JP3362402B2 - High temperature induced lignin peroxidase gene - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lignin peroxidase (hereinafter abbreviated as LPO) gene, a recombinant plasmid containing the gene, and a transformant containing the recombinant plasmid. Since LPO acts on lignin to lower its molecular weight or decompose it, it can be used for various steps in the production of paper pulp from a lignocellulosic material such as wood. That is, it can be used for lowering or degrading the molecular weight of lignin in the pulping process, pulp bleaching process, wastewater treatment process and the like. Furthermore, in the field of so-called cellulosic biomass utilization, it can be used for the purpose of enhancing saccharification efficiency by decomposing lignin as a pretreatment for saccharification of cellulose.

[0002]

2. Description of the Related Art LPO is an enzyme that decomposes lignin and is known as Phanerocaete chrysosporium (Phaner).
Ochaete chrysosporium), containing heme as a prosthetic group,
It was revealed that the molecular weight is about 42,000, that hydrogen peroxide is required for the enzymatic action, and that the phenolic hydroxyl group at the 4-position of the lignin model compound acts on the compound that has become a methoxyl group. [FEB
S Lett. 169, 247 (1984)].

Recently, LPO is a white-rot fungus other than Fanellocaete chrysosporium, for example, Arakawakawatake (C).
oriolus hirsutus) [Japanese Patent Application Laid-Open No. 64-2
7468], Yakeiro (Bjerkander)
a adustus) [see Japanese Patent Laid-Open No. 303483], Coriolus versico
lor) [Acta Chem. Scand. , B4
1, 766 (1987)], Koganesurokotake
(Phlebia radiata) [Bioche
m. J. , 254, 877 (1988)], etc., but there are few strains for which specific production of LPO has been confirmed, and purified examples are other than the above strains. unknown.

Several LPO isozymes can be found in the culture of any of these bacteria, and it can be considered that various enzymes are involved in lignin degradation. In any of the strains of Fanelocaete chrysosporium, Kawaratake, Kogaresurokotake and Yakeirotake, wild-type strains were used for LPO.
When producing L., there were restrictions such as suppressing the nitrogen source concentration in the medium to a low concentration and increasing the oxygen partial pressure in the incubator, and it was difficult to mass-produce LPO.

Attempts have so far been made to obtain a mutant strain which produces LPO under a high nitrogen source concentration and under an oxygen partial pressure at the same level as in the atmosphere [Japanese Patent Laid-Open No. 64-27469, Japanese Laid-Open Patent Publication No. Hei 2-27469].
-72875] has not yet been put to practical use. Also,
Among LPO-producing bacteria, the gene has been isolated from Fanelocaete chrysosporium [Nature, 3
26, 520 (1987)], Kawaratake [B. B.
R. C. , 179, 428 (1991)], Yakeirotake [Japanese Patent Application No. 3-59284] and Koganesurokotake [Gene, 85, 343, (1989)], and industry utilizing the LPO gene of these bacteria. No production has been done yet.

[0006] LPO can also produce LPO by static culture in the same manner as the above strains (Japanese Patent Laid-Open No. 64-27468), but its production is small, and the production method is further improved for practical use. Is desired.

[0007]

Therefore, the present invention is an LP
It is intended to provide a useful LPO gene for a practical production method of O.

[0008]

[Means for Solving the Problems] The present inventors have succeeded in cloning a novel LPO gene from a cDNA and chromosomal DNA library prepared based on mRNA isolated from LPO-producing Pseudomonas aeruginosa cells. Then, the present invention has been completed. That is, the present invention provides a lignin peroxidase gene consisting of DNA encoding the amino acid sequence of SEQ ID NO: 4. As a specific example thereof, the present invention provides a lignin peroxidase gene derived from a chromosome containing a structural gene having the base sequence of SEQ ID NO: 1 and a promoter region thereof, and further provides a lignin peroxidase gene (cDNA having the base sequence of SEQ ID NO: 2). )I will provide a. Further, the present invention provides a recombinant vector containing the lignin peroxidase gene having the nucleotide sequence of SEQ ID NO: 1 or 2 or encoding the amino acid sequence of SEQ ID NO: 4.

Further, the present invention is a transformant containing the above recombinant vector. Hereinafter, the present invention will be described in detail. Structural Gene The structural gene of LPO of the present invention is 2 in SEQ ID NO: 5.
87 th of the base sequence or 22 th of SEQ ID NO: 2, from A TG to 1735-th TC T A TG (M
From et) consisting 1125 th TC T (Ser) to the nucleotide sequence. This base sequence encodes 368 amino acids, and encodes a leader sequence of 26 amino acids from the N terminus and a 342 amino acid lignin peroxidase, respectively.

The chromosomal DNA (SEQ ID NO: 1) contains 6 introns (348 in the structural gene according to the GT-AG rule).
~ 406th, 615th to 674th, 810th to 864th, 1412 to 1468th, 1544 to 1600th, 1666 to 1772th), TATAAGT near the 200th is TATA-box, and 90th.
CGATTGGCT in the vicinity of the tenth or CTATTGGGA in the vicinity of the 105th is considered to be a so-called CAAT-box.

The asparagine at position 103 from the N-terminal amino acid of the mature protein, valine, is considered to be an N-glycosylated amino acid residue. Proximal histidine coordinated by heme iron found in peroxidase is considered to be histidine at position 179, and distalhistidine involved in the decomposition of hydrogen peroxide is considered to be histidine at position 48. These upstream and downstream amino acid sequences are different from each other. It had a high homology with the peroxidase (see Table 1).

[0012]

[Table 1]

The LPO gene of the present invention is
As shown in Table 2, it differs from the LPO H8 gene of Chrysosporium in various points, and its homology is 70% in the nucleotide sequence and 60% in the amino acid sequence.
It is completely different from a gene. Further, as can be seen from Table 1, it is a novel LPO gene having a different amino acid sequence from known peroxidase.

