WO2003070939A1 - Fragment d'adn ayant une activite de promoteur - Google Patents

Fragment d'adn ayant une activite de promoteur Download PDF

Info

Publication number
WO2003070939A1
WO2003070939A1 PCT/JP2003/002057 JP0302057W WO03070939A1 WO 2003070939 A1 WO2003070939 A1 WO 2003070939A1 JP 0302057 W JP0302057 W JP 0302057W WO 03070939 A1 WO03070939 A1 WO 03070939A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
seq
sequence
promoter
enzyme
Prior art date
Application number
PCT/JP2003/002057
Other languages
English (en)
Japanese (ja)
Inventor
Akira Tsukamoto
Seiji Nakagame
Mari Kabuto
Original Assignee
Oji Paper Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oji Paper Co., Ltd. filed Critical Oji Paper Co., Ltd.
Priority to AU2003211681A priority Critical patent/AU2003211681A1/en
Priority to JP2003569832A priority patent/JP4016948B2/ja
Publication of WO2003070939A1 publication Critical patent/WO2003070939A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes

Definitions

  • the present invention relates to a novel DNA fragment having a promoter activity obtained from basidiomycetes, particularly Coriolus hirsutus, a recombinant DNA containing the same, a vector containing the DNA fragment or the recombinant DNA, and a host containing the vector.
  • the present invention relates to a cell, a method for high production of a lignin-degrading enzyme using the host cell, and a method for treating wood chips using the enzyme.
  • E. coli is often not suitable as a host.
  • Escherichia coli produces useful polypeptides derived from higher organisms such as humans, it is not produced as an active enzyme protein.
  • problems such as the difficulty in purifying the product.
  • methods for producing lower eukaryotic yeast as a host have been actively studied, but a new problem of low productivity has arisen. Therefore, for production of polypeptides, eukaryotes such as Aspergillus spp.
  • Basidiomycetes belong to eukaryotes, but are thought to be more closely related to animal cells than yeast (TL Smith, Proc. Natl. Acad. Sci. USA, 86, 7063 (1989)). Lignin-degrading ability is strong ⁇ Coriolus hirstus is a basidiomycete belonging to the genus Coriolus, and a host-vector system was developed by the present inventors. The production of oxidase has been successful (Japanese Patent Application Laid-Open No. 6-054691).
  • the promoter region used is an amino acid synthase gene. Because of the promoter region of the phenoloxidase gene involved in lignin degradation, which is involved in the degradation of the lignin peroxidase gene lignin peroxidase, the wild type strain IF04917 can be used to produce the lignin peroxidase production medium ( Low carbon and low nitrogen sources), and there were problems such as the fact that lignin peroxidase was obtained as a secondary metabolite and required a long time for gene expression. In addition, it has been reported that promoters used in other species, such as filamentous fungi, for enzyme protein production by genetic recombination do not function in Basidiomycetes (A. Lorna, et. ., Curr. Genet., 16, 35 (1989)).
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that basidiomycetes, particularly Coriolus. Focusing on the cellulose degrading enzyme gene, DNA fragments encoding various cellulose degrading enzyme genes were cloned from the chromosomal DNA restriction enzyme fragment of Coriolus hirustas, and the promoter region upstream of this gene was sequenced. It has been found that the promoter region is effective for expression of a gene encoding a useful polypeptide in a host / vector system.
  • a structural gene containing a manganese peroxidase gene, a high-temperature inducible lignin peroxidase gene Japanese Patent Laid-Open Publication No. 5-260978
  • a signal peptide of a laccase gene cloned from Coriolus hilustus.
  • a host cell oretin-potassium rubamoyltransferase-deficient Coriolus hirustas mutant, to obtain a manganese peroxidase, lignin peroxidase or laccase high-producing strain.
  • the invention has been completed.
  • the present invention provides the following (1) to (12).
  • a recombinant DNA comprising a DNA fragment containing a promoter sequence of a cellulolytic enzyme gene derived from a basidiomycete, and a gene encoding a lignin degrading enzyme, wherein the gene is transcribed and linked to the DNA fragment.
  • a method for producing lignin-degrading enzyme comprising the step of producing lignin-degrading enzyme.
  • a recombinant DNA comprising a DNA fragment containing a promoter sequence of a basidiomycete-derived cellulolytic enzyme gene and a gene encoding a lignin degrading enzyme, wherein the gene is transcribed and linked to the DNA fragment ,
  • a step of preparing a vector containing, and a step of performing transformation using the vector to prepare a host cell that produces high lignin degrading enzyme comprising the step of treating.
  • the promoter sequence of the cellulolytic enzyme gene is promoted by the cellobiose dehydrogenase gene, cellobiohydrolase I gene, cellobio hydrolase II gene, endorcanase gene, or jS-dalcosidase gene.
  • a DNA fragment containing the promoter sequence of the cellulolytic enzyme gene was isolated and contained a celloose dehydrogenase gene promoter sequence having any one of the following nucleotide sequences (a) to (c): The method according to (1), (2) or (3), which is a DNA fragment.
  • nucleotide sequence having one or more nucleotides deleted, substituted or added in SEQ ID NO: 1 or 2, and having cellobiose dehydrogenase gene promoter activity;
  • the DNA fragment containing the promoter sequence of the cellulolytic enzyme gene is isolated and contains the cellobiohydrolase I gene promoter sequence having any one of the following nucleotide sequences (a) to (c): The method according to (1), (2) or (3), wherein the fragment is a DNA fragment.
  • the DNA fragment containing the promoter sequence of the cellulolytic enzyme gene is an isolated DNA fragment containing the cellobiohydrolase II gene promoter sequence having any one of the following nucleotide sequences (a) to (c): The method according to (1), (2) or (3).
  • nucleotide sequence having a nucleotide sequence in which one or more nucleotides have been deleted, substituted or added in SEQ ID NO: 49, and which has cellobiohydrolase II gene promoter activity.
  • a lignin degrading enzyme is selected from the group consisting of a manganese peroxidase gene, a lignin peroxidase gene, and a laccase gene.
  • FIG. 1 is a diagram showing the structure of a manganese peroxidase-expressing plasmid PCHCBHI31PMP described in Example 13.
  • FIG. 2 is a diagram showing the structure of the laccase expression plasmid pCHCBHI31PLAC described in Example 16.
  • FIG. 3 shows the structure of the lignin peroxidase expression vector P CHCBHI31PLiP described in Example 19.
  • FIG. 4 is a diagram showing the structure of a manganese peroxidase expression plasmid PCHCBHI27PMP described in Example 23.
  • FIG. 5 is a diagram showing the structure of the laccase expression plasmid pCHCBHI27PLAC described in Example 26.
  • Figure 6 is a diagram showing the lignin peroxide O Kishida over peptidase expression vector P structure CHCBHI27PLiP described in Example 2 9.
  • FIG. 7 is a diagram showing the structure of manganese peroxidase expression plasmid PCHCBHI26PMP described in Example 33.
  • Figure 8 is a diagram showing a structure of a laccase expression plasmid P CHCBHI26PLAC described in Example 3 6.
  • FIG. 9 is a diagram showing the structure of the lignin peroxidase expression vector pCHCBHI26PLiP described in Example 39.
  • FIG. 10 shows the structure of the manganese peroxidase expression plasmid pCHCBHIIPMP described in Example 43.
  • FIG. 11 is a diagram showing the structure of the laccase expression plasmid pCHCBHIIPLAC described in Example 46.
  • FIG. 12 shows the structure of the lignin peroxidase expression plasmid pCHCBHIIPLiP described in Example 49. Description of Sequence Listing
  • SEQ ID NO: 3 shows a probe for screening a clone containing a cellobiose dehydrogenase gene derived from Coriolus' Hilstas IF04917 strain.
  • SEQ ID NO: 4 shows a sense primer for synthesizing a 2.3 kbp DNA fragment containing a cellobiose dehydrogenase gene promoter region derived from C. hinoretus IF04917 by PCR.
  • SEQ ID NO: 5 was obtained by synthesizing a 2.3 kbp DNA fragment containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirustas IF04917 by PCR. Shows the sense primer for synthesizing a 2.2 kbp DNA fragment containing the oxidase structural gene by PCR.
  • SEQ ID NO: 7 shows an antisense primer for synthesizing, by PCR, a 2.2 kbp DNA fragment containing a manganese peroxidase structural gene portion derived from C. hirstus IF04917.
  • SEQ ID NO: 8 shows a sense primer for synthesizing, by PCR, a 1.7 kbp DNA fragment containing a lignin peroxidase structural gene portion derived from Coriolus hirustas IF04917.
  • SEQ ID NO: 9 shows a sense primer for synthesizing, by PCR, a DNA fragment containing a cellobiose dehydrogenase gene promoter region derived from Coriolus hirstus IF04917.
  • SEQ ID NO: 10 shows an antisense primer for synthesizing, by PCR, a DNA fragment containing a cellobiose dehydrogenase gene promoter region derived from Coriolus hirustas IF04917.
  • SEQ ID NO: 11 shows a sense primer for synthesizing, by PCR, a DNA fragment containing a laccase structural gene portion derived from Coriolus hirustas IF04917.
  • SEQ ID NO: 12 shows an antisense primer for synthesizing, by PCR, a DNA fragment containing a laccase structural gene portion derived from Coriolus' Hilstas IF04917.
  • SEQ ID NO: 16 shows a probe for screening a clone containing a plasmid PCHCBHI31 gene derived from Coriolus' Hilstas IF04917 strain.
  • the base “y” indicates thymidine or cytidine, and the base “r” indicates guanosine or adenosine.
  • SEQ ID NO: 17 shows a sense primer for synthesizing a 2.3 kbp Ncol fragment containing a plasmid pCHCBHI31 gene promoter region derived from Coriolus coriolus' Hilstas IF04917 strain by PCR.
