride to a solution containing the target peptide. Detect whether the solution after the reaction contains aromatic rubonic acid (reaction product) such as 2-naphthoic acid by GC-MS. As a result, if it can be confirmed that the aromatic aldehyde has been converted to an aromatic carboxylic acid, the target peptide has an aromatic compound dehydrogenase activity.
一方、本発明に係る遺伝子は、上述したぺプチドをコ一ドする遺伝子であって、 例えば配列番号 2の塩基配列からなる。 また、 本発明に係る遺伝子は、 配列番号 2の塩基配列に対して 6 0 %以上の相同性、 好ましくは 7 0 %以上の相同性、 よ り好ましくは 8 0 %以上の相同性を有する塩基配列からなり、 上述した芳香族化 合物デヒ ドロゲナーゼ活性を有するぺプチドをコードする DNAからなるものであ つても良い。 ここで、 相同性とは、 配列解析ソフトウェアである DNASIS (日立ソ フトウェアエンジニアリング） 等を用いて、 例えば、 マキシムマッチング法のコ マンドを実行することにより求められる数値である。 その際のパラメータは、 デ フォルトの設定 （初期設定） とする。  On the other hand, the gene according to the present invention is a gene that codes for the above-described peptide, and comprises, for example, the base sequence of SEQ ID NO: 2. In addition, the gene according to the present invention has a nucleotide sequence having a homology of 60% or more, preferably 70% or more, more preferably 80% or more of the nucleotide sequence of SEQ ID NO: 2. It may comprise a sequence and may comprise a DNA encoding a peptide having the above-mentioned aromatic compound dehydrogenase activity. Here, the homology is a numerical value obtained, for example, by executing a command of the Maxim matching method using DNASIS (Hitachi Software Engineering) which is sequence analysis software. The parameters at that time shall be the default settings (initial settings).
一方、 ここで、 芳香族アルコールデヒドロゲナ一ゼ活性とは、 ヒ ドロキシメチ ル基を分子内に有する芳香族アルコール (芳香族ヒ ドロキシメチル化合物) を芳 香族アルデヒ ド酸に酸化する活性を意味する。 配列番号 5のアミノ酸配列からな る芳香族アルコールデヒ ドロゲナーゼは、 2-キシレン資化細菌口ドコッカス ·ォ パカス (Rhodococcus opacus) TKN14株から単離することができる。 なお、 配列 番号 5のアミノ酸配列をコードする塩基配列を配列番号 4に示す。 なお、 ロドコ ッカス 'ォパカス (Rhodococcus opacus) TKN14株は、 株式会社海洋バイオテク ノロジ一からカルチャーコレクション番号 「MBIC 05572」 として入手すること力 S できる。 したがって、 上記芳香族アルコールデヒ ドロゲナーゼをコードする遺伝 子 （配列番号 4の塩基配列） は、 入手した口ドコッカス 'ォパカス (Rhodococcus opacus) TKN14株から定法に従って取得することができる。 また、 上記芳香族ァ ルコールデヒ ドロゲナーゼをコードする遺伝子は、 配列番号 4の塩基配列情報に 基づいて化学合成により取得することもできる。 On the other hand, the aromatic alcohol dehydrogenase activity means an activity of oxidizing an aromatic alcohol (aromatic hydroxymethyl compound) having a hydroxymethyl group in the molecule to an aromatic aldehyde. The aromatic alcohol dehydrogenase comprising the amino acid sequence of SEQ ID NO: 5 can be isolated from the 2-xylene-utilizing bacterium Rhodococcus opacus TKN14. The base sequence encoding the amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 4. Rhodococcus opacus TKN14 strain can be obtained from Marine Biotechnology Co., Ltd. under the culture collection number “MBIC 05572”. Therefore, the gene encoding the above-mentioned aromatic alcohol dehydrogenase (base sequence of SEQ ID NO: 4) can be obtained from the obtained mouthdococcus opacus TKN14 strain according to a conventional method. The gene encoding the above aromatic alcohol dehydrogenase is represented by the nucleotide sequence information of SEQ ID NO: 4. It can also be obtained by chemical synthesis based on this.
また、 本発明に係るペプチドは、 芳香族化合物アルコールデヒ ドロゲナーゼ活 性を有している限り、 配列番号 5に示すァミノ酸配列において 1若しくは複数個 のアミノ酸が付加、 欠失若しくは置換されたアミノ酸配列からなるものであって も良い。 ここで、 複数個のアミノ酸とは、 例えば 1〜 5 0個のアミノ酸、 好まし くは 1〜3 5個のァミノ酸、 より好ましくは 1〜2 0個のアミノ酸、 最も好まし くは 1〜1 0個のアミノ酸を意味する。  In addition, as long as the peptide according to the present invention has aromatic compound alcohol dehydrogenase activity, one or more amino acids in the amino acid sequence shown in SEQ ID NO: 5 are added, deleted or substituted. It may consist of an array. Here, the plurality of amino acids means, for example, 1 to 50 amino acids, preferably 1 to 35 amino acids, more preferably 1 to 20 amino acids, most preferably 1 to Means 10 amino acids.
さらに、 本発明に係るペプチドは、 芳香族化合物デヒ ドロゲナーゼ活性を有し ている限り、 配列番号 4の塩基配列からなる DNA又はそれと相補的な DNAに対し て、 ストリンジェントな条件でハイブリダイズする DNAによりコードされるもの であっても良い。 ここで、 ストリンジェントな条件とは、 いわゆる特異的なハイ ブリツドが形成され、 非特異的なハイブリッドが形成されない条件をいう。 ス ト リンジ ントな条件下としては、例えば、相同性が高い D N A同士（例えば 6 0 % 以上の相同性を有する D N A同士）がハイブリダィズし、それより相同性が低い D N A同士がハイブリダイズしない条件を挙げることができる。 具体的に、 ストリ ンジェントな条件としては、 通常のサザンハイブリダィゼーションにおける洗い の条件である 60°C、 1 X SSC、 0. 1%SDS、 好ましくは、 0. 1 X SSC、 0. 1%SDSに相当 する塩濃度でハイブリダィズする条件が挙げられる。  Furthermore, the peptide according to the present invention hybridizes under stringent conditions to DNA consisting of the base sequence of SEQ ID NO: 4 or DNA complementary thereto as long as it has aromatic compound dehydrogenase activity. It may be one encoded by DNA. Here, the stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. As stringent conditions, for example, DNAs with high homology (for example, DNAs having homology of 60% or more) hybridize and DNAs with lower homology do not hybridize with each other. Can be mentioned. Specifically, stringent conditions include washing conditions in normal Southern hybridization, 60 ° C, 1 X SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1 A condition for hybridizing at a salt concentration corresponding to% SDS is mentioned.
さらにまた、 本発明に係るぺプチドが芳香族化合物アルコールデヒ ドロゲナー ゼ活性を有しているか否かは、 例えば、 対象のペプチドを含む溶液に 2 ヒドロキ シメチルナフタレン等の芳香族ヒドロキシメチル化合物 （基質） を添加し、 反応 後の溶液に 2 -ナフタルデヒド等の芳香族アルデヒ ド （反応産物） が含まれるか否 かを GC - MSによって検出する。 その結果、 芳香族アルコールが芳香族アルデヒ ド へ変換されていることが確認できれば、 対象のぺプチドは芳香族化合物アルコー ルデヒ ドロゲナーゼ活性を有することとなる。  Furthermore, whether or not the peptide according to the present invention has an aromatic compound alcohol dehydrogenase activity is determined by, for example, determining whether the peptide containing the peptide of interest has an aromatic hydroxymethyl compound such as 2-hydroxymethylnaphthalene ( Substrate) is added, and GC-MS detects whether the solution after the reaction contains aromatic aldehydes (reaction products) such as 2-naphthalaldehyde. As a result, if it can be confirmed that the aromatic alcohol has been converted to an aromatic aldehyde, the subject peptide will have an aromatic alcohol dehydrogenase activity.
一方、本発明に係る遺伝子は、上述したぺプチドをコ一ドする遺伝子であって、 例えば配列番号 4の塩基配列からなる。 また、 本発明に係る遺伝子は、 配列番号 On the other hand, the gene according to the present invention is a gene that codes for the above-mentioned peptide, and consists of the base sequence of SEQ ID NO: 4, for example. The gene according to the present invention is SEQ ID NO:
4の塩基配列に対して 6 0 %以上の相同性、 好ましくは 7 0 %以上の相同性、 よ り好ましくは 8 0 %以上の相同性を有する塩基配列からなり、 上述した芳香族化 合物アルコールデヒ ドロゲナーゼ活性を有するぺプチドをコ一ドする DNAからな るものであっても良レ、。ここで、相同性とは、配列解析ソフトウェアである DNASIS (日立ソフトウェアエンジニアリング） 等を用いて、 例えば、 マキシムマツチン グ法のコマンドを実行することにより求められる数値である。 その際のパラメ一 タは、 デフォルトの設定 （初期設定） とする。 It consists of a base sequence having 60% or more homology, preferably 70% or more homology, more preferably 80% or more homology with respect to the base sequence of 4. It may be composed of DNA that encodes a peptide having compound alcohol dehydrogenase activity. Here, the homology is a numerical value obtained by, for example, executing a command of the Maxim matching method using DNASIS (Hitachi Software Engineering), which is sequence analysis software. The parameters at that time are the default settings (initial settings).
本発明に係る遺伝子は、 通常使用されているベクターに連結（挿入）することに より得られる組換えベクターとすることができる。 本発明に係る遺伝子を挿入す るためのベクターは、宿主中で複製可能なものであれ.ば特に限定されず、例えば、 プラスミ ド醒、 ファージ DNAなどが挙げられる。  The gene according to the present invention can be a recombinant vector obtained by ligation (insertion) into a commonly used vector. The vector for inserting the gene according to the present invention is not particularly limited as long as it can be replicated in the host, and examples thereof include plasmid emergence and phage DNA.
プラスミ ド DNAとしては、 pBR322、 pBR325、 pUC118、 pUC119などの大腸菌宿主 用プラスミ ド、 pUB110、 pTP5 などの枯草菌用プラスミ ド、 YEpl3、 YEp24、 YCp50 などの酵母宿主用プラスミ ド、 ρΒΙ221、 ρΒΠ21などの植物細胞宿主用プラスミ ド などが挙げられ、 ファージ DNAとしては； ファージなどが挙げられる。 さらに、 レトロウィルス又はワクシニアウィルスなどの動物ウィルス、 バキュ口ウィルス などの昆虫ウィルスベクターを用いることもできる。  The plasmid DNA includes plasmids for E. coli hosts such as pBR322, pBR325, pUC118, and pUC119, plasmids for Bacillus subtilis such as pUB110 and pTP5, plasmids for yeast hosts such as YEpl3, YEp24, and YCp50, ρΒΙ221, ρΒΠ21, etc. Examples include plasmids for plant cell hosts, and examples of phage DNA include: phages. Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.
ベクターに本発明に係る遺伝子を挿入するには、 まず、 本発明に係る遺伝子を 有する精製された DNAを適当な制! ¾酵素で切断し、 適当なベクター DNAの制限酵 素部位又はマルチクローニングサイ トに挿入してベクターに連結する方法などが 採用される。  In order to insert the gene according to the present invention into a vector, first, the purified DNA having the gene according to the present invention is cleaved with an appropriate restriction enzyme, and a restriction enzyme site or a multicloning site of an appropriate vector DNA is cut. A method of inserting into a vector and linking to a vector is employed.
本発明に係る遺伝子は、 その遺伝子の機能が発揮されるようにベクターに組み 込むことができる。 すなわちベクターは、 プロモーター、 本発明に係る遺伝子、 所望によりェンハンサーなどのシスエレメント、 スプライシングシグナル、 ポリ Α付加シグナル、 選択マーカー、 リボソーム結合配列 （SD配列） 等を含むように 調製することができる。 なお、 選択マーカーとしては、 例えば、 アンピシリン耐 性遺伝子、 ネオマイシン耐性遺伝子及びジヒ ドロ葉酸還元酵素遺伝子などを使用 することができる。  The gene according to the present invention can be incorporated into a vector so that the function of the gene is exhibited. That is, the vector can be prepared so as to contain a promoter, a gene according to the present invention, a cis element such as an enhancer, a splicing signal, a polyaddition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like. As the selection marker, for example, ampicillin resistance gene, neomycin resistance gene, dihydrofolate reductase gene and the like can be used.
本発明に係る遺伝子を含む組換えベクターを、 当該遺伝子が発現し得るように 宿主中に導入することにより形質転換体を得ることができる。 ここで、 宿主とし ては、 本発明の遺伝子を発現できるものであれば特に限定されるものではない。 例えば、 エツシエリ ヒア · コリ (Escherichia coli)などのエツシェリヒァ属、 チル,ス ·ズプチリス (Baci llus subti l is)などのバチルス属、 シュ一ドモナス ·プ チダ（Pseudomonas putida)などのシユードモナス属、 リゾビゥム · メ リロティ (Rhizobium mel iloti)などのリゾビゥム属に属する細菌が挙げられ、 サッカロマ イセス ·セレビシェ (Saccharomyces cerevis iae)、 シゾサッカロマイセス ·ポン ベ (Schizosaccharomyces pombeノ、 ヒ ヒ ノ ' · ノヽス 卜 リ ス (Pichia pastoris)なとの 酵母が挙げられ、 シロイヌナズナ、 タバコ、 トウモロコシ、 イネ、 ニンジンなど から株化した植物細胞ゃ該植物から調製したプロ トプラストが挙げられ、 COS 細 胞、 CH0細胞などの動物細胞が挙げられ、. あるいは Sf9、 Sf21 などの昆虫細胞が 挙げられる。 A transformant can be obtained by introducing a recombinant vector containing the gene of the present invention into a host so that the gene can be expressed. Here, the host is not particularly limited as long as it can express the gene of the present invention. For example, Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, Syudomonas such as Pseudomonas putida, Rhizobium me Examples include bacteria belonging to the genus Rhizobium, such as Rhizobium mel iloti, and Saccharomyces cerevis iae, Schizosaccharomyces pombe, and Hihi no ichi ris. yeast, etc., plant cells established from Arabidopsis thaliana, tobacco, corn, rice, carrots, etc., protoplasts prepared from these plants, animal cells such as COS cells, CH0 cells, etc. Or insect cells such as Sf9 and Sf21.
大腸菌などの細菌を宿主とする場合は、 本発明の組換えベクターが該細菌中で 自律複製可能であると同時に、 プロモーター、 リボゾーム結合配列、 本発明の遺 伝子、 転写終結配列により構成されていることが好ましい。 また、 プロモーター を制御する遺伝子が含まれていてもよい。  When a bacterium such as E. coli is used as a host, the recombinant vector of the present invention can autonomously replicate in the bacterium, and at the same time, is composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Preferably it is. Moreover, the gene which controls a promoter may be contained.
プロモーターとしては、 大腸菌などの宿主中で発現できるものであればいずれ を用いてもよい。例えば trpプロモーター、 lacプロモーター、 PLプロモーター、 PRプロモーターなどの大腸菌由来のものや T7プロモーターなどのファージ由来 のものが用いられる。 さらに、 tac プロモーターなどのように人為的に設計改変 されたプロモーターを用いてもよい。  Any promoter can be used as long as it can be expressed in a host such as E. coli. For example, those derived from Escherichia coli such as trp promoter, lac promoter, PL promoter, PR promoter, and those derived from phage such as T7 promoter are used. In addition, artificially designed and modified promoters such as the tac promoter may be used.
細菌への組換えベクターの導入方法としては、 細菌に DNAを導入する方法であ れぱ特に限定されるものではない。例えばカルシウムイオンを用いる方法 [Cohen, S. N. , et al . ： Proc. Natl . Acad. Sci .， USA, 69 : 2110—2114 (1972) ]、 エレク ト口 ポレーシヨン法などが挙げられる。  The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions [Cohen, SN, et al .: Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972)], and an electroporation method.