[0014]

[Table 2]

The LPO gene of the present invention can be obtained as follows from a chromosomal DNA library of Pseudomonas aeruginosa or a cDNA library synthesized from mRNA. Preparation of chromosomal DNA library A medium capable of proliferating Pleurotus cornucopiae, for example glucose / peptone medium (glucose 2%, polypeptone 0.5
%, Yeast extract 0.2%, KH ₂ PO ₄ 0.1%, Mg
SO ₄ . 7H ₂ 00.5%), the bacterial cells cultured were collected by filtration,
Freeze in liquid nitrogen.

Crush the cells in a mortar, etc.
Et al. [Proc. Natl. Acad. Sci. U
SA, 81, 1470 (1984)] and the like.
Chromosomal DNA is prepared by the extraction method described in 1. Chromosome DN
A is cleaved with an appropriate restriction enzyme that can be introduced into a vector, ligated to the vector, introduced into E. coli, and transformed to obtain chromosome D.
Create an NA library. In this case, the host vector system to be used is not particularly limited, but preferably Escherichia coli is used as a host and a cosmid vector (pHC79 etc.) is used.
To use. Chromosome D, depending on the type of vector used
In some cases, it is better to cut NA with an appropriate restriction enzyme, and then fractionate it by an ordinary method such as agarose gel electrophoresis or sucrose density gradient centrifugation, so that the lengths are appropriately adjusted.

Preparation of cDNA Library Preparation of a cDNA library from mRNA containing LPO gene of Pleurotus cornucopiae is carried out as follows.
Although any strain may be used as Arakawakawatake, for example, IFO4917 strain can be used. C of the fungus body
The cells suitable for preparing the DNA library may be those during LPO production, preferably JP-A-2-30348.
It can be obtained by culturing with the method shown in 3 added with the modification of changing the culturing temperature from 28 ° C to 37 ° C. The cells thus obtained are immediately frozen in liquid nitrogen, crushed using, for example, a mortar, a Waring blender, etc., and a conventional method for extracting RNA from plant cells [Sambrook et al.,
"Molecular Cloning A Labo
rally Manual / 2nd Edition
(1989)] to extract RNA. R obtained
The mRNA in NA was concentrated and purified through an affinity column such as oligo dT cellulose, and then a portion of this was used to synthesize cDNA, which was ligated to an E. coli cloning vector, for example, λgt10, through an appropriate linker or adapter. Then, a cDNA library can be prepared.

These operations are generally known methods,
For example, the above-mentioned "Molecular Cloning A"
Laboratory Manual ". Alternatively, a commercially available mRNA extraction kit, cd
It can be easily carried out by appropriately using kits such as NA synthesis kit, cDNA cloning kit, and ligation kit.

Isolation of LPO gene from library
And determination of its nucleotide sequence The lignin peroxidase gene as a DNA probe can be prepared according to the examples of Japanese Patent Application No. 4-52673. In addition, E. coli E. coli containing the gene. coli
JM109 / pUCLP0G4 can be obtained from the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, by the Micro Engineering Research Institute No. 12678 (FERM P
-12678). Restriction enzyme B of the lignin peroxidase gene of Pleurotus cornucopiae
The 2.2 kbp fragment obtained by digestion with amHI was nick translated [Sambrook et al., "M.
olecular Cloning A Labora
tory Manual / 2nd Edition (1
989)] and labeled with ³² P as a probe, and can be selected by colony or plaque hybridization from the aforementioned chromosomal DNA library or cDNA library. In addition, L isolated by the above method
The nucleotide sequence of the PO gene is determined by the method of Sanger et al. [Pr
oc. Natl. Acad. Sci. USA, 74, 5
463, (1977)].

A clone selected from the chromosomal DNA library is used as an E. coli vector pBluescriptIIs.
A strain obtained by subcloning into E. coli E. coli. col
iXL-1 Blue / pBSLPOG7 has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microindustry Research Institute No. 12683 (FERM P-12683).
In addition, a strain obtained by synthesizing cDNA from mRNA and subcloning a clone selected from the cDNA library prepared with λgt10 into an E. coli vector pBluescriptIIsk +, Escherichia coli E. coli. coli XL
-1 Blue / pBSLPOC7 is available at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Micro Engineering Research Institute No. 12680 (FERM
P-12680).

Recombinant DNA, transformant LP represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2
The O gene can be incorporated into an appropriate vector, for example, a plasmid vector to obtain an expression recombinant vector. In this case, the vector is not particularly limited, and may be λ phage, cosmid vector, pUC, pBl.
uescript etc. are used appropriately. Furthermore, yeast and Bacillus subtilis vectors are also used. In addition, a transformant can be obtained by introducing the above recombinant plasmid into a desired host cell. Escherichia coli, Bacillus subtilis and yeast are used as host cells.

[0022]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1. Preparation of cDNA library Agarella chinensis is cultivated under the same conditions as those described in JP-A-64-27468 except that the culturing temperature is changed from 28 ° C to 37 ° C, and after culturing and washing, freezing in liquid nitrogen and Waring It was crushed with a blender. Equipment used below,
Reagents can be prepared by conventional methods [Sanbrook et al., "Mol.
electrical Cloning A Laborato
ry Manual / 2nd "Edition (198
9)] Diethylpyrocarbonate-treated product was used. Total RNA was extracted from about 5 g of the crushed cells using an RNA extraction kit manufactured by Amersham, according to its manual.

