JP6527708B2 - Novel microorganism and method for producing 2,3-dihydroxynaphthalene using the same - Google Patents

Description

The present invention is a novel microorganism, and a method for producing 2,3-dihydroxynaphthalene useful as a raw material of an epoxy resin or a synthetic raw material of a dye or medicine using the microorganism as a raw material of inexpensive 3-hydroxy-2-naphthoic acid About.

It is known that 2,3-dihydroxy naphthalene is useful as a raw material of epoxy resin, diazo type copying material, thermosensitive recording medium, toner for electrostatic charge development, and other basic medicines. As an industrial production method of 2,3-dihydroxynaphthalene, a method of heating sodium 2,3-dihydroxynaphthalene-6-sulfonate obtained by chemical synthesis from 2-naphthol in dilute sulfuric acid is known. There is a problem that the production cost is high because the synthesis reaction in the stage is required [Non-patent document 1].

Regarding the production of dihydroxynaphthalene using microorganisms, it is known that 1,2-dihydroxynaphthalene can be produced from naphthalene as a raw material using E. coli expressing naphthalene dioxygenase and naphthalene 1,2-dihydrodiol dehydrogenase [Non-patent document 2]. It is known that microorganisms which decompose anthracene metabolize anthracene via 3-hydroxy-2-naphthoic acid and 2,3-dihydroxynaphthalene [Non-patent document 3]. However, the enzyme that converts 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene has not been isolated. Although Bacillus thermophilus Leo borane scan (Bacillus thermoleovorans) is known to be metabolized via the 2,3-dihydroxynaphthalene-naphthalene, the enzymes that produce 2,3-dihydroxynaphthalene not isolated [ Non Patent Literature 4]. In addition, there is no report on a method for producing 2,3-dihydroxynaphthalene suitable for industrial production utilizing enzymes involved in these metabolic pathways.

FUNDAMENTAL PROCESSES OF DYE CHEMISTRY, HANS EDUARD FIERZ-DAVID and Louis BLANGEY, INTERSCIENCE PUBLISHERS LTD., LONDON (1949) Tetrahedron Letters, Vol. 38, No. 35, p. 6267-6270 (1997) Biodegradation, Vol. 3, p .. 351-368 (1992) Ap.l. Environ. Microbiol., Vol. 66, No. 2, p .. 518-523 (2000) J. Ind. Microbiol. Biotechnol. Vol. 23, p. 284-293 (1999) Biochemical and Biophysical Research Communications, Vol. 327, p. 656-6621 (2005) J. Bacteriol., Vol. 181, No. 9, p..2675-2682 (1999) Appl. Environ. Microbiol., Vol. 67, No. 6, p. 2507-2514 (2001)

An object of the present invention is to provide a method for producing 2,3-dihydroxynaphthalene from inexpensive 3-hydroxy-2-naphthoic acid as a raw material.

As a result of conducting earnest studies to solve the above problems, the present inventors found that Sphingomonas yanoikuyae B1 (a non-patent document 5), which is a gram-negative bacterium having anthracene assimilation ability, has anthracene. It has been found that upon metabolism, 2,3-dihydroxynaphthalene is produced.

Subsequently, Sphingomonas yanoi yae obtained as accession number NZ_JGVR00000000.1 from the GenBank (GenBank; hereinafter abbreviated as GB) database of the National Center for Biotechnology Information (hereinafter, NCBI). The genome sequence information of strain B1 was analyzed, and a protein having homology with the aromatic ring hydroxyl dioxygenase large subunit shown in the amino acid sequence shown in SEQ ID NO: 2 (GB accession number KFD 28101.1 of NCBI) (hereinafter referred to as DNA shown in SEQ ID NO: 1 which codes for oxygenase, large subunit protein), and aromatic ring hydroxyl dioxygenase small subunit of the amino acid sequence shown in SEQ ID NO: 6 (GB accession number KFD 28100.1 of NCBI) A protein having homology (hereinafter referred to as oxygena The DNA shown in SEQ ID NO: 5 encoding the small subunit protein) was cloned. These two types of proteins have homology with the large subunit protein and small subunit protein of aromatic ring hydroxyl dioxygenase, respectively, but have monooxygenase reaction of hydroxylating the 2-position of 1-hydroxy-2-naphthoic acid. Since it is known to carry out, it will be called aromatic ring hydroxylase, not aromatic ring hydroxyl dioxygenase [Non-patent document 6].

Subsequently, a strain expressing the two types of proteins was constructed using recombinant DNA technology, using Escherichia coli ( Escherichia coli ; hereinafter referred to as "E. coli" as appropriate) K12 strain as a host. When the strain was cultured and 3-hydroxy-2-naphthoic acid was added, it was found that the strain produced 2,3-dihydroxynaphthalene. Furthermore, in order to increase the production amount of 2,3-dihydroxynaphthalene, it encodes a ferredoxin protein shown in the amino acid sequence (Accession No. KFD 28099.1 of GB of NCBI) shown in SEQ ID NO: 10 of Sphingomonas yanoi yae B1 strain. After cloning the DNA shown in SEQ ID NO: 9 and the DNA shown in SEQ ID NO: 14 encoding SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 14 (GB accession number KFD 28072.1 of NCBI) shown in SEQ ID NO: 13 An E. coli strain was constructed in which proteins were expressed together with oxygenase, large subunit protein and oxygenase, small subunit protein. When this E. coli strain was cultured and 3-hydroxy-2-naphthoic acid was added, it was found that a significant amount of 2,3-dihydroxynaphthalene was produced. From these results, these two proteins were known to be involved in the conversion of 1-hydroxy-2-naphthoic acid to 1,2-dihydroxynaphthalene, but, unexpectedly, 3-hydroxy-2 It has been found that it has the function of converting naphthoic acid to 2,3-dihydroxynaphthalene, and the present invention has been completed.