  • SEQ ID NO: 18 shows an antisense primer for PCR-synthesizing a 2.3 kbp Ncol fragment containing a plasmid pCHCBHI31 gene promoter region derived from Coriolus coriolus' Hilstas IF04917 strain.
  • SEQ ID NO: 19 shows a sense primer for synthesizing, by PCR, a 2.2 kbp Ncol fragment containing a mangan peroxidase structural gene portion derived from Coriolus genus Coriolus hirustas IF04917.
  • SEQ ID NO: 20 synthesizes a 2.2 kbp Ncol fragment containing the mangan peroxidase structural gene derived from Coriolus genus Coriolus hirustas IF0 4917 by PCR.
  • the following shows a sense primer for PCR-based synthesis of a 2.4 kbp XhoI-EcoRI fragment containing the derived laccase structural gene.
  • SEQ ID NO: 22 shows an antisense primer for synthesizing a 2.4 kbp Xhol-EcoRI fragment containing a laccase structural gene portion derived from Coriolus genus Coriolus hirustus IF04917 by PCR.
  • SEQ ID NO: 23 shows a sense primer for synthesizing a 2.2 kbp Ncol fragment containing a lignin peroxidase structural gene portion derived from Coriolus genus Coriolus hirustas IF04917 by PCR.
  • SEQ ID NO: 24 synthesizes a 2.2 kbp Ncol fragment containing the lignin peroxidase structural gene portion derived from Coriolus coriolus' Hilstas IF0 4917 strain by PCR.
  • SEQ ID NO: 28 is derived from Coriolus Hilstas IF0 4917 strain. 1 shows a probe for screening for a clone containing the cellohydrohydrolase II gene. The base "i" indicates inosine.
  • SEQ ID NO: 29 shows a sense primer for synthesizing, by PCR, a 3.1 kbp Ncol fragment containing a cellohydrolase'II (CBHII) gene promoter region derived from C. hirstus IF04917.
  • CBHII cellohydrolase'II
  • SEQ ID NO: 30 is a cellophyllohydrolase gene promoter region derived from Coriolus hirustas IF04917 strain.3.1 kbp Ncol fragment is synthesized by PCR.
  • SEQ ID NO: 31 is derived from Coriolus hirustas IF04917 strain. Shown below are the sense primers for synthesizing a 2.2 kbp Ncol fragment containing the manganese peroxidase structural gene by PCR.
  • SEQ ID NO: 32 is a fragment of a 2.2 kbp Ncol fragment containing a manganese peroxidase structural gene portion derived from C. coli hirustas IF04917. Shows the sense primer.
  • SEQ ID NO: 33 shows a sense primer for PCR-synthesizing a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from Coriolus' Hilstas IF04917.
  • SEQ ID NO: 34 shows an antisense primer for PCR-synthesizing a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from Coriolus hirutas IF04917.
  • SEQ ID NO: 35 shows a sense primer for PCR-synthesizing a 2.2 kbp Ncol fragment containing a lignin peroxidase structural gene portion derived from Coriolus hirustas IF04917.
  • SEQ ID NO: 36 shows an antisense primer for PCR-based synthesis of a 2.2 kbp Ncol fragment containing a lignin peroxidase structural gene portion derived from Coriolus' Hilstas IF04917 strain.
  • SEQ ID NO: 40 shows a probe for screening a clone containing the cellobio hydrolase II gene derived from C. hirstus IF04917.
  • the base “y” represents thymidine or cytidine, and the base “r” represents guanosine or adenosine.
  • SEQ ID NO: 41 represents a sense primer for synthesizing a 1.1 kbp Ncol fragment containing a cellohydrohydrase II (CBHI26) gene promoter region derived from Coriolus hirutas IF04917 by PCR.
  • CBHI26 cellohydrohydrase II
  • SEQ ID NO: 42 was obtained by PCR using a 1.1 kbp Ncol fragment containing the cellobio hydrolase II (CBHI26) gene promoter region derived from Coriolus hirustas IF0 4917.
  • SEQ ID NO: 43 was obtained from Coriolus hirutas IF0 4917 strain. Shown below are the sense primers for synthesizing a 2.2 kbp Ncol fragment containing the manganese peroxidase structural gene portion by PCR.
  • SEQ ID NO: 44 shows an antisense primer for synthesizing, by PCR, a 2.2 kbp Ncol fragment containing a manganese peroxidase structural gene portion derived from Coriolus hirstas IF04917.
  • SEQ ID NO: 45 shows a sense primer for PCR-synthesizing a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from Coriolus' Hilstas IF04917.
  • SEQ ID NO: 46 shows an antisense primer for synthesizing, by PCR, a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from C. holorum IF0 4917 strain.
  • SEQ ID NO: 47 shows a sense primer for synthesizing, by PCR, a 2.2 kbp Ncol fragment containing a lignin peroxidase structural gene portion derived from Coriolus hirustas IF04917.
  • SEQ ID NO: 48 shows an antisense primer for synthesizing, by PCR, a 2.2 kbp Ncol fragment containing a lignin peroxidase ⁇ "ze structural gene portion derived from Coriolus' Hilstas IF04917.
  • SEQ ID NO: 52 shows a probe for screening a clone containing the cellobio hydrolase II gene derived from Coriolus hirustas IF04917.
  • the base "i" indicates inosine.
  • SEQ ID NO: 53 shows a sense primer for synthesizing, by PCR, a 3.1 kbp Ncol fragment containing the cellobio hydrolase II gene promoter region derived from Coriolus' Hilstas IF04917.
  • SEQ ID NO: 54 is for synthesizing a 3.1 kbp Ncol fragment containing the cellobio hydrolase II gene promoter region derived from Coriolus hirustas IF04917 by PCR.
  • SEQ ID NO: 55 is derived from Coriolus hirustas IF04917. Shown below are the sense primers for synthesizing a 2.2 kbp Ncol fragment containing the manganese peroxidase structural gene by PCR.
  • SEQ ID NO: 56 shows an antisense primer for synthesizing, by PCR, a 2.2 kbp Ncol fragment containing a manganese peroxidase structural gene portion derived from C. hirstus IF04917.
  • SEQ ID NO: 57 is a sense primer for PCR-synthesizing a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from Coriolus hiltus IF04917. Show one.
  • SEQ ID NO: 58 shows an antisense primer for synthesizing a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from Coriolus hirutas IF04917 by PCR.
  • SEQ ID NO: 59 shows a sense primer for synthesizing, by PCR, a 2.2 kbp Ncol fragment containing a lignin peroxidase structural gene portion derived from Coriolus hirustas IF04917.
  • SEQ ID NO: 60 shows an antisense primer for synthesizing, by PCR, a 2.2 kbp Ncol fragment containing a lignin peroxidase structural gene portion derived from C. hirstus IF04917.
  • the present invention in a first aspect, comprises a DNA fragment comprising a promoter sequence of a basidiomycete-derived cellulose-degrading enzyme gene, and a gene encoding a lignin-degrading enzyme; A step of preparing a vector containing the recombinant DNA linked to the fragment; a step of performing transformation using the vector to prepare a host cell that produces high lignin-degrading enzyme; A method for producing a lignin-degrading enzyme, the method comprising culturing a host cell in the presence of cellulose to produce a lignin-degrading enzyme.
  • the first step of the method comprises: a DNA fragment containing a promoter sequence of a basidiomycete-derived cellulose-degrading enzyme gene; and a gene encoding a lignin-degrading enzyme, wherein the DNA fragment is transcribed.
  • the promoter sequence of the cellulose-degrading enzyme gene is not particularly limited as long as it is a promoter sequence of an enzyme capable of degrading cellose, and is a cellobiose dehydrogenase gene, a cellobiohydrolase I gene, a cellobiohydrolase II gene. , The endoglucanase gene, or the promoter sequence of the / 3-dalcosidase gene.
  • the basidiomycete any bacterium can be used as long as it is a genus having the ability to degrade cellulose. In particular, Coriolus hirsutus sp.
  • DNA fragment containing the promoter sequence of the cellulolytic enzyme gene for example, DNA fragments shown in the following 1 to 3 can be used.
  • nucleotide sequence having a nucleotide sequence complementary to the nucleotide sequence of (a) and a nucleotide sequence that hybridizes under stringent conditions, and having a cellobiose dehydrogenase gene promoter activity .
  • nucleotide sequence having one or more nucleotides deleted, substituted or added in SEQ ID NO: 1 or 2, and having cellobiose dehydrogenase gene promoter activity;
  • the isolated DNA fragment containing the cellobiose dehydrogenase gene promoter sequence described in 1 above can be obtained by the following procedure.
  • chromosomal DNA In order to prepare chromosomal DNA from Coriolus hilstas, an ordinary method for extracting chromosomal DNA such as the method of Yelton et al. [Proc. Natl. Acad. Sci. USA, 81, 1470 (1984)] can be used. Next, the obtained chromosomal DNA is treated with an appropriate restriction enzyme such as Sau3AI, partially digested, and fractionated by sucrose gradient ultracentrifugation to obtain a fragment of 10 kbp to 25 kbp. obtain. The DNA fragment obtained above is ligated to phage DNA treated with a restriction enzyme that generates the same cohesive end. As the phage DNA, for example, EMB L3 [A-M, Frishauf et al., J. Mol.
  • ⁇ phage DNA is used.
  • the obtained DNA fragment-linked phage is packaged in vitro to obtain a chromosome DNA library.
  • a commonly used cloning vector preferably an Escherichia coli cloning vector, for example, pUC-based vector ⁇ UC18 [C. Yanisch-Perron et al., Gene, 33, 103 (1985)] or the like is used. be able to.
  • Cloning vectors are not limited to those exemplified above, and may be commercially available or known ones described in the literature.
  • the purified celliobiose dehydrogenase of Coriolus hirstus was completely digested with lysyl endopeptidase and subjected to amino acid sequencing.
  • a synthetic DNA probe prepared based on the nucleotide sequence deduced from this amino acid sequence a clone containing the promoter region of the cellopsidase hydrogenase gene is selected by plaque hybridization.