From 2 mg of the total RNA, an mRNA Purification Kit manufactured by Pharmacia was used,
A poly (A) RNA fraction was prepared according to the manual. Next, double-stranded cDNA was synthesized from 5 μg of poly (A) RNA using a cDNA synthesis kit manufactured by Amersham Co. according to its manual. Using the cDNA cloning kit manufactured by Amersham, from the synthesized double-stranded cDNA, c at λgt10 according to its manual
A DNA library was prepared as follows. cDNA
The EcoRI adapter was ligated to and unreacted adapter was removed by gel filtration column.
The fraction containing the A fragment was removed. Further, the above-mentioned cDNA fragment fraction was treated with T4 DNA kinase, recovered by phenol / chloroform treatment and ethanol precipitation, air-dried,
It was dissolved in 10 μl of TE solution.

Λgt10 arm 1 μg, cDNA100
ligation buffer containing ng and 2.5 units of T4 DNA ligase was reacted for 16 hours at 16 ° C. to ligate the arm and cDNA,
Packaged in vitro. Packaged recombinant λ
Phage were infected with E. coli NM514 and plaqued on LB agar plates. As a result, 5 × 10 ⁷ plaques appear per 1 μg of insert cDNAc
A DNA library could be obtained.

Example 2. L from cDNA library
Isolation of the PO cDNA gene The LPO gene from the above cDNA library can be isolated by a conventional method [Sambrook et al., "Molecular".
r Cloning A Laboratory Man
ual / 2nd "Edition (1989)] plaque hybridization.

That is, one plate (diameter 90 mm)
Scatter the cDNA library so that 1000 plaques will appear on the top. When the plaque is about 0.5 mm to 1 mm in size, place the nylon membrane on the plate for 2 minutes and transfer the plaque to the membrane, followed by Amersham The denaturing operation was performed once, the neutralization operation was performed twice, and the cells were washed with 2 × SSC and air-dried according to the manual of the blotting membrane of 1.
Fixed to the membrane. The probe used for plaque hybridization was the chromosome LPO gene (pUCLPOG4) 2.2 kbp isolated in Japanese Patent Application No. 4-52673.
Was prepared as follows. Recombinant plasmid 1 obtained by culturing Escherichia coli JM109 / pUCLPOG4 in which the gene has been cloned and obtaining the cells by the alkaline extraction method
0 μg was completely digested with the restriction enzyme BamHI, and the 2.2 kbp LPOG4 gene fragment was excised by 1% agarose gel electrophoresis. The BamHI fragment (2.2 kbp)
1 μg was labeled with [α- ³² P] dCTP according to the manual using a multilabeling kit manufactured by Amersham according to the manual.

Subsequently, 5 ml of prehybridization solution (5 × SS) was used for each nylon membrane.
C, 1 × Denhardt's solution, 1% SDS, 100 μg / ml
It was shaken in salmon-denatured DNA) at 65 ° C. overnight. After the completion of pre-hybridization, 2 ml of hybridization solution (5 x SSC, 1 x Denhardt's solution,
1% SDS, 100 μg / ml salmon denatured DNA),
Further, a DNA probe was added so that the concentration was 1 × 10 ⁶ cpm / ml, and hybridization was carried out at 50 ° C. for 24 hours. After hybridization, the membrane is 2 × SSC containing 1% SDS and 0.1 × S according to a conventional method.
After washing several times with SC at different times, after drying, positive clones were selected by autoradiography using an intensifying screen. Phage DNA was prepared from the isolated positive clones by liquid culture by a conventional method, cleaved with restriction enzyme BamHI, and fractionated by 1% agarose gel electrophoresis to obtain a 1.3 kbp cDNA fragment. The fragment was pBluescript manufactured by Stratagene in accordance with a conventional method.
Subcloned into the BamHI site of IISK +,
E. coli XL-1 Blue was transformed. Subcloned DNA was prepared in large quantities and subjected to ultracentrifugation (50,000 rp).
m, 16 hr, 20 ° C.) and the nucleotide sequence was determined. The nucleotide sequence is determined by Takara Shuzo's deletion kit and United States Biochemica.
It was carried out using a Sequenase kit manufactured by I. The results are as shown in SEQ ID NO: 2.

The fragment after cutting with BamHI was used as St
Ratagene plasmid pBluescri
It was ligated to the BamHI site of ptIISK +, and E. coli E. Strain E. coli obtained by transformation into E. coli XL-1 Blue. coli XL-1 Blue / pBSLPOC7
Is Microbiology Research Institute, Microbial Industry Research Institute, AIST 1268
Deposited as No. 0 (FERM P-12680).

Example 3. Creation of chromosomal gene library
Agar piece of 5mm diameter from an agar plate culture of manufacturing Coriolus hirsutus (IFO4917 strain) punched with a cork borer, glucose peptone medium (2% glucose, 0.5% of polypeptone, 0.2% yeast extract, KH ₂ PO ₄ 0 .1
%, MgSO ₄ .7H ₂ 00.05%, pH 4. with phosphoric acid.
(Prepared in 5) 200 ml was inoculated and cultivated with shaking at 28 ° C for 7 days. After collecting the bacterial cells by filtration, the bacterial cells were washed with 1 L of sterilized water and frozen with liquid nitrogen. 5 g of this frozen microbial cell was crushed using a mortar, a Waring blender and the like. Transfer the crushed cells to a centrifuge tube, and use Lysis buffer (100 mM Tris (pH8), 100 mM EDTA, 100 mM NaCl).
Furthermore, 10 ml of proteinase K was added to 100 μg / ml) and the mixture was incubated at 55 ° C. for 3 hours. After incubation, perform phenol extraction and chloroform extraction, and gradually add ethanol to the aqueous layer to add D
When NA was deposited, the chromosomal DNA was wound up and dissolved in a TE solution.