酵母を宿主とする場合は、 例えばサッカロマイセス 'セレピシェ、 シゾサッカ ロマイセス .ボンべ、 ピヒア . ノ ストリスなどが用いられる。 この場合、 プロモ 一ターとしては酵母中で発現できるものであれば特に限定されず、 例えば gal l プロモーター、 gal l O プロモーター、 ヒートショックタンノヽ。ク質プロモーター、 When yeast is used as a host, for example, Saccharomyces cereviche, Schizosaccharomyces bomb, Pichia nostris, etc. are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast. For example, gal l promoter, gal l O promoter, heat shock tannomi. Quality promoter,
MF CK Iプロモーター、 PH05プロモーター、 PGKプロモーター、 GAPプロモーター、MF CK I promoter, PH05 promoter, PGK promoter, GAP promoter,
ADHプロモーター、 A0X1プロモーターなどが挙げられる。 酵母への組換えベクターの導入方法としては、 酵母に DNAを導入する方法であ れば特に限定されず、 例えばエレク ト口ポレーシヨン法 [Becker, D. M.， et al. ： Methods. Enzymol. , 194 : 182-187 (1990) ]、 スフエロプラス ト法 [Hinnen, A. et al. : Proc. Natl. Acad. Sc i.， USA, 75 : 1929-1933 (1978) ]、 酢酸リチウム法 [Itoh， H. ： J. Bacteriol.， 153 : 163— 168 (1983) ]などが挙げられる。 Examples include ADH promoter and A0X1 promoter. The method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, the electric mouth population method [Becker, DM, et al .: Methods. Enzymol., 194: 182-187 (1990)], Spheroplast method [Hinnen, A. et al .: Proc. Natl. Acad. Sci., USA, 75: 1929-1933 (1978)], lithium acetate method [Itoh, H. : J. Bacteriol., 153: 163-168 (1983)].
植物細胞を宿主とする場合は、例えばシロイヌナズナ、タバコ、 トウモロコシ、 イネ、 ニンジンなどから株化した細胞ゃ該植物から調製したプロ トプラストが用 いられる。 この場合、 プロモーターとしては植物中で発現できるものであれば特 に限定されず、 例えば力リフラヮーモザィクウィルスの 35S RNAプロモーター、 rd29A遣伝子プロモーター、 rbcSプロモーターなどが挙げられる。  When plant cells are used as hosts, for example, cells established from Arabidopsis thaliana, tobacco, corn, rice, carrots, etc., protoplasts prepared from the plants are used. In this case, the promoter is not particularly limited as long as it can be expressed in a plant, and examples thereof include a power reframer mosaic virus 35S RNA promoter, an rd29A gene promoter, and an rbcS promoter.
植物への組換えベクターの導入方法としては、 Abel らのポリエチレングリコー ノレを用いる方法 [Abel, H. , et al. ： Plant J. 5： 421-427 (1994) ]やエレク トロポレー ション法などが挙げられる。 '  As a method for introducing a recombinant vector into a plant, a method using polyethylene glycolate by Abel et al. [Abel, H., et al .: Plant J. 5: 421-427 (1994)] and an electroporation method are available. Can be mentioned. '
動物細胞を宿主とする場合は、 サル細胞 COS- 7、 Vero、 チャイニーズハムスタ 一卵巣細胞 （CH0細胞）、 マウス L細胞、 ラット GH3、 ヒ ト FL細胞などが用いられ る。 プロモーターとして SR aプロモーター、 SV40プロモーター、 LTRプロモータ 一、 CMV プロモーターなどが用いられ、 また、 ヒ トサイ トメガロウィルスの初期 遺伝子プロモーターなどを用いてもよい。 動物細胞への組換えベクターの導入方 法としては、 例えばエレク ト口ポレーシヨン法、 リン酸カルシウム法、 リポフエ クション法などが挙げられる。  When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CH0 cells), mouse L cells, rat GH3, and human FL cells are used. As the promoter, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, etc. may be used, and a human megalovirus early gene promoter may also be used. Examples of methods for introducing a recombinant vector into animal cells include the electoral position method, the calcium phosphate method, and the lipofection method.
昆虫細胞を宿主とする場合は、 Sf9細胞、 Sf21細胞などが用いられる。 昆虫細 胞への組換えベクターの導入方法としては、 例えば、 リン酸カルシウム法、 リポ フエクション法、 エレク トロポレーション法などが用いられる。  When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into an insect cell, for example, a calcium phosphate method, a lipofection method, an electroporation method and the like are used.
以上のようにして得られた形質転換体を、 ヒ ドロキシメチル基又はアルデヒ ド 基を有する芳香族化合物を含む培地で培養することによって、培養物及び/又は形 質転換体から芳香族カルボン酸を得ることができる。 本発明に係る芳香族アルデ ヒ ドデヒ ドロゲナーゼは、 宿主自身が有している芳香族ヒ ドロキシメチル化合物 を芳香族アルデヒ ドに酸化する芳香族アルコールデヒ ドロゲナーゼ活性を高める ことができる。 したがって、 芳香族ヒ ドロキシメチル化合物を含む培地で形質転 換体を培養することによって、 宿主自身の芳香族アルコールデヒドロゲナーゼ活 性を高め、結果として、培養物及び/又は形質転換体から芳香族カルボン酸を得る ことができる。 An aromatic carboxylic acid is obtained from the culture and / or transformant by culturing the transformant obtained as described above in a medium containing an aromatic compound having a hydroxymethyl group or an aldehyde group. be able to. The aromatic aldehyde dehydrogenase according to the present invention can increase the activity of an aromatic alcohol dehydrogenase that oxidizes an aromatic hydroxymethyl compound possessed by the host itself into an aromatic aldehyde. Therefore, transformation in a medium containing aromatic hydroxymethyl compounds By culturing the recombinant, the host's own aromatic alcohol dehydrogenase activity is increased, and as a result, an aromatic carboxylic acid can be obtained from the culture and / or the transformant.
また、 本発明に係る芳香族アルコールデヒドロゲナーゼは、 宿主自身が有して いる芳香族アルデヒ ドデヒ ドロゲナーゼ或いは宿主に導入した芳香族アルデヒ ド デヒ ドロゲナーゼの基質となる芳香族アルデヒドを産生することができる。 した がって、 芳香族ヒドロキシメチル化合物を含む培地で形質転換体を培養すること によって、培養物及び/又は形質転換体から芳香族カ ボン酸を得ることができる。 特に、 本発明に係る芳香族アルデヒ ドデヒドロゲナーゼ遗伝子及ぴ芳香族アル コールデヒ ドロゲナーゼ遺伝子を共発現するように形質転換した微生物によれば、 各酵素の作用によって培地に含まれる芳香族ヒドロキシメチル化合物を効率良く 芳香族力ルボン酸に変換することができる。  In addition, the aromatic alcohol dehydrogenase according to the present invention can produce an aromatic aldehyde that is possessed by the host itself or an aromatic aldehyde that becomes a substrate for the aromatic aldehyde dehydrogenase introduced into the host. Therefore, aromatic carboxylic acid can be obtained from the culture and / or transformant by culturing the transformant in a medium containing an aromatic hydroxymethyl compound. In particular, according to the microorganism transformed to co-express the aromatic aldehyde dehydrogenase gene and the aromatic alcohol dehydrogenase gene according to the present invention, the aromatic hydroxymethyl compound contained in the medium by the action of each enzyme Can be efficiently converted into aromatic rubonic acid.
基質となるヒ ドロキシメチル基を有する芳香族化合物と、 反応産物である芳香 族カルボン酸との対応を図 1 a及ぴ図 1 bに示す。 また、 基質となるアルデヒ ド 基を有する芳香族化合物と、 反応産物である芳香族カルボン酸との対応を図 4に 示す。  The correspondence between the aromatic compound having a hydroxymethyl group as a substrate and the aromatic carboxylic acid as a reaction product is shown in FIGS. 1a and 1b. Figure 4 shows the correspondence between an aromatic compound having an aldehyde group as a substrate and an aromatic carboxylic acid as a reaction product.
なお、 図 1 a及び 1 bにおいて 1A〜19Aは基質を示し、 1B〜19Bはそれぞれ 1A 〜19Aに対応する反応産物を示している。 1A〜19A及び 1B〜19Bの化合物名は以下 の通りである ：  In FIGS. 1 a and 1 b, 1A to 19A represent substrates, and 1B to 19B represent reaction products corresponding to 1A to 19A, respectively. The compound names of 1A-19A and 1B-19B are as follows:
1A、 1, 4-ジヒ ドロキシメチルナフタレン； 1B、4 -ヒ ドロキシメチル- 1-ナフ トェ酸； 2A、 2-ヒ ドロキシメチル- 6 -メチノレナフタレン； 2B、 6 -メチル- 2-ナフ トェ酸； 3A、 1 -ヒ ドロキシメチルナフタレン ； 3B、 1 -ナフ トェ酸； 4A、 2 -ヒ ドロキシメチルナ フタレン ； 4B、 2-ナフトェ酸； 5A、 4-メチルベンジルアルコール； 5B、 4 -メチル 安息香酸； 6A、 3 -メチルベンジルアルコール； 6B、 3-メチル安息香酸； 7A、 2-メ チルベンジルアルコール； 7B、 2-メチル安息香酸； 8A、 ベンジルアルコール； 8B、 安息香酸； 9A、 キシレン- α -ひ ' -ジオール； 9Β、 4-ヒ ドロキシメチル安息香酸；1A, 1,4-dihydroxymethylnaphthalene; 1B, 4-hydroxymethyl-1-naphthoic acid; 2A, 2-hydroxymethyl-6-methylenonaphthalene; 2B, 6-methyl-2-naphthoic acid; 3A, 1-hydroxymethylnaphthalene; 3B, 1-naphthoic acid; 4A, 2-hydroxymethylnaphthalene; 4B, 2-naphthoic acid; 5A, 4-methylbenzyl alcohol; 5B, 4-methylbenzoic acid; 6A 3B-Methylbenzyl alcohol; 6B, 3-Methylbenzoic acid; 7A, 2-Methylbenzyl alcohol; 7B, 2-Methylbenzoic acid; 8A, Benzyl alcohol; 8B, Benzoic acid; 9A, Xylene-α-Hin ' -Diol; 9Β, 4-hydroxymethylbenzoic acid;
10Α、 4 -ヒ ドロキシベンジルアルコール； 10Β、 4 -ヒ ドロキシ安息香酸； 11Α、 3 - ヒ ドロキシベンジルアルコール； 11Β、 3-ヒ ドロキシ安息香酸； 12Α、 2-ヒ ドロキ シベンジルアルコール； 12Β、 2 -ヒ ドロキシ安息香酸； 13Α、 4 -メ トキシベンジル アルコール； 13B、 4 -メ トキシ安息香酸； 14A、 3-メ トキシベンジルアルコール ；10Α, 4-Hydroxybenzyl alcohol; 10Β, 4-Hydroxybenzoic acid; 11Α, 3-Hydroxybenzyl alcohol; 11Β, 3-Hydroxybenzoic acid; 12Α, 2-Hydroxybenzyl alcohol; 12Β 2-Hydroxybenzoic acid; 13Α, 4-Methoxybenzyl Alcohol; 13B, 4-methoxybenzoic acid; 14A, 3-methoxybenzyl alcohol;
14B、 3-メ トキシ安息香酸； 15Α、 .2 -メ トキシベンジルアルコール； 15B、 2-メ トキ シ安息香酸； 16A、 4-ェチルベンジルアルコール； 16B、 4-ェチル安息香酸； 17A、15B, .2-Methoxybenzyl alcohol; 15B, 2-Methoxybenzoic acid; 16A, 4-Ethylbenzyl alcohol; 16B, 4-Ethylbenzoic acid; 17A, 14B, 3-Methoxybenzoic acid;
4 -ィソプロピルべンジルアルコール； 17B、 4 -ィソプロピル安息香酸； 18A、 3-ク ロロべンジルアルコール ； 18B、 3-ク口口安息香酸； 19A、 3_フエニルプロピルァ ルコール； 19B、 3-フエニルプロピオン酸。 17-B, 4-Isopropyl benzoic acid; 18A, 3-chlorobenzil alcohol; 18B, 3-mouth benzoic acid; 19A, 3_phenylpropyl alcohol; 19B, 3 -Phenylpropionic acid.
同様に図 2において、 1A〜6Aは基質を示し、 .1B〜6Bはそれぞれ 1A〜6Aに対応 する反応産物を示している。 1A〜6A及び 1B〜6Bの化合物名は以下の通りである： Similarly, in FIG. 2, 1A to 6A represent substrates, and .1B to 6B represent reaction products corresponding to 1A to 6A, respectively. The compound names 1A-6A and 1B-6B are as follows:
1A、 1 -ナフタルデヒ ド ； 1B、 1-ナフ トェ酸 ； 2A、 2-ナフタルデヒ ド ； 2B、 2 -ナフ トェ酸 ； 3A、 4-メチルベンズアルデヒ ド； 3B、 4 -メチル安息香酸； 4A、 3-メチル ベンズァノレデヒ ド； 4B、 3-メチル安息香酸； 5A、 2-メチルベンズアルデヒ ド； 5B、1A, 1-naphthalaldehyde; 1B, 1-naphthoic acid; 2A, 2-naphthalaldehyde; 2B, 2-naphthoic acid; 3A, 4-methylbenzaldehyde; 3B, 4-methylbenzoic acid; 4A, 3 -Methyl benzanolide; 4B, 3-methylbenzoic acid; 5A, 2-methylbenzaldehyde; 5B,
2-メチル安息香酸； 6A、 ベンズアルデヒ ド ； 6B、 安息香酸。 2-methylbenzoic acid; 6A, benzaldehyde; 6B, benzoic acid.
上述した形質転換体を用いた芳香族カルボン酸の製造法においては、 図 1 a、 図 1 b及び図 2に示したヒ ド口キシメチル基又はアルデヒ ド基を有する芳香族化 合物を含む培地を使用する。 ここで、 当該芳香族化合物の培地における濃度は、 特に限定されないが、 例えば 10 mM、 好ましくは 2 mM、 より好ましくは 0. 5 mM とする。 '  In the method for producing an aromatic carboxylic acid using the transformant described above, a medium containing an aromatic compound having a hydroxymethyl group or an aldehyde group shown in FIG. 1a, FIG. 1b and FIG. Is used. Here, the concentration of the aromatic compound in the medium is not particularly limited. For example, the concentration is 10 mM, preferably 2 mM, more preferably 0.5 mM. '
基本となる培地としては、 上述した形質転換体の宿主に適したものを使用する ことができる。 例えば、 宿主として大腸菌を使用した場合、 LB培地を使用するこ とができる。 また一般的には、 宿主が資化し得る炭素源、 窒素源、 無機塩類など を含有し、 形質転換体の培養を効率的に行うことができる培地であれば、 天然培 地、 合成培地のいずれを用いてもよい。  As a basic medium, a medium suitable for the host of the transformant described above can be used. For example, when E. coli is used as the host, LB medium can be used. In general, any medium that contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host and that can efficiently culture transformants can be used. May be used.
例えば、 炭素源としては、 グルコース、 フラク トース、 スクロース、 デンプン などの炭水化物、 酢酸、 プロピオン酸などの有機酸、 エタノール、 プロパノール などのアルコール類が用いられる。 窒素源としては、 アンモニア、 塩化アンモニ ゥム、 硫酸アンモニゥム、 酢酸アンモニゥム、 リン酸アンモユウムなどの無機酸 若しくは有機酸のアンモニゥム塩又はその他の含窒素化合物のほか、 ペプトン、 肉エキス、 コーンスティープリカ一などが用いられる。 無機物としては、 リン酸 第一カリウム、 リン酸第二カリウム、 リン酸マグネシウム、 硫酸マグネシウム、 塩化ナトリウム、 硫酸第一鉄、 硫酸マンガン、 硫酸銅、 炭酸カルシウムなどが用 いられる。 さらに、 培地中には、 必要に応じてアンピシリンゃテトラサイクリン などの抗生物質を添加してもよい。 For example, as the carbon source, carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salt of organic acid, and other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, etc. Is used. Inorganic substances include potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, Sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used. Furthermore, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
培養は、 形質転換体の宿主にも依るが通常、 振盪培養又は通気攪拌培養などの 好気的条件下、 30〜37°Cで 6〜24時間行う。 培養期間中、 pHは 7. 0〜7. 5に保持 する。 pHの調整は、 無機又は有機酸、 アルカリ溶液などを用いて行う。  Culturing is usually performed at 30 to 37 ° C for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and agitation culture, depending on the host of the transformant. During the incubation period, the pH is maintained at 7.0 to 7.5. The pH is adjusted using inorganic or organic acid, alkaline solution, etc.
プロモーターとして誘導性のプロモーターを含む組換えベクターで形質転換し た微生物を培養する場合には、 必要に応じてィンデユーザーを培地に添加しても よい。 例えば、 Lac プロモーターを用いた発現ベクターで形質転換した微生物を 培養するときにはイソプロピル - jS - D-チォガラク トピラノシド（IPTG)などを、 trp プロモーターを用いた発現べクタ一で形質転換した微生物を培養するときにはィ ンドールァクリル酸（IAA)などを培地に添加してもよレ、。  When culturing a microorganism transformed with a recombinant vector containing an inducible promoter as a promoter, an indeuser may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the Lac promoter, when culturing a microorganism transformed with isopropyl-jS-D-thiogalactopyranoside (IPTG) or the like with an expression vector using the trp promoter. Indoleacrylic acid (IAA) may be added to the medium.