  • the DNA fragment containing the promoter sequence of the cellobiose dehydrogenase gene is isolated from the selected clone, and a restriction map is created and sequenced.
  • the fragment containing the cellobiose dehydrogenase gene described above was inserted into an appropriate cloning vector (for example, a pUC-based vector such as pUC19), and then transferred to Sanger et al. Natl. Acad. Sci. USA, 74, 5463 (1977)).
  • an appropriate cloning vector for example, a pUC-based vector such as pUC19
  • SEQ ID NO: 1 or 2 was determined.
  • the DNA fragment containing the cellobiose dehydrogenase gene promoter sequence can be obtained from the fragment containing the cellobiose dehydrogenase gene by PCR (polymerase chain reaction).
  • PCR polymerase chain reaction
  • a primer for PCR a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases, based on the base sequence represented by SEQ ID NO: 1 or its complementary sequence is sensed. It can be used as a primer or an antisense primer.
  • a sense primer shown in SEQ ID NO: 4 and an antisense primer shown in SEQ ID NO: 5 can be used. (See Example 3). It can also be obtained by chemical synthesis according to a conventional method.
  • the promoter sequence or promoter region according to the present invention has a function of regulating the transcription of a structural gene, and a functional nucleotide sequence substantially conserved in a eukaryotic promoter is motif-modified (TATA, CCAA). , GC box, etc.). Therefore, the nucleotide sequence represented by SEQ ID NO: 1 is one specific example of such a promoter sequence, and as long as it has promoter activity, it hybridizes under stringent conditions to a sequence complementary to the nucleotide sequence. Soy sequences are also within the scope of the present invention.
  • stringent conditions refers to, for example, a sodium concentration of 15 to 90 O mM, preferably 15 to 15 O mM, and a temperature of 37 to 80 ° C, preferably It means the condition at 50 to 65 ° C.
  • site-specific mutagenesis techniques such as oligonucleotide site-specific mutagenesis and cassette mutagenesis (for example, based on the nucleotide sequence represented by SEQ ID NO: 1 or 2 Short Protocols In Molecular Biology, Third Edition, John Wiley & Sons, Inc.).
  • Escherichia coli JM109 / pCHCDHl and Escherichia coli JM109 / pCHCDH2 which contain genomic DNA fragments containing the cellulomotor dehydrogenase gene motor sequence derived from Coriolus hirstas, were released on February 8, 2002.
  • National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (Japan Deposited at Tsukuba East 1-chome 1-Chome No. 6), Central Ibaraki Prefecture, Japan, and have been assigned accession numbers FERM BP-8278 and FERM BP-8279, respectively.
  • DNAs having the nucleotide sequence represented by SEQ ID NO: 1 contained in these deposited strains are also included in the present invention.
  • a chromosome gene library was prepared from Choriolas hirstas.
  • a synthetic DNA probe prepared from the obtained chromosomal gene library based on the base sequence of the cellobiohydrolase I gene isolated from other species (for example, Phanerochaete chrysosporiura).
  • a clone containing both the cellobio-hydrolase I gene and the promoter region is selected by plaque hybridization using E. coli.
  • the DNA fragment containing the gene of interest is isolated from the selected clone and sequenced.
  • the sequence was determined by inserting the above-mentioned fragment containing the cellobiohydrolase I chromosomal gene into an appropriate closing vector (for example, a pUC-based vector such as pUC19), and using the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)).
  • an appropriate closing vector for example, a pUC-based vector such as pUC19
  • the gene having this sequence is a genomic gene consisting of 4624 base pairs (SEQ ID NO: 14) containing the cellobiohydrolase I gene derived from Choriolus and a cellobiohydrolase I gene promoter region upstream thereof.
  • the promoter region exists at base numbers 1-2293 (SEQ ID NO: 13), and TATAAA is found at base numbers 2202-2207.
  • the cellobiohydrolase I structural gene exists at base numbers 2294 to 3775, and is composed of three exons and two introns (intervening sequences). Specifically, intron 1 exists at 2586-2633, and intron 2 exists at 2965-23022. Further, base numbers 3776 to 4624 are a 3 'untranslated region including a terminator.
  • the amino acid sequence deduced from the sequence analysis was found to be the amino acid sequence shown in SEQ ID NO: 15 consisting of 457 amino acid residues.
  • the DNA fragment containing the cellobiohydrolase I gene promoter region can be obtained by PCR (polymerase chain reaction) from the above-mentioned fragment containing the cellobiohydrolase I chromosomal gene.
  • PCR polymerase chain reaction
  • a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence shown in SEQ ID NO: 13 or its complementary sequence is used as a sense primer or an antisense primer.
  • the sense primer shown in SEQ ID NO: 17 and the antisense primer shown in SEQ ID NO: 18 can be used (see Example 15). It can also be obtained by chemical synthesis according to a conventional method.
  • the base sequence shown in SEQ ID NO: 13 is one specific example of such a promoter sequence. As long as the base sequence has promoter activity, deletion, substitution, addition, etc. of one or more bases in the base sequence can be performed. Sequences containing mutations or modifications are also within the scope of the invention. Such mutation or modification can be performed by a well-known site-specific mutation, such as oligonucleotide site-directed mutagenesis or cassette mutation based on the nucleotide sequence shown in SEQ ID NO: 13 (for example, Short protocols In Molecular Biology, Third Edition, John Wiley & Sons, Inc.).
  • the sequence of the second DNA fragment was determined in the same manner as in the case of the first DNA fragment described above. As a result, the nucleotide sequence shown in SEQ ID NO: 26 was determined.
  • the gene having this sequence is a genomic gene consisting of Coriolus-derived cellobiohydrolase I gene and a 4170 base pair upstream of which contains a cellobiohydrolase I gene promoter region.
  • the promoter region is present at nucleotides 1 to 2182 (SEQ ID NO: 25), and TATAAA is found at nucleotides 2099 to 2104.
  • the cellobiohydrolase I gene is present at base numbers 211-3667, and is composed of three exons and two introns (intervening sequences).
  • intron 1 exists at 2475 to 2535
  • intron 2 exists at 2862 to 2917
  • base numbers 3668 to 4170 are a 3 'untranslated region containing a terminator.
  • the amino acid sequence deduced from the sequence analysis was found to be the amino acid sequence shown in SEQ ID NO: 27 consisting of 457 amino acid residues. Was.
  • the second DNA fragment containing the cellobiohydrolase I gene promoter region can be obtained by PCR (polymerase chain reaction) from the above fragment containing the cellobiohydrolase I chromosomal gene.
  • PCR polymerase chain reaction
  • a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence shown in SEQ ID NO: 25 or its complementary sequence is used as a sense primer or an antisense primer.
  • a sense primer shown in SEQ ID NO: 29 and an antisense primer shown in SEQ ID NO: 30 can be used (see Example 25).
  • the nucleotide sequence shown in SEQ ID NO: 25 is one specific example of such a promoter sequence. As long as it has promoter activity, deletion, substitution, addition, etc. of one or more nucleotides in the nucleotide sequence can be performed. Sequences containing mutations or modifications are also within the scope of the invention. Such a mutation or modification can be performed based on the nucleotide sequence shown in SEQ ID NO: 1 by well-known site-specific mutagenesis techniques (eg, Short protocols In Molecular Biology, Third Edition, John Wiley & Sons, Inc.).
  • the third DNA fragment was sequenced as in the case of the first DNA fragment above.
  • the base sequence shown in SEQ ID NO: 38 was determined.
  • the gene having this sequence is a genomic gene consisting of 39.16 base pairs including a cellohydrohydrase I gene derived from Coriolus and a cellohydrohydrase I gene promoter region upstream thereof.
  • the promoter region exists at base numbers 1 to 1086 (SEQ ID NO: 37), and TATAAA is found at base numbers 1001 to 1006.
  • the cellobiohydrolase I structural gene is present at base numbers 1087-1259, and is composed of three exons and two introns (intervening sequences).
  • intron 1 is present at 1379-1444, and intron 2 is present at 1771-1829. Furthermore, base numbers 2580 to 3916 are terminators. 3 'untranslated region containing It was ascertained that the amino acid sequence deduced from the sequence analysis was the amino acid sequence shown in SEQ ID NO: 39 consisting of 456 amino acid residues.
  • the third DNA fragment containing the cellobiohydrolase I gene promoter region can be obtained from the above-mentioned fragment containing the cellobiohydrolase I chromosomal gene by PCR (polymerase chain reaction).
  • PCR polymerase chain reaction
  • a primer for PCR a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence shown in SEQ ID NO: 37 or its complementary sequence is a sense primer or an antisense primer.
  • the sense primer shown in SEQ ID NO: 41 and the antisense primer shown in SEQ ID NO: 42 can be used (see Example 25).
  • the base sequence shown in SEQ ID NO: 37 is one specific example of such a promoter sequence, and one or more bases may be deleted, substituted, added, etc. in the base sequence as long as it has one promoter activity. Sequences containing mutations or modifications of are also within the scope of the invention. Such mutations or modifications can be performed using well-known site-directed mutagenesis techniques such as oligonucleotide site-directed mutagenesis or cassette mutation based on the nucleotide sequence shown in SEQ ID NO: 37 (for example, Short protocols In Molecular Biology, Third Edition, John Wiley & Sons, Inc.).
  • the isolated DNA fragment containing the cellobiohydrolase II gene promoter sequence described in 3. above can be obtained by the following procedure.
  • a chromosomal DNA library was obtained in the same manner as described in 1. above.
  • a synthetic DNA prepared from the obtained chromosomal DNA library based on the base sequence of the cellobiohydrolase II gene isolated from other species (for example, Phanerochaete chrysosporium).
  • a plaque hybridization is performed to select a clone containing the cellohydrohydrase II gene.
  • the DNA fragment containing the seropiohydrolase II gene is isolated from the selected clone and sequenced.