That is, the present invention relates to the following (1) to (8).
(1) A microorganism which expresses a ferredoxin protein and a ferredoxin reductase protein and has a property in which the ability to metabolize 2,3-dihydroxynaphthalene is deficient or attenuated, and which comprises the following (a), (b), (c) ), (D) or (e), as well as the DNAs shown in (f), (g), (h), (i) or (j) below; And a microorganism having the ability to produce 2,3-dihydroxynaphthalene from (a) a DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4.
(B) contains a sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and from 3-hydroxy-2-naphthoic acid to 2,3-dihydroxy acid DNA encoding a protein having a function involved in conversion to naphthalene.
(C) A function comprising an amino acid sequence showing 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 4 and involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene A DNA encoding a protein having
(D) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 3
(E) hybridises under stringent conditions with DNA consisting of a sequence complementary to the sequence of all or part of the base sequence shown in SEQ ID NO: 1 or 3, and from 3-hydroxy-2-naphthoic acid , DNA encoding a protein having a function involved in conversion to 3-dihydroxynaphthalene.
(F) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 6 or 8.
(G) contains a sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 6 or 8, and from 3-hydroxy-2-naphthoic acid to 2,3-dihydroxy acid DNA encoding a protein having a function involved in conversion to naphthalene.
(H) A function comprising an amino acid sequence showing 67% or more identity with the amino acid sequence shown in SEQ ID NO: 6 or 8, and involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene A DNA encoding a protein having
(I) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 7
(J) hybridises under stringent conditions with DNA consisting of a sequence complementary to the sequence of all or part of the base sequence shown in SEQ ID NO: 5 or 7 and from 3-hydroxy-2-naphthoic acid , DNA encoding a protein having a function involved in conversion to 3-dihydroxynaphthalene.
(2) The microorganism according to (1) above, which expresses the protein by introducing a recombinant DNA encoding the ferredoxin protein and / or the ferredoxin reductase protein.
(3) The above (1) or (2), wherein the ferredoxin protein is a protein encoded by the DNA shown in (k), (l), (m), (n) or (o) below: Microorganisms described.
(K) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 10 or 12.
(L) A DNA encoding a protein having an electron transfer function, which comprises a sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 10 or 12.
(M) A DNA comprising an amino acid sequence showing 38% or more identity to the amino acid sequence shown in SEQ ID NO: 10 or 12 and encoding a protein having an electron transfer function.
(N) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 9 or 11.
(O) a DNA which hybridizes under stringent conditions with a DNA consisting of a sequence complementary to all or part of the base sequence shown in SEQ ID NO: 9 or 11 and which encodes a protein having an electron transfer function .
(4) The ferredoxin reductase protein is a protein encoded by the DNA shown in the following (p), (q), (r), (s) or (t): The microorganism according to any one of the above.
(P) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 14 or 16.
(Q) A DNA encoding a protein comprising a sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 14 or 16 and having a function of reducing ferredoxin protein.
(R) A DNA which comprises an amino acid sequence showing 33% or more identity to the amino acid sequence shown in SEQ ID NO: 14 or 16 and which encodes a protein having ferredoxin reducing activity.
(S) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 13 or 15.
(T) A protein having the function of hybridizing under stringent conditions with DNA consisting of a sequence complementary to all or part of the sequence of the nucleotide sequence shown in SEQ ID NO: 13 or 15 and reducing ferredoxin protein DNA to encode.
(5) The microorganism according to any one of (1) to (4), which does not have a DNA encoding an enzyme protein which metabolizes 2,3-dihydroxynaphthalene inherently.
(6) Any one of the above (1) to (4), which has been modified such that the ability to metabolize 2,3-dihydroxynaphthalene is deficient or diminished using mutagenesis or recombinant DNA technology Microorganisms.
(7) By reacting the microorganism according to any one of the above (1) to (6) with 3-hydroxy-2-naphthoic acid in a medium containing 3-hydroxy-2-naphthoic acid, A process for producing 2,3-dihydroxynaphthalene, which comprises producing and accumulating 2,3-dihydroxynaphthalene in a culture medium and collecting 2,3-dihydroxynaphthalene from the culture medium.
(8) A culture of the microorganism according to any one of (1) to (6) or a treated product of the culture and 3-hydroxy-2-naphthoic acid mixed in an aqueous medium, A process for producing 2,3-dihydroxynaphthalene comprising forming and accumulating 2,3-dihydroxynaphthalene in a medium and collecting 2,3-dihydroxynaphthalene from the medium.

According to the present invention, there is provided a microorganism which expresses ferredoxin protein and ferredoxin reductase protein and has a property of deficient or diminished ability to metabolize 2,3-dihydroxynaphthalene, which comprises 3-hydroxy-2-naphthoic acid Using microorganisms that express two types of proteins (oxygenase, large subunit protein and oxygenase, small subunit protein) involved in conversion to 2,3-dihydroxynaphthalene, 2-hydroxy-2-naphthoic acid is used as a raw material2 It is possible to provide a method for producing 3, 3-dihydroxy naphthalene inexpensively.

The present invention will be described in detail below.

As the oxygenase / large subunit protein of the present invention, for example, a protein having the amino acid sequence of SEQ ID NO: 2 derived from Sphingomonas yanoi yae B1 strain, or a sequence derived from Novosphingobium aromaticivorans F199 strain A protein having the amino acid sequence of No. 4 (GB Accession No. AAD 04009.1 of NCBI) can be mentioned. Furthermore, the oxygenase / large subunit protein of the present invention comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 or 4, and 3-hydroxy-2- A protein having a function involved in the conversion of naphthoic acid to 2,3-dihydroxynaphthalene can be mentioned. In addition, since the amino acid sequence identity between the protein of SEQ ID NO: 2 and the protein of SEQ ID NO: 4 is 80%, the amino acid sequence of SEQ ID NO: 2 or 4 and 80 of the large subunit protein of the present invention % Consisting of an amino acid sequence having an identity of at least 90%, particularly preferably at least 95%, and a function involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene It is possible to give a protein having

Here, "the function involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene" refers to a microorganism that expresses ferredoxin protein and ferredoxin reductase protein, the oxygenase / large subunit protein. It means a function capable of producing 2,3-dihydroxynaphthalene from 3-hydroxy-2-naphthoic acid when expressed together with an oxygenase / small subunit protein.

Examples of the oxygenase / small subunit protein used in the present invention include a protein having the amino acid sequence of SEQ ID NO: 6 derived from Sphingomonas yanoi yae B1 strain, or SEQ ID NO: 8 derived from Novosphingobium aromatiiboranes F199 strain. And a protein having the amino acid sequence of (Accession No. AAD 04008.1 of NCBI GB). Moreover, the oxygenase / small subunit protein used in the present invention is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 6 or 8 and 3-hydroxy- A protein having a function involved in the conversion of 2-naphthoic acid to 2,3-dihydroxynaphthalene can be mentioned. Further, since the amino acid sequence identity between the protein of SEQ ID NO: 6 and the protein of SEQ ID NO: 8 is 67%, the amino acid sequence of SEQ ID NO: 6 or 8 and 67 of the present invention are % Consisting of an amino acid sequence having an identity of at least 90%, particularly preferably at least 95%, and a function involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene It is possible to give a protein having

Here, "the function involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene" refers to a microorganism that expresses ferredoxin protein and ferredoxin reductase protein, the oxygenase / small subunit protein. It means a function capable of producing 2,3-dihydroxynaphthalene from 3-hydroxy-2-naphthoic acid when expressed together with an oxygenase / large subunit protein.

The aromatic ring hydroxyl dioxygenase contains non-heme iron of the active center and Rieske type clusters for receiving electrons from the electron transfer system. In the case of an aromatic ring hydroxylase comprising the oxygenase / large subunit protein and the oxygenase / small subunit protein of the present invention, the reduction reaction by the ferredoxin reductase protein which receives electrons from NAD (P) H and the ferredoxin reductase protein The ferredoxin protein that receives electrons by this constitutes the electron transfer system. Then, the electrons received by the ferredoxin protein are transferred to the Rieske-type cluster, and are used for the oxidation reaction.

As the ferredoxin protein used in the present invention, for example, a protein having the amino acid sequence of SEQ ID NO: 10 derived from Sphingomonas yanoi yae B1 strain, or the amino acid sequence of SEQ ID NO: 12 derived from Novosphingobium aromatiiensis F199 strain ( There is a protein having GB accession number AAD 04007.1) of NCBI. In addition, the ferredoxin protein used in the present invention comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 10 or 12, and A protein having an electron transfer function to oxygenase can be mentioned. The identity of the amino acid sequence between the protein of SEQ ID NO: 10 and the protein of SEQ ID NO: 12 is 82%. Moreover, as shown in the below-mentioned Example, electron transfer can be performed with respect to the aromatic ring hydroxylase of this invention also with the ferredoxin protein which E. coli K-12 strain has. Among the ferredoxin proteins possessed by E. coli K-12, the protein having high homology in the amino acid sequence to the protein of SEQ ID NO: 10 and the protein of SEQ ID NO: 12 has ferredkin having the amino acid sequence of GB accession No. NP — 417035.1 Although it is a protein, its identity is 40% and 38%, respectively. Therefore, the ferredoxin protein used in the present invention comprises an amino acid sequence having 38% or more identity, preferably 82% or more, particularly preferably 90% or more with the amino acid sequence of SEQ ID NO: 10 or 12 And the protein which has a function which transmits an electron to the aromatic ring hydroxyl dioxygenase of this invention can be mentioned. Generally, not only one type of ferredoxin protein that transmits electrons to the riske-type cluster of the aromatic ring hydroxyl dioxygenase but also a ferredoxin protein having low homology with each other has an electron transfer function to the riske-type cluster of the aromatic ring hydroxyl dioxygenase It is known to have For example, when the oxygenase / large subunit protein and the oxygenase / small subunit protein of the present invention are expressed in E. coli K12 strain, it is confirmed that 2,3-dihydroxynaphthalene is formed from 3-hydroxy-2-naphthoic acid is made of.