なお、 培養後、 本発明に係るペプチドが菌体内又は細胞内に生産される場合に は、 菌体又は細胞を破碎することにより当該べプチドを抽出することもできる。 また、 当該ペプチドが菌体外又は細胞外に生産される場合には、 培養液をそのま ま使用するカ 遠心分離などにより菌体又は細胞を除去する。 その後、 タンパク 質の単離精製に用いられる一般的な生化学的方法、例えば硫酸アンモ-ゥム沈殿、 ゲノレクロマトグラフィー、 イオン交換クロマトグラフィー、 ァフィ二ティーク口 マトグラフィーなどを単独で又は適宜組み合わせて用いることにより、 前記培養 物中から本発明のぺプチドを単離精製することもできる。  When the peptide according to the present invention is produced in cells or cells after culturing, the peptide can also be extracted by destroying the cells or cells. In addition, when the peptide is produced outside the cells or cells, the cells or cells are removed by centrifuging using the culture solution as it is. Then, general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, genomic chromatography, ion exchange chromatography, affinity mouth matography, etc., alone or in combination as appropriate. By using it, the peptide of the present invention can be isolated and purified from the culture.
以下、 実施例により本発明について具体的に説明する。 しかしながら、 本発明 の技術的範囲は以下の実施例により限定的に解釈されるものではない。 なお、 実 施例 1〜1 0では本発明に係る芳香族アルデヒ ドデヒ ドロゲナーゼについて説明 し、 実施例 1 1〜1 8では本発明に係る芳香族アルコールデヒ ドロゲナーゼにつ いて説明する。  Hereinafter, the present invention will be specifically described by way of examples. However, the technical scope of the present invention is not limited to the following examples. Examples 1 to 10 describe the aromatic aldehyde dehydrogenase according to the present invention, and Examples 11 to 18 describe the aromatic alcohol dehydrogenase according to the present invention.
〔実施例 1〕 1， 4 -ジメルナフタレン資化菌の分離と解析  [Example 1] Isolation and analysis of 1,4-dimernaphthalene assimilating bacteria
100 mlの無機培地 （W培地） に石油噴出源から採集したサンプル 0. 1 %、 1, 4- ジメルナフタレン 0. 2%を添加して 30°Cで振盪培養した。 W培地の組成を以下に 示す。 K P04 (0. 85 g/1) , Na2HP04 X 12H20 (4. 90 g/1)， (NH4) 2S04 (0. 50 g/1)， MgS04 X7H20 (0.10 g/1), FeS04X7H20 (9.50 mg/l), MgO (10.75mg/l), CaC03 (2.00 mg/l) ZnS04 X 7H20 (l.44 mg/l), MnS04 X 4H20 (1.12 mg/1), CuS04 X 5H20 (0.25 mg/1), CoS04 X 7H20 (0.28 mg/1), H3B04(0.06 mg/1), and cone. HC1 (5.13 X 10— 2 ml/1) 0 増殖した細菌を基質の 1， 4 -ジメルナフタレンを含む新しい W培地に 10%植菌し、 上と同じ条件で培養した。 この操作 10回をく り返して集積培養を行った。最後に 菌を寒天プレートに撒き、 シングルクローンを選択した。 その結果、 14丽 61株が 得られた。 14DN61株の 16S rDNAの塩基配列を決定し、 データーベース上で検索 し 7ことこ >スフィンコモナス (Sphingomonas) (Lararaee, L.， Lawrence, J. R.， and Greer, C. W.， Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclof op-methyl-degrading biof ilm consortium. Can. J. Microbiol. 46(2), 133-142, 2000； Pinyakong, 0.， Habe, H. , Yoshida, T.， Nojiri,、 H. and Omori, T.， Identirication of three novel salicylate 1 -hydroxylases involved in the phenanthrene degradation of Sphingobium sp. strain P2. Biochem. Biophys. Res. Commun. 301(2)， 350 - 357， 2003； Wang, Y. and Lau, P. C. , Sequence and expression of an isocitrate dehydrogenase encoding gene from a polycyclic aromatic hydrocarbon oxidizer, Sphingomonas yanoikuyae Bl. Gene 168(1), 15 - 21, 1996) と 99%以上の相同性 を示した。従って、 14DN61株はスフィンゴモナス (Sphingomonas)属と同定した。 また、 14DN61 株は 2， 6 -ジメルナフタレン、 アントラセン、 フエナンスレン、 £ - キシレンを資化することも確認された。 To 100 ml of inorganic medium (W medium), 0.1% of a sample collected from an oil ejection source and 0.2% of 1,4-dimernaphthalene were added and cultured at 30 ° C with shaking. The composition of W medium is shown below. K P0 4 (0. 85 g / 1), Na 2 HP0 4 X 12H 2 0 (4. 90 g / 1), (NH 4 ) 2 S0 4 (0. 50 g / 1), MgS0 4 X7H 2 0 (0.10 g / 1), FeS0 4 X7H 2 0 (9.50 mg / l), MgO (10.75 mg / l), CaC0 3 (2.00 mg / l) ZnS0 4 X 7H 2 0 (l.44 mg / l), MnS0 4 X 4H 2 0 (1.12 mg / 1), CuS0 4 X 5H 2 0 (0.25 mg / 1), CoS0 4 X 7H 2 0 (0.28 mg / 1), H 3 B0 4 (0.06 mg / 1), and cone. HC1 (5.13 X 10— 2 ml / 1) 0 The inoculated bacteria were inoculated 10% in a new W medium containing the substrate 1,4-dimernaphthalene and cultured under the same conditions as above. . This operation was repeated 10 times to perform enrichment culture. Finally, the bacteria were plated on an agar plate and a single clone was selected. As a result, 14 61 strains were obtained. 14DN61 strain 16S rDNA base sequence was determined and searched in the database. 7 Koto> Sphingomonas (Lararaee, L., Lawrence, JR, and Greer, CW, Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclof op-methyl-degrading biofilm consortium. Can. J. Microbiol. 46 (2), 133-142, 2000; Pinyakong, 0., Habe, H., Yoshida, T., Nojiri, H. and Omori, T., Identirication of three novel salicylate 1-hydroxylases involved in the phenanthrene degradation of Sphingobium sp. strain P2. Biochem. Biophys. Res. Commun. 301 (2), 350-357, 2003; Wang, Y. and Lau, PC, Sequence and expression of an isocitrate dehydrogenase encoding gene from a polycyclic aromatic hydrocarbon oxidizer, Sphingomonas yanoikuyae Bl. Gene 168 (1), 15-21, 1996) showed 99% or more homology. Therefore, the 14DN61 strain was identified as the genus Sphingomonas. The 14DN61 strain was also confirmed to assimilate 2,6-dimernaphthalene, anthracene, phenanthrene, and £ -xylene.
〔実施例 2〕 遺伝子操作実験  [Example 2] Gene manipulation experiment
プラスミ ドの調製、 制限酵素処理、 ライゲーシヨン反応、 形質転換などの通常 の遺伝子操作実験は、 Sambrookらの Molecular Cloning (Sambrook, J., Fritsch, Routine genetic engineering experiments such as plasmid preparation, restriction enzyme treatment, ligation reaction, transformation, etc. are performed by Sambrook et al. In Molecular Cloning (Sambrook, J., Fritsch,
E. F. , and Maniatis, T.， 1989， "Molecular cloning -a laboratory manual" ， 2nd edition, または、 Sambrook, J.', Russell, D. W. , 2001， "Molecular cloning 一 a laboratory manual", Third edition, Cold Spring Harbor Laboratory Press,EF, and Maniatis, T., 1989, "Molecular cloning -a laboratory manual", 2nd edition, or Sambrook, J. ', Russell, DW, 2001, "Molecular cloning a laboratory manual", Third edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N. Y. ) に示された方法、 または、 試薬のプロトコールに示 された方法により行った。 Cold Spring Harbor, N. Y.) or the method indicated in the reagent protocol.
〔実施例 3〕 Sphingomonas属 14DN61株からの染色体 DNAの調製 実施例 1で取得した Sphingomonas属 14DN61株を 300 mlの Tryptic Soy Broth (TSB)培地 （Difco) で 30°C、 2日間培養した。 菌体を集菌後、 STE緩衝液 （100 mM NaCl, 10 mM Tris · HC1, 1 mM EDTA, pH 8. 0 ) で二回洗浄し、 68°Cで 15分間熱 処理をした後、 5 mg/ml のリゾチ一ム （Sigma) と 100 i g/mlの RNAase (Sigma) を含む I液 （50 mM グルコース、 25 mM Tris · HC1， 10 mM EDTA, pH 8. 0)に懸濁 した。 37°Cで一時間ィンキュベートした後、 10 mg/mlになるように Protenase K (Sigma)を加え、 37°Cで 10分間ィンキュベートした。 さらに最終濃度が 1% にな るように N- Lauroylsarcosin · Naを添加し、 転倒混和により穏やかに完全に混合 した後 37°Cで 3時間ィンキュベートした。 さらにフエノール/クロロホルム抽出 を数回行った後、 2 倍量のエタノールをゆつく りと添加しながら、 析出してきた 染色体 DNAをガラス棒で巻きつけ、 70% エタノールでリンスした後、 2 ml の TE 緩衝液 （10 mM Tris · HC1， 1 mM EDTA, pH 8. 0) に溶解して、 染色体 DNA溶液と した。 ' [Example 3] Preparation of chromosomal DNA from Sphingomonas sp. Strain 14DN61 The Sphingomonas sp. Strain 14DN61 obtained in Example 1 was cultured in 300 ml of Tryptic Soy Broth (TSB) medium (Difco) at 30 ° C. for 2 days. After collecting the cells, the cells were washed twice with STE buffer (100 mM NaCl, 10 mM Tris · HC1, 1 mM EDTA, pH 8.0), heat-treated at 68 ° C for 15 minutes, then 5 mg It was suspended in solution I (50 mM glucose, 25 mM Tris · HC1, 10 mM EDTA, pH 8.0) containing / ml lysozyme (Sigma) and 100 ig / ml RNAase (Sigma). After incubating at 37 ° C for 1 hour, Protenase K (Sigma) was added to 10 mg / ml, and incubated at 37 ° C for 10 minutes. Furthermore, N-Lauroylsarcosin · Na was added so that the final concentration would be 1%, and the mixture was gently mixed thoroughly by inversion and then incubated at 37 ° C for 3 hours. After several phenol / chloroform extractions, slowly add 2 volumes of ethanol, wind up the precipitated chromosomal DNA with a glass rod, rinse with 70% ethanol, and then add 2 ml of TE. It was dissolved in a buffer solution (10 mM Tris · HC1, 1 mM EDTA, pH 8.0) to obtain a chromosomal DNA solution. '
〔実施例 4〕 コスミ ドライブラリーの作製  [Example 4] Preparation of cosmid library
Sphingomonas属 14DN61株の染色体 DNAを約 30— 42 kbの長さの DNA断片が回 収できるように^ 3AIによって部分的に切断し、フォスファターゼ（CIAP、TAKARA 社製） 処理した。 DNAの Sau3AIによる切断パターンの確認は TAEァガロースゲル を用いた電気泳動で行った。 スミドベクターとして SuperCos 1 (Stratagene) を用い、 ^ I消化した後、 フォスファターゼ処理を行った。 さらに、 このべクタ 一 DNAを ^ HIで消化した後、上記の染色体 DNAの Sau3AI部分分解物とのライゲ ーシヨン反応 （Ligation High T0Y0B0社製） を行った。 次に、 in vitroパッケ一 ジング（LAMBDA INN， Wako社製）を行った後、大腸菌 (Escherichia coli) XLl- Blue MR株への感染を行った。 感染した大腸菌はアンピシリンを含む LBプレートによ つて選択し、 約 1， 000個の大腸菌クローンを用意した。  The chromosomal DNA of Sphingomonas genus 14DN61 was partially cleaved with 3AI so that a DNA fragment of about 30-42 kb in length could be collected and treated with phosphatase (CIAP, manufactured by TAKARA). The cleavage pattern of DNA by Sau3AI was confirmed by electrophoresis using a TAE agarose gel. SuperCos 1 (Stratagene) was used as a smid vector, digested with ^ I, and then treated with phosphatase. Furthermore, this vector DNA was digested with ^ HI, and then subjected to a ligation reaction (Ligation High T0Y0B0) with the Sau3AI partial degradation product of the above chromosomal DNA. Next, after in vitro packaging (LAMBDA INN, manufactured by Wako), infection with Escherichia coli XLl-Blue MR strain was performed. Infected E. coli were selected by LB plates containing ampicillin, and about 1,000 E. coli clones were prepared.
〔実施例 5〕 芳香族アルデヒドデヒ ドロゲナーゼ遺伝子の単離  [Example 5] Isolation of aromatic aldehyde dehydrogenase gene
Sphingomonas属 14DN61株は 1, 4-ジメチルナフタレンをカテコールまで分解し た後、 カテコールジォキシゲナーゼによって環解裂すると予想した。 目的とする 遺伝子は力テコ^"ルジォキシゲナーゼ遺伝子の周辺にある可能性が考えられる。 カテコールジォキシゲナーゼによって分解されたカテコールは黄色になるため、 容易にスク リーニングできることから.、 まずこの遺伝子の単離を試みた。 LBプレ 一ト上にある約 1， 000個の大腸菌クローンのうち数百個を用いて、 50 mMのカテ コールの水溶液をスプレーし、 37°Cにて 30分間を置いた。 その結果、 一つのクロ ーン （pMA13 - 10) が黄色になった。 しかしながら、 pMA13- 10は 1，4 -ジヒ ドロキシ メチルナフタレンの変換能を持っていなかつたため、 pMA13 - 10と重なるクローン (pMA9-54)をライプラリーから新たにコロニーハイブリダィゼーション法で単離 した。 Sphingomonas sp. Strain 14DN61 was expected to cleave 1,4-dimethylnaphthalene to catechol and then cleave it with catechol dioxygenase. The target gene may be in the vicinity of the force lever gene. The catechol degraded by catechol dioxygenase turns yellow, Because of the ease of screening, we first attempted to isolate this gene. A few hundred out of about 1,000 E. coli clones on the LB plate were used to spray an aqueous solution of 50 mM catechol and left at 37 ° C for 30 minutes. As a result, one clone (pMA13-10) turned yellow. However, since pMA13-10 did not have the ability to convert 1,4-dihydroxymethylnaphthalene, a clone overlapping pMA13-10 (pMA9-54) was newly isolated from the library by colony hybridization.