  • the DNA fragment containing the cellobiohydrolase II gene described above can be inserted into a cloning vector (for example, a pUC-based vector such as pUC19), and the method can be carried out according to the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)).
  • a cloning vector for example, a pUC-based vector such as pUC19
  • the gene having this sequence is a genomic gene consisting of 49 3 base pairs including the structural gene of cellohydrohydrase II gene derived from C. hirstus, the upstream 3 ′ untranslated region including a promoter region and a terminator. .
  • the promoter region exists at base numbers 1 to 3121 (SEQ ID NO: 49), and TATAAA is found at base numbers 3059 to 3064.
  • the structural gene for cellobiohydrolase II exists at base numbers 3122 to 4822 and is composed of 7 exons and 6 introns (intervening sequences). Specifically, exon 1 is 3 1 2 2 to 3 21, exon 2 is 3280 to 3 346, exon 3 is 3 402 to 3 549, exon 4 is 3605 to 4220, and exon 5 is 42 75 to 4 Exon 6 is located at 4425-4676, and exon 7 is located at 4736-4822, respectively.
  • intron 1 is 3222 to 3279
  • intron 2 is 3347 to 3401
  • intron 3 is 35 50 to 3604
  • intron 4 is 4221 to 4274
  • intron 5 is 4364 to 4424
  • intron 6 is 4677 to 4735, respectively.
  • base numbers 482 to 4933 are a 3 'untranslated region containing a terminator.
  • the amino acid sequence deduced from the sequence analysis was found to be the amino acid sequence shown in SEQ ID NO: 51 consisting of 453 amino acid residues.
  • a DNA fragment containing the base sequence of the promoter region of the cellobiohydrolase II gene can be obtained from the fragment containing the cellobiohydrolase II gene by PCR (polymerase chain reaction).
  • PCR polymerase chain reaction
  • a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence represented by SEQ ID NO: 49 and its complementary sequence is a sense primer, antisense It can be used as a primer.
  • a sense primer shown in SEQ ID NO: 53 and an antisense primer shown in SEQ ID NO: 55 can be used (see Example 3).
  • the base sequence represented by SEQ ID NO: 49 is one specific example of such a promoter sequence, and as long as it has promoter activity, a sequence complementary to the base sequence can be obtained under stringent conditions.
  • Hybridizing sequences are also within the scope of the invention.
  • the stringent condition means that the homology with the nucleotide sequence shown in SEQ ID NO: 49 is 70% or more, preferably 80% or more, more preferably 90% or more, particularly 95% or more.
  • site-specific mutagenesis techniques such as oligonucleotide site-directed mutagenesis and cassette mutagenesis (for example, based on the nucleotide sequence represented by SEQ ID NO: 49) Short protocols In Molecular Biology, Third Edition, John Wiley & Sons, Inc.).
  • the gene encoding a lignin-degrading enzyme is not particularly limited as long as it encodes an enzyme capable of degrading lignin, and examples thereof include a manganese peroxidase gene, a lignin peroxidase gene, and a laccase gene.
  • These genes can also be obtained by well-known genomic cloning, cDNA cloning, or PCR based on sequences registered in gene punk, sequences described in literatures, and the like. Alternatively, as for the deposited genes, those that can be obtained by requesting a sale can be used.
  • “transcribable” means that transcription of a gene encoding a lignin degrading enzyme into mRNA under the action of a promoter in a host.
  • the gene coding for lignin-degrading enzyme is linked downstream of the DNA fragment having the promoter sequence of the cellulolytic enzyme gene, and is transcribed into mRNA by the action of the promoter.
  • the DNA fragment containing the promoter sequence of the cellulose degrading enzyme gene and the gene encoding lignin degrading enzyme should be ligated using an appropriate DNA ligase after the introduction of restriction sites, blunting or sticky terminating as necessary.
  • Clouni Recombinant DNA techniques including cloning, ligation, and PCR, are described in, for example, J. Sambrook et al., Molecular loning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, and short Protocols In Molecular Biology, Third Edition, A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
  • the type of vector is not particularly limited, but is selected according to the type of host transformed with this vector.
  • a vector capable of autonomous replication in a prokaryotic or eukaryotic host cell or homologous recombination in a chromosome can be used. Plasmids, viruses, including phages, and cosmids.
  • the vector may appropriately contain a selection marker, an origin of replication, a terminator, a polylinker, an enhancer, a ribosome binding site, and the like.
  • Various vectors for prokaryotes and eukaryotes such as bacteria, fungi, yeasts, animals, and plants are commercially available, or described in the literature.
  • a lignin-degrading enzyme-producing host cell transformed with the vector defined above is prepared.
  • host cells include not only fungi including basidiomycetes, fungi, and yeasts, but also other eukaryotic cells (animal cells, plant cells, insect cells, algae, etc.) and prokaryotic cells (bacterium, cyanobacteria, etc.). At least, any host cell can be used as long as it can exhibit promoter activity in the expression of a gene encoding lignin-degrading enzyme.
  • preferred host cells are basidiomycetes, and particularly preferred is Coriolus hirstus.
  • an auxotrophic mutant 0JI-1078 (FERM BP-4210) deficient in ornithine force rubamoyltransferase activity of Coriolus hilus stas described in Examples below is used.
  • an Escherichia coli recombinant strain JM109 / pUCRl Japanese Patent Application Laid-Open No. 6-0 549 691; FERM BP-4201 containing an ordinin force rubamoinole transferase gene isolated from Coriolus hirustus was used.
  • JM109 / pUCRl Japanese Patent Application Laid-Open No. 6-0 549 691; FERM BP-4201 containing an ordinin force rubamoinole transferase gene isolated from Coriolus hirustus was used.
  • Can be used as a selection marker gene an Escherichia coli recombinant strain JM109 / pUCRl (Japanese Patent Application Laid-
  • Transformation methods include calcium chloride ZPEG method, calcium phosphate method, acetic acid
  • Examples of the method include, but are not limited to, the lithium method, the electoral portation method, the protoplast method, the spheroplast method, the lipofection method, and the agglomerate method.
  • the transformed lignin-degrading enzyme-producing host cell is cultured in the presence of cellulose to produce lignin-degrading enzyme.
  • Ligninolytic enzymes are produced in secreted form when expressed and translated in the form of a fusion with the signal peptide, and can be isolated directly from the medium in this case, but the polypeptide is not secreted.
  • the cells When produced in a form, the cells are separated, and the cells are destroyed by a treatment such as sonication or homogenizing to obtain an extract, from which the polypeptide can be isolated.
  • Isolation and purification include solvent extraction, salting out, desalting, organic solvent precipitation, ultrafiltration, ion exchange, hydrophobic interaction, HPLC, gel filtration and affinity chromatography, electrophoresis, and chromatofocusing. Such methods can be performed alone or in combination.
  • manganese peroxidase a lignin peroxidase gene portion, or a laccase gene portion is ligated downstream of the promoter region of the present invention
  • manganese peroxidase; Lignin peroxidase or laccase can be produced in large quantities.
  • Each gene is not particularly limited, and various genes having known sequences can be used.
  • AIST Advanced Industrial Science and Technology
  • a DNA fragment comprising a promoter sequence of a basidiomycete-derived cellulolytic enzyme gene, and a gene encoding a lignin degrading enzyme
  • the step of preparing the vector and the step of performing transformation using the vector to prepare a lignin-degrading enzyme-high producing host cell are as defined in the first embodiment.
  • the processing can be performed, for example, as follows.
  • the resulting lignin-degrading enzyme-producing host cells are cultured on a potato dextrose agar medium, stored at a low temperature (4 ° C), and cut out from this plate to obtain a gnorecose.peptone medium (2% glucose, Poripe Easthampton 0.5%, yeast extract 0 ⁇ 2%, KH 2 P0 4, MgS0 4 0. 05%, 3 0 0 ml volume containing p H4 with phosphoric acid. 5 for preparation) one by 1 0 0 m 1 Inoculate an Erlenmeyer flask and culture with shaking at 28 ° C and 100 rpm for 1 week. After culturing, the host cells are filtered off and washed with sterile water to remove the remaining medium.
  • a gnorecose.peptone medium 2% glucose, Poripe Easthampton 0.5%, yeast extract 0 ⁇ 2%, KH 2 P0 4, MgS0 4 0. 05%, 3 0 0
  • the obtained lignin-degrading enzyme-producing host cells were ground together with sterilized water for 15 sec with a Warinda blender, and the dry weight of the host cells was reduced to 1 g per 1 kg of absolutely dry eucalyptus wood chip. Inoculate so that After inoculation, agitate well so that the host cells spread throughout. Incubate for 1 week at 28 ° C with aeration. Saturated steam is aerated as needed so that the water content of the wood chips is 40 to 65%. Ventilation should be performed so that the amount of air flow per chip is 0.00 Olv vm.
  • the obtained chromosomal DNA100OOug is partially digested with the restriction enzyme Sau3AI, fractionated by 5-20% sucrose density gradient ultracentrifugation (30,000 rpm, 18 hours), and sectioned into 20-40 kbp fragments. Collected. This fragment segment was ligated to Toyobo's phage; LEMBL3-Bam arm using T4 DNA ligase, and the resulting phage DNA was packaged using STRATAGENE's Gigapack Gold, and then Escherichia coli P2392 strain (the above Gigapack Gold). The chromosomal DNA library was infected.
  • Example 2 Isolation of a DNA fragment containing a promoter sequence of cellobiose dehydrogenase gene from a chromosomal gene library
  • a clone containing the promoter region of the mouth biose dehydrogenase gene was selected by plaque hybridization. This series of operations was performed according to a conventional method (Sambrook et al., Molecular Cloning A Laboratory Manual / 2nd Edition (1989)).
  • the probe used for the plaque hybridization was obtained by labeling a synthetic oligomer having the following sequence at the 3 'end with fluorescein using an oligo DNA labeling kit manufactured by Amersham. As a result, four positive clones could be selected from about 40,000 plaques.