The ferredoxin reductase protein to be used in the present invention is, for example, a protein having the amino acid sequence of SEQ ID NO: 14 derived from Sphingomonas yanoi yae B1 strain, or the amino acid of SEQ ID NO: 16 derived from Novosphingium aromatensis Vol. A protein having the sequence (GB accession number AAD03978.1 of NCBI) can be mentioned. Further, the ferredoxin reductase protein used in the present invention comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 14 or 16, and the aromatic ring water of the present invention There can be mentioned proteins having an electron transfer function to oxidative dioxygenase. The identity of the amino acid sequence between the protein of SEQ ID NO: 14 and the protein of SEQ ID NO: 16 is 78%. In addition, as shown in Examples described later, electron transfer can be performed to the aromatic ring hydroxylase of the present invention even with a ferredoxin reductase protein possessed by E. coli K-12 strain. Among the ferredoxin proteins possessed by E. coli K-12, a protein having high amino acid sequence homology to the protein of SEQ ID NO: 14 and the protein of SEQ ID NO: 16 is ferredoxin having the amino acid sequence of GB accession number NP — 417037.1 Reductase protein, but its identity is 33% and 34% respectively. Therefore, the ferredoxin reductase protein used in the present invention is an amino acid sequence having an identity of 33% or more, preferably 78% or more, particularly preferably 90% or more, with the amino acid sequence of SEQ ID NO: 14 or 16 And proteins having the function of transferring electrons to the aromatic ring hydroxylase of the present invention.

It consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the above-mentioned oxygenase / large subunit protein or oxygenase / small subunit protein, and from 3-hydroxy-2-naphthoic acid to 2,3- A protein having a function involved in conversion to dihydroxynaphthalene, and a protein having an electron transfer function consisting of an amino acid sequence having a deletion, substitution or addition of several amino acids in the above-mentioned ferredoxin protein or ferredoxin reductase protein , Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) Current Protocols in Moreki Abbreviated as Ra Biology), Nucleic Acids Res., 10, 6487 (1982), Proc. Natl.
Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Res., 13,
4431 (1985), Proc. Natl. Acad. Sci., USA, 82, 488 (1985), etc., using, for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12 or the like. , SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 13 which encode a deletion, substitution or addition at a specific position in a protein consisting of the amino acid sequence represented by It can be obtained by introducing site-specific mutations into the DNA represented by 15. The number of one or several amino acids deleted or substituted or added is particularly limited as long as the function or electron transfer function involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene is maintained. Preferably, it is within the number of differences from the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, preferably 1 to 20, more preferably 1 to 10, One to five are particularly preferred.

Examples of the DNA encoding the oxygenase / large subunit protein of the present invention include a DNA having a base sequence represented by SEQ ID NO: 1 or 3.
Examples of the DNA encoding the oxygenase / small subunit protein of the present invention include a DNA having the base sequence represented by SEQ ID NO: 5 or 7.
Examples of the DNA encoding the ferredoxin protein used in the present invention include a DNA having a base sequence represented by SEQ ID NO: 9 or 11.
Examples of the DNA encoding the ferredoxin reductase protein used in the present invention include a DNA having a base sequence represented by SEQ ID NO: 13 or 15.

In the DNA encoding the oxygenase / large subunit protein or the oxygenase / small subunit protein of the present invention, the function associated with the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene is not lost A DNA in which a mutation such as a substitution mutation, a deletion mutation, or an insertion mutation has been introduced, for example, all or part of the DNAs represented by SEQ ID NOs: 1, 3, 5, or 7 as a probe, and stringent by hybridization. Also included are DNA that hybridizes under conditions.

In the DNA encoding the ferredoxin protein used in the present invention, mutations such as substitution mutation, deletion mutation, insertion mutation and the like have been introduced within the range that does not lose the electron transfer function to the aromatic ring hydroxyl dioxygenase of the present invention. Also included are DNAs, for example, all or part of the DNA represented by SEQ ID NO: 9 or 11 as a probe, which hybridizes under stringent conditions by the hybridization method.

The DNA encoding the ferredoxin reductase protein used in the present invention is a DNA in which mutations such as substitution mutation, deletion mutation, insertion mutation, etc. have been introduced within a range not losing the function of reducing the ferredoxin protein used in the present invention. For example, a DNA that hybridizes under stringent conditions by the hybridization method using all or part of the DNA represented by SEQ ID NO: 13 or 15 as a probe is also included.

Here, specifically, a DNA that hybridizes under stringent conditions is a hybridization performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which the DNA is immobilized. Wash the filter at 65 ° C. in an SSC solution of a concentration between 0.1 and 2 times the concentration (the composition of the 1 × SSC solution is 150 mM NaCl, 15 mM sodium citrate) Means DNA that can be identified by The experimental method of hybridization is described in Molecular Cloning, A Laboratory Manual, 2nd Edition (Sambrook, Fritsch, Maniatis), Cold. Spring Harbor Laboratory Press, 1989.].

The microorganism of the present invention is a microorganism which expresses ferredoxin protein and ferredoxin reductase protein and has a property of deficient or attenuated ability to metabolize 2,3-dihydroxynaphthalene, and encodes an oxygenase large subunit protein. Any microorganism is used, as long as it is a microorganism that retains the DNA encoding the DNA and the DNA encoding the oxygenase / small subunit protein and has the ability to convert 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene. Can. That is, the microorganism described in the above (1) can be used. The microorganism having the above-mentioned properties may be a transformant obtained by introducing one or more of the DNAs described in (1) into a host cell using recombinant technology. The ferredoxin protein and / or ferredoxin reductase protein described in the above (1) may be a transformant in which the protein is expressed by introducing a recombinant DNA encoding the protein.

The microorganism of the present invention has DNAs encoding the oxygenase / large subunit protein of the present invention, the oxygenase / small subunit protein, and the ferredoxin protein and the ferredoxin reductase protein, respectively, as in the case of the Sphingomonas yanoi yae B1 strain. In addition to the method of expressing the protein by introducing the recombinant DNA encoding the protein, mutations may be introduced into the genomic DNA to increase the expression amount of these proteins, or The ability to convert 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene can also be improved by replacing the natural promoter of the genomic DNA encoding the protein of (1) with a strong promoter.

The microorganism of the present invention has a property in which the ability to metabolize 2,3-dihydroxynaphthalene is deficient or attenuated. Twelve E. coli strains etc. originally lack the ability of wild type strains to metabolize 2,3-dihydroxynaphthalene, but this property is a prerequisite for the microorganism of the present invention. On the other hand, when the ability to metabolize 2,3-dihydroxynaphthalene, as in the case of Sphingomonas yanoi yae B1 strain, the DNA encoding an enzyme protein involved in the degradation or modification of 2,3-dihydroxynaphthalene is used. By introducing a mutation, it is necessary to eliminate or attenuate the ability to metabolize 2,3-dihydroxynaphthalene. As the attenuation, the ability of the microorganism to metabolize 2,3-dihydroxynaphthalene is preferably reduced to 10% or less before the modification, and preferably to 1% or less before the modification. Alternatively, mutation is introduced into a promoter involved in transcription of a DNA encoding an enzyme protein involved in the degradation or modification of 2,3-dihydroxynaphthalene using recombinant technology or the like, or into a translation initiation region of the protein, or The antisense RNA method may be modified to delete or attenuate the expression of the protein or to delete or reduce the ability to metabolize 2,3-dihydroxynaphthalene by gene disruption.

When the microorganism of the present invention is a transformant, a recombinant DNA is prepared according to the method described in Molecular Cloning 2nd Edition, and is obtained by transforming a host cell using the recombinant DNA. Can. In the following, the cloning of DNA and the method for preparing transformants are described in detail. Other transformants can be obtained by the same method.