100 μg of the obtained chromosomal DNA was partially digested with the restriction enzyme Sau3AI and fractionated by 10-40% sucrose density gradient ultracentrifugation (30,000 rpm, 18 hours) to obtain a DNA fragment fraction of 30-50 kbp. collected. Next, 20 μg of the cosmid vector pHC79 was sufficiently digested with a restriction enzyme BamHI20 unit, treated with alkaline phosphatase, extracted with phenol, extracted with chloroform, precipitated with ethanol, and then dissolved in a TE buffer solution. 30 partially digested with the vector DNA and Sau3AI
Chromosomal DNA fragments of ˜50 kbp were mixed at a molar ratio of 3: 1, and the ligation reaction was carried out overnight at 16 ° C. with T4 DNA ligase, followed by packaging with an in vitro packaging kit manufactured by Amersham. It was a library.

Example 4. From chromosomal DNA library
Isolation of LPO gene of LPO gene was carried out by conventional colony hybridization [Samb
“Molecular Cloning” by Brook et al.
A Laboratory Manual / 2nd "
Edition (1989)]. Infecting the chromosomal DNA library with E. coli DH1,
Scatter so that about 500 recombinant Escherichia coli appear on an LB plate with a diameter of 90 mm containing 50 μg / ml ampicillin, and grow until the recombinant E. coli grows to a diameter of about 1 mm.
Cultured at 7 ° C. Next, the colonies grown on the agar medium were transferred to a nylon membrane, placed on the LB agar medium containing 200 mg / ml of chloramphenicol with the surface on which the colony was transferred, and incubated at 37 ° C. for 24 hours. . Subsequently, the denaturing operation with an alkaline denaturing solution was performed once, the neutralizing operation with a neutralizing solution was performed twice, and further 2 according to the manual of the blotting membrane manufactured by Amersham.
After washing with × SSC and air-drying, the recombinant DNA was immobilized on the membrane by UV irradiation.

Subsequently, as in the case of the plaque hybridization described above, 5 ml per one nylon membrane was used.
Prehybridization solution (5 x SSC, 1 x
Denhardt's solution, 1% SDS, 100 μg / ml salmon-denatured DNA) was shaken overnight at 65 ° C. After completion of prehybridization, 2 ml of hybridization solution (5 x SSC, 1 x Denhardt's solution, 1% SD)
S, 100 μg / ml salmon denatured DNA), and
A synthetic DNA probe was added so that the concentration was 1 × 10 ⁶ cpm / ml, and hybridization was carried out at 50 ° C. for 24 hours. After hybridization, the membrane is 2 × SSC containing 1% SDS and 0.1 × SS according to a conventional method.
After washing several times with C at different times, after drying, a positive colony to which a DNA probe described later hybridizes was detected by autoradiography using an intensifying screen.

The above probe was prepared as follows. 10 μg of the plasmid (pBSLPOC7) containing the high temperature inducible lignin peroxidase gene isolated in Example 2 was digested with a restriction enzyme BamHI, and a 1.3 kbp DNA fragment was separated, extracted and purified by 1% agarose gel electrophoresis. 1 μg of the BamHI 1.3 kbp fragment was subjected to [α-32P] d using a multilabeling kit manufactured by Amersham.
Radiolabeled by CTP according to the manual.

Positive colonies were isolated and cultured in 3 ml of LB medium containing 50 μg / ml of ampicillin, and a plasmid was prepared according to the alkaline extraction method. The above plasmid DNA was completely digested with two kinds of restriction enzymes BamHI and EcoRI, subjected to 1% agarose gel electrophoresis, and then subjected to ordinary Southern hybridization method using nylon membrane. Sambrook et al., "Molebrook".
color Cloning A Laborator
y Manual / 2nd "Edition (198
9)] was used to detect the LPO gene.

The DNA fragment (3.5 kbp) detected by Southern hybridization was excised by agarose gel electrophoresis, and then pBluescriptIISK +
After ligation to the vector, E. coli XL-IBlue strain was transformed. The resulting E. coli transformant strain E. E. coli XL-1Blue / pBSLPOG7 has been deposited at the Institute for Microbial Industry, Institute of Industrial Technology, as Microindustrial Research Institute No. 12683 (FERM P-12683).

The entire nucleotide sequence of the LPO gene is the TAKARA kilosequencing deletion kit manufactured by Takara Shuzo Co., Ltd. and the United States Biochemical
The procedure was performed according to the respective manuals using a Sequenase kit manufactured by the company. The results are shown in SEQ ID NO: 1 and SEQ ID NO: 3. <Structure of LPO gene> From the comparison between the high temperature inducible LPO gene derived from the chromosomal DNA represented by SEQ ID NO: 1 and SEQ ID NO: 3 and the gene derived from the mRNA of SEQ ID NO: 2, the presence and expression of an intron on the chromosomal DNA It was revealed that there are CATT and TATAbox regions that are involved in the regulation of Escherichia coli and a leader sequence involved in secretion. These are collectively shown in SEQ ID NO: 5.

The intron on the chromosome is 34 of SEQ ID NO: 5.
8-406th, 615-674th, 810-864
Position, 1412 to 1468th, 1544 to 1600th, and 1666 to 1772th). The 84th to 92nd 5'-CGATT
GGCT-3 'or 101-109th 5'-C
TATTGGGA-3 'is CAATbox, 197-2
The 03rd 5'-TATAAGT-3 'is TATAbo
Considered to be x. Furthermore, as an arrangement similar to the heat shock element, 113'-126th 5'-TACG
AACGTACTCG-3 'is recognized.

The 26 amino acid sequence starting from the 287th methionine to arginine is the leader sequence, and an open reading frame encoding 342 amino acids from the amino-terminal valine to the carboxyl-terminal serine is found as a mature protein. The 103rd aspartic acid of the mature protein is Asn-X-Se.
It is considered to be N-glycosylated from the sequence of r. In addition, proximal histidine coordinated by heme iron, which is common to many peroxidases, is seen at position 179, and distalhistidine involved in the decomposition of hydrogen peroxide is seen at position 48. It had a relatively high homology with that of peroxidase. However, the sequence was completely different from the known LPO gene sequence and was a novel sequence.