  • Recombinant phage DNA prepared from a positive clone in accordance with a conventional method was digested with various restriction enzymes, and subjected to Southern hybridization using the synthetic DNA described above. As a result, clones hybridizing to the probe as DNA bands of 7.2 kbp and 5.3 kbp were found in the fragment obtained by digestion with the restriction enzyme XhoI.
  • the PCR method was performed using two primers shown in SEQ ID NOs: 4 and 5 below from plasmid pCHCDHl containing the promoter region of cellobiose dehydrogenase gene derived from Coriolus hirstus obtained in Example 2. As a result, a 2.2 kbp fragment (fragment 1) having BamHI to bluntend of the cellobiose dehydrogenase promoter region was amplified.
  • Primer 1 5'-GGATCCGAGGATCGCAACCGCG-3 '(SEQ ID NO: 4)
  • E. coli vector pUC18 is cut with restriction enzymes BamHI to EcoRI, the above DNA fragments 1 and 2 are mixed, ligated with T4 DNA ligase, and then mixed with E. coli JM1. 09 strains were transformed, and a plasmid into which the DNA fragments of fragment 1 and fragment 2 were simultaneously inserted was isolated from the ampicillin-resistant transformants and named pCDHPMN.
  • Primer 1 5'-CTTTCAAGACACTCGCCTTCG-3 '(SEQ ID NO: 6)
  • Primer 2 5,-GAATTCGCATGTAGGTCCGCG-3, (SEQ ID NO: 7)
  • SMY medium 1% sucrose, 1% malt extract, 0.4% yeast extract
  • SMY medium 1% sucrose, 1% malt extract, 0.4% yeast extract
  • a piece of agar with a diameter of 5 mm was punched out from a plate agar medium of Coriolus hilstas OJI-107 strain with a cork borer, inoculated into an SMY medium, and incubated at 28 ° C for 7 days. Pre-culture). However, they were shaken once or twice a day to break up the hypha.
  • the liquid culture mycelia were collected by filtration with a nylon mesh (pore size 3 0 Ai m), and washed with osmoregulation solution (0. 5M Mg S 0 4, 5 0m 1 maleate buffer (p H 5. 6)).
  • the cells were suspended in lml of cell wall degrading enzyme solution per 100mg of wet cells, Protoplasts were released by incubation at 28 ° C for 3 hours with gentle shaking.
  • the following commercially available enzyme preparations were used in combination as cell wall lytic enzymes.
  • 5 mg of cellulase ONOZUKA RS manufactured by Yakult Co., Ltd. and 1 Omg of Yata 1ase were dissolved in 1 mg of the above osmotic pressure adjusting solution and used as an enzyme solution. .
  • the mycelial fragments and protoplasts remaining on the nip mesh are removed using the above osmotic pressure adjusting solution. Washed twice.
  • the resulting protoplast suspension was centrifuged (1, OOO kXg, 5 minutes), the supernatant was removed, and the suspension was resuspended with 4 ml of 1 M sucrose (20 mM MOPS buffer, pH 6.3). After turbidity, centrifugation was repeated, and the well was washed twice with the above 1M sucrose solution.
  • the precipitate was suspended in 500 ⁇ l of a 1 M sorbitol solution (20 mM MES, pH 6.4) to which 40 mM calcium chloride was added, to give a protoplast solution. This solution was stored at 4 ° C.
  • Protoplast concentrations were determined by direct microscopy using a hemocytometer. All centrifugation was performed in a swing rotor at 1,000 X g for 5 minutes at room temperature. d. Transformation
  • Example 5 Production of manganese peroxidase by transformant Oxygen bleaching after hardwood pulp (LOKP) ⁇ peptone medium (LOKP 1%, Poripepu tons 0.5%, yeast extract 0. 2% KH 2 P_ ⁇ 4, Mg S 0 4 0. 0 5%, phosphorus 5 ml of the agar of 50 mm 2 of the transformed strain obtained in Example 4 above was inoculated into a 300 ml Erlenmeyer flask containing 100 ml of each (prepared to pH 4.5 with acid). Then, the cells were cultured with shaking at 28 ° C. and 100 rpm. Six days later, the obtained culture solution was centrifuged to obtain a supernatant.
  • Enzyme activity was 0.5 M sodium phosphate buffer (pH 5.5) 50 ⁇ l, enzyme solution 34 5 ⁇ l, 10 mM hydrogen peroxide 5 ⁇ l, 1 mM MnSO 4 1 Of the Mn (III) malonic acid complex resulting from the reaction by recording the increase in absorbance at 270 nm over time.
  • the enzymatic activity of the malic acid complex was observed at ⁇ 1 Zm1 / min on the sixth day after the start of the culture.
  • the enzyme activity unit was defined as an activity that increases the Mn (III) malonic acid complex by 1 ⁇ mo 1 per minute. On the other hand, this activity was not observed in the culture supernatant of the OJ1-1708 strain containing no donor DNA cultured under the same conditions.
  • the structural gene region of the lignin peroxidase chromosomal gene (Japanese Patent Laid-Open No. 5-260978; PBSLP0G7; FERM P-12683) was ligated downstream of the promoter of the cellobiose dehydrogenase gene from Coriolus hirstas to obtain an expression plasmid.
  • the plasmid p CHCDHl containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirstus obtained in Example 2 was used to obtain P from the plasmid p CHCDHl using the two primers shown in SEQ ID NOS: 4 and 5 in Example 3.
  • fragment 3 a 2.2 kbp fragment having BamHI to bluntend in the promoter region was amplified.
  • the lignin peroxidase structural gene portion was removed by using the plasmid pBSL POG7 as a template and a DNA fragment was prepared by the primer extension method using the primer shown in SEQ ID NO: 8 below. Cleavage with III yielded a 1.7 kbp DNA fragment (fragment 4) consisting of the lignin peroxidase structural gene portion.
  • E. coli vector was digested with pUC18 by restriction enzymes EcoRI and HindiII, the two DNA fragments of fragments 5 and 6 were mixed, and ligated with T4 DNA ligase.
  • the strain 09 was transformed, and a plasmid into which the DNA fragments of the above fragments 3 and 4 were simultaneously inserted was isolated from the transformed ampicillin-resistant transformants and named pCDHP LP.
  • a protoplast solution of C. hirstus was obtained.
  • the plasmid 2 mu g prepared in about 1 0 6/1 00 ⁇ 1 of protoplasts solution to Example 6 was added at cyclic or linear.
  • a plasmid pUCRl having an onolenitine canolebamoinoretransferase gene derived from Coriolus hinorestas was added in an amount of 0.2 ⁇ , and the mixture was cooled on ice for 30 minutes.
  • an equal volume of a PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled for 30 minutes.
  • the mixture was mixed with a minimal soft agar medium (agar 1%) containing 0.5M sucrose and leucine, and wound on a plate.
  • the plate was cultured at 28 for 4 days to obtain a transformant. Further, DNA was prepared from the above transformant, and it was confirmed by Southern hybridization that the desired lignin peroxidase gene expression plasmid was incorporated.
  • Oxygen bleaching after hardwood pulp (LOKP) ⁇ peptone medium (LOKP 1%, Poripepu tons ⁇ . 5% yeast extract 0. 2% KH 2 P_ ⁇ 4, Mg S_ ⁇ 4 0.05%, in phosphate 5 ml of the agar of the transformant 50 mm 2 obtained in the above Example 7 was inoculated into a 300 ml Erlenmeyer flask containing 100 ml of each prepared at pH 4.5. The cells were cultured at 100 rpm with rotary shaking. Six days later, the obtained culture was centrifuged to obtain a supernatant.
  • the activity was measured using 8 mM veratryl alcohol 25 ⁇ l, 0.5% sodium tartrate buffer ( ⁇ 3.0) 50 / ⁇ 1, enzyme solution 400 ⁇ l, 2.7 mM hydrogen peroxide 25 ⁇ l. Mix well and measure the absorbance of veratraldehyde at 110 nm over time, and add veratryl alcohol to the culture supernatant. On day 5 from the start of the stationary culture, the activity of the enzyme to be converted to the aldehyde was observed in the range of 3 ⁇ 0 to 500 units Zm1. Here, the enzyme activity unit is veratramulaldehyde per minute.
  • the activity to increase 1 ⁇ 1 was defined as 1 unit. On the other hand, this activity was not observed in the culture supernatant of the OJ1-1070 strain containing no donor DNA cultured under the same conditions.
  • PCR was performed using the plasmid p CHCDHl obtained in Example 2 as a template and the two primers shown in SEQ ID NOS: 9 and 10 below, and BamHI treatment was performed. A DNA fragment having I to bluntend (fragment 7) was obtained.
  • Primer 1 5'-GGATCCGAGGATCGCAACCGCG-3 '(SEQ ID NO: 9)
  • Primer 2 5-CATGGCGGGCAGTCGTAGTGT-3 '(SEQ ID NO: 10)
  • plasmid OJ-POG-E1 (Accession No. BP-2793) containing a laccase gene derived from Coriolus hirustas was amplified by PCR using two primers shown in SEQ ID NOS: 11 and 12 below.
  • Primer 2 5'-GAATTCCCGGGGACGTATACG-3, (SEQ ID NO: 12)
  • Plasmid pTALAC was digested with a restriction enzyme XhoI, and then blunted with a modifying enzyme K1enowfragment, followed by digestion with a restriction enzyme EcoRI to obtain a laccase structural gene portion (fragment 6).
  • a protoplast solution of Coriolus hirustus was used. I got The plasmid 2 g prepared in about 1 0 6 Z 1 0 0 mu 1 of protoplasts solution to Example 9 was added with cyclic or linear. Further, as a selectable marker, 0.2 ⁇ g of a plasmid p UCR1 having a gene encoding a chorenorhintin canolebamoinoretransferase gene derived from Coriolus hinorestas was added, and the mixture was cooled on ice for 30 minutes.