The above-mentioned Sphingomonas x japonicum B1 strain or the novosphingobium aromatiivolans F199 strain is cultured by a known method generally used for culturing Sphingomonas bacteria or Novosphingobium bacteria. After cultivation, chromosomal DNA of the microorganism is isolated and purified by known methods (for example, the method described in Current Protocols in Molecular Biology). Using synthetic DNA from this chromosomal DNA, oxygenase / large subunit protein and oxygenase having functions involved in conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene by hybridization method or PCR method etc. · A fragment containing DNA encoding a small subunit protein, and a fragment containing DNA encoding a ferredoxin protein having a function of transferring electrons to aromatic ring hydroxyl dioxygenase, and a ferredoxin reductase having a function of reducing ferredoxin protein A fragment containing DNA encoding a protein can be obtained.

The synthetic DNA is a nucleotide sequence represented by SEQ ID NO: 1 or 3 of a DNA encoding an oxygenase / large subunit protein derived from Sphingomonas • Yanoi yae B1 strain or Novosphingobium • aromaticiboranes F199 strain; The nucleotide sequence represented by SEQ ID NO: 5 or 7 of the DNA encoding the small subunit protein of the oxygenase derived from Monas japonica B1 strain or the novosphingobium aromatiivolans F199 strain, and Sphingomonas japonica B1 strain or The base sequence represented by SEQ ID NO: 9 or 11 of the DNA encoding the ferredoxin protein from Novosphingobium aromatiivolans strain F199, and Sphingomonas yanoi yae B1 strain or novosphingobium Can be designed based on the nucleotide sequence shown in SEQ ID NO: 13 or 15 of the DNA encoding the ferredoxin reductase protein from Loma tee grain lance F199 strain.

As a vector to which the above DNA is ligated, any vector capable of autonomous replication in E. coli K12 can be used, and any vector such as a plasmid vector or a phage vector can be used. Specifically, pUC19 [Gene, 33, 103 (1985) ], PUC18, pBR322, pHelix 1 (Roche Diagnostics), ZAP Express (Stratagene, Strategies, 5, 58 (1992)), pBluescript II SK (+) [Stratagene, Nucleic Acids] Res., 17, 9494 (1989)], pUC118 (manufactured by Takara Bio Inc.), etc. can be used.

E. coli (Escherichia coli) used as a host of the recombinant DNA obtained by ligating the DNA obtained above to the vector may be any microorganism belonging to Escherichia coli, but specifically, E. coli MG1655 (Science, 277, 1453 (1997)), a strain derived from E. coli K-12, E. coli XL1-Blue MRF '(Stratagene, Strategies, 5, 81 (1992) ], E. coli C600 (Genetics, 39, 440 (1954)), E. coli JM 105 (Gene, 38, 275 (1985)), E. coli JM109, E. coli BL21 and the like.

Rhodococcus (Rhodococcus) genus, Acinetobacter (Acinetobacter) spp., Bradyrhizobium (Bradyrhizobium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Sinorhizobium (Sinorhizobium) genus, Burebibakuteri um (Brevibacterium) genus, Novo sphingolipids bi um (Novosphingobium) genus or Ralstonia (Ralstonia) genus, in Sphingomonas (Sphingomonas) microorganism belonging to the genus, when introducing the DNA is self replicable among these microorganisms Use a vector. Preferably, recombinant DNA can be introduced into the host microorganism using a shuttle vector capable of autonomous replication in both of the microorganism and E. coli K12 strain.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the above-mentioned host cell, for example, a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], electroporation [Nucleic Acids Res., 16, 6127 (1988)], and the like.

Recombinant DNA can be extracted from the transformant obtained as described above, and the base sequence of the DNA of the present invention contained in the recombinant DNA can be determined. For determination of the base sequence, a commonly used base sequence analysis method such as dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or 3730xl type DNA analyzer (manufactured by Applied Biosystems), etc. The following sequence analyzer can be used.

Alternatively, the target DNA can also be prepared by chemical synthesis using a Perceptive Biosystems 8905 DNA synthesizer or the like based on the base sequence of the DNA determined above.

The transformant expressing the oxygenase / large subunit protein, the oxygenase / small subunit protein, the ferredoxin protein and / or the ferredoxin reductase protein described above expresses the above DNA in a host cell using the following method Obtained by

When using a DNA encoding the above-mentioned protein, a DNA fragment of appropriate length containing a portion encoding the protein of the present invention can be prepared, if necessary. In addition, the production rate of the protein can also be improved by substituting a base so that the base sequence of the portion encoding the protein is an optimal codon for host expression. A transformant expressing the DNA of the present invention is inserted into the downstream of the promoter of an appropriate expression vector to produce a recombinant DNA, and the recombinant DNA is used as a host compatible with the expression vector. It can be obtained by introduction into cells.

As a host for expressing the protein of the present invention, any host can be used as long as it can express a target gene, such as bacteria, yeast, animal cells, insect cells, plant cells and the like. Preferably, a microorganism which expresses ferredoxin protein and ferredoxin reductase protein and has the property of deficient or attenuated the ability of 2,3-dihydroxynaphthalene to metabolise can be used. More preferably, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodoshdomonas, Sinorhizobium, Brevibacterium, Novosphingobium, Ralstonia having the above properties It is possible to cite bacteria of the genus or sphingomonas. More preferably, E. coli K12 strain can be mentioned.

By using the microorganism of the present invention prepared by expressing the oxygenase / large subunit protein of the present invention and the oxygenase / small subunit protein of the present invention in such a host microorganism, it is possible to obtain Dihydroxynaphthalene can be produced. That is, 2,3-dihydroxynaphthalene can be obtained by adding the microorganism of the present invention to a medium containing 3-hydroxy-2-naphthoic acid and reacting it with 3-hydroxy-2-naphthoic acid.

In addition, when such a microorganism of the present invention is grown to introduce 3-hydroxy-2-naphthoic acid into cells to produce 2,3-dihydroxynaphthalene, 3-hydroxy-2-naphthoic acid It is preferable to use a microorganism having the transport (intake) ability of

As an expression vector, a vector capable of autonomous replication in the host cell or capable of being integrated into a chromosome, and containing a promoter at a position at which the DNA of the present invention can be transcribed is used.

When a prokaryote such as bacteria is used as a host cell, a recombinant DNA comprising the DNA of the present invention can be autonomously replicated in prokaryotes, and at the same time, a promoter, ribosome binding sequence, DNA of the present invention, transcription Preferably, the termination sequence is a recombinant DNA composed of: A gene that controls a promoter may be included.

As a vector for introducing and expressing a protein of the present invention or a fusion protein of the protein and a fusion protein of the protein and another protein into a microorganism such as E. coli, a so-called multicopy vector is preferable, and ColE1-derived replication A plasmid having a starting point, such as a plasmid of pUC series or a plasmid of pBR322 series or derivatives thereof may be mentioned. Here, the "derivative" means that the plasmid has been modified by base substitution, deletion, insertion, addition and / or inversion and the like. The term "modification" as used herein also includes modification by mutation or UV irradiation or the like, or modification by natural mutation or the like. More specifically, as a vector, for example, pUC19 [Gene, 33, 103 (1985)], pUC18, pBR322, pHelix1 (Roche Diagnostics), pKK233-2 (Amersham Pharmacia Biotech) ), PSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen), pET-3 (Novagen) pBluescript II SK (+), pBluescript II KS (+) (Stratagene) , PSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.), and the like can be used.

Any promoter may be used as long as it can be expressed in host cells such as E. coli. For example, promoters such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, etc. derived from E. coli such as T7 promoter and phage, etc., and artificially designed and modified as tac promoter, lacT7 promoter And Pm promoter controlled by XylS protein of P. putida TOL plasmid can be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 5 to 18 bases). In the recombinant DNA of the present invention, a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to place the transcription termination sequence immediately below the structural gene.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the above-mentioned host cell, for example, a method using calcium ion [Proc. Natl. Acad. Sci., USA, 69, 2110] (1972)], electroporation [Nucleic Acids Res., 16, 6127 (1988)], conjugation and transmission [JGC Ottow, Ann. Rev. Microbiol., Vol. 29, p. 80 (1975)], cells And fusion methods (MH Gabor, J. Bacteriol., Vol. 137, p. 1346 (1979)) and the like.