[0039]

INDUSTRIAL APPLICABILITY The present invention provides novel chromosomal LPO gene and cDNA LPO gene, their recombinant plasmids and transformants. As a result, lignin peroxidase can be produced using the gene recombination technique. Furthermore, it is considered that the temperature induces the production of substances by the transformation of other proteins using the promoter region involved in the expression of the lignin peroxidase chromosome gene induced by high temperature as a host.

[0040]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1718 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: genomic DNA origin Organism name: Coriolus hirsutus Share name: IFO4917 Sequence features 1-1449 P CDS 1-137 P sig peptide 138-1449 P mat peptide 62-120 intron 329-388 intron 524-578 intron 1126-1182 intron 1258-1314 intron 1380-1486 intron 504-506 P N-glycosylation site 279-281 P distal histidine 787-789 P proximal histidine   Array:   ATGGCGTTCA AGGCTCTTCT CTCCGCCGTC TCCCTCATAG CGGCCTTCCA 50   GGGCGCGAGC GGTACGGGGC CTCACCTTCA CTGCCCTAAC ACCCCAGATC 100   GTCACGCCCA GCACCGCTAG CTGCATTGAC GAAACGCGTC GCCTGCCCCG 150   ACGGCAAGAA CACCGCGACG AACGCGGCGT GCTGCCAGCT GTTCGCGATC 200   CGCGACGACA TCCAGGAGAA CCTCTTCCAC GGCGCGCAGT GCGGCGAGAA 250   CGCGCACGAG TCCCTCCGCA TCACCTTCCA CGACGCGATC AGCTTCTCGC 300   CCGCCCTCGA GGCCAAGGGC CAGTTTGGGT AGGTCGCCCC TTCTCCGCTG 350   CGGAGAGAGG TGGATAGTTC TCTGACGGCA CTTTGCAGCG GCGGAGGTGC 400   AGACGGCTCT ATCGCGATTT TCCCTGAGAT CGAGACCSSG TTCCACGCCA 450   ACATCGGCCT CGACGAGATC GTCGCTGAGC AGAAGCCGCT CATCGCGCGC 500   CACAACATCT CGCACGCCGA CTTGTGATGC TCTGCACTAC ACCTCTGCGT 550   TGTTTCGGAC TCACCGCCTC CACCCCAGTA TCATGTTTGC GGGTGCCCTC 600   GGTGCATCCA ACTGCCCCGG CGCGCCCCGC CTCGACTTCT TCCTCGGCCG 650   CAAGGACGCC ACCCGCCCGG CCCCGGACGG GCTGGTCCCT GAGCCGTTCG 700   ACACGCTCGA GGACGTGTTC GCGCGTCTTG CCGACGCGTC GAGCGGCGAG 750   TTCGACGAGA TCCTGACGGT GTGGCTGCTG ACCGCGCACA CGATCGCGGC 800   GGCGGACCAC CTGGACGAGA CGATCCCGGG CACGCCGATG GACTCGACGC 850   CGCACATCTG GGACACGCAG TTCTTCATCG AGACGCAGCT GCGCGGGACG 900   GCGTTCCCGG GCAAGGGCGG GAACCACGGC GAGGTGATGT CGCCGCTCAA 950   GGGCGAGATC CGGCTGCAGA CGGACCACCT GCTCGCGCGC GACTCGCGCA 1000   CGGCGTGCGA GTGGCAGTCG TTCGTGAACA ACCAGCAGAA GGCGCAGGAC 1050   ATGTTCGCGT TCGTGTTCCA CGACCTGTCG ATGCTCGGCC AGGACCCGGA 1100   CAGTCTCATC GACTGCAGCG AGCTGGTGCG TGTTTCCTGC GCGACGCGCA 1150   GGTCGCGAGG GCTGACTGGC TGCGATGCAC AGATTCCGCA GCCCGCCCCG 1200   GTCATCGGAA CGGCCCACTT CCCCGCCGGC CTCAGCAACA AGGACATCGA 1250   GCAGGCGGTG CGCACGGTCT CCCCACTTTT CCATTTGCTG TTACTGAGTG 1300   CAGAATGCGT ATAGTGCGCG GACACCCCCT TCCCCACGCT CCCGACCCAG 1350   CCTGGCCCGA AGACCACCGT CGCCCCGGTG TGAGCTTCCC ACTATCCACT 1400   ACTCTTTACA CCCACACTCA TGCGTTCATT CCGTAGCCCC ATCAACTCTT 1450   GAACGCCTAA GGCGGTCGTC TAGGTAAACG GTAGACAGTA CACGGTTAAA 1500   CGTCGATTTG CGGAACTGGC CTTGAATTTC TTTACGTCTT CTGGTCTCGC 1550   GGCATTTTGC CCACGAGAGG GACGTACTCT TGTTGTATTA TCACGGGTGA 1600   ATGTAACATT CAATGTCGCG GTGTATAGAG TGCTACTATC TGCTTTGCGT 1650   GTATTCGCTC TTTCCGAAGC TTCTGAGTGG CAGAGACGAC ATACGTACTA 1700   CTCTGTACAT CGAGTCGA 1718