コスミ ド pMA9- 54を含む大腸菌 JM109を 3 mlのアンピシリン 100 μ g/mlを含 む LB培地 （1 %トリプトン、 0. 5% イース トエキス、 0. 5% NaCl ) で 30°Cにて 12 時間培養を行った後、 菌体を遠心操作により回収後、 アンピシリン 100 g/ml 0. 2% (w/v) グルコース、 0. 01 % ( /v) チアミン、 1 mMの IPTGを含む 3 ml の M9培地 （Na2HP04， 6 g/L ; KH2P04, 3 g/L ; NaCl, 0. 5 g/L ; NH4C1， 1 g/L ; MgS04, 1 mM ; CaCl2， 0. 1 mM ; FeS04, 0. 01 mM) で菌体を溶かした。 この菌液にさらに 1 , 4- ジヒ ドロキシメチルナフタレンの 100 raM DMS0溶液を最終濃度 0. 1 mMとなるよう に添加し、 30PCで ·2日間共培養を行った。 この培養液 100 lに対し酢酸ェチル 150 μ 1、 3Ν塩酸 10 μ 1 を加えボルテックス攪拌を行い、 15, 000 rpmで 5分間 遠心し、上清の酢酸ェチル相を回収後乾固した。 次に、 トリメチルシリル化剤 （ジ 一エルサイエンス（株）） でトリメチルシリル化してガスクロマトグラフィ一マス スぺク トル （GC-MS) 用サンプルとした。 GC- MSは島津の model QP5050A, カラム は J&W Sc i ent ifi c社製の DB- 5 column (30 x 0. 25mm) を使用した。 カラム温度 は最初 50°Cで 5分間を保った後、 300°Cまで 10°Cノ分の速度で昇温し、 300°Cで 4 分間を保った ( 1サンプルにっき計 34 分の測定条件）。 注入温度と検出温度は 300°Cであった。分析結果を図 3に示す。 1，4 -ジヒ ドロキシメチルナフタレンの変 換産物として、保持時間 25. 1分のピークが検出され、その分子量は 346であった。 この分子量から計算して 4 -ヒ ドロキシメチ^^- 1 -ナフトェ酸と推定した。 この化 合物の詳細な同定は 〔実施例 6〕 で述べる。 以上の結果から、 クローン （pMA9- 54) が有するコスミ ド PMA9-54内には、 1，4-ジ七 ドロキシメチルナフタレンを 4 -ヒ ド 口キシメチル- 1 -ナフトェ酸に変換するデヒ ドロゲナーゼ遺伝子がコードされて いると考えられた。 〔実施例 6〕 1， 4-ジヒドロキシメチルナフタレン変換遺伝子のサブクローニング とシークェンス解析 Escherichia coli JM109 containing cosmid pMA9-54 in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) containing 3 ml of ampicillin for 12 hours at 30 ° C After culturing, the cells were collected by centrifugation, then ampicillin 100 g / ml 0.2% (w / v) glucose, 0.01% (/ v) thiamine, 3 ml containing 1 mM IPTG M9 medium (Na 2 HP0 4 , 6 g / L; KH 2 P0 4 , 3 g / L; NaCl, 0.5 g / L; NH 4 C1, 1 g / L; MgS0 4 , 1 mM; CaCl 2 , The cells were dissolved in 0.1 mM; FeS0 4 , 0.01 mM). The bacterial solution further 1, 4 dihydric mud xylene was added 100 RAM DMS0 solution of methyl naphthalene to a final concentration of 0. 1 mM, was-2 days co-culture with 30 P C. To 100 l of this culture solution, 150 μl of ethyl acetate and 10 μl of 3% hydrochloric acid were added, vortexed and centrifuged for 5 minutes at 15,000 rpm, and the supernatant ethyl acetate phase was recovered and dried. Next, it was trimethylsilylated with a trimethylsilylating agent (DG Science Co., Ltd.) to prepare a sample for gas chromatography / mass spectrum (GC-MS). The GC-MS used was a Shimadzu model QP5050A, and the column used was a DB-5 column (30 x 0.25 mm) manufactured by J & W Scientific. The column temperature was initially maintained at 50 ° C for 5 minutes, and then increased to 300 ° C at a rate of 10 ° C nomin, and maintained at 300 ° C for 4 minutes. ). The injection temperature and detection temperature were 300 ° C. Figure 3 shows the analysis results. As a conversion product of 1,4-dihydroxymethylnaphthalene, a peak with a retention time of 25.1 minutes was detected, and its molecular weight was 346. Calculated from this molecular weight, it was estimated to be 4-hydroxymethyl ^^-1-naphthoic acid. Detailed identification of this compound is described in [Example 6]. Based on the above results, the clone PMA9-54 has a dehydrogenase that converts 1,4-diseptyloxymethylnaphthalene into 4-hydroxymethyl-1-naphthoic acid. The gene was thought to be encoded. [Example 6] Subcloning and sequence analysis of 1,4-dihydroxymethylnaphthalene conversion gene
PMA9-54を Kpnlで切断し、 その 7 - kb ]^1断片を大腸菌ベクター pBluescript II KS+に挿入し、 プラスミ ド pMA7K5および、 これと揷入断片が逆方向のプラスミ ド pMA lを構築した。 これらの JM109形質転換体の 4 -ヒ ドロキシメチル- 1-ナフ トェ酸生成活性を実施例 5に記載した方法により調べたところ、 両方とも酵素活 性が確認された。 次に、 pMA7K5 と pMA7Kl を様々な制限酵素で切断し、 図 4に示 すように様々なサブクローンを構築した。 これらのクローンの 4-ヒ ドロキシメチ ル -1-ナフトェ酸生成活性を調べた結果、酵素活性を示す最小断片を持つプラスミ ドは pNB2. 18Nであった。 pNB2. 18Nは 7- kb Kpnl断片中の 2. 18- kb Notト BamHI断 片を pBluescript II SK+の Notl-BamHI部位に挿入して作製したプラスミドであ る。  PMA9-54 was cleaved with Kpnl, and the 7-kb] ^ 1 fragment was inserted into the E. coli vector pBluescript II KS + to construct plasmid pMA7K5 and plasmid pMAl in which the inserted fragment was reversed. When the 4-hydroxymethyl-1-naphthoic acid producing activity of these JM109 transformants was examined by the method described in Example 5, both were confirmed to have enzyme activity. Next, pMA7K5 and pMA7Kl were cleaved with various restriction enzymes, and various subclones were constructed as shown in FIG. As a result of examining the 4-hydroxymethyl-1-naphthoic acid producing activity of these clones, the plasmid having the smallest fragment showing the enzyme activity was pNB2.18N. pNB2.18N is a plasmid prepared by inserting the 2.18-kb Not BamHI fragment in the 7-kb Kpnl fragment into the Notl-BamHI site of pBluescript II SK +.
次に、 7-kb ΚβηΙ断片のシークェンス解析を行った。シークェンス反応は Takara Next, the sequence analysis of the 7-kb ΚβηΙ fragment was performed. The sequence response is Takara
PGR Thermal Cycler MP (宝酒造社製)、 BigDye Terminator Cycle Sequencing Ready Reaction Kit Ver. 2. 0 (Appl ied Biosystems 社製） を用い、 ジメチルスルホキ シド (DMS0) を 5 %添加の条件で、 96°C 5分、 92°C 10秒、 50。C 5秒、 60°CPGR Thermal Cycler MP (Takara Shuzo), BigDye Terminator Cycle Sequencing Ready Reaction Kit Ver. 2.0 (Applied Biosystems), 96 ° C with 5% dimethylsulfoxide (DMS0) 5 minutes, 92 ° C 10 seconds, 50. C 5 seconds, 60 ° C
4分、 25サイクルで行った。反応生成物は Appl ied Biosystems 3700 DNA Analyzer4 minutes, 25 cycles. The reaction product is Applied Biosystems 3700 DNA Analyzer
(Appl ied Biosystems社製）により解析した。また、解析の結果得られた 6, 921 bp の塩基配列を配列番号 1に示す。 遺伝子配列の相同性の解析、 0RFの検索を NCBI ホームページよりそれぞれ BLAST 2. 0 (gapped BLAST) , 0RF finderで行った。(Applied Biosystems). Further, the base sequence of 6,921 bp obtained as a result of the analysis is shown in SEQ ID NO: 1. The homology analysis of gene sequences and the search for 0RF were performed with the BLAST 2.0 (gapped BLAST) and 0RF finder from the NCBI website.
2. 18- kb Notl-BamHI断片の塩基配列からは唯一のオープンリーディングフレーム2. The only open reading frame from the 18-kb Notl-BamHI fragment base sequence
(0RF) しか見つからず、 この 0RFが 1， 4 -ジヒ ドロキシメチルナフタレンから 4 - ヒ ドロキシメチル -1 -ナフトェ酸への変換に関与していると考えられた。この遺伝 子にコ ー ドされた推定ア ミ ノ酸配列はゲノム解析で明らかにされた Spningomonas aromaticivorans plasraid pNLl の/"ノレァヒ ドアヒ ドロケナ1— li(0RF) was found, and this 0RF was thought to be involved in the conversion of 1,4-dihydroxymethylnaphthalene to 4-hydroxymethyl-1-naphthoic acid. The putative amino acid sequence encoded by this gene was revealed by genome analysis of Spningomonas aromaticivorans plasraid pNLl / "Norehi Adahi Drokena 1 — li
(XylC, accession number, T31265)、 Sphingomonas chungbukens is DJ77 (PhnN, access ion number, AF073359— 5)のべンズアルデヒ ドデヒ ドロゲナーゼと 90%の 相同性を示した （以後、 本発明者が取得した この 0RFを phnN、 コードされる酵 素を PhnNと呼称する）。 なお、 データーベース上で公開された上記の二酵素遺伝 子の機能などは解明されていなかった。 また、 PhnNの推定アミノ酸配列はよく研 究されている Pseudomonasputidaの T0Lプラスミ ドの XylBあるいは XylCとの類 似性は認められなかった。 14DN61株の (配列番号 1中の塩基番号 3222— 4721) の塩基配列及びコードされるアミノ酸配列をそれぞれ配列番号 2及び配列番号 3 に示す。データーベース上で PhnNの推定アミノ酸配列とある程度の相同性を持ち ながら機能も明らかにされてレ、る酵素は Xanthobacter autotrophicus GJ10のク ロロァセタノレデヒ ドヒ ドロゲナーゼ （AldB) (Bergeron, H.， Labbe, D. ， Turmel, C. and Lau, P. C. , し丄 oning, sequence and expression of a l inear-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase - encoding genes in Xanthobacter autotrophicusGJlO. Gene 207 (1)， 9-18， 1998) 、 Cytophaga sp. KUC-1 のァノレデヒ ドデヒ ドロゲナーゼ (Alddh) (Yamanaka, Y. , Kazuoka, Τ. , Yoshida, Μ. ， Yamanaka, Κ. , Oikawa, Τ. and Soda, Κ. ， Thermostable aldehyde dehydrogenase from psychrophile, Cytooha_ga sp. KUC-1： enzymologi cal characteri stics and functional properti es. Biochera. Biophys. Res. Commun. 298 (5)， 632-637， 2002)、 Al cal igenes eutrophaのァセタルデヒ ドヒ ドロゲナー セ ( AocD ) ( Pri ef ert, H.， Kruger, N.， j endrossek, D.， Scnmidt, B. , and Steinbuchel, A. , Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcal igenes eutrophus. J. Bacteriol. 174 (3) , 899-907, 1992) などのアルデヒドデヒ ドロゲナーゼが挙 げられる。 また、 プロパン代謝に関わるアルコールデヒドロゲナーゼ （Adh3) と の相同性も見つ力つた (Kotani, T.， Yamamoto, T.， Yurimoto, H. , Sakai, Y. and Kato, N. , Propane monooxygenase and NAD+ - dependent secondary alcohol dehydrogenase in'propane metabol ism by Gordonia sp. stra in TY-5. J. Bacreriol . 185 (24)， 7120-7128, 2003)。 これらの酵素は捕酵素として NAD+に依存する。 PhnN とこれらの酵素のァミノ酸配列を DNA解析ソフ ト GENETYX-MACでァライメントし た結果、 NAD+結合部位と思われる PhnNのァミノ酸配列 216、 218、 222番の Gly、 活性中心と思われる 260番の Glu、 294番の Cys、 が保存されていた。 以上の結果 から今回単離された hnN酵素遺伝子は NAD+依存性デヒドロゲナーゼであること が示唆された。 また、 SphinRomonas chungbukensis DJ77の acetaldehyde dehydrogenase遺伝 子 ( hnl ) 、 4 - hydroxy - 2 - oxovalerate aldorase 遺伝子 ( phnj ) 、 及ぴ 4-oxalosrotonate decarboxylase遺伝子 （E_hnK) (Hwang, S. , Kim, S. J. , Kim, C. K.， Kim, Y.， Kim, S. J. and Kim, Y. C.， The phnl.T genes encoding acetaldehyde dehydrogenase (acylating) and 4-hydroxy-2~oxovalerate aldolase in Pseudomonas sp. DJ77 and their evolutionary impl ications. Biochem. Biophys. Res. Commun. 256 (3) , 469-473， 1999) とそれぞれ 77%、 90%、 80%の相同性を持つ配列も見つかった （我々の遺伝子も同様にそれぞれ phnl, phnj及び hnKと名づけた）。 (XylC, accession number, T31265), Sphingomonas chungbukens is DJ77 (PhnN, access ion number, AF073359—5) showed a 90% homology with the Bende aldehyde dehydrogenase (hereinafter this inventor acquired this 0RF). phnN, the encoded enzyme is called PhnN). The above-mentioned two-enzyme genetics published on the database The function of the child has not been elucidated. In addition, the deduced amino acid sequence of PhnN was not similar to the well-studied T0L plasmid of Pseudomonasputida with XylB or XylC. The nucleotide sequence of the 14DN61 strain (base numbers 3222 to 4721 in SEQ ID NO: 1) and the encoded amino acid sequence are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The enzyme, which has a certain degree of homology with the deduced amino acid sequence of PhnN in the database and whose function has been clarified, is the chloroaceta nodehyde hydrogenase (AldB) (Bergeron, H., Xanthobacter autotrophicus GJ10). Labbe, D., Turmel, C. and Lau, PC, Shion oning, sequence and expression of al inear-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase-encoding genes in Xanthobacter autotrophicusGJlO. Gene 207 (1), 9-18, 1998), Cytophaga sp. KUC-1 Anoledehydehydrogenase (Alddh) (Yamanaka, Y., Kazuoka, Τ., Yoshida, Μ., Yamanaka, Κ., Oikawa, Τ. And Soda, Κ., Thermostable aldehyde dehydrogenase) from psychrophile, Cytooha_ga sp. KUC-1: enzymologi cal characteri stics and functional properti es. Biochera. Biophys. Res. Commun. 298 (5), 632-637, 2002), Al cal igenes eutropha, ) (Pri ef ert, H., Kruger, N., j endrossek, D., Scnmidt, B., and Steinbuchel, A., Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcal igenes eutrophus. J. Bacteriol. 174 (3), 899-907, Aldehyde dehydrogenase such as 1992). In addition, homology with alcohol dehydrogenase (Adh3) involved in propane metabolism was also observed (Kotani, T., Yamamoto, T., Yurimoto, H., Sakai, Y. and Kato, N., Propane monooxygenase and NAD + -dependent secondary alcohol dehydrogenase in propane metabol ism by Gordonia sp. stra in TY-5. J. Bacreriol. 185 (24), 7120-7128, 2003). These enzymes depend on NAD + as scavengers. PhnN and the amino acid sequences of these enzymes were aligned with DNA analysis software GENETYX-MAC. As a result, PhnN's amino acid sequences 216, 218 and 222 were considered to be NAD + binding sites. Glu, No. 294 Cys, was preserved. These results suggest that the hnN enzyme gene isolated this time is a NAD + -dependent dehydrogenase. In addition, the acetaldehyde dehydrogenase gene (hnl), 4-hydroxy-2-oxovalerate aldorase gene (phnj), and 4-oxalosrotonate decarboxylase gene (E_hnK) (Hwang, S., Kim, SJ, Kim, CK) of SphinRomonas chungbukensis DJ77 , Kim, Y., Kim, SJ and Kim, YC, The phnl.T genes encoding acetaldehyde dehydrogenase (acylating) and 4-hydroxy-2 ~ oxovalerate aldolase in Pseudomonas sp. DJ77 and their evolutionary impl ications. Biochem. Biophys. Res Commun. 256 (3), 469-473, 1999) and 77%, 90%, and 80% homologous sequences were also found respectively (our genes were also named phnl, phnj and hnK, respectively) .