It is also possible to produce a microorganism in which the production amount of any one or more of the above-mentioned proteins is enhanced as compared with a wild strain using recombinant DNA technology or the like to increase the production amount of 2,3-dihydroxynaphthalene. Specifically, a promoter having higher transcription activity than a natural promoter is used as a promoter for expressing a gene encoding any of the above-mentioned proteins, or as a terminator for terminating transcription of a gene encoding the protein For example, a terminator having a transcription termination activity stronger than that of a natural terminator is used, or a high copy number vector is used as an expression vector, and chromosomal integration by homologous recombination and the like can be mentioned.

The 2,3-dihydroxynaphthalene-producing microorganism of the present invention obtained as described above is preferably cultured in a medium containing 0.1 mM to 1 M of 3-hydroxy-2-naphthoic acid, and 2, 2,3-Dihydroxynaphthalene can be produced by producing and accumulating 3-dihydroxynaphthalene and collecting it from the culture. The method of culturing the microorganism of the present invention in a culture medium can be carried out according to a conventional method used for culturing a microorganism.

Cultivation of the microorganism of the present invention can be carried out in a conventional nutrient medium containing a carbon source, nitrogen source, inorganic salt, various vitamins and the like, and carbon sources include, for example, sugars such as glucose, sucrose, fructose and the like, ethanol, Alcohols such as methanol, organic acids such as citric acid, malic acid and succinic acid, waste molasses and the like are used. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like are used alone or in combination. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition to this, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and biotin can be added to the medium. As a raw material for producing 2,3-dihydroxy naphthalene, 3-hydroxy-2-naphthoic acid is added.

The culture is usually performed under aerobic conditions such as aeration and agitation, shaking and the like. The culture temperature is not particularly limited as long as the microorganism of the present invention can grow, and the pH in the middle of the culture is also not particularly limited as long as the microorganism of the present invention can grow. PH adjustment during culture can be performed by adding an acid or an alkali.

The 2,3-dihydroxynaphthalene-producing microorganism of the present invention is cultured, and then the culture of the microorganism or a treated product of the culture is added to an aqueous medium containing 3-hydroxy-2-naphthoic acid. It is also possible to form and accumulate 2,3-dihydroxynaphthalene therein, and to collect 2,3-dihydroxynaphthalene from the medium.

As a processed product of the culture, one obtained by immobilizing the microorganism of the present invention on a carrier may be used. In such a case, cells recovered from the culture or washed with an appropriate buffer, for example, a phosphate buffer (pH 6 to 10) of about 0.02 to 0.2 M or the like can be used. In addition, crushed products obtained by crushing the cells collected from the culture by means of ultrasonic waves, pressing, etc., extracts containing the protein of the present invention obtained by extracting the crushed products with water or the like, The extract is further subjected to treatment such as ammonium sulfate salting out, column chromatography, etc. What is obtained by immobilizing the partially purified component etc. of the protein of the present invention obtained in the present invention on a carrier is also used for producing 2,3-dihydroxynaphthalene of the present invention. It can be used.

Immobilization of these cells, disrupted cells, extract or purified enzyme is carried out by immobilizing cells etc. on a suitable carrier such as acrylamide monomer, alginic acid or carrageenan according to a commonly used method known per se. It can be done by the method of

The aqueous medium used for the reaction can be an aqueous solution containing 3-hydroxy-2-naphthoic acid or a suitable buffer, for example, a phosphate buffer (pH 6 to 10) of about 0.02 to 0.2 M or so. To the aqueous medium, it is also possible to add 0.05 to 2.0% (w / v) of toluene, xylene, a nonionic surfactant and the like, when it is necessary to further increase the material permeability of the cell membrane of the cells.

The concentration of 3-hydroxy-2-naphthoic acid as a starting material for the reaction in an aqueous medium is suitably about 0.1 mM to 1 M. The enzyme reaction temperature and pH in the above aqueous medium are not particularly limited, but generally 10 to 60 ° C., preferably 15 to 50 ° C. are appropriate, and the pH in the reaction solution is usually 5 to 10, preferably about 6 to 9 It can be done. Moreover, adjustment of pH can be performed by adding an acid or an alkali. The enzyme used in the invention can be obtained by collecting the cell extract as it is or from it by centrifugation, filtration or the like and suspending it in water or a buffer. The enzyme thus obtained is reacted in the presence of 3-hydroxy-2-naphthoic acid, but the concentration of 3-hydroxy-2-naphthoic acid in the reaction solution can be such that the activity of the enzyme is not inhibited. It is advantageous to make it as high as possible. The reaction may be performed by any method of standing, stirring and shaking. Alternatively, the enzyme may be immobilized on a suitable support and packed into a column, and a solution containing 3-hydroxy-2-naphthoic acid may be flowed. The reaction is usually performed at 10 to 60 ° C., preferably 15 to 50 ° C., at a pH of 5 to 9, preferably at a pH of 6 to 9.

Moreover, when an antioxidant or a reducing agent is added to the above aqueous medium at the time of reaction, the production yield of 2,3-dihydroxynaphthalene may be further improved. Antioxidants / reductants include ascorbic acid, isoalkorbic acid, cysteine, sulfites such as sodium sulfite and sodium bisulfite, and thiosulfates such as sodium thiosulfate. The addition concentration varies depending on the type of antioxidant / reducing agent, but it is desirable to add it at a concentration that does not inhibit the formation of 2,3-dihydroxynaphthalene, usually 0.001 to 5% (W / V), preferably 0.005 to 1 %.

Moreover, when the oxidizing agent is added to the above aqueous medium at the time of reaction, the production yield of 2,3-dihydroxynaphthalene may be further improved. Examples of the oxidizing agent include nitrates such as sodium nitrite and potassium nitrite, metal salts such as ferric chloride, halogens, peroxy acids and the like, with preference given to sodium nitrite and ferric chloride. Although the addition concentration varies depending on the type of oxidizing agent, it is desirable to add it at a concentration that does not inhibit the formation of 2,3-dihydroxynaphthalene, and is usually 0.001 to 0.05% (W / V), preferably 0.005 to 0.02%.

The 2,3-dihydroxynaphthalene from the culture solution or reaction solution after completion of the culture can be isolated and purified by extraction with an organic solvent such as ethyl acetate. In addition, after removing insoluble components such as cells from the culture solution by centrifugation etc. as necessary, for example, a method using activated carbon, a method using an ion exchange resin, a crystallization method, a distillation method, a precipitation method, etc. The 2,3-dihydroxy naphthalene can be collected by the method alone or in combination.

Hereinafter, the method of the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

Example 1 Production of 2,3-dihydroxynaphthalene in Escherichia coli K-12 strain not expressing foreign ferredoxin protein and ferredoxin reductase protein

(1) Isolation and Purification of Chromosomal DNA Obtained from the Japan Collection of Microorganisms operated by the RIKEN BioResource Center, Sphingobium yanoi yae B1 strain (strain No. JCM30198) and provided from the apan Collection of Microorganisms Culture was performed according to the information. Subsequently, chromosomal DNA was isolated and purified from the cultured cells using a DNA purification kit (illustra bacteria genomicPrep Mini Spin Kit; manufactured by GE Healthcare).