SEQ ID NO: 2 Sequence length: 1330 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA origin Organism name: Coriolus hirsutus Share name: IFO4917 Sequence features 22-1125 P CDS 22-99 P sig peptide 100-1125 P mat peptide   Array:   CTTACAGTGT ACTAGAACCT CATGGCGTTC AAGGCTCTTC TCTCCGCCGT 50   CTCCCTCATA GCGGCCTTCC AGGGCGCGAG CGCTGCATTG ACGAAACGCG 100   TCGCCTGCCC CGACGGCAAG AACACCGCGA CGAACGCGGC GTGCTGCCAG 150   CTGTTCGCGA TCCGCGACGA CATCCAGGAG AACCTCTTCC ACGGCGCGCA 200   GTGCGGCGAG AACGCGCACG AGTCCCTCCG CATCACCTTC CACGACGCGA 250   TCAGCTTCTC GCCCGCCCTC GAGGCCAAGG GCCAGTTTGG CGGCGGAGGT 300   GCAGACGGCT CTATCGCGAT TTTCCCTGAG ATCGAGACCG CGTTCCACGC 350   CAACATCGGC CTCGACGAGA TCGTCGCTGA GCAGAAGCCG CTCATCGCGC 400   GCCACAACAT CTCGCACGCC GACTTTATCA TGTTTGCGGG TGCCCTCGGT 450   GCATCCAACT GCCCCGGCGC GCCCCGCCTC GACTTCTTCC TCGGCCGCAA 500   GGACGCCACC CGCCCGGCCC CGGACGGCCT GGTCCCTGAG CCGTTCGACA 550   CGCTCGAGGA CGTGTTCGCG CGTCTTGCCG ACGCGTCGAG CGGCGAGTTC 600   GACGAGATCC TGACGGTGTG GCTGCTGACC GCGCACACGA TCGCGGCGGC 650   GGACCACCTG GACGAGACGA TCCCGGGCAC GCCGATGGAC TCGACGCCGC 700   ACATCTGGGA CACGCAGTTC TTCATCGAGA CGCAGCTGCG CGGGACGGCG 750   TTCCCGGGCA AGGGCGGGAA CCACGGCGAG GTGATGTCGC CGCTCAAGGG 800   CGAGATCCGG CTGCAGACGG ACCACCTGCT CGCGCGCGAC TCGCGCACGG 850   CGTGCGAGTG GCAGTCGTTC GTGAACAACC AGCAGAAGGC GCAGGACATG 900   TTCGCGTTCG TGTTCCACGA CCTGTCGATG CTCGGCCAGG ACCCGGACAG 950   TCTCATCGAC TGCAGCGAGC TGATTCCGCA GCCCGCCCCG GTCATCGGCA 1000   CGGCCCACTT CCCCGCCGGC CTCAGCAACA AGGACATCGA GCAGGCGTGC 1050   GCGGACACCC CCTTCCCCAC GCTCCCGACC CAGCCTGGCC CGAAGACCAC 1100   CGTCGCCCCG GTCCCCATCA ACTCTTGAAC GCCTAAGGCG GTCGTCTAGG 1150   TAAACGGTAG ACAGTACACG GTTAAACGTC GATTTGCGGA ACTGGCCTTG 1200   AATTTCTTTA CGTCTTCTGG TCTCGCGGCA TTTTGCCCAC GAGAGGGACG 1250   TACTCTTGTT GTATTATCAC GGGTGAATGT AACATTCAAT GTCGCGGTGT 1300   AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1330

SEQ ID NO: 3 Sequence length: 286 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: genomic DNA origin Organism name: Coriolus hirsutus Share name: IFO4917 Sequence features 84-92 CAAT-BOX 101-109 CAAT-BOX 197-203 TATA-BOX 113-126 heat shock element   Array:   AGATGTGGAC CGGGCCCGCG CGGCGCGGTG TAGGTCGTCC CCCACAGTGT 50   ACCGGCGGCA GTGTCGTCGG AATGCCTGCG CTGCGATTGG CTCGCGCACG 100   CTATTGGGAC CGTACGAACG TACTCGTCCT CCGGGAGTAC GGTGCAGCAC 150   TTCGCGCACC GTTCGTTTTC GTGTTCCGAG TGCGATGTCA GAGGAGTATA 200   AGTAGGGCGA AACGATCGCC GAGTGGCCTC ACGAGGCCTC CTCGGCTTCT 250   CCTCTGGGGA TCTCTCTTAC AGTGTACTAG AACCTC 286

SEQ ID NO: 4 Sequence length: 368 Sequence type: Amino acid origin Organism name: Coriolus hirsutus Share name: IFO4917 Sequence features 1-368 P CDS 1-26 P sig peptide 26-368 P mat peptide   Array:   Met Ala Phe Lys Ala Leu Leu Ser Ala Val Ser Leu Ile Ala Ala                     5 10 15   Phe Gln Gly Ala Ser Ala Ala Leu Thr Lys Arg Val Ala Cys Pro                    20 25 30   Asp Gly Lys Asn Thr Ala Thr Asn Ala Ala Cys Cys Gln Leu Phe                    35 40 45   Ala Ile Arg Asp Asp Ile Gln Glu Asn Leu Phe His Gly Ala Gln                    50 55 60      Cys Gly Glu Asn Ala His Glu Ser Leu Arg Ile Thr Phe His Asp                    65 70 75   Ala Ile Ser Phe Ser Pro Ala Leu Glu Ala Lys Gly Gln Phe Gly                    80 85 90   Gly Gly Gly Ala Asp Gly Ser Ile Ala Ile Phe Pro Glu Ile Glu                    95 100 105   Thr Ala Phe His Ala Asn Ile Gly Leu Asp Glu Ile Val Ala Glu                   110 115 120   Gln Lys Pro Leu Ile Ala Arg His Asn Ile Ser His Ala Asp Phe                   125 130 135   Ile Met Phe Ala Gly Ala Leu Gly Ala Ser Asn Cys Pro Gly Ala                   140 145 150   Pro Arg Leu Asp Phe Phe Leu Gly Arg Lys Asp Ala Thr Arg Pro                   155 160 165   Ala Pro Asp Gly Leu Val Pro Glu Pro Phe Asp Thr Leu Glu Asp                   170 175 180   Val Phe Ala Arg Leu Ala Asp Ala Ser Ser Gly Glu Phe Asp Glu                   185 190 195   Ile Leu Thr Val Trp Leu Leu Thr Ala His Thr Ile Ala Ala Ala                   200 205 210   Asp His Leu Asp Glu Thr Ile Pro Gly Thr Pro Met Asp Ser Thr                   215 220 225   Pro His Ile Trp Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg                   230 235 240   Gly Thr Ala Phe Pro Gly Lys Gly Gly Asn His Gly Glu Val Met                   245 250 255   Ser Pro Leu Lys Gly Glu Ile Arg Leu Gln Thr Asp His Leu Leu                   260 265 270      Ala Arg Asp Ser Arg Thr Ala Cys Glu Trp Gln Ser Phe Val Asn                   275 280 285   Asn Gln Gln Lys Ala Gln Asp Met Phe Ala Phe Val Phe His Asp                   290 295 300   Leu Ser Met Leu Gly Gln Asp Pro Asp Ser Leu Ile Asp Cys Ser                   305 310 315   Glu Leu Ile Pro Gln Pro Ala Pro Val Ile Gly Thr Ala His Phe                   320 325 330   Pro Ala Gly Leu Ser Asn Lys Asp Ile Glu Gln Ala Cys Ala Asp                   335 340 345   Thr Pro Phe Pro Thr Leu Pro Thr Gln Pro Gly Pro Lys Thr Thr                   350 355 360   Val Ala Pro Val Pro Ile Asn Ser                   365