  • Enzyme activity was 50 ⁇ l of 1 M sodium acetate buffer (pH 4.0), 5 mM 2,2 — azino—b ios (3—ethilbenzthiazoline — 6-su1 fonate) (ABTS) 501
  • the enzyme solution 4001 was added, and the ABTS oxide generated as a result of the reaction was recorded by increasing the absorbance at 420 nm over time. It was visually observed at 40 units Z mL.
  • the enzyme activity unit is defined as the amount of enzyme required to oxidize 1 ⁇ mol ABTS per minute.
  • only 5 nit / ml was observed in the culture supernatant of the 0JI-17078 strain containing no donor DNA cultured under the same conditions.
  • a chromosomal gene library was obtained in the same manner as in Example 1. From the obtained library, a clone containing the cellobiohydrolase I gene was selected by plaque hybridization. This different operation was performed according to a conventional method [Sambrook et al., "Molecular Cloning A Laboratory Manual / 2nd Edition (1989)"].
  • the probe used for plaque hybridization was a synthetic oligomer having the sequence of the following SEQ ID NO: 16 labeled with fluorescein at the 3 ′ end using an oligo DNA labeling kit manufactured by Amersham.
  • base Y indicates base T or C
  • base R indicates base G or A.
  • Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above-mentioned synthetic DNA. As a result, in the fragment obtained by digestion with the restriction enzymes EcoRI and BamHI, a clone that hybridized to 4.6 kbp as a single DNA band was observed.
  • the above-mentioned DNA fragment 4.6 kbp was cut out by agarose gel electrophoresis, subcloned into the EcoRI-BamHI site of the E. coli vector pUC19, and transformed into the E. coli JM109 strain to obtain a plasmid pCHCBHI31.
  • the nucleotide sequence of the subcloned DNA fragment was determined.
  • the nucleotide sequence was determined using Applied Biosystems BigDye Terminator Cycle Sequencing using ⁇ R) ⁇ , and electrophoresis was performed using Applied Biosystems DNA soil protein enhancer PRISM 310 to analyze the nucleotide sequence.
  • the nucleotide sequence is shown in SEQ ID NO: 14.
  • the cellobio hydrolase I gene derived from C. hirstus was separated by two introns.
  • the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 15.
  • Example 13 Construction of a manganese peroxidase gene expression vector by the cellobiohydrolase I gene promoter After digesting the plasmid P CHCBHI31 containing the promoter region of the cellobio hydrolase I gene derived from Coriolus hirustas with the restriction enzyme Ncol, blunting is performed using the modifying enzyme Klenow fragment, self-ligating, and the Ncol site is eliminated. The obtained Ncol site disappearance plasmid was amplified to a promoter region of 2.3 kbp by PCR using the following two primers (1, 2), and then to pUC18 Smal site using T4 DNA ligase. After ligation, plasmid pCHCBHI31P was obtained.
  • Primer 1 5'-GGATCCGTTCACACACTGAC-3 '(SEQ ID NO: 17)
  • Primer 1 —— 2 5 -CCATGGCGGTCAGCTGGGGCTG-3 '(SEQ ID NO: 18)
  • plasmid pBSMPOGl (Accession No. FERM P-14933) containing the manganese peroxidase gene derived from Coriolus hiltus was subjected to PCR using the two primers (3, 4) shown below by PCR. Amplify a partial Ncol about 2.2 kb fragment.
  • Primer 3 5 -CCATGGCTTTCAAGACACTCG-3 '(SEQ ID NO: 19)
  • Primer 4 5 -CCATGGGGAAGGACTGGTGAG-3 '(SEQ ID NO: 20)
  • the obtained PCR fragment was inserted using a TA cloning kit manufactured by In Vitrogen to obtain pTAMP.
  • the obtained pTAMP was digested with a restriction enzyme Ncol, about 2.2 kb was cut out, and inserted into the Ncol site of PCHCBHI31P to obtain a plasmid pCHCBHI31PMP (FIG. 1).
  • Example 14 Production of Transformant Producing High Manganese Peroxidase Protoplasts were prepared and purified in the same manner as in Example 4. Next, transformation was performed as follows.
  • the mixture was mixed with a minimal soft agar medium (agar 1%) containing 0.5 M sucrose and leucine and spread on a plate.
  • the plate was cultured at 28 ° C for 4 days to obtain a transformant.
  • DNA was prepared from the above transformant, and it was confirmed that the desired manganese peroxidase expression plasmid was incorporated. Confirmed by Southern hybridization.
  • Example 1 4 obtained in pulp 'peptone broth 50 ml of the transformed strain Erlenmeyer flask 500ml volume (bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1. 5 g / l, MgS0 4 ⁇ 7 ⁇ 2 0 0. 5 g / l, thiamine hydrochloride 2 mg / l, MnS0 4 ⁇ 5H 2 0 48 mg / l, adjusted to pH 5. 0 with phosphoric acid) to the 50 mm 2 agar Five pieces were inoculated and cultured at 28 ° C for 6 days with shaking. Six days later, the resulting culture was centrifuged to obtain a supernatant.
  • Enzyme activity 0. 5 M Ma port phosphate Natoriumu buffer (pH 5. 5) 50 ⁇ 1 , thoroughly mixing the enzyme solution 345 ⁇ 1, 10 mM hydrogen peroxide 5 ⁇ 1, 1 mM MnS0 4 100, The increase in the absorbance at 270 nm of the ⁇ (III) malonic acid complex generated as a result of the reaction was performed over time, and the enzymatic activity of the Mn (III) malonic acid complex was cultivated in the culture supernatant. It was observed at 3.5 ⁇ mol / ral / min on the 6th day after initiation. Where the enzyme activity unit is Mn per minute
  • the plasmid pCHCBHI31P obtained in Example 13 was digested with a restriction enzyme Ncol (manufactured by Takara Shuzo Co., Ltd.), and then smoothed using Klenow fragment. Then, it was digested with the restriction enzyme EcoRI to obtain the expression vector.
  • Ncol manufactured by Takara Shuzo Co., Ltd.
  • 0J-P0G-E1 (FERM BP-2793) was prepared using the two primers (5, 6) shown below.
  • Primer 5 5'-CTCG AGGTTCC AGTCTCTG- 3 '(SEQ ID NO: 21)
  • the obtained PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALAC.
  • Example 1 7 obtained transformant strain 500 ml 50 ml of the pulp 'peptone broth Erlenmeyer flask volume (bleached pulp 30 g / l, peptone 10 g / l, ⁇ , ⁇ 0 4 1.5 g / l, MgS0 4 ⁇ 7 ⁇ 2 00.5 g / l, thiamine hydrochloride 2mg / l, CuS0 4 ⁇ 5H 2 0100 mg / l, agar pieces 50 mm 2 to adjustment) to pH 5.0 with phosphoric acid and five inoculated, 28
  • the cells were cultured under shaking at 6 ° C for 6 days. Six days later, the resulting culture was centrifuged to obtain a supernatant.
  • Enzyme activity was 50 ⁇ l of 1 M sodium acetate buffer (pH 4.0), 5 mM ABTS (2,2'-azino-bis (3-ethilbenzthiazoline-6-sulfonate) 501, 400 ⁇ l of enzyme solution And add the ABTS oxide resulting from the reaction over time to increase the absorbance at 420 nm.
  • the enzyme activity was found in the culture supernatant on the 5th day from the start of the culture, at 35 units / ml.
  • enzyme activity unit is defined as the amount of enzyme required to oxidize 1 ⁇ mol ABTS per minute.
  • only 5 units / ml was found in the culture supernatant of Choriolas hirstus OJI-1078 strain (FERM BP-4210) containing no donor DNA cultured under the same conditions.
  • the plasmid pCHCBHI31P obtained in Example 13 was digested with a restriction enzyme Ncol (Takara Shuzo) to obtain an expression vector.
  • the plasmid pBSLP0G7 / E. Coli JM109 (FERM P-12683) containing the high temperature inducible lignin peroxidase gene derived from Coriolus hirustas was amplified by PCR using the following two primers (7, 8). .
  • Primer 8 5, -CCATGGAAGCTTCGGAAAGAGC-3 '(SEQ ID NO: 24)
  • the obtained PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALiP.
  • Plasmid pTALiP was digested with restriction enzyme Ncol to obtain a part of the gene for the structure of the lydanine peroxidase, and the above-mentioned treated cellobiohydrolase I expression vector pCHCBHI31P The resulting plasmid was named pCHCBHI31PLiP (FIG. 3).
  • arginine-requiring Coriolus hirustas 0JI-1078 strain (FERM BP-4210) is transformed using the pCHCBHI31PLiP obtained in Example 19, an orditin canoleba derived from Coriolus hi / restus is used as a selection marker.
  • a transformant was obtained by simultaneously introducing the plasmid pUCRl carrying the Moire transferase gene (eg, PEG method or electoral poration method).
  • the target transformant could be obtained irrespective of whether the DNA that could be provided for the transformation was circular or linear. The transformation conditions are described below.
  • Example 2 0 obtained in 50 ml of pulp Erlenmeyer flask Transformants 500 ml capacity.
  • Peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1 . 5 g / l, MgS0 4 ⁇ 7 ⁇ 2 0 0. 5 g / l, thiamine hydrochloride 2 mg / l, 5 mM veratryl alcohol, agar pieces 50 mm 2 to adjustment) to pH 4.
  • 5 with phosphoric acid 5 Inoculated individually, and cultured at 28 ° C for 6 days under shaking. Six days later, the resulting culture was centrifuged to obtain a supernatant.
  • Enzyme activity was determined by adding 100 ⁇ l of 1 M sodium tartrate buffer (pH 3.0), 25 ⁇ l of 8 mM veratryl alcohol, 350 ⁇ l of enzyme solution ⁇ 5.4 ⁇ m 25 ⁇ l of 4 mM hydrogen peroxide, and the result of the reaction.