〔実施例 7〕 4 -ヒ ドロキシメチル- 1-ナフトェ酸の構造決定  [Example 7] Structure determination of 4-hydroxymethyl-1-naphthoic acid
4 -ヒ ドロキシメチル- 1 -ナフ トェ酸の構造は以下のように決定した。 pNB2. 18N を含む大腸菌 JM109を 1 Lのアンピシリン 100 g/mlを含む LB培地で 30°Cにて 12時間培養を行った後、菌体を遠心操作により回収後、アンピシリン 100 g/ml、 0. 2% (w/v) グルコース、 0. 01% (w/v) チアミン、 1 mMの IPTGを含む M9培地 1 Lで菌体を溶かした。 この菌液にさらに 1， 4-ジヒ ドロキシメチルナフタレンの 100 mM DMS0溶液を最終濃度 0. 1 mMとなるように添加し、 30°Cで 2日間共培養を 行った。 この培養液を塩酸で PH3にした後、 1 Lの酢酸ェチルで二回抽出を行つ た。 酢酸ェチル相をエバポレーターで乾固の状態にし、 丽 R分析を行った。 iH NMR および 13C NMRの測定結果は以下の通り。 ¾ 丽 R (測定機器： Varian IN0VA 750、 750MHz 溶媒：重メタノール、 外部標準としてテトラメチルシランを 0 ppmとし た。） δ 5. l l (s)、 δ 7. 56 (t， J=7. 5Hz)、 7. 58 (t， J=7. 5Hz)、 δ 7. 62 (d， J=7. 5Hz)、 δ 8. 11 (d, J=7. 5Hz)、 δ 8. 15 (d， J=7. 5Hz) , δ 8. 92 (d, J=7. 5Hz) 0 13C蘭 R (測 定機器： Varian IN0VA 500、 125MHz、 溶媒：重メタノール、 外部標準としてテト ラメチルシランを O ppmとした。） δ 63. 2, δ 124. 2、 δ 124. 8、 5 127. 3、 δ 127. 5、 δ 128. 1、 5 128. 8、 δ 130. 8、 δ 132. 7、 δ 132. 8、 δ 143. 4、 δ 171. 1。 また、 本化 合物をトリメチルシリル化後、 GC-MS にて分子量を測定したところ分子量 346を 検出した。 以上、 匪 R、 13C NMR, GC-MS の測定結果より本化合物が 4 -ヒ ドロキ シメチル- 1 -ナフトェ酸 （4- hydroxymethyl - 1- naphthoic aci d) であることが明ら かとなつた。 〔実施例 8〕 pNB2. 18Nを含む大腸菌 JM109形質転換体の細胞抽出物を用いた酵 素活性測定 The structure of 4-hydroxymethyl-1-naphthoic acid was determined as follows. pNB2. After 12 hours incubation at 30 ° C in LB medium containing ampicillin 100 g / ml of E. coli JM109 1 L containing 18N, after recovered by centrifugation of the bacteria, ampicillin 100 g / m l, The cells were dissolved in 1 L of M9 medium containing 0.2% (w / v) glucose, 0.01% (w / v) thiamine and 1 mM IPTG. To this bacterial solution, a 100 mM DMS0 solution of 1,4-dihydroxymethylnaphthalene was added to a final concentration of 0.1 mM, and co-cultured at 30 ° C for 2 days. After the culture solution P H3 with hydrochloric acid, having conducted twice extracted with acetic Echiru of 1 L. The ethyl acetate phase was brought to dryness with an evaporator and subjected to 丽 R analysis. The measurement results of iH NMR and 13 C NMR are as follows. ¾ 丽 R (Measuring instrument: Varian IN0VA 750, 750MHz Solvent: Heavy methanol, Tetramethylsilane was set to 0 ppm as external standard) δ 5. ll (s), δ 7. 56 (t, J = 7.5 Hz ), 7.58 (t, J = 7.5 Hz), δ 7.62 (d, J = 7.5 Hz), δ 8. 11 (d, J = 7.5 Hz), δ 8. 15 (d, J = 7.5 Hz), δ 8. 92 (d, J = 7.5 Hz) 0 13 C orchid R (measuring instrument: Varian IN0VA 500, 125 MHz, solvent: deuterated methanol, tetramethylsilane as external standard with O ppm) Δ 63.2, δ 124.2, δ 124.8, 5 127.3, δ 127.5, δ 128.1, 5 128.8, δ 130.8, δ 132.7, δ 132 8, δ 143.4, δ 171.1. In addition, after the trimethylsilylation of this compound, the molecular weight was measured by GC-MS, and a molecular weight of 346 was detected. As described above, it was revealed from the measurement results of 匪 R, 13 C NMR, and GC-MS that this compound was 4-hydroxymethyl-1-naphthoic acid (4-hydroxymethyl-1-naphthoic acid). [Example 8] Measurement of enzyme activity using cell extract of E. coli JM109 transformant containing pNB2.18N
ρΝΒ2· 18Nを含む大腸菌 JM109形質転換体を 3 mlのアンピシリン 100 μ g/ralを 含む LB培地で 30°Cにて 12時間培養を行った後、 200 ml のアンピシリン 100 μ g/mlを含む LB培地に植菌し、 37°Cにて 0D (600 nm) が 0. 5になるように培養し た。 次に、 I mMの IPTGを添加し、 4時間を誘導した。 菌体を遠心操作により回収 後、 10 ml,の 50 mMの Tris- HC1 (pH 7. 5) に溶かした。 菌体は大岳製作所のフレ ンチプレス （l，500 MPa) で二回破壊し、 15000 rpmで遠心後上清を細胞抽出物と した。 細胞抽出物のたんぱく質の濃度測定は Bradford (Bradford, M. M. , A rapid and sens itive method for the quantitation of microgram quantities of protein utilizing the priciple of protein-dye binding. Anal. Biochem. 72， 248-254, 1976) の方法にしたがって行った。  E. coli JM109 transformant containing ρΝΒ2 · 18N was cultured at 30 ° C for 12 hours in LB medium containing 3 ml of ampicillin 100 μg / ral, and then LB containing 200 ml of ampicillin 100 μg / ml. The medium was inoculated and cultured at 37 ° C so that 0D (600 nm) was 0.5. Next, I mM IPTG was added and induced for 4 hours. The cells were collected by centrifugation and then dissolved in 10 ml of 50 mM Tris-HC1 (pH 7.5). The cells were disrupted twice with a Otake Seisakusho punch press (l, 500 MPa), centrifuged at 15000 rpm, and the supernatant was used as the cell extract. Protein concentrations in cell extracts are measured by Bradford (Bradford, MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the priciple of protein-dye binding. Anal. Biochem. 72, 248-254, 1976) It went according to the method of.
アルデヒ ドデヒドロゲナーゼ活性の測定は 1 ml の 50 raM Tris-HCl (pH 7. 5) に最終濃度 50 μ Mの NAD+、 1 mgの pNB2. 18Nの大腸菌 JM109形質転換体の細胞抽 出物、 基質として 50 μ Μの 2-ナフタルデヒドを添加した。 30°Cにて 1時間反応 させた後、 〔実施例 5〕 の方法に従って酢酸ェチルで抽出し、 GC-MSで反応産物を 解析した。 その結果、 2-ナフタルデヒ ドからの反応産物として 2-ナフトェ酸およ ぴ 2-ヒドロキシメチルナフタレンが検出された。同様の実験で補酵素の NAD+を添 加しない場合、 約 20%の 2-ナフタエ酸しか検出されなかった。 このことは PhnN が NAD+に依存していることを示唆し、 〔実施例 6〕 で記述した PhnNの推定アミノ 酸配列に NAD+結合部位が保存されていることと一致した。 また、 2-ヒドロキシメ チルナフタレンが検出されたことはアルコールデヒ ドロゲナーゼの逆方向の酵素 活性によるものと考えられる。  Aldehyde dehydrogenase activity was measured using 1 ml of 50 raM Tris-HCl (pH 7.5) in a final concentration of 50 μM NAD +, 1 mg of pNB2.18N E. coli JM109 transformant as a cell extract and substrate. 50 μΜ 2-naphthalaldehyde was added. After reacting at 30 ° C for 1 hour, extraction was performed with ethyl acetate according to the method of [Example 5], and the reaction product was analyzed by GC-MS. As a result, 2-naphthoic acid and 2-hydroxymethylnaphthalene were detected as reaction products from 2-naphthalaldehyde. In the same experiment, when no coenzyme NAD + was added, only about 20% 2-naphthoic acid was detected. This suggests that PhnN is dependent on NAD +, which is consistent with the conserved NAD + binding site in the predicted amino acid sequence of PhnN described in [Example 6]. The detection of 2-hydroxymethylnaphthalene is thought to be due to the enzyme activity in the reverse direction of alcohol dehydrogenase.
アルコールデヒ ドロゲナーゼ活性の測定は 1 ml のグリシン/ NaOHバッファー (pH 9. 4) に最終濃度 50 μ Mの NAD+、 1 mgの ρΝΒ2· 18Nの大腸菌 JM109形質転換 体の細胞抽出物、 基質として 50 の 2 -ヒドロキシメチルナフタレンを添加し た。 30°Cにて 1時間反応させた後、 '〔実施例 5〕 の方法に従って酢酸ェチルで抽出 し、 GC- MS で反応産物を解析した。 その結果、 2-ヒ ドロキシメチルナフタレンか らの反応産物として 2 -ナフトェ酸が検出された。 以上の実験結果により、 PhnN を発現した大腸菌はアルコールデヒ ドロゲナーゼとアルデヒ ドデヒ ドロゲナーゼ 両方の酵素活性を持つことが証明された。 Alcohol dehydrogenase activity was measured using 1 ml glycine / NaOH buffer (pH 9.4) in a final concentration of 50 μM NAD +, 1 mg ρΝΒ2 · 18N E. coli JM109 transformant cell extract, as substrate 50 2-hydroxymethylnaphthalene was added. After reacting at 30 ° C for 1 hour, extraction with ethyl acetate was performed according to the method of [Example 5], and the reaction product was analyzed by GC-MS. As a result, 2-naphthoic acid was detected as a reaction product from 2-hydroxymethylnaphthalene. Based on the above experimental results, PhnN It was proved that Escherichia coli expressing the enzyme activity of both alcohol dehydrogenase and aldehyde dehydrogenase.
〔実施例 9〕 PhnNタンパク質の精製  [Example 9] Purification of PhnN protein
Eimil遺伝子を含む 1. 8 kb Bgin- Hindlll断片を pTrcHisAの Bglll-HindlHサ イ トに揷入して、 プラスミ ド pTrcNlを作製した。 pTrcNl を含む大腸菌 JM109形 質転換体の細胞抽出物は〔実施例 8〕 に従って調製した。細胞抽出物を Amersharm Biosciences社の HisTrapTM HPカラムで精製した。 吸着バッファ一は 20 mM リン 酸、 30 mMィミダゾール、 0. 5 M NaCl、 溶出バッファ一は 20 mM リン酸、 500 mM ィミダゾール、 0. 5 M NaClを用いた。 溶出したサンプルは 20 mM Tris-HCl (pH8) を用レヽて透析し、 Amersharm Biosciences社の Mono Q 5/5 HRで更こ精製した。 吸着バッファーは 20 mM Tris-HCl (pH8)、 溶出バッファ一は吸着バッファーに 1 M NaClを添加したものを使用した。溶出したサンプルは SDS- PAGEで確認した結果、 ほぼ単一パンドであった。  A 1.8 pkb Bgin-Hindlll fragment containing the Eimil gene was inserted into the Bglll-HindlH site of pTrcHisA to produce plasmid pTrcNl. A cell extract of E. coli JM109 transformant containing pTrcNl was prepared according to [Example 8]. The cell extract was purified on Amersharm Biosciences HisTrap ™ HP column. The adsorption buffer was 20 mM phosphate, 30 mM imidazole, 0.5 M NaCl, and the elution buffer was 20 mM phosphate, 500 mM imidazole, 0.5 M NaCl. The eluted sample was dialyzed against 20 mM Tris-HCl (pH 8) and further purified with Mono Q 5/5 HR manufactured by Amersharm Biosciences. The adsorption buffer was 20 mM Tris-HCl (pH 8), and the elution buffer was 1 M NaCl added to the adsorption buffer. As a result of SDS-PAGE confirming the eluted sample, it was almost a single panda.
精製酵素を用いたアルコールデヒ ドロゲナーゼ活性の測定は、 2-ヒドロキシメ チルナフタレンを基質として 〔実施例 8〕 の方法に従って行った。 その結果、 酵 素活性は検出されなかった。 また、 精製酵素を用いたアルデヒ ドデヒ ドロゲナー ゼ活性の測定は、 2 -ナフタルデヒ ドを基質として 〔実施例 8〕 の方法に従って行 つた。 その結果、 酵素活性は検出された。 以上の結果から、 PhnNはアルデヒ ドデ ヒ ドロゲナーゼであること、および 遺伝子を発現した大腸菌形質転換体によ る芳香族アルコールからカルボン酸への変換は、 大腸菌の持つアルコールデヒド ロゲナーゼと PhnN のアルデヒ ドデヒ ドロゲナーゼの共同作用で行われたことが 明かとなった。  The alcohol dehydrogenase activity was measured using the purified enzyme according to the method of [Example 8] using 2-hydroxymethylnaphthalene as a substrate. As a result, no enzyme activity was detected. In addition, aldehyde dehydrogenase activity using a purified enzyme was measured according to the method of [Example 8] using 2-naphthalaldehyde as a substrate. As a result, enzyme activity was detected. Based on the above results, PhnN is an aldehyde dehydrogenase, and the conversion of aromatic alcohols to carboxylic acids by E. coli transformants expressing the gene indicates that the alcohol dehydrogenase of Ph. It was revealed that this was done by the joint action of drogenase.
〔実施例 1 0〕 アルコールおよびアルデヒド芳香族化合物の変換反応  [Example 10] Conversion reaction of alcohol and aldehyde aromatic compound
pNB2. 18Nを含む大腸菌 JM109を 3 mlのアンピシリン 100 g/mlを含む LB培 地で 30°Cにて 12時間培養を行った後、 菌体を遠心操作により回収後、 アンピシ リン 100 ^ g/ml, 0. 2% (w/v) グルコース、 0. 01% (w/v) チアミン、 1 mMの IPTG を含む M9培地 3 mlで菌体を溶かした。 この菌体液を基質の数と同じよう 用意 した。 菌体液にそれぞれの基質 100 mM DMS0溶液を基質の最終濃度が 0. 5 mMにな るように添加し栓つき試験管に移し、 蓋を密閉して 30°Cで二日間培養を行った。 この培養液それぞれ 100 μ 1を酢酸ェチルで実施例 5に記載した方法で抽出し、 酢酸ェチル相を GC/MSにて実施例 5に記載の条件で分析を行った。 市販の標準品 とのマスパターンおよび保持時間の比較から、 2-ヒ ドロキシメチル- 6-メチルナフ タレンから 6 -メチル -2-ナフトェ酸、 1- 2 -ヒ ドロキシメチルナフタレンからそ れぞれ 1-， 2 -ナフトェ酸、 2 -， 3 -， 4 -メチルベンジルアルコールからそれぞれ 2-, 3 -， 4 -メチル安息香酸、 ベンジルアルコールから安息香酸、 キシレン- α - α ' - ジオールから 4 -ヒ ドロキシメチル安息香酸、 2-, 3 -， 4 -ヒ ドロキシベンジルアル コールからそれぞれ 2 -， 3 -， 4-ヒ ドロキシ安息香酸、. 2-， 3 -， 4 -メ トキシベンジ ルアルコールからそれぞれ 2 -， 3-, 4 -メ トキシ安息香酸、 4 -ェチルベンジルアル コールから 4 -ェチル安息香酸、 4 -ィソプロピルべンジルアルコールから 4- ソプ 口ピル安息香酸、 3-クロ口べンジルアルコールから 3-クロ口安息香酸、 3-フエ二 ルプロピルアルコールから 3 -フエニルプロピオン酸への変換反応が確認された。 ただし、 最初の変換産物の 6-メチル -2-ナフトェ酸のみ市販品が無かったため、 Bramucc i, M.ら (背京技 と同じ文献： Bramucc i, M.， Singh, M.， and Nagaraj an, V.， Biotransformat i on οι — xyl ene and 2, o-dimethylnaphthal ene by xyl ene monooxygenase cl oned from a ^phingomonas i so丄 ate. Ap l . Microbiol . Biotechnol . 59 (6) , 679-684， 2002) が報告したもののマススぺク トルと比較し て同定した。 pNB2. E. coli JM109 containing 18N was cultured for 12 hours at 30 ° C in LB medium containing 3 ml of ampicillin 100 g / ml, and the cells were collected by centrifugation, and then ampicillin 100 ^ g / The cells were dissolved in 3 ml of M9 medium containing ml, 0.2% (w / v) glucose, 0.01% (w / v) thiamine and 1 mM IPTG. This bacterial cell solution was prepared as many as the number of substrates. Each substrate 100 mM DMS0 solution was added to the bacterial cell solution so that the final concentration of the substrate was 0.5 mM, transferred to a test tube with a stopper, and the lid was sealed and cultured at 30 ° C for 2 days. 100 μl of each culture was extracted with ethyl acetate by the method described in Example 5, and the ethyl acetate phase was analyzed by GC / MS under the conditions described in Example 5. From the comparison of mass patterns and retention times with commercially available standards, 2-hydroxymethyl-6-methylnaphthalene, 6-methyl-2-naphthoic acid, and 1-2-hydroxymethylnaphthalene, 1- 2-, 3-naphthoic acid, 2-, 3-, 4-methylbenzyl alcohol, 2-, 3-, 4-methylbenzoic acid, benzyl alcohol, benzoic acid, xylene-α-α'-diol, 4-hydroxymethyl Benzoic acid, 2-, 3-, 4-hydroxybenzyl alcohol 2-, 3-, 4-hydroxybenzoic acid, 2-, 3-, 4-methoxybenzyl 2-, respectively. 3-, 4-Methoxybenzoic acid, 4-Ethylbenzyl alcohol to 4-Ethylbenzoic acid, 4-Isopropyl Benzyl alcohol to 4-Sop pill benzoic acid, 3-Chloro Benzyl alcohol to 3 -Black mouth benzo , 3 from 3-phenylene Le propyl alcohol - conversion reaction to phenylalanine acid was confirmed. However, because there was no commercial product of only the first conversion product, 6-methyl-2-naphthoic acid, Bramucc i, M. et al. (Same literature as SKYKYO: Bramucc i, M., Singh, M., and Nagaraj an , V., Biotransformat i on οι — xyl ene and 2, o-dimethylnaphthal ene by xyl ene monooxygenase cl oned from a ^ phingomonas i so 丄 ate. Ap l. Microbiol. Biotechnol. 59 (6), 679-684, 2002 ), But compared with the mass spectrum of what was reported.