SEQ ID NO: 5 Sequence length: 2004 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: genomic DNA origin Organism name: Coriolus hirsutus Share name: IFO4917 Sequence features 84-92 CAAT-BOX 101-109 CAAT-BOX 197-203 TATA-BOX 113-126 heat shock element 287-1735 P CDS 287-423 P sig peptide 424-1735 P mat peptide 348-406 intron 615-674 intron 810-864 intron 1412-1468 intron 1544-1600 intron 1666-1772 intron 790-792 P N-glycosylation site 565-567 P distal histidine 1073-1075 P proximal histidine   Array:   AGATGTGGAC CGGGCCCGCG CGGCGCGGTG TAGGTCGTCC CCCACAGTGT 50   ACCGGCGGCA GTGTCGTCGG AATGCCTGCG CTGCGATTGG CTCGCGCACG 100   CTATTGGGAC CGTACGAACG TACTCGTCCT CCGGGAGTAC GGTGCAGCAC 150   TTCGCGCACC GTTCGTTTTC GTGTTCCGAG TGCGATGTCA GAGGAGTATA 200   AGTAGGGCGA AACGATCGCC GAGTGGCCTC ACGAGGCCTC CTCGGCTTCT 250   CCTCTGGGGA TCTCTCTTAC AGTGTACTAG AACCTC ATG GCG TTC AAG GCT 301                                           Met Ala Phe Lys Ala                                                             Five   CTT CTC TCC GCC GTC TCC CTC ATA GCG GCC TTC CAG GGC GCG AGC 346   Leu Leu Ser Ala Val Ser Leu Ile Ala Ala Phe Gln Gly Ala Ser                    10 15 20   G GTACGGGGCC TCACCTTCAC TGCCCTAACA CCCCAGATCG TCACGCCCAG 397   A   CACCGCTAG CT GCA TTG ACG AAA CGC GTC GCC TGC CCC GAC GGC AAG 444             la Ala Leu Thr Lys Arg Val Ala Cys Pro Asp Gly Lys                             25 30   AAC ACC GCG ACG AAC GCG GCG TGC TGC CAG CTG TTC GCG ATC CGC 489   Asn Thr Ala Thr Asn Ala Ala Cys Cys Gln Leu Phe Ala Ile Arg        35 40 45   GAC GAC ATC CAG GAG AAC CTC TTC CAC GGC GCG CAG TGC GGC GAG 534   Asp Asp Ile Gln Glu Asn Leu Phe His Gly Ala Gln Cys Gly Glu        50 55 60   AAC GCG CAC GAG TCC CTC CGC ATC ACC TTC CAC GAC GCG ATC AGC 579   Asn Ala His Glu Ser Leu Arg Ile Thr Phe His Asp Ala Ile Ser        65 70 75   TTC TCG CCC GCC CTC GAG GCC AAG GGC CAG TTT GG GTAGGTCGCC 624   Phe Ser Pro Ala Leu Glu Ala Lys Gly Gln Phe Gl        80 85 9   CCTTCTCCGC TGCGGAGAGA GGTGGATAGT TCTCTGACGG CACTTTGCAG C GGC 678                                                          y Gly                                                          0   GGA GGT GCA GAC GGC TCT ATC GCG ATT TTC CCT GAG ATC GAG ACC 723   Gly Gly Ala Asp Gly Ser Ile Ala Ile Phe Pro Glu Ile Glu Thr                95 100 105   GCG TTC CAC GCC AAC ATC GGC CTC GAC GAG ATC GTC GCT GAG CAG 768   Ala Phe His Ala Asn Ile Gly Leu Asp Glu Ile Val Ala Glu Gln               110 115 120   AAG CCG CTC ATC GCG CGC CAC AAC ATC TCG CAC GCC GAC TT 809   Lys Pro Leu Ile Ala Arg His Asn Ile Ser His Ala Asp Ph               125 130 13   GTGATGCTCT GCACTACACC TCTGCGTTGT TTCGGACTCA CCGCCTCCAC 859   CCCAG T ATC ATG TTT GCG GGT GCC CTC GGT GCA TCC AAC TGC CCC 904         e Ile Met Phe Ala Gly Ala Leu Gly Ala Ser Asn Cys Pro         5 140 145      GGC GCG CCC CGC CTC GAC TTC TTC CTC GGC CGC AAG GAC GCC ACC 949   Gly Ala Pro Arg Leu Asp Phe Phe Leu Gly Arg Lys Asp Ala Thr       150 155 160   CGC CCG GCC CCG GAC GGG CTG GTC CCT GAG CCG TTC GAC ACG CTC 994   Arg Pro Ala Pro Asp Gly Leu Val Pro Glu Pro Phe Asp Thr Leu       165 170 175   GAG GAC GTG TTC GCG CGT CTT GCC GAC GCG TCG AGC GGC GAG TTC 1039   Glu Asp Val Phe Ala Arg Leu Ala Asp Ala Ser Ser Gly Glu Phe       180 185 190   GAC GAG ATC CTG ACG GTG TGG CTG CTG ACC GCG CAC ACG ATC GCG 1084   Asp Glu Ile Leu Thr Val Trp Leu Leu Thr Ala His Thr Ile Ala       195 200 205   GCG GCG GAC CAC CTG GAC GAG ACG ATC CCG GGC ACG CCG ATG GAC 1129   Ala Ala Asp His Leu Asp Glu