  • the resulting veratrialdehyde was recorded by recording the increase in absorbance at 310 nm over time, and the enzymatic activity was observed in the above culture supernatant at 0.8 units / ml on the 5th day from the start of culture.
  • the enzyme activity unit is defined as the amount of enzyme required to produce 1 / z mol of veratrialdehyde per minute.
  • no lignin peroxidase activity was observed in the culture supernatant of Choriolus' Hilstas 0JI-1078 strain (FERM BP-4210) containing no donor DNA cultured under the same conditions.
  • a clone was selected from the chromosomal gene library.
  • the probe used for plaque hybridization was the following SEQ ID NO: 28 A synthetic oligomer having a sequence is labeled at its 3 ′ end with fluorescein using an oligo DNA labeling kit manufactured by Amersham.
  • base Y indicates base T or C
  • base R indicates base G or A.
  • Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above-mentioned synthetic DNA. As a result, in the fragment obtained by digestion with the restriction enzyme Sail, a clone hybridizing at 4.2 kbp as a single DNA band was observed.
  • the above DNA fragment 4.2 kbp was excised by agarose gel electrophoresis, subcloned into the Sail site of the E. coli vector PUC19, and transformed into the E. coli JM109 strain to obtain a plasmid pCHCBHI27.
  • the nucleotide sequence of the subcloned DNA fragment was determined.
  • the nucleotide sequence was determined using the BigDye Terminator Cycle Sequencing kit manufactured by Applied Biosystems, and the iL and fj levels were compared using Applied Biosystems.
  • Electrophoresis was performed using a DNA sequencer PRISM 310 manufactured by the company, and the nucleotide sequence was analyzed.
  • the nucleotide sequence is shown in SEQ ID NO: 26.
  • the cellobio hydrolase I gene derived from C. hinorestas was divided by two introns.
  • the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 27.
  • the following two primers (1, 2) were used from plasmid PCHCBHI27, which contains the promoter region of the C. hirstus cellobiohydrolase I gene. Ligation was performed using T4 DNA ligase to obtain plasmid pCHCBHI27P.
  • Primer 1 5'-GATCTCCGCCTTGGCCCTCG-3 '(SEQ ID NO: 29)
  • Primer 2 5,-CCATGGCCGAAGCCGGGGTC-3 '(SEQ ID NO: 30)
  • a plasmid containing the manganese peroxidase gene from Coriolus hirustus Rasmid pBSMPOGl (Accession No. FERM P-14933) is amplified by PCR using the following two primers (3, 4) to amplify an approximately 2.2 kb fragment of the manganese peroxidase structural gene portion Ncol.
  • Primer 3 5'-CCATGGCTTTCAAGACACTCG-3 '(SEQ ID NO: 31)
  • Primer 4 5 -CCATGGGGAAGGACTGGTGAG-3 '(SEQ ID NO: 32)
  • the obtained PCR fragment was inserted using TA Cloning kit manufactured by In Vitrogen, and was used as pTAMP.
  • the obtained pTAMP was digested with a restriction enzyme Ncol, about 2.2 kb was cut out, and inserted into the Ncol site of PCHCBHI27P to obtain a plasmid pCHCBHI27PMP (FIG. 4).
  • Protoplasts were prepared and purified in the same manner as in Example 4. Then, transformation was performed as follows.
  • PUCRl containing the onolenitine canolebamoinoletransferase gene derived from it was quenched with 0.2 ⁇ ug, cooled on ice for 30 minutes, and then an equal volume of PEG solution (50% PEG 3400, After adding 20 mM MOPS (pH 6.4)), the mixture was ice-cooled for 30 minutes, then mixed with a minimal soft agar medium (agar 1%) containing 0.5 M sucrose and leucine, and spread on a plate. The plate was cultured for 4 days at 28 ° C. to obtain a transformant.DNA was prepared from the transformant to confirm that the desired manganese peroxidase expression plasmid was incorporated. By Zan Hybridization It was confirmed.
  • Example 2 4 obtained in pulp-peptone broth 50 ml of the transformed strain Erlenmeyer flask 500ml volume (bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1. 5 g / l, MgS0 4 ' 7H 2 0 0. 5 g / l, thiamine hydrochloride 2 mg / l, MnS0 4 ⁇ 5H 2 0 48 mg / l, adjusted to pH 5. 0 with phosphoric acid) to 50 mm 2 Were inoculated and cultured at 28 ° C for 6 days with shaking. Six days later, the resulting culture was centrifuged to obtain a supernatant.
  • Enzyme activity 0. 5 M Ma port phosphate sodium buffer (pH 5. 5) 50 ⁇ 1 , the enzyme solution 345 ⁇ 1, 10 mM hydrogen peroxide 5 ⁇ 1, 1 mM MnS0 4 100; ul sufficiently mixed And the result of the reaction Carried out by and Jill Mn (III) recorded child chasing the absorbance increase of 270 n m malonate complex time, in the culture supernatant Mn (III) malonate complex enzyme activity initiated culture 6 At day 8.5, it was observed at 8.5 ⁇ mol / ml / min.
  • the enzyme activity unit was defined as the activity of increasing 1 mol of the Mn (III) malonic acid complex in 1 minute.
  • Choriolas hirstas 0JI-1078 strain (FERM
  • the plasmid pCHCBHI27P obtained in Example 23 was digested with a restriction enzyme Ncol (manufactured by Takara Shuzo Co., Ltd.), and then smoothed using Klenow fragment. Then, it was digested with the restriction enzyme EcoRI to obtain the expression vector.
  • Ncol manufactured by Takara Shuzo Co., Ltd.
  • 0J-P0G-E1 (FERM BP-2793) was prepared using the following two primers (5, 6).
  • Primer 5 5, -CTCGAGGTTCCAGTCTCTG-3, (SEQ ID NO: 33)
  • the obtained PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALAC.
  • the plasmid pCHCBHI27PLAC 2 ⁇ g was created manufactured in Example 2 6 to about 10 6 cells / 100 il concentration of protoplasts solution 100 was added in cyclic or linear, the pUCRl as a selection marker to further 0. 2 ⁇ The mixture was chilled and ice-cooled for 30 minutes.
  • Example 2 7 obtained transformant strain 500 ml 50 ml of the pulp-peptone liquid medium Erlenmeyer flask volume (bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1 . 5 g / l, MgS0 4 ⁇ 7 ⁇ 2 0 0. 5 g / l, thiamine hydrochloride 2 mg / l, CuS0 4 ⁇ 5H 2 0 100 mg / l, 50 to adjustment) to pH 5.
  • 0 with phosphate mm Five agar pieces of No. 2 were inoculated and cultured under shaking at 28 ° C. for 6 days. Six days later, the resulting culture was centrifuged to obtain a supernatant.
  • Enzyme activity was 50 ⁇ l of 1 M sodium acetate buffer (pH 4.0), 50 ⁇ l, 5 mM ABTS (2,2 azino-bis (3-ethilbenzthiazoline-6-sulfonate) 50 ⁇ l, 400 ⁇ l of enzyme solution Add 1 and record the increase in absorbance at 420 nm over time for the ABTS oxide resulting from the reaction.
  • Enzyme activity was found in the above culture supernatant at 57 days / ml on the 5th day from the start of culture.
  • the unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 ⁇ ABTS per minute, while the choriolus' Hilstas 0JI without donor DNA cultured under the same conditions. -Only 5 units / ml was found in the culture supernatant of the 1078 strain (FERM BP-4210).
  • the plasmid pCHCBHI27P obtained in Example 23 was digested with a restriction enzyme Ncol (Takara Shuzo) to obtain an expression vector.
  • a plasmid PBSLP0G7 / E. Coli JM109 (FERM P-12683) containing a high temperature inducible lignin peroxidase gene derived from Coriolus hiltus was amplified by PCR using the following two primers (7, 8). .
  • Primer 7 5'-CCATGGCGTTCAAGGCTCTTC-3 '(SEQ ID NO: 35)
  • Primer 8 5, -CCATGGAAGCTTCGGAAAGAGC-3, (SEQ ID NO: 36)
  • the obtained PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALiP.
  • Plasmid pTALiP was digested with a restriction enzyme Ncol to obtain a part of the gene for the structure of the lydanine peroxidase, which was introduced into the above-described cellohydrohydrase I expression vector pCHCBHI27P.
  • the obtained plasmid was named pCHCBHI27PLiP (FIG. 6).
  • Plasmid PCHCBHI27PLiP 2 was created manufactured in Example 2 9 protoplasts solution of about 10 6 cells / 100 / l concentration to 100 ⁇ ⁇ ; the ug added in cyclic or linear, the pUCRl as a selection marker to further 0.2 / ig was added, and the mixture was ice-cooled for 30 minutes.
  • Example 31 Production of lignin peroxidase by a transformant
  • the transformant obtained in Example 30 above was placed in a 500-ml Erlenmeyer flask in a 50-ml pulp / peptone liquid medium (bleached pulp 30).
  • Enzyme activity was measured by adding 1 ⁇ M sodium tartrate buffer (pH 3.0) 100 ⁇ l, 8 mM veratryl alcohol 25 ⁇ l, enzyme solution 350 ⁇ l, 5.4 mM hydrogen peroxide 251, and adding vera Tolualdehyde was measured by recording the increase in absorbance at 310 nm over time, and enzyme activity was found in the above culture supernatant at 1.5 units / ml on the 5th day from the start of culture.
  • the unit of enzyme activity is defined as the amount of enzyme required to produce 1 ⁇ of benzyl alcohol per minute.
  • clones were selected from one chromosomal gene library.
  • the probe used for plaque hybridization was a synthetic oligomer having the sequence of the following SEQ ID NO: 40 labeled with fluorescein at the 3 'end using an oligo DNA labeling kit manufactured by Amersham.
  • base Y indicates base T or C
  • base R indicates base G or A.