(2) Construction of an E. coli strain expressing an aromatic ring hydroxylase derived from Sphingobium japonica B1 strain According to the GBBI database of NCBI, bphA2c gene which encodes an oxygenase / small subunit protein derived from Sphingobium japonica B1 strain ( Primers for obtaining DNA comprising the sequence of bphA2c gene and bphA1c gene (SEQ ID NO: 17) by obtaining the sequence data of SEQ ID NO: 3 and the bphA1c gene (SEQ ID NO: 1) encoding oxygenase / large subunit protein via the Internet And 18) designed. The synthesis of each PCR primer was outsourced to Fasmac. Preparation of synthetic DNAs such as PCR primers was performed by outsourcing to Fasmac unless otherwise stated. Using the chromosomal DNA obtained above as a template and the DNA primers of SEQ ID NOS: 17 and 18, PrimeDNA DNA polymerase (Takara Bio Inc.) was used to amplify DNA containing the bphA2c gene and the bphA1c gene according to the attached instruction. . Subsequently, the DNA containing the bphA2c gene and the bphA1c gene obtained above is inserted between the PacI site and the NotI site of the E. coli expression vector pUXPEaLT19 (see JP-A-2014-50373) to obtain the bphA2c gene and The plasmid pUXPEaLT_bphA21c expressing the bphA1c gene was obtained. The transformed strain pUXPEaLT_bphA21c / JM109 was constructed by introducing the plasmid pUXPEaLT_bphA21c obtained above into Escherichia coli JM109 strain using a calcium ion transformation method.

(3) Construction of E. coli Strain Expressing E. coli lacZ Gene The β-galactosidase gene (hereinafter abbreviated as lacZ gene) of E. coli MG1655 strain, which is a gene not related to the hydroxylation of 3-hydroxy-2-naphthoic acid It was decided to use for control experiment. As a base sequence of lacZ gene, a sequence of accession number JF 300162 from base number 1 to 3075 is obtained from NCBI GB database, and PCR primers (SEQ ID NOs: 19 and 20) for cloning lacZ gene based on this sequence. Was designed and synthesized. E. coli MG1655 strain obtained from E. coli from National Institute of Genetics, National BioResource Project, LB liquid medium (10 g / l tryptone (Difco), 5 g / l dry yeast extract (Difco), 10 g / l chloride Sodium] and shake culture overnight at 37 ° C. Based on the collected bacterial cells, E. coli chromosomal DNA was prepared using illustra bacteria genomicPrep Mini Spin Kit (manufactured by GE healthcare).

Using the chromosomal DNA obtained in the above (1) as a template, DNA of the lacZ gene was amplified using PrimeSTAR DNA polymerase and PCR primers (SEQ ID NOS: 19 and 20) according to the attached instruction. The PCR amplification product was purified, digested with restriction enzymes PacI and NotI, and inserted between the PacI site and the NotI site of E. coli expression vector pUXPEaLT19 to obtain plasmid pUXPEaLT_lacZ expressing the lacZ gene. Next, the plasmid pUXPEaLT_lacZ was transformed into E. coli JM10.
The transformed strain pUXPEaLT_lacZ / JM109 was constructed by introducing into 9 strains using a calcium ion-based transformation method.

(4) Production of 2,3-dihydroxynaphthalene using the above-mentioned Escherichia coli strain The pUXPEaLT_bphA21c / JM109 strain constructed above and the pUXPEaLT_lacZ / JM109 strain were cultured overnight in 0.5 ml of LB liquid medium containing 100 mg / L of ampicillin respectively. Then, the culture solution was inoculated 1% in 4 ml of LB liquid medium containing 100 mg / L of ampicillin, and cultured at 32 ° C. After adding m-toluic acid to a final concentration of 1 mM in the logarithmic growth phase, culture was continued at 32 ° C. for 3 hours. After washing the cultured cells twice with 10 mM potassium phosphate buffer (pH 7.0), 100 μl of 10 mM potassium phosphate buffer containing 2% glucose and 1 mM 3-hydroxy-2-naphthoate Suspended in The cell concentration was adjusted to be 10.0 at an absorbance of 600 nm.

This cell suspension was allowed to react for 18 hours at 30 ° C. and 1000 rpm. The reaction solution was centrifuged (room temperature, 1 minute, 15000 × g), 50 μl of the supernatant was transferred to a new 1.5 mL tube, 5 μl of 1N hydrochloric acid and 250 μl of ethyl acetate were added and mixed well. After centrifugation, 200 μl of the supernatant was taken and transferred to a new 1.5 mL tube. The dried product obtained by placing this upper layer solution in a vacuum drier was dissolved in 2% acetonitrile. The organic compounds in this solution were analyzed under the separation conditions of high performance liquid chromatography (HPLC) shown in Table 1 using an LC-TOF mass spectrometer (LCT, Inc. LCT Premier XE). The retention time of high performance liquid chromatography and the mass value from the mass spectrometer were combined to identify 2,3-dihydroxynaphthalene produced as compared with the standard 2,3-dihydroxynaphthalene.

As a result, as shown in Table 2, about 8 μM of 2,3-dihydroxynaphthalene was detected in pUXPEaLT_bphA21c / JM109 strain. On the other hand, 2,3-dihydroxynaphthalene was not detected in pUXPEaLT_lacZ / JM109 strain expressing the lacZ gene. Therefore, 2,3-dihydroxynaphthalene is produced from 3-hydroxy-2-naphthoic acid by expressing bphA2c gene and bphA1c gene in E. coli K12 strain not expressing foreign ferredoxin protein and ferredoxin reductase protein It became clear.

Example 2 Production of 2,3-dihydroxynaphthalene in Escherichia coli expressing oxygenase large subunit protein, oxygenase small subunit protein, ferredoxin protein and ferredoxin reductase protein

(1) Construction of an E. coli strain expressing three types of proteins involved in the production of 2,3-dihydroxynaphthalene derived from Sphingobium japonica B1 strain bphA3 gene encoding the ferredoxin protein of Sphingobium yanoicaya B1 strain (GB Ac The base sequence data (SEQ ID NO: 9) of session No. JPOU01000022.1 base No. 151095 to 151421) is acquired from the GBBI database of NCBI via the Internet, and a primer (SEQ ID NO: 21) for amplifying DNA containing bphA3 gene Designed and synthesized. Subsequently, using the chromosomal DNA obtained in Example 1 (1) as a template and using the DNA primers of SEQ ID NOS: 18 and 21 according to the attached instruction, PrimeStar DNA polymerase (purchased from Takara Bio) is used to obtain bphA3 gene , DNA containing the bphA2c gene and the bphA1c gene was amplified.

The DNA containing the bphA3 gene, bphA2c gene and bphA1c gene obtained above is inserted between the PacI site and NotI site of the E. coli expression vector pUXPEaLT19 to obtain the plasmid pUXPEaLT_bphA321c expressing the bphA3 gene, bphA2c gene and bphA1c gene The
The transformed strain pUXPEaLT_bphA321c / JM109 was constructed by introducing the plasmid pUXPEaLT_bphA321c obtained above into the E. coli JM109 strain using a calcium ion transformation method.

(2) Construction of an E. coli strain that expresses four types of proteins involved in 2,3-dihydroxynaphthalene production from Sphingobium japonica B1 strain bphA4 gene encoding the ferredoxin reductase protein of Sphingobium janoia B1 strain ( The nucleotide sequence data (SEQ ID NO: 11) of the complementary strand of base numbers 124178 to 125404 of JPOU01000022.1 is obtained from the GBBI database of NCBI via the Internet, and a primer (SEQ ID NO: 22) for amplifying DNA containing bphA4 gene 23) designed and synthesized. Subsequently, using the chromosomal DNA obtained in Example 1 (1) as a template and DNA primers of SEQ ID NOs: 22 and 23, amplification was performed using PrimeStar DNA polymerase in accordance with the attached instruction. After adding an A residue to the 3 'end of the amplified DNA fragment by Taq DNA polymerase, the amplified DNA fragment is purified by gel electrophoresis and incorporated into pT7Blue-T vector (Novagen) to construct plasmid pT7_bphA4_B1. did.

The DNA encoding ferredoxin reductase protein is excised from plasmid pT7_bphA4_B1 with restriction enzyme SwaI and restriction enzyme NotI, and the DNA is inserted between the PmeI site and NotI site of plasmid pUXPEaLT_bphA321c obtained above to obtain bphA3 gene, bphA2c gene , And the plasmid pUXPEaLT_bphA321c_A4 expressing the bphA1c gene and the bphA4 gene. Subsequently, a transformant pUXPEaLT_bphA321c_A4 / JM109 was constructed by introducing plasmid pUXPEaLT_bphA321c_A4 into E. coli JM109 strain using a calcium ion transformation method.