また、 pNB2. 18Nを含む大腸菌 JM109は、 1-， 2-ナフタルデヒ ドからそれぞれ 1 -， 2-ナフトェ酸、 2- 3 -， 4 -メチノレベンズアルデヒ ドからそれぞれ 2-， 3-, 4-メチ ル安息香酸、 ベンズアルデヒ ドから安息香酸への変換反応も示した。 以上の生成 産物はべクター pBluescr ipt I I SKのみを含む大腸菌 JM109を用いた変換実験で は、 ほとんど検出されなかった。 大腸菌内で合成された PhnN酵素が、 毒性のある 芳香族アルデヒ ド化合物を高率よくカルボン酸に変換できることによって、 元々 大腸菌が有していたアルコールデヒ ドロゲナーゼ活性も高められたと考えられた。 以上の結果から、今回単離された PhnN酵素は基質特異性の広いアルデヒ ドデヒ ド ロゲナーゼ活性を持っていることが明らかとなった。 しかも、 PhnN酵素は大腸菌 等の微生物と相性がよく、 遺伝子を発現した大腸菌の形質転換体はべンジル アルコールのような単環化合物からヒ ドロキシメチルナフタレンのような多環化 合物に至る非常に広い範囲の芳香族化合物をカルボン酸に変換できたことから、 本酵素は新規でかつ産業上有用な酵素だと考えられた。 In addition, E. coli JM109 containing pNB2.18N is 1-, 2-naphthoaldehyde from 1-, 2-naphthoic acid, 2-3-, 4-methinolevene aldehyde from 2-, 3-, 4-, respectively. A conversion reaction from methylbenzoic acid and benzaldehyde to benzoic acid was also shown. These products were hardly detected in the conversion experiment using E. coli JM109 containing only vector pBluescript II SK. The PhnN enzyme synthesized in E. coli was able to convert toxic aromatic aldehyde compounds to carboxylic acids at a high rate, and the alcohol dehydrogenase activity originally possessed by E. coli was thought to be enhanced. From the above results, it was revealed that the PhnN enzyme isolated this time has aldehyde dehydrogenase activity with a wide substrate specificity. Moreover, the PhnN enzyme is compatible with microorganisms such as Escherichia coli, and the transformant of E. coli expressing the gene is a polycyclic compound such as benzyl alcohol from a monocyclic compound such as benzyl alcohol. Since a very wide range of aromatic compounds leading to the compound could be converted to carboxylic acid, this enzyme was considered to be a novel and industrially useful enzyme.
〔実施例 1 1〕 遺伝子操作実験 [Example 1 1] Gene manipulation experiment
以下に説明するプラスミ ドの調製、 制限酵素処理、 ライゲーシヨン反応、 形質 転換などの通常の遺伝子操作実験は、 Sambrookらの Molecular Cloning (Sambrook, J.， ritsch, E. , and Mamatis, T.， 1989， Molecular cloning -a laboratory manual , 2nd edition、または、 Sambrook, J. , Russel l, D. W. , 2001, "Molecular cloning -a laboratory manual , Third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. ) に示された方法、 または、 核実験系に使用し た試薬に添付されたプロ トコールに示された方法により行った。  Routine genetic manipulation experiments such as plasmid preparation, restriction enzyme treatment, ligation reaction, transformation, etc., described below, are described in Sambrook et al., Molecular Cloning (Sambrook, J., ritsch, E., and Mamatis, T., 1989). , Molecular cloning -a laboratory manual, 2nd edition, or Sambrook, J., Russel, DW, 2001, "Molecular cloning -a laboratory manual, Third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) Or the method shown in the protocol attached to the reagent used in the nuclear test system.
〔実施例 1 2〕 Rhodococcus opacus TKN1 株からの染色体 DNAの調製 [Example 1 2] Preparation of chromosomal DNA from Rhodococcus opacus TKN1 strain
目的とする遺伝子群の供給源となった土壌細菌口 ドコッカス · ォパカス (Rhodococcus opacus) TKN14株は、 岩手県釜石市の土壌から、 £-キシレンで優 占化する 2ーキシレン資化細菌として、発明者らが所属する（株）海洋バイオテクノ ロジー研究所により釜石土壌より単離されたものである （特開 2002 - 238551号公 報）。 本 TKN14株は、 16S rDNAや gyrB遺伝子の配列の分析により、 ロドコッカス 属細菌に分類されたものである。 一般に土壌中で、 nrキシレンや £ -キシレンと比 ベて、 2ーキシレンは非常に分解しにくい物質として知られており、 2—キシレン分 解細菌や分解系遺伝子の知見はほとんど存在していなかった。なお、本発明者は、 本 TKN14菌が、 2-キシレンの分解において、そのベンゼン環の直接酸化ではなく、 その側鎖のメチル基の酸化により代謝することを確かめていた。 R. opacus TKN14 株は、 独立行政法人産業技術総合研究所特許生物寄託センターに寄託されている (受託番号： FERM P- 18195、 受託日 ：平成 13年 2月 2日）。 なお、 本菌株は、 当 初 「TKN14株」 ではなく、 「KN14株」 と呼んでいたため、 前記寄託センターにおい ては、 ["Rhodococcus sp. KN - 14」 という名称で寄託されている。 ロドコッカス ' ォパカス (Rhodococcus opacus) TKN14株は、 株式会社海洋バイオテクノロジー からカルチャーコレクション番号 「MBIC 05572」 として入手することができる。 R. opacus TKN14株を 300 ml の Tryptic Soy Broth (TSB) 培地（Difco)で 30°C、 2日間培養した。 菌体を集菌後、 STE緩衝液 （lOO mM NaCl, 10 mM Tris - HCl, 1 mM EDTA, pH 8. 0 ) で二回洗浄し、 68°Cで 15分間熱処理をした後、 5 rag/ml のリゾ チーム （Sigma) と 100 μ g/mlの RNase (Sigma)を含む I液 （50 mM グルコース、 25 mM Tris · HCl, 10 mM EDTA, pH 8. 0)に懸濁した。 37。Cで一時間インキュベー トした後.、 10mg/mlになるように Protenase K (Sigma)を加え、 37。Cで 10分間ィ ンキュベートした。 さらに最終濃度が 1% になるように N-Lauroyl sarcosin . Na を添加し、転倒混和により穏やかに完全に混合した後 37°Cで 3時間ィンキュベー トした。 さらにフエノール/クロ口ホルム抽出を数回行った後、 2倍量のエタノー ルをゆっく りと添加しながら、 析出してきた染色体 DNAをガラス棒で巻きつけ、 70% ェタノールでリンスした後、 2 mlの TE緩衝液 （10 mM Tri s · HCl, 1 mM EDTA, pH 8. 0) に溶解して、 染色体 DNA溶液とした。 Soil Bacteria Molecula, the source of the target genes, Rhodococcus opacus TKN14 is a 2-xylene-utilizing bacterium that is dominated by £ -xylene from soil in Kamaishi City, Iwate Prefecture. It was isolated from Kamaishi soil by the Marine Biotechnology Research Institute, Inc. to which they belong (Publication No. 2002-238551). This TKN14 strain was classified as a Rhodococcus bacterium by analysis of the 16S rDNA and gyrB gene sequences. Generally, 2-xylene is known to be extremely difficult to degrade in soil compared to nr-xylene and £ -xylene, and there was little knowledge of 2-xylene-degrading bacteria and degradation genes. . The present inventor has confirmed that the TKN14 bacteria are metabolized not by direct oxidation of the benzene ring but by oxidation of the methyl group of the side chain in the degradation of 2-xylene. R. opacus TKN14 strain has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Accession number: FERM P-18195, accession date: February 2, 2001). Since this strain was initially called “KN14 strain” instead of “TKN14 strain”, it was deposited under the name [“Rhodococcus sp. KN-14” at the Deposit Center. Rhodococcus opacus TKN14 strain can be obtained from Marine Biotechnology Co., Ltd. under the culture collection number “MBIC 05572”. The R. opacus TKN14 strain was cultured in 300 ml of Tryptic Soy Broth (TSB) medium (Difco) at 30 ° C. for 2 days. After collecting the cells, they were washed twice with STE buffer (lOO mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0), heat-treated at 68 ° C for 15 minutes, and then 5 rag / The suspension was suspended in solution I (50 mM glucose, 25 mM Tris · HCl, 10 mM EDTA, pH 8.0) containing ml lysozyme (Sigma) and 100 μg / ml RNase (Sigma). 37. Incubate for 1 hour at C. Then add Protenase K (Sigma) to 10 mg / ml 37. Incubated at C for 10 minutes. Furthermore, N-Lauroyl sarcosin.Na was added to a final concentration of 1%, and the mixture was gently mixed thoroughly by inversion and then incubated at 37 ° C for 3 hours. After several phenol / chloroform extractions, slowly add 2 volumes of ethanol, wind up the precipitated chromosomal DNA with a glass rod and rinse with 70% ethanol. It was dissolved in ml of TE buffer (10 mM Tris · HCl, 1 mM EDTA, pH 8.0) to obtain a chromosomal DNA solution.
〔実施例 1 3〕 コスミ ドライブラリーの作製 [Example 1 3] Preparation of cosmid library
TKN14株の染色体 DNAを約 30-42 kbの長さの DNA断片が回収できるように Chromosomal DNA of TKN14 strain can be recovered with a DNA fragment of about 30-42 kb in length
Sau3AIによって部分的に切断し、 フォスファターゼ （CIAP、 Takara社製） 処理し た。 DNAの §^3ΑΙによる切断パターンの確認は ΤΑΕァガロースゲルを用いた電気 泳動で行った。 コスミ ドベクターとして SuperCos 1 (Stratagene社製） を用い、 ^il消化した後、 フォスファターゼ処理を行った。 さらに、 このベクター DNAを BamHIで消化した後、上記の染色体 DNAの Sau3AI部分分解物とのライゲーション 反応（Ligat ion High T0Y0B0社製）を行った。次に、 in vi troパッケージング（LAMBDA INN, Wako社製） を行った後、 大腸菌 (Escherichia col i) XL1- Blue MR株への感 染を行った。感染した大腸菌はアンピシリンを含む LBプレートによって選択した。 〔実施例 1 4〕 PCR法により TKN14株の xylB遺伝子の部分配列増幅 Partially cleaved with Sau3AI and treated with phosphatase (CIAP, manufactured by Takara). Confirmation of the DNA cleavage pattern by § ^ 3 行 っ was performed by electrophoresis using a garose gel. SuperCos 1 (Stratagene) was used as a cosmid vector, digested with ^ il, and then treated with phosphatase. Furthermore, this vector DNA was digested with BamHI, and then ligated with the Sau3AI partial degradation product of the chromosomal DNA (Ligat ion High T0Y0B0). Next, in vitro packaging (LAMBDA INN, manufactured by Wako) was performed, followed by infection with Escherichia coli XL1-Blue MR strain. Infected E. coli were selected by LB plates containing ampicillin. [Example 14] Amplification of partial sequence of xylB gene of TKN14 strain by PCR method
3つのアルコールデヒ ドロゲナーゼ遺伝子、 Ac inetobacter sp. ADP1株の areB Three alcohol dehydrogenase genes, Ac inetobacter sp. ADP1 strain areB
(Jones, R. M. . , Col l ier, L. S. , Neidl e, E. L. , and Wi l l iams, P. A.， areABC genes determine the catabol i sm of ary丄 esters in Ac inetobacter sp. ADP1, J.(Jones, R. M.., Col l ier, L. S., Neidl e, E. L., and Wi l l iams, P. A., areABC genes determine the catabol i sm of ary 丄 esters in Ac inetobacter sp. ADP1, J.
Bacteriol. 181, 4568-4575, 1999)、 Pseudomonas put ida TOLプラスミ ド p請 0 の xylB (Harayaraa, S.， Rekik, M. , Wubbolts, M.， Rose, K.， Leppik, R. A. , and Timmis, K. N, Characterization of five genes in the upper-pathway operon of TOL plasmid pWWO from Pseudomonas putida and identification of the gene products. J. BacterioL 171， 5048 - 5055， 1989)、 Acinetobacter calcoaceticus T) xylB (Gillooly, D. J. , Robertson, A. G.， and Fe son, C. A.， Molecular characterization of benzyl alcohol dehydrogenase and benza丄 dehyde dehydrogenase II of Acinetobacter calcoaceticus. Biochem. J. 330, 1375 - 1381， 1998)のァライメントから相同性の高い部分を用いて .2つのプライマー（xylB2F ; 5 ' -GTV YTW GGN CAY GAR GG - 3 ' 及び、 ylB3R； 5， - CAA ASG ARG MYT GSS CAA A - 3 ' ) を設計した （図 5)。 Bacteriol. 181, 4568-4575, 1999), Pseudomonas put ida TOL xylB (Harayaraa, S., Rekik, M., Wubbolts, M., Rose, K., Leppik, RA, and Timmis, K. N, Characterization of five genes in the upper-pathway operon of TOL plasmid pWWO from Pseudomonas putida J. BacterioL 171, 5048-5055, 1989), Acinetobacter calcoaceticus T) xylB (Gillooly, DJ, Robertson, AG, and Fe son, CA, Molecular characterization of benzyl alcohol dehydrogenase and benza 丄 dehyde dehydrogenase II of Acinetobacter calcoaceticus. Biochem. J. 330, 1375-1381, 1998) using a highly homologous part. Two primers (xylB2F; 5'-GTV YTW GGN CAY GAR GG-3 'and ylB3R 5;-CAA ASG ARG MYT GSS CAA A-3 ') was designed (Fig. 5).
TKN14株の染色体 DNAを鍚型にし、 設計したプライマーで PCRを行った。 その 結果 214 bpの断片が増幅し TAクローニングによりベクターにクローニングした。 クローニングした 214 bpの断片の塩基配列はベクターの T7プロモーターを用い て決定した。 決定した塩基配列を配列番号 6に示す。  The chromosomal DNA of strain TKN14 was made into a saddle shape, and PCR was performed with the designed primers. As a result, a 214 bp fragment was amplified and cloned into a vector by TA cloning. The base sequence of the cloned 214 bp fragment was determined using the T7 promoter of the vector. The determined base sequence is shown in SEQ ID NO: 6.
〔実施例 1 5〕 214 bp増幅 DNA断片のプローブの作製 [Example 15] Preparation of 214 bp amplified DNA fragment probe
TKN14株の 2LZiSJt伝子の全長を含むクローンを遺伝子ライブラリ一から単離す るため、 実施例 1 4でクローニングした 214 bp増幅 DNA断片を含むベクターの T7 promoter配列から T7 RNA polymeraseによりプローブ配列を RNAに転写して プローブを下記のように作製した。鎵型 DNA溶液 14 μ 1に Sol. 1 (Transcription buffer, 2.0 1 ； DIG RNA labelling mixture, 2.0 μ ΐ ; T7 RNA polymerase, 1.0 1) を 6 μ ΐ加えた。 37°Cで 90分加温した。 この溶液 2 1を電気泳動し転写 産物を確認した。 上の溶液の残り 18 μ 1に DNase l 1 μ 1、 RNase Inhibitor 1 t lを加え、 37°Cで 30分間加温した。 DEPC処理水 80 μ 1、3Μ酢酸ナトリゥム（ρΗ5.2) 10 μ 1、100%エタノール 250 μ 1を加え - 80°Cで 1一 2時間エタノール沈澱した。 15000 rpm、 30分間遠心し、 上澄みを捨て冷 100%エタノールで沈澱を洗浄した。 沈澱を乾燥させ、 DEPC処理水 20 1に溶解させた。 プローブのラベルは PCR DIG プローブ合成キット （ロシュ社製） を用いて行った。 〔実施例 1 6〕 コロニーハイブリダイゼーションぉよぴサブクローニング コロニーをメンプレンにトランスファーした後、 メンブレンを 80°Cで 5分間乾 燥した。 乾燥後のメンブレンをアルミに包み 80°Cで 2. 5時間固定した。 メンプレ ン 100 cm2にっき 20 ml のプレハイブリ溶液をメンブレンとバックに入れた後バ ッグを密閉し、 37°Cで 2時間プレハイプリを行つた。 プローブをハイブリ溶液で 希釈しプレハイ溶液を捨てたハイプリバッグにプローブを含むハイプリ溶液を加 え、 37°Cで 3時間ハイプリを行った。ハイプリ終了後メンブレンを 0. 2 X SSC (SSC の組成； 0. 3 クェン酸 3ナトリウム、 3 M 塩化ナトリ.ゥム） で 2回洗浄し、 ハイ プリされなかったプローブを除去した。メンブレンを 0. 1 X SSCで洗浄し洗浄に使 用したバッファ一で 1分間平衡化しプロッキング溶液で 60分間振盪した。抗体希 釈液を入れ室温で 30分間浸透した後洗浄バッファー 100 mlで 15分間 2回洗浄を 行った。 検出用のバッファーを入れ 2分間メンプレンを平衡化させ、 検出パッフ ァ一で 100倍に希釈した CSPDをメンプレンにアプライした。メンプレンを気泡が 入らないようにハイプリバッグに入れシールし 37°Cで 15〜30分加温した。 加 S 後のメンブレンを暗室内で X線フィルム上に置き感光用の下線とのフタを締めて 10〜； 15秒間感光させた。感光したフィルムを現像し目的のバンドの検出を行った。 以上のようにシグナルを示す一つのクローンを単離した （18-34と命名）。 To isolate a clone containing the full length of the 2LZiSJt gene of TKN14 from the gene library, the probe sequence was converted to RNA from the T7 promoter sequence of the vector containing the 214 bp amplified DNA fragment cloned in Example 14 by T7 RNA polymerase. The probe was prepared as follows. 6 μΐ of Sol. 1 (Transcription buffer, 2.0 1; DIG RNA labeling mixture, 2.0 μΐ; T7 RNA polymerase, 1.0 1) was added to 14 μ 1 of the vertical DNA solution. Heated at 37 ° C for 90 minutes. The solution 21 was electrophoresed to confirm the transcript. DNase l 1 μ 1 and RNase Inhibitor 1 tl were added to the remaining 18 μ 1 of the above solution and heated at 37 ° C. for 30 minutes. DEPC treated water 80 μ1, 3Μ sodium acetate (ρΗ5.2) 10 μ1, 100% ethanol 250 μ1 was added, and ethanol precipitation was performed at −80 ° C. for 1-2 hours. After centrifugation at 15000 rpm for 30 minutes, the supernatant was discarded and the precipitate was washed with cold 100% ethanol. The precipitate was dried and dissolved in DEPC-treated water 201. Probe labeling was performed using a PCR DIG probe synthesis kit (Roche). [Example 16] Subcloning of colony hybridization After transferring the colonies to membrane, the membrane was dried at 80 ° C for 5 minutes. The dried membrane was wrapped in aluminum and fixed at 80 ° C for 2.5 hours. Menpure down 100 cm 2 diary 20 ml of pre-hybridization solution was sealed bar Tsu grayed After placing the membrane and back, KoTsuta 2 hours Purehaipuri at 37 ° C. The probe solution containing the probe was added to the hybrid bag where the probe was diluted with the hybrid solution and the pre-high solution was discarded, and the sample was subjected to high-pressure at 37 ° C for 3 hours. After completion of the hybridization, the membrane was washed twice with 0.2 X SSC (SSC composition; 0.3 sodium trisodium citrate, 3 M sodium chloride) to remove the probe that had not been amplified. The membrane was washed with 0.1 X SSC, equilibrated with the buffer used for washing for 1 minute, and shaken with the prokking solution for 60 minutes. The antibody diluted solution was added and permeated at room temperature for 30 minutes, and then washed twice with 100 ml of washing buffer for 15 minutes. The detection buffer was added and the membrane was equilibrated for 2 minutes, and CSPD diluted 100-fold with the detection buffer was applied to the membrane. Membrene was sealed in a high-ply bag so that air bubbles would not enter, and heated at 37 ° C for 15-30 minutes. After the addition, the membrane was placed on an X-ray film in a dark room, and the cover with the underline for exposure was tightened for 10 to 15 seconds. The exposed film was developed and the target band was detected. One clone showing the signal was isolated as described above (named 18-34).