Thr Ile Pro Gly Thr Pro Met Asp       210 215 220   TCG ACG CCG CAC ATC TGG GAC ACG CAG TTC TTC ATC GAG ACG CAG 1174   Ser Thr Pro His Ile Trp Asp Thr Gln Phe Phe Ile Glu Thr Gln       225 230 235   CTG CGC GGG ACG GCG TTC CCG GGC AAG GGC GGG AAC CAC GGC GAG 1219   Leu Arg Gly Thr Ala Phe Pro Gly Lys Gly Gly Asn His Gly Glu       240 245 250   GTG ATG TCG CCG CTC AAG GGC GAG ATC CGG CTG CAG ACG GAC CAC 1264   Val Met Ser Pro Leu Lys Gly Glu Ile Arg Leu Gln Thr Asp His       255 260 265   CTG CTC GCG CGC GAC TCG CGC ACG GCG TGC GAG TGG CAG TCG TTC 1309   Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys Glu Trp Gln Ser Phe       270 275 280      GTG AAC AAC CAG CAG AAG GCG CAG GAC ATG TTC GCG TTC GTG TTC 1354   Val Asn Asn Gln Gln Lys Ala Gln Asp Met Phe Ala Phe Val Phe       285 290 295   CAC GAC CTG TCG ATG CTC GGC CAG GAC CCG GAC AGT CTC ATC GAC 1399   His Asp Leu Ser Met Leu Gly Gln Asp Pro Asp Ser Leu Ile Asp       300 305 310   TGC AGC GAG CTG GTGCGTGTTT CCTGCGCGAC GCGCAGGTCG CGAGGGCTG 1450   Cys Ser Glu Leu       315   ACTGGCTGCG ATGCACAG ATT CCG CAG CCC GCC CCG GTC ATC GGA ACG 1498                       Ile Pro Gln Pro Ala Pro Val Ile Gly Thr                               320 325   GCC CAC TTC CCC GCC GGC CTC AGC AAC AAG GAC ATC GAG CAG GCG 1543   Ala His Phe Pro Ala Gly Leu Ser Asn Lys Asp Ile Glu Gln Ala           330 335 340   GTGCGCACGG TCTCCCCACT TTTCCATTTG CTGTTACTGA GTGCAGAATG 1593   CGTATAG TGC GCG GAC ACC CCC TTC CCC ACG CTC CCG ACC CAG CCT 1639           Cys Ala Asp Thr Pro Phe Pro Thr Leu Pro Thr Gln Pro                   345 350 355   GGC CCG AAG ACC ACC GTC GCC CCG GT GTGAGCTTCC CACTATCCAC 1685   Gly Pro Lys Thr Thr Val Ala Pro Va                   360   TACTCTTTAC ACCCACACTC ATGCGTTCAT TCCGTAG C CCC ATC AAC TCT 1735                                            l Pro Ile Asn Ser                                              365   TGAACGCCTA AGGCGGTCGT CTAGGTAAAC GGTAGACAGT ACACGGTTAA 1785   ACGTCGATTT GCGGAACTGG CCTTGAATTT CTTTACGTCT TCTGGTCTCG 1835   CGGCATTTTG CCCACGAGAG GGACGTACTC TTGTTGTATT ATCACGGGTG 1885   AATGTAACAT TCAATGTCGC GGTGTATAGA GTGCTACTAT CTGCTTTGCG 1935   TGTATTCGCT CTTTCCGAAG CTTCTGAGTG GCAGAGACGA CATACGTACT 1985   ACTCTGTACA TCGAGTCGA 2004

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 9/08 C12R 1:19) (72) Inventor Yukio Kita 1-10-6 Shinonome, Koto-ku, Tokyo Oji Paper Co., Ltd. Central Research Laboratory (56) Reference JP-A-1-27468 (JP, A) Biochemi, Vol. 74, No. 2, P.I. 177-182 RW Old, Translated by Motoaki Anai, "Principle of Gene Manipulation" 3rd Edition, Baifukan, April 20, 1987, P. 115 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/53 GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneS eq

Claims (8)

(57) [Claims] 1. A lignin peroxidase gene comprising a DNA encoding the amino acid sequence represented by SEQ ID NO: 4. 2. A lignin peroxidase structural gene represented by the nucleotide sequence of SEQ ID NO: 1. 3. A lignin peroxidase gene represented by the nucleotide sequence of SEQ ID NO: 2. 4. A control region gene having a promoter activity which is induced by high temperature and which is represented by the nucleotide sequence of SEQ ID NO: 3. 5. A recombinant vector containing the nucleotide sequence according to claim 1. 6. A recombinant vector containing the control region gene according to claim 4. 7. A transformant containing the recombinant vector according to claim 5. 8. A transformant containing the recombinant vector according to claim 6.