(3) Production of 2,3-dihydroxynaphthalene using the above-mentioned E. coli strain With regard to pUXPEaLT_bphA321c / JM109 strain, pUXPEaLT_bphA321c_A4 / JM109 strain and pUXPEaLT_lacZ / JM109 strain constructed above, using 3-hydroxy-2-naphthoic acid as a raw material 2 The evaluation of the productivity of 3, 3-dihydronaphthalene was carried out. The productivity evaluation was performed in the same manner as in Example 1.
As a result, as shown in Table 3, in the pUXPEaLT_bphA321c / JM109 strain and the pUXPEaLT_BphA321c_A4 / JM109 strain, production of 290 μM and 830 μM 2,3-dihydronaphthalene was confirmed, respectively. On the other hand, 2,3-dihydronaphthalene was not detected in pUXPEaLT_lacZ / JM109 strain expressing the lacZ gene.

Example 3 Production of 2,3-dihydroxynaphthalene using aromatic ring hydroxylase from novosphingobium aromaticiboranes strain F199

(1) Isolation / Purification of Chromosomal DNA of Novosphingobium Aromasticiboranes F199 Strain The Novosphingobium Aromasticibolus F199 strain was obtained from the American Type Culture Collection (ATCC 700278). Subsequently, chromosomal DNA was isolated and purified from the cells obtained by culturing the Novosphingobium aromatiiboranes F199 strain.

(2) Construction of an E. coli strain that expresses three types of proteins involved in 2,3-dihydroxynaphthalene production derived from Novosphingobium aromatiiboranes F199 strain Ferredoxin derived from Novosphingobium aromatiiboranes F199 strain A DNA region (bphA3 gene (SEQ ID NO: 11) encoding a protein, bphA2c gene (SEQ ID NO: 7) encoding an oxygenase / small subunit protein, and a bphA1c gene (SEQ ID NO: 3) encoding an oxygenase / large subunit protein (SEQ ID NO: 3) Obtain nucleotide sequence data of GB accession number CP000676.1 (nucleotide numbers 33613 to 35714) via the Internet, and use primers (SEQ ID NOs: 24 and 25) for amplifying DNA containing bphA3 gene, bphA2c gene and bphA1c gene Designed and synthesized. A DNA containing the bphA3 gene, bphA2c gene and bphA1c gene derived from Novosphingobium aromatiivolans strain F199 was amplified by the PCR method using primers of SEQ ID NOS: 24 and 25 using the chromosomal DNA obtained above as a template I did.

A plasmid expressing the bphA3 gene, bphA2c gene and bphA1c gene by inserting the thus obtained DNA containing the bphA3 gene, bphA2c gene and bphA1c gene between the PacI site and NotI site of the E. coli expression vector pUXPEaLT19 Obtained pUXPEaLT_bphA 321 c_F199. The transformed strain pUXPEaLT_bphA321c_F199 / JM109 was constructed by introducing the plasmid pUXPEaLT_bphA321c_F199 into E. coli JM109 strain using a calcium ion transformation method.

(3) Construction of an E. coli strain expressing four types of proteins involved in 2,3-dihydroxynaphthalene production derived from novosphingobium and aromaticiboranes strain F199 Novosphingobium and aromatiiboranes from the GBBI database of NCBI To obtain bphA4 gene (SEQ ID NO: 15 of the complementary strand of base number 4755-5981 of GB accession No. CP000676.1), which is a ferredoxin reductase gene derived from strain F199, via the Internet to amplify bphA4 The primers (SEQ ID NOS: 26 and 27) were designed and synthesized The bphA4 gene was prepared using the chromosomal DNA obtained in Example 1 (1) as a template and the primers of SEQ ID NOS: 26 and 27 as attached. It was amplified using PrimeStar DNA polymerase (purchased from Takara Bio) according to Taq DNA polymerase. After addition of A residues at the 3 'end of the amplified DNA fragments by Ze, the amplified DNA fragment was purified by gel electrophoresis, a plasmid was constructed pT7_bphA4_F199 by incorporating into pT7Blue-T vector (Novagen).

The DNA encoding the leptidase enzyme protein is excised from plasmid pT7_bphA4_F199 with restriction enzyme SwaI and restriction enzyme NotI, and inserted between the PmeI site and NotI site of plasmid pUXPEaLT_bphA321c_F199 obtained above to obtain bphA3 gene, bphA2c gene, bphA1c gene , And a plasmid pUXPEaLT_bphA321c_A4_F199 expressing the bphA4 gene was obtained.
The transformed strain pUXPEaLT19_bphA321c_A4_F199 / JM109 was constructed by introducing the plasmid pUXPEaLT_bphA321c_A4_F199 obtained above into the E. coli JM109 strain using a calcium ion transformation method.

(4) Production of 2,3-dihydroxynaphthalene using the above transformant strain pUXPEaLT_BphA321c_F199 / JM109 strain constructed above, pUXPEaLT_bphA321c_A4_F199 / JM109 strain, pUXPEaLT_lacZ / JM109 strain and pUXPEaLT_lacZ / JM109 strain: An evaluation of the productivity of 2,3-dihydronaphthalene from acid was conducted. The productivity evaluation was performed in the same manner as in Example 1.

As a result, as shown in Table 4, production of 190 μM and 310 μM 2,3-dihydroxynaphthalene was confirmed in the pUXPEaLT_BphA321c_F199 / JM109 strain and the pUXPEaLT_BphA321c_A4_F199 / JM109 strain, respectively. On the other hand, 2,3-dihydroxynaphthalene was not detected in pUXPEaLT_lacZ / JM109 strain expressing the lacZ gene. Therefore, it was revealed that 2,3-dihydroxynaphthalene was produced from 3-hydroxy-2-naphthoate by the expression of the bphA2c gene and the bphA1c gene of the novosphingobium aromaticivorus F199 strain.

Example 4 Production of 2,3-dihydroxynaphthalene in Corynebacterium bacteria expressing oxygenase large subunit protein, oxygenase small subunit protein, ferredoxin protein and ferredoxin reductase protein

(1) Construction of expression vector pECsf for Corynebacterium bacteria As a shuttle vector for Escherichia coli-Corynebacterium used for holding foreign genes etc. in a plasmid state in Corynebacterium glutamicum ( Corynebacterium g / Lutamicum ) ATCC13032 strain The pHCG100 constructed based on the plasmid pHM1519 carried by Corynebacterium glutamicum ATCC 13032 was used. The construction method of plasmid pHCG100 and the information of Corynebacterium glutamicum ATCC13032 strain are disclosed in JP-A-2014-188282.

In order to add PacI site, NotI site, transcription termination sequence and SfiI site to plasmid pHCG100, two synthetic DNAs represented by SEQ ID NOS: 28 and 29 were prepared. These synthetic DNAs are ligated by ligating the DNA fragment of BamHI-PvuII (size: about 3.2 kb) derived from plasmid pHCG100 and the DNA fragment of ClaI and BamH derived from plasmid pHCG100 (size: about 1.7 kb). The plasmid pHCGPN was obtained.

Two synthetic DNAs represented by SEQ ID NOS: 30 and 31 were synthesized in order to add SwaI site, transcription termination sequence and SbfI site to the plasmid pHCGPN obtained above. These synthetic DNAs were inserted between the BamHI and PacI sites of plasmid pHCGPN to obtain plasmid pECPNsf.

Subsequently, in order to add an XhoI site, a transcription termination site and an SfiI site to plasmid pECPNsf, two synthetic DNAs represented by SEQ ID NOS: 32 and 33 were synthesized. These synthetic DNAs were inserted between the BamHI site and the SwaI site of plasmid pECPNsf to obtain an expression vector pECsf for Corynebacterium bacteria.