大腸菌の陽性クローン 18-34から、プラスミ ド（P18-34と命名） DNAを調製し、 Accl、 BamHI、 Sac I, S^hlでそれぞれ切断し 0· 6%ァガロースゲルで電気泳動した 後 （図 6 )。 メンブランに転写し、 214 bpの増幅 DNA断片めプローブでサザンハ ィプリダイゼーシヨンを行った。 その結果、 3. 9 kbの Sphl断片が xylB遺伝子を コードしていることが明らかとなった。 この 3. 9 kb の Sphl断片をゲルから切り 出し pUC18の^ ilサイ トに挿入してプラスミ ド（pXylB- F14と命名）を作製した。 〔実施例 1 7〕 TKN14株の xylB遺伝子のシークェンス解析  Plasmid (named P18-34) DNA was prepared from E. coli positive clone 18-34, cleaved with Accl, BamHI, Sac I, S ^ hl and electrophoresed on a 6% agarose gel (Fig. 6). ). The membrane was transferred to a membrane, and Southern hybridization was performed with a 214 bp amplified DNA fragment probe. As a result, it was revealed that the 3.9 kb Sphl fragment encodes the xylB gene. This 3.9 kb Sphl fragment was excised from the gel and inserted into the pUC18 il site to prepare a plasmid (named pXylB-F14). [Example 17] Sequence analysis of xylB gene of TKN14 strain
プラスミ ド pXylB- F14の 3. 9 kb 断片のシークェンス解析を行った。 シー クェンシング反応は Takara PCR Thermal Cycler MP (宝酒造社製）、 BigDyeIt was Sequence analysis of 3. 9 kb fragment of the plasmid pXylB- F14. The sequencing reaction is Takara PCR Thermal Cycler MP (Takara Shuzo), BigDye
Terminator Cycle Sequencing Ready Reaction Kit ver. 2. 0 (Appl ied Biosystems 社製） を用い、 ジメチルスルホキシド （DMS0) を 5 %添加の条件で、 96°C 5分、 92°C 10秒、 50°C 5秒、 60°C 4分、 25サイクルで行った。反応生成物は Appl ied Biosystems 3700 DNA Analyzer (Appl ied Biosystems 社製） により解析した。 また、 解析の結果得られた 3， 874 bpの塩基配列を配列番号 7に示す。 遺伝子配列 の相同性の解析、 0RFの検索を NCBIホームページよりそれぞれ BLAST 2. 0 (gapped BLAST) , ORF f inderで行った。 3， 874 bpの塩基配列から二つの 0RFが見つかつ た。 一つの 0RF (配列番号 4 ) がコードするアミノ酸配列 （配列番号 5 ) はこれ まで知られた XylB [前述した、 P. putidaの T0L.プラスミ ドにコードされる XylB や Acinetobacter属由来の XylB (AreB) ]のアミノ酸 K列と 33〜44%の相同性（同 一性： identity) を持っていたので、 xylB遺伝子と命名した。 Using Terminator Cycle Sequencing Ready Reaction Kit ver. 2.0 (Applied Biosystems) and adding 5% dimethyl sulfoxide (DMS0), 96 ° C for 5 minutes, The test was performed at 92 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes, and 25 cycles. The reaction product was analyzed with Applied Biosystems 3700 DNA Analyzer (Applied Biosystems). In addition, SEQ ID NO: 7 shows the 3,874 bp nucleotide sequence obtained as a result of the analysis. The homology analysis of the gene sequence and the search for 0RF were carried out from the NCBI homepage using BLAST 2.0 (gapped BLAST) and ORF binder, respectively. Two 0RFs were found from the 3,874 bp base sequence. The amino acid sequence (SEQ ID NO: 5) encoded by one 0RF (SEQ ID NO: 4) is the known XylB [XylB encoded by the T0L. Plasmid of P. putida and XylB derived from the genus Acinetobacter (AreB )] Has 33-44% homology (identity) with the amino acid K sequence of)], so it was named xylB gene.
R. opacus TKN14株の xylB遺伝子産物は、 触媒機能が異なる 2種のアルコール デヒドロゲナーゼとも相同性を有した。すなわち、 TKN14株の XylBは、 Pseudomonas sp. Ml株の j3 - myrcene (モノテルペン；イソプレノイ ドの一種） 代謝系のアルコ 一ノレデヒ ドロケナーセ (MyrB) ( Iurescia, S.， Marconi, A. M.， Tofani, D. , Gambacorta, A.， Peterno, A.， Devirgi l ns, C, van der Werf , M. , and Zennaro. , E.， Identif icat ion and sequenc ing of β -myrcene catabo丄 ism genes from Pseudomonas sp. Strain Ml, Appl . Environ. Microbiol. 65， 2871-2876, 1999)、 及び、 Pseudomonas属の〈- terpineol (モノテルペン；イソプレノィ ドの一種） 代 謝に関わるアルコールデヒ ドロゲナーゼ遺伝子 (terpD) (Peterson, J. A. , Lu, J. — Y. , Gei ssel soder, J. , Graham-Lorence, S， . Carmona, C. , Wi tney, F. , and Lorence, M. C.， Cytochrome P~450terp. Isolat ion and purif icat ion of the protein and cloning and sequenc ing of its operon. J. Biol . Chem. 267, 14193-14203, 1992) と 49%の相同性 （同一性） を持っていた。  The xylB gene product of R. opacus TKN14 was also homologous to two alcohol dehydrogenases with different catalytic functions. In other words, XylB of TKN14 strain is an alcoholic monoaldehydro dankenase (MyrB) (Iurescia, S., Marconi, AM, Tofani, D. , Gambacorta, A., Peterno, A., Devirgi l ns, C, van der Werf, M., and Zennaro., E., Identif icat ion and sequencing of β -myrcene catabo 丄 ism genes from Pseudomonas sp. Strain Ml, Appl. Environ. Microbiol. 65, 2871-2876, 1999), and the alcohol dehydrogenase gene (terpD) (Peterson, JA) involved in the metabolization of <-terpineol (monoterpene; isoprenoid) of the genus Pseudomonas , Lu, J. — Y., Gei ssel soder, J., Graham-Lorence, S,. Carmona, C., Witney, F., and Lorence, MC, Cytochrome P ~ 450terp. Isolat ion and purif icat ion of the protein and cloning and sequencing of its operon. J. Biol. Chem. 267, 14193-14203, 1992) and 49% homology (identity) .
以上の結果から、 opacus TKN14由来の xylB遺伝子にコードされる芳香族ァ ルコールデヒ ドロゲ^ "一ゼ（配列番号 5 )は既存のものとは 49%以下の相同性（同 —性） しか有さない新規なタンパク質であることが明らかとなった。 本 TKN14株 の XylB、及びこれまで報告されている芳香族アルコールデヒ ドロゲナーゼの系統 樹を図 7に示した。 驚いたことに系統樹上では、 TKN14株の XylBは、 これまでよ く研究されてきた芳香族化合物代謝に関わる T0L プラスミ ド上にコードされる Based on the above results, the aromatic alcohol dehydration ^ “Ize” (SEQ ID NO: 5) encoded by the xylB gene derived from opacus TKN14 has no more than 49% homology (similarity) with the existing one. The phylogenetic tree of XylB of this TKN14 strain and the aromatic alcohol dehydrogenase reported so far are shown in Fig. 7. Surprisingly, in the phylogenetic tree, XylB of TKN14 is encoded on the T0L plasmid involved in aromatic metabolism that has been well studied so far.
XylBのグループとは分かれ、ィソプレノィ ドの代謝に関わるアルコールデヒドロ ゲナーゼである MyrB, TerpDと同じグループに分類されることが示された。 した がって、 今回単離された TKN14株由来の liL遺伝子 （配列番号 4 ) は全く新規な 芳香族アルコールデヒ ドロゲナーゼ遺伝子であると言える。 Alcohol dehydro, which is separate from the XylB group, is involved in isoprenoid metabolism It was shown that it is classified into the same group as MyrB and TerpD which are geneases. Therefore, it can be said that the liL gene (SEQ ID NO: 4) derived from the TKN14 strain isolated this time is a completely new aromatic alcohol dehydrogenase gene.
一方、 もう一つの 0RF (配列番号 8 ) がコードするアミノ酸配列 （配列番号 7 ) .は、 Pseudomonas属のシ卜クロム P~450terp (Peterson, J. A. , Lu, J. - Y.， Geisselsoder, J.， Graham -] Lorence, S， . Carmona, C. , Witney, F.， and Lorence, M. C.， Cytochrome P~450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J. Biol. Chem. 267, 14193 - 14203， 1992) と 46%の相同性 （同一性） を持っていた。  On the other hand, the amino acid sequence (SEQ ID NO: 7) encoded by another 0RF (SEQ ID NO: 8) is Pseudomonas genus Cychrome P ~ 450terp (Peterson, JA, Lu, J.-Y., Geisselsoder, J. , Graham-] Lorence, S,. Carmona, C., Witney, F., and Lorence, MC, Cytochrome P ~ 450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J. Biol. Chem. 267 , 14193-14203, 1992) and 46% homology (identity).
〔実施例 1 8〕 ill ^遺伝子を持つ大腸菌の芳香族アルコールの変換活性 xylB遺伝子を含む 1， 844 bp Xhol- Pstl断片を 3. 9 - kb S^hl DNA断片から切り 出して pBluescript KS IIの Xhol-Pstlサイ トに挿入し、 pXylB8を作製した （図 8 )。 また、 xylB遺伝子を含む 1. 7 kbの Xhol- Sphl断片を、 実施例 6で調整した プラスミ ド ρΝΒ2· 18Nの Xhol-Sphlサイ トに挿入して pPhnN- XylBを作製した （図 8 )。 プラスミ ド pNB2. 18Nは、 hnN遺伝子を含む 2· 18 - kb Notl-BamHI断片を pBluescript II SK+の Notl - BamHI部位に揷入されたプラスミ ドである。 本実施例 で作製したプラスミ ド PPhnN_XylBを持つ大腸菌は、芳香族アルデヒ ドからカルボ ン酸への変換がスムーズになり、 カルボン酸の生成量を定量することによって xylB遺伝子産物の活性が評価できると考えられる。 なお、 本実施例では上述した ようにして xylB遺伝子を取得したが、 xylB遺伝子は配列番号 4の塩基配列情報 に基づいて化学合成により取得することもできる。 [Example 18] Aromatic alcohol conversion activity of Escherichia coli with ill ^ gene A 1,844 bp Xhol-Pstl fragment containing the xylB gene was excised from the 3.9-kb S ^ hl DNA fragment and pBluescript KS II It was inserted into the Xhol-Pstl site to prepare pXylB8 (Fig. 8). In addition, a 1.7 kb Xhol-Sphl fragment containing the xylB gene was inserted into the Xhol-Sphl site of plasmid ρΝΒ2 · 18N prepared in Example 6 to prepare pPhnN-XylB (FIG. 8). The plasmid pNB2.18N is a plasmid in which a 2 · 18-kb Notl-BamHI fragment containing the hnN gene is inserted into the Notl-BamHI site of pBluescript II SK +. Escherichia coli with the plasmid P PhnN_XylB prepared in this example has a smooth conversion from aromatic aldehyde to carboxylic acid, and the xylB gene product activity can be evaluated by quantifying the amount of carboxylic acid produced. Conceivable. In the present example, the xylB gene was obtained as described above, but the xylB gene can also be obtained by chemical synthesis based on the nucleotide sequence information of SEQ ID NO: 4.
プラスミ ド pPhnNおよび pPhnN- XylB Cのいずれか一方を含む大腸菌 JM101を 10 ml のアンピシリン 100 Ai g/mlを含む LB培地で 30°Cにて 12時間培養をそれぞ れ行った後、菌体を遠心操作により回収後、アンピシリン 100 g/ml , 0. 2% (w/v) グルコース、 0. 01 % (w/v) チアミン、 1 mMの IPTGを含む M9培地（Na2HP04， 6 g/L ;E. coli JM101 containing either plasmid pPhnN or pPhnN-XylB C was cultured in LB medium containing 10 ml of ampicillin 100 Aig / ml at 30 ° C for 12 hours. After recovery by centrifugation, ampicillin 100 g / ml, 0.2% (w / v) glucose, 0.01% (w / v) thiamine, M9 medium containing 1 mM IPTG (Na 2 HP0 4 , 6 g / L;
KH2P04, 3 g/L ; NaCl, 0. 5 g/L ; NH4C1， 1 g/L; MgS04, 1 raM ; CaCl2， 0. 1 mM; FeS04，KH 2 P0 4 , 3 g / L; NaCl, 0.5 g / L; NH 4 C1, 1 g / L; MgS04, 1 raM; CaCl 2 , 0.1 mM; FeS0 4 ,
O. Ol raM) 3 mlで菌体を溶かした。 この菌体液を基質の数と同じように用意した。 菌体液にそれぞれの基質 100 mM DMS0溶液を基質の最終濃度が 0. 5 mMになるよう に添加し栓つき試験管に移し、 蓋を密閉して 30°Cで二日間培養を行った。 この培 養液それぞれ 200 μ 1を等量にメタノールと混ぜて遠心分離後、 HPLCの分析に供 した。 HPLCは Watersの Al 1 iance、カラムはォクタデシルシリカ逆相カラム（length. 10 cm; diameter, 4.6 mm, Waters)。 流速は 1 ml/ min0 サンプノレ注入後溶媒 A .(0.1% phosphate in water)で 2分間流した後、 溶媒 B (0.1% phosphate in acetonitrile) で 70%までのグラジェントをかけた。 70%の溶媒 B で 5分間流し た。 検出には 230から 280 nmのマキシマルを測定した。 O. Ol raM) The cells were dissolved in 3 ml. This bacterial cell solution was prepared in the same manner as the number of substrates. Add 100 mM DMS0 solution of each substrate to the bacterial solution so that the final concentration of the substrate is 0.5 mM. The sample was transferred to a test tube with a stopper, sealed, and cultured at 30 ° C for 2 days. 200 μ1 of each culture solution was mixed with methanol in equal amounts, centrifuged, and subjected to HPLC analysis. HPLC is Waters Al 1 iance, column is octadecyl silica reverse phase column (length. 10 cm; diameter, 4.6 mm, Waters). The flow rate was 1 ml / min 0 after the injection of sampnore, followed by flowing for 2 minutes with solvent A (0.1% phosphate in water) and then applying a gradient of up to 70% with solvent B (0.1% phosphate in acetonitrile). It was flushed with 70% solvent B for 5 minutes. For detection, a maximum of 230 to 280 nm was measured.