(2) Cloning of gap Promoter In order to express four types of proteins involved in 2,3-dihydroxynaphthalene production in Corynebacterium glutamicum ATCC13032 strain, it was decided to clone the gap promoter of this strain.

The genomic sequence information of Corynebacterium glutamicum ATCC13032 strain is obtained via the Internet as NCBI • GB accession number NC — 006958, and then the promoter region of the gap gene of the strain (hereinafter abbreviated as gap promoter; 480 of base numbers 1685102 to 1685581 Based on the base sequence (SEQ ID NO: 34) of the region of bp), primers (SEQ ID NO: 35 and 36) were designed and synthesized to amplify the gap promoter region. The chromosomal DNA obtained above was used as a template using DNA primers of SEQ ID NOS: 35 and 36, and amplified according to the attached instruction. The DNA of this gap promoter region was digested with restriction enzymes SnaBI and PacI, and then inserted between the SnaBI site and PacI site of plasmid pECsf to obtain plasmid pECsf_gapS.

(3) Construction of 2,3-dihydroxynaphthalene-producing plasmid DNAs encoding the bphA3 gene, bphA2c gene, bphA1c gene and bphA4 gene cut out from the plasmid pUXPEaLT_bphA321c_A4 obtained in Example 2 (2) with restriction enzymes PacI and NotI Was inserted between the PacI site and the NotI site of the plasmid pECsfD_gap obtained above, to obtain the plasmid pECsf_gapS_bphA321c_bphA4 expressing the bphA3 gene, bphA2c gene, bphA1c gene and bphA4 gene.

The transformant pECsf_gapS_bphA321c_bphA4 / ATCC13032 was constructed by introducing the plasmid pECsf_gapS_bphA321c_bphA4 obtained above into the Corynebacterium glutamicum ATCC13032 strain using the transformation method by electroporation.

By inserting the DNA encoding the lacZ gene excised from the plasmid pUXPEaLT_lacZ obtained in Example 1 (3) with the restriction enzymes PacI and NotI, between the PacI site and the NotI site of the plasmid pECsf_gapS obtained above , The plasmid pECsf_gapS_lacZ expressing the lacZ gene was obtained.

The transformant pECsf_gapS_lacZ / ATCC13032 was constructed by introducing the plasmid pECsf_gapS_lacZ obtained above into the Corynebacterium glutamicum ATCC13032 strain using the transformation method by electroporation.

(4) Production of 2,3-dihydroxynaphthalene in Corynebacterium glutamicum strain ATCC13032 CGXII medium containing 50 mg / L kanamycin (ammonium sulfate 20 g / L, urea 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate · heptahydrate 0.25 g / L, calcium chloride 10 mg / L, sulfuric acid Ferrous iron heptahydrate 10 mg / L, manganese sulfate pentahydrate 10 mg / L, zinc sulfate heptahydrate 1 mg / L, copper sulfate 0.2 mg / L, nickel chloride hexahydrate After culture with 0.5 ml of 0.02 mg / L of biotin, 0.2 mg / L of biotin, 20 g / L of glucose, 30 mg / L of protocatechuate, pH 7.0) for 16 hours, CGXII containing 50 mg / L of kanamycin Medium 4
The cells were inoculated 1% to ml and cultured at 32 ° C. for 9 hours. A cell suspension was prepared by the same method as in Example 1 (4) so that the cell concentration was 20.0 at an absorbance of 600 nm, and the production amount of 2,3-dihydroxynaphthalene was measured.

As a result, as shown in Table 5, in the pECsf_gapS_bphA 321 c_bphA4 / ATCC 13032 strain, production of about 145 μM of 2,3-dihydroxynaphthalene was confirmed. On the other hand, 2,3-dihydroxynaphthalene was not detected in pECsf_gapS_lacZ / ATCC 13032 strain expressing the lacZ gene.

Claims (6)

Using the mutagenesis treatment or recombinant DNA techniques, 2,3 ability to metabolize dihydroxynaphthalene a microorganism modified to deficient or attenuated, express ferredoxin protein and the ferredoxin reductase protein, One or of (a), below shown in (b), (c), (d) or DNA shown in (e), and the following (f), (g), (h), (i) or (j) 1. A microorganism carrying DNA and capable of producing 2,3-dihydroxynaphthalene from 3-hydroxy-2-naphthoic acid (a) A DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4.
(B) contains a sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and from 3-hydroxy-2-naphthoic acid to 2,3-dihydroxy acid DNA encoding a protein having a function involved in conversion to naphthalene.
(C) A function comprising an amino acid sequence showing 90 % or more identity to the amino acid sequence shown in SEQ ID NO: 2 or 4 and involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene A DNA encoding a protein having
(D) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 3
(E) hybridises under stringent conditions with DNA consisting of a sequence complementary to the sequence of all or part of the base sequence shown in SEQ ID NO: 1 or 3, and from 3-hydroxy-2-naphthoic acid , DNA encoding a protein having a function involved in conversion to 3-dihydroxynaphthalene.
(F) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 6 or 8.
(G) contains a sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 6 or 8, and from 3-hydroxy-2-naphthoic acid to 2,3-dihydroxy acid DNA encoding a protein having a function involved in conversion to naphthalene.
(H) A function comprising an amino acid sequence showing 90 % or more identity with the amino acid sequence shown in SEQ ID NO: 6 or 8 and involved in the conversion of 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene A DNA encoding a protein having
(I) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 7
(J) hybridises under stringent conditions with DNA consisting of a sequence complementary to the sequence of all or part of the base sequence shown in SEQ ID NO: 5 or 7 and from 3-hydroxy-2-naphthoic acid , DNA encoding a protein having a function involved in conversion to 3-dihydroxynaphthalene.   2. The microorganism according to claim 1, wherein the microorganism is modified to express a ferredoxin protein and / or a recombinant DNA encoding a ferredoxin reductase protein so that the protein is expressed. The microorganism according to claim 2, wherein the ferredoxin protein is a protein encoded by the DNA shown in the following (k), (l), (m), (n) or (o).
(K) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 10 or 12.
(L) A DNA encoding a protein having an electron transfer function, which comprises a sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 10 or 12.
(M) A DNA comprising an amino acid sequence showing 90 % or more identity to the amino acid sequence shown in SEQ ID NO: 10 or 12 and encoding a protein having an electron transfer function.
(N) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 9 or 11.
(O) a DNA which hybridizes under stringent conditions with a DNA consisting of a sequence complementary to all or part of the base sequence shown in SEQ ID NO: 9 or 11 and which encodes a protein having an electron transfer function . The microorganism according to claim 2, wherein the ferredoxin reductase protein is a protein encoded by the DNA shown in the following (p), (q), (r), (s) or (t).
(P) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 14 or 16.
(Q) A DNA encoding a protein comprising a sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 14 or 16 and having a function of reducing ferredoxin protein.
(R) A DNA comprising an amino acid sequence showing 90 % or more identity to the amino acid sequence shown in SEQ ID NO: 14 or 16 and encoding a protein having ferredoxin reducing activity.
(S) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 13 or 15.
(T) A protein having the function of hybridizing under stringent conditions with DNA consisting of a sequence complementary to all or part of the sequence of the nucleotide sequence shown in SEQ ID NO: 13 or 15 and reducing ferredoxin protein DNA to encode. By reacting the microorganism according to any one of claims 1 to 4 with 3-hydroxy-2-naphthoic acid in a medium containing 3-hydroxy-2-naphthoic acid, -A process for producing 2,3-dihydroxynaphthalene, which comprises producing and accumulating dihydroxynaphthalene and collecting 2,3-dihydroxynaphthalene from the culture medium. A culture of the microorganism according to any one of claims 1 to 4 or a treated product of the culture and 3-hydroxy-2-naphthoic acid are mixed in an aqueous medium to obtain -A process for producing 2,3-dihydroxynaphthalene, which comprises forming and accumulating dihydroxynaphthalene and collecting 2,3-dihydroxynaphthalene from the medium.