結果を表 1に示す。 .  The results are shown in Table 1. .
表 1 芳香族アルコールからカルボン酸への変換 生産物量（ zM) Table 1 Conversion of aromatic alcohols to carboxylic acids Amount of product (zM)
pPhnNを含む大 pPhnN-XylBを 基質 生産物 腸菌 . 含む大腸菌  Large pPhnN-XylB containing pPhnN Substrate Product Enterobacteriaceae
2-Hydroxymethyl  2-Hydroxymethyl
naphthalene 2 - Naphthoic acid 98.4 493.1naphthalene 2-Naphthoic acid 98.4 493.1
Benzyl slcoho丄 Benzoic acid 30.3 355.6 o一 Methy丄 benzy丄 o-MethylbenzoiG Benzyl slcoho 丄 Benzoic acid 30.3 355.6 o Methy 丄 benzy 丄 o-MethylbenzoiG
alcohol acid 5.8 20,0 m-Methylbenzyl m-Methylbenzoic alcohol acid 5.8 20,0 m-Methylbenzyl m-Methylbenzoic
alcohol acid 24.5 240.6alcohol acid 24.5 240.6
^-Methylbenzyl £-Methylbenzoic ^ -Methylbenzyl £ -Methylbenzoic
alcohol acid 10.1 226.5alcohol acid 10.1 226.5
Xylene— a—ひ'— diol 4-Hydroxymethyl Xylene—a— ひ '—diol 4-Hydroxymethyl
benzoic acid 3,4 179.8 o-Hydroxybenzyl o-Hydroxybenzoic  benzoic acid 3,4 179.8 o-Hydroxybenzyl o-Hydroxybenzoic
alcohol acid 3.7 123.7 m-Hydroxybenzyl m~Hydroxybenzoic alcohol acid 3.7 123.7 m-Hydroxybenzyl m ~ Hydroxybenzoic
alcohol acid 0.0 32.0 g-Hydroxybenzyl £-Hydroxybenzoic alcohol acid 0.0 32.0 g-Hydroxybenzyl £ -Hydroxybenzoic
alcohol acid 1. 4 352. 5 o-Methoxybenzyl o-Methoxybenzoic alcohol acid 1. 4 352. 5 o-Methoxybenzyl o-Methoxybenzoic
alcohol acid 18. 4 227. 0J2 - Methoxybenzyl m-Methoxybenzo ic alcohol acid 18. 4 227. 0J2-Methoxybenzyl m-Methoxybenzo ic
alcohol acid 11. 1 557. 3alcohol acid 11. 1 557. 3
^-Methoxybenzyl £-Methoxybenzoic ^ -Methoxybenzyl £ -Methoxybenzoic
alcohol acid . 17. 3 514. 3 m-Chlorobenzyl m-Chlorobenzoic alcohol acid. 17. 3 514. 3 m-Chlorobenzyl m-Chlorobenzoic
alcohol acid 27. 4 233. 4alcohol acid 27. 4 233. 4
Cinnamyl a lcohol Cinnamic acid 314, 8 581. 8Cinnamyl a lcohol Cinnamic acid 314, 8 581. 8
Vani llyl alcohol Vani l l ic acid 6. 3 241. 4 プラスミ ド pPhnN-XylBを持つ E. col i JM101は、 2-ヒ ドロキシメチルナフタレン から 2 -ナフドエ酸、 ベンジルアルコールから安息香酸、 2 -， 3 -， 4-メチルベンジ ルアルコールからそれぞれ 2 -， 3-, 4 -メチル安息香酸、 キシレン- α -ひ， -ジォ ールから 4-ヒ ドロキシメチル安息香酸、 2-， 3-， 4-ヒ ドロキシベンジルアルコー ルからそれぞれ 2-, 3- 4-ヒ ドロキシ安息香酸、 2-, 3- 4 -メ トキシペンジノレア ノレコールからそれぞれ 2 -， 3-, 4-メ トキシ安息香酸、 3-クロ口べンジルアルコー ルから 3 -クロ口安息香酸、 シナミルアルコールから桂皮酸、 ヴァニルアルコール からバニリン酸への変換反応が確認された。 カルボン酸の生成量はプラスミ ド pPhnNを持つ E. col i JM101のよりはるかに高いことから、 XylBが機能した結果 であることは明らかとなった。 産業上の利用可能性 Vani llyl alcohol Vani ll ic acid 6. 3 241.4 E. coli JM101 with the plasmid pPhnN-XylB is 2-hydroxymethylnaphthalene to 2-naphthoic acid, benzyl alcohol to benzoic acid, 2-, 3 2-, 3-, 4-methyl benzoic acid from-, 4-methyl benzyl alcohol, xylene-α-, 4-diol to 4-hydroxymethyl benzoic acid, 2-, 3-, 4-hydride, respectively 2-, 3--4-hydroxybenzoic acid from roxybenzyl alcohol, 2--, 3--4-hydroxybenzoinoleol, 2--, 3-, 4-methoxybenzoic acid, 3-chloro mouth respectively Conversion reactions from benzyl alcohol to 3-chlorobenzoic acid, cinnamyl alcohol to cinnamic acid, and vanyl alcohol to vanillic acid were confirmed. Since the amount of carboxylic acid produced was much higher than that of E. coli JM101 with the plasmid pPhnN, it was clear that XylB was a functioning result. Industrial applicability
本発明により、 単環化合物から多環化合物に至る非常に広い範囲の芳香族化合 物をカルボン酸に変換できる芳香族デヒ ドロゲナーゼ活性を有する新規なぺプチ ド及び当該ペプチドをコードする遺伝子を提供することができる。 また、 本発明 により、 ヒ ドロキシメチル基また アルデヒ ド基を有する芳香族化合物を芳香族 カルボン酸に変換できる有用な形質転換体を提供することができる。 さらに、 本 発明により、 上記形質転換体を利用した、 ヒ ドロキシメチル基またはアルデヒ ド 基を有する芳香族化合物から芳香族カルボン酸を製造する方法を提供することが できる。 According to the present invention, a novel peptide having an aromatic dehydrogenase activity capable of converting a very wide range of aromatic compounds from monocyclic compounds to polycyclic compounds into carboxylic acids, and a gene encoding the peptide are provided. can do. Further, according to the present invention, an aromatic compound having a hydroxymethyl group or an aldehyde group is converted into an aromatic group. Useful transformants that can be converted into carboxylic acids can be provided. Furthermore, the present invention can provide a method for producing an aromatic carboxylic acid from an aromatic compound having a hydroxymethyl group or an aldehyde group, using the transformant.
本明細書で引用した全ての刊行物、 特許および特許出願をそのまま参考として 本明細書にとり入れるものとする。  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

請求の範囲 The scope of the claims
1 . 以下の （a )、 （b )、 又は （c ) に示すペプチド： 1. Peptides shown in the following (a), (b), or (c):
( a ) 配列番号 3記載のアミノ酸配列からなるぺプチド、 (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3,
( b ) 配列番号 3記載のアミノ酸配列において 1もしくは複数個のアミノ酸が付 カロ、 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物デ'ヒ ドロ ゲナーゼ活性を有するペプチド、  (b) a peptide comprising an amino acid sequence in which one or a plurality of amino acids are appended, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 3, and having an aromatic compound dehydrogenase activity,
( c ) 配列番号 2記載の塩基配列からなる DNA又はそれと相補的な DNAとストリ ンジヱントな条件下でハイブリダィズする DNAがコードする細菌由来のぺプチド であって、 芳香族化合物デヒ ドロゲナーゼ活性を有するペプチド。  (c) a peptide derived from a bacterium encoded by DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or DNA complementary thereto and hybridized under stringent conditions, and has an aromatic compound dehydrogenase activity peptide.
2 . 以下の （a )、 （b )、 又は （c ) に示すペプチドをコードする遺伝子： ( a ) 配列番号 3記載のァミノ酸配列からなるぺプチド、  2. A gene encoding a peptide shown in the following (a), (b), or (c): (a) a peptide comprising an amino acid sequence described in SEQ ID NO: 3,
( b ) 配列番号 3記載のアミノ酸配列において 1もしくは複数個のアミノ酸が付 カロ、 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物デヒ ドロ ゲナーゼ活性を有するぺプチド、  (b) a peptide having an aromatic compound dehydrogenase activity, comprising an amino acid sequence in which one or a plurality of amino acids are appended, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 3, and
( c ) 配列番号 2記載の塩基配列からなる DNA又はそれと相補的な DNAとストリ ンジェントな条件下でハイブリダィズする DNAがコードする細菌由来のぺプチド であって、 芳香族化合物デヒ ドロゲナーゼ活性を有するペプチド。  (c) A peptide derived from a bacterium encoded by a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO: 2 or a complementary DNA thereof, and having an aromatic compound dehydrogenase activity peptide.
3 . 請求項 2に記載'の遺伝子を導入して得られる形質転換体であって、 ヒ ド 口キシメチル基またはアルデヒ ド基を有する芳香族化合物を芳香族カルボン酸に 変換できる形質転換体。  3. A transformant obtained by introducing the gene according to claim 2 and capable of converting an aromatic compound having a hydroxymethyl group or an aldehyde group into an aromatic carboxylic acid.
4 . 形質転換体が大腸菌であることを特徴とする請求項 3に記載の形質転換 体。 ，  4. The transformant according to claim 3, wherein the transformant is Escherichia coli. ,
5 . 請求項 3又は 4に記載の形質転換体を、 ヒドロキシメチル基またはアル デヒ ド基を有する芳香族化合物を含む培地で培養して培養物又は菌体から芳香族 カルボン酸を得ることを特徴とする、 芳香族カルボン酸の製造法。  5. The transformant according to claim 3 or 4 is cultured in a medium containing an aromatic compound having a hydroxymethyl group or an aldehyde group to obtain an aromatic carboxylic acid from the culture or the cells. A method for producing an aromatic carboxylic acid characterized by the following.
6 . 得られた芳香族カルボン酸が、 4 -ヒ ドロキシメチル- 1 -ナフトェ酸、 6 - メチル -2 -ナフトェ酸、 1 -ナフトェ酸、 2 -ナフトェ酸、 4 -メチル安息香酸、 3-メチ ル安息香酸、 2 -メチル安息香酸、 安息香酸、 4 -ヒ ドロキシメチル安息香酸、 4-ヒ ドロキシ安息香酸、 3-ヒ ドロキシ安息香酸、 2 -ヒ ドロキシ安息香酸、 4 -メ トキシ 安息香酸、 3 -メ トキシ安息香酸、 2-メ トキシ安息香酸、 4 -ェチル安息香酸、 4 -ィ ソプロピル安息香酸、 3 -クロ口安息香酸及ぴ 3 -フ ニルプロピオン酸からなる群 から選ばれる少なくとも 1種の芳香族カルボン酸であることを特徴とする請求項 5に記載の芳香族カルボン酸の製造法。 6. The resulting aromatic carboxylic acid is 4-hydroxymethyl-1-naphthoic acid, 6-methyl-2-naphthoic acid, 1-naphthoic acid, 2-naphthoic acid, 4-methylbenzoic acid, 3-methyl. Benzoic acid, 2-Methylbenzoic acid, Benzoic acid, 4-Hydroxymethylbenzoic acid, 4-Hydroxy Dorokishi benzoic acid, 3-arsenate Dorokishi acid, 2 - arsenate Dorokishi benzoic acid, 4 - main butoxy benzoic acid, 3 - main butoxy benzoic acid, 2-main butoxy benzoic acid, 4 - Echiru benzoic acid, 4 - I an isopropyl benzoic 6. The method for producing an aromatic carboxylic acid according to claim 5, wherein the method is at least one aromatic carboxylic acid selected from the group consisting of an acid, 3-chlorobenzoic acid and 3-phenylpropionic acid. .
7 . 以下の （a )、 （b )、 又は ( c ) に示すぺプチド：  7. Peptides shown in (a), (b) or (c) below:
( ) 配列番号 5記載のアミノ酸配列からなるぺプチド、  () A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5,
( ) 配列番号 5記載のァミノ酸配列において 1もしくは複数個のァミノ酸が付 力 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物デヒ ドロ ゲづ "一ゼ活性を有するぺプチド、  () Consisting of an amino acid sequence in which one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 5 have been deleted or substituted, and an aromatic compound dehydrogenase "peptide having a single activity,
( c ) 配列番号 4記載の塩基配列からなる DNA又はそれと相補的な DNAとストリ ンジェントな条件下でハイブリダイズする DNAがコードする細菌由来のぺプチド であって、 芳香族アルコールデヒ ドロゲナーゼ活性を有するぺプチド。  (c) a bacterially-derived peptide encoded by a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or a DNA that hybridizes with the complementary DNA under stringent conditions, and has an aromatic alcohol dehydrogenase activity. Peptides that have.
8 . 以下の （a )、 （b )、 又は （c ) に示すペプチドをコードする遺伝子： ( a ) 配列番号 5記載のアミノ酸配列からなるペプチド、  8. A gene encoding a peptide shown in the following (a), (b), or (c): (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5,
( b ) 配列番号 5記載のアミノ酸配列において 1もしくは複数個のアミノ酸が付 カロ、 欠失もしくは置換されたアミノ酸配列からなり、 かつ芳香族化合物デヒ ドロ ゲナーゼ活性を有するぺプチド、  (b) a peptide having an aromatic compound dehydrogenase activity, comprising an amino acid sequence in which one or a plurality of amino acids are appended, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 5, and
( c ) 配列番号 4記載の塩基配列からなる DNA又はそれと相補的な DNAとストリ ンジェントな条件下でハイブリダィズする DNAがコードする細菌由来のぺプチド であって、 芳香族アルコールデヒ ドロゲナーゼ活性を有するペプチド。  (c) a peptide derived from a bacterium encoded by DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or DNA complementary thereto and hybridized under stringent conditions and having aromatic alcohol dehydrogenase activity peptide.
' 9 . 請求項 8に記載の遺伝子を導入して得られる微生物であって、 ヒドロキ シメチル基を有する芳香族化合物を芳香族カルボン酸に変換できる形質転換体。  '9. A microorganism obtained by introducing the gene according to claim 8, which is capable of converting an aromatic compound having a hydroxymethyl group into an aromatic carboxylic acid.
1 0 . 微生物が大腸菌であることを特徴とする請求項 9に記載の形質転換体。 1 1 . 請求項 9又は 1 0に記載の形質転換体を、 ヒドロキシメチル基を有す る芳香族化合物を含む培地で培養して培養物又は菌体から芳香族カルボン酸を得 ることを特徴とする、 芳香族カルボン酸の製造法。  10. The transformant according to claim 9, wherein the microorganism is Escherichia coli. 1 1. The transformant according to claim 9 or 10 is cultured in a medium containing an aromatic compound having a hydroxymethyl group, and an aromatic carboxylic acid is obtained from the culture or the cells. A process for producing an aromatic carboxylic acid.
1 2 . 得られた芳香族カルボン酸が、 2-ナフトェ酸、 安息香酸、 2-, 3-， 4- メチル安息香酸、 4-ヒ ドロキシメチル安息香酸、 2 -， 3 -， 4-ヒ ドロキシ安息香酸、 2- 3 -， 4-メ トキシ安息香酸、 3 -クロ口安息香酸、 桂皮酸及ぴバニリン酸からな る群から選ばれる少なく とも 1種の芳香族カルボン酸であることを特徴とする請 求項 1 1に記載の芳香族カルボン酸の製造法。 1 2. The resulting aromatic carboxylic acid is 2-naphthoic acid, benzoic acid, 2-, 3-, 4-methylbenzoic acid, 4-hydroxymethylbenzoic acid, 2-, 3-, 4-hydroxybenzoic acid. acid, Claims characterized in that it is at least one aromatic carboxylic acid selected from the group consisting of 2- 3- , 4-methoxybenzoic acid, 3-chlorobenzoic acid, cinnamic acid and vanillic acid Item 11. A method for producing an aromatic carboxylic acid according to Item 11.
PCT/JP2005/004163 2004-08-12 2005-03-03 Novel aromatic dehydrogenase gene and process for producing aromatic carboxylic acid WO2006016432A1 (en)

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