  • Recombinant phage DNA prepared from a positive clone in accordance with a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above-mentioned synthetic DNA. As a result, the fragments obtained by digestion with the restriction enzymes Pstl and Nhel A clone hybridizing to 3.9 kbp was observed as a single DNA band.
  • the nucleotide sequence is shown in SEQ ID NO: 38.
  • the cellobio hydrolase I gene derived from C. hinorestas was divided by two introns.
  • the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 39.
  • Plasmid pCHCBHI26 which contains the promoter region of the cellobio hydrolase I gene derived from Coriolus hirstas, is digested with the restriction enzyme Ncol, blunt-ended using the kenow fragment, and self-ligated using T4 DNA ligase to generate the Ncol site. The disappeared plasmid PCBCBHI26-Ncol was obtained. Next, the above plasmid was amplified to a promoter region of 1.1 kbp by PCR using the two primers (1, 2) shown below, and ligated to the Smal site of pUC18 using T4 DNA ligase. pCHCBHI26P was obtained.
  • Primer 1 5 '-CTGCAGGGCGCGCTATTAGG-3' (SEQ ID NO: 41)
  • Primer ⁇ 2 5'-CCATGGCGAAGAGGACGTGC-3 '(SEQ ID NO: 42)
  • plasmid pBSMPOGl (Accession No. FERM P-14933) containing the manganese peroxidase gene derived from Coriolus hiltus was subjected to PCR using the two primers (3, 4) shown below by PCR. Amplify a partial Ncol about 2.2 kb fragment.
  • Primer 4 5 -CCATGGGGAAGGACTGGTGAG-3 '(SEQ ID NO: 44)
  • the obtained PCR fragment was inserted using a TA cloning kit manufactured by In Vitrogen to obtain pTAMP.
  • the obtained pTAMP was digested with a restriction enzyme Ncol, about 2.2 kb was cut out, and inserted into the Ncol site of PCHCBHI26P to obtain a plasmid pCHCBHI26PMP (FIG. 7).
  • Example 34 Coriolus hinoretus (Coriolus hirsutus) transgenic method
  • Protoplasts were prepared and purified in the same manner as in Example 4. Next, transformation was performed as follows.
  • the mixture was mixed with a minimal soft agar medium (agar 1%) containing 0.5 M sucrose and leucine and spread on a plate.
  • the plate was cultured at 28 ° C for 4 days to obtain a transformant.
  • DNA was prepared from the above transformant, and it was confirmed by Southern hybridization that the desired manganese peroxidase expression plasmid was incorporated.
  • Example 3 4 obtained in transformed pulp-peptone broth 50 ml of the transformed strain Erlenmeyer flask 500ml volume (bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1. 5 g / l, MgS0 4 ⁇ 7 ⁇ 2 ⁇ 0. 5 g / l, thiamine hydrochloride 2 mg / l, MnS0 4 ⁇ 5H 2 0 48 mg / l, adjusted to pH 5. 0 with phosphoric acid) to 50 mm 2 Were inoculated and cultured at 28 ° C for 6 days with shaking. Six days later, the resulting culture was centrifuged to obtain a supernatant.
  • Enzyme activity 0. 5 M Ma port phosphate sodium buffer (pH 5. 5) 50 ⁇ 1 , the enzyme solution 345 1, 10 mM hydrogen peroxide 5 ⁇ 1, 1 mM thoroughly mixed MnS0 4 100 ⁇ ⁇
  • the increase in absorbance at 270 nm of the ⁇ (III) malonic acid complex generated as a result of the reaction was performed over time, and the enzymatic activity of the Mn (III) malonic acid complex in the culture supernatant was confirmed. On the 6th day after the start of the culture, it was observed at 5.5 ⁇ mo 1 / ml / mi ⁇ .
  • the enzyme activity unit was defined as the activity to increase the Mn (III) malonic acid complex by 1 ⁇ per minute for 1 cut.
  • a choriolus' Hilstas 0JI-1078 strain (FERM This activity was not observed in the culture supernatant of BP-4210).
  • the plasmid pCHCBHI26P obtained in Example 33 was digested with the restriction enzyme Ncol (manufactured by Takara Shuzo Co., Ltd.), and then blunted using Klenow fragment. Then, it was digested with the restriction enzyme EcoRI to obtain the expression vector.
  • Ncol manufactured by Takara Shuzo Co., Ltd.
  • 0J-P0G-E1 (deposit number BP-2793) was amplified by PCR using the following two primers (5, 6).
  • Primer 5 5'-CTCGAGGTTCCAGTCTCTG-3 '(SEQ ID NO: 45)
  • Primer 6 5'-GAATTCCCGGGGACGTATACG-3 '(SEQ ID NO: 46)
  • the resulting PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALAC.
  • the plasmid pTALAC was digested with the restriction enzyme Xhol, and then blunt-ended using the modification enzyme klenow fragment. Thereafter, the digestion was carried out with EcoRI restriction enzyme to obtain a laccase structural gene portion, which was introduced into the above-described cellobiohydrolase I expression vector pCHCBHI26P.
  • the obtained plasmid was named PCHCBHI26PLAC (FIG. 8).
  • arginine-requiring Coriolus hirustas 0JI-1078 strain (FERM BP-4210) is transformed using the pCHCBHI26PLAC obtained in Example 36, a plasmid (O.I. Transformants were obtained by simultaneously introducing (pUCRl) (PEG method or electoral poration method).
  • pUCRl PEG method or electoral poration method
  • the target transformant could be obtained irrespective of whether the DNA that can be provided for the transformation was cyclic or linear.
  • the transformation conditions are described below.
  • a PEG solution 50% PEG 3400, 20 mM MOPS (pH 6.4)
  • Example 3 50 ml of Erlenmeyer flask 500 ml of volume transformants obtained in 7 pulp-peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1 . 5 g / l, MgS0 4 ⁇ 7 ⁇ 2 0 0. 5 g / l, thiamine hydrochloride 2 mg / l, CuS0 4 ⁇ 5H 2 0 100 mg / l, 50 to adjustment) to pH 5.
  • 7 pulp-peptone liquid medium bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1 . 5 g / l, MgS0 4 ⁇ 7 ⁇ 2 0 0. 5 g / l, thiamine hydrochloride 2 mg / l, CuS0 4 ⁇ 5H 2 0 100 mg / l, 50 to adjustment
  • Enzyme activity was 1 M sodium acetate buffer (pH 4.0) 50 ⁇ l, 5 mM ABTS (2, 2'-azino-bis (3-ethilbenzthiazoline-6-sulfonate) 501, enzyme solution Add 400 ⁇ l and record the ABTS oxide resulting from the reaction by increasing the absorbance at 420 nm over time.
  • Enzyme activity in the above culture supernatant is 52 units / where the enzyme activity unit is defined as the amount of enzyme required to oxidize 1 mol of ABTS per minute, while Coriolus hilstas without donor DNA cultured under the same conditions. Only 5 units / ml was observed in the culture supernatant of strain 0JI-1078 (FERM BP-4210).
  • the plasmid pCHCBHI26P obtained in Example 33 was digested with a restriction enzyme Ncol (manufactured by Takara Shuzo) to obtain an expression vector.
  • a plasmid PBSLP0G7 / E. Coli JM109 (FERM P-12683) containing the high temperature inducible lignin peroxidase gene derived from Coriolus hirustas was amplified by PCR using the following two primers (7, 8). .
  • Primer 7 5'-CCATGGCGTTCAAGGCTCTTC-3 '(SEQ ID NO: 47)
  • Plasmid pTALiP was digested with a restriction enzyme Ncol to obtain a lignin peroxidase structural gene portion, which was introduced into the above-described cellobiohydrolase I expression vector pCHCBHI26P.
  • the obtained plasmid was named pCHCBHI26PLiP (FIG. 9).
  • the plasmid PCHCBHI26PUP 2 ⁇ g was created manufactured in Example 3 9 to about 10 6 cells / 100 concentration protoplasts solution 100 1 was added at cyclic, or linear, the pUCRl as a selection marker to further 0. 2 ⁇ ug addition!] And ice-cooled for 30 minutes.
  • Example 4 0 obtained in pulp 'peptone broth 50 ml of the transformed strain in an Erlenmeyer flask 500 ml of volume (bleached pulp 30 g / l, peptone 10 g / l, KH 2 P0 4 1. 5 g / l, MgS0 4 ⁇ 7 ⁇ 2 0 0. 5 g / l, 5 pieces of agar pieces 50 mm 2 in thiamine hydrochloride 2 mg / l, 5raM veratryl alcohol Honoré, adjusted to pH 4. 5 with phosphoric acid) Inoculate and incubate at 28 ° C for 6 days with shaking Nourished. Six days later, the resulting culture was centrifuged to obtain a supernatant.
  • Enzyme activity was measured by adding 1 ⁇ M sodium tartrate buffer (pH 3.0) 100 ⁇ 8 mM veratolinol alcohol 25 ⁇ 1, enzyme solution 350 ⁇ 1, 5.4 mM hydrogen peroxide 25 ⁇ ⁇
  • the resulting verataldehyde was recorded by recording the increase in absorbance at 310 nm over time, and the enzyme activity was found in the above culture supernatant at 1.0 unit / ml on the 5th day from the start of the culture.
  • the enzyme activity unit is defined as the amount of enzyme required to produce 1 ⁇ of veratrialdehyde per minute.
  • the culture supernatant of Choriolus' Hilstas 0JI-1078 strain (FE thigh BP-4210), which did not contain donor DN, cultured under the same conditions, did not show any ligne peroxidase activity.
  • a chromosomal gene library was obtained in the same manner as in Example 1. From the obtained library, a clone containing the promoter region of the cellobio hydrolase II gene was selected by plaque hybridization. This series of procedures was carried out by a conventional method (sambrook et al., Molecular Straining A Laboratory Manual / 2nd Edition (1989; ⁇ Z). The probe used for plaque hybridization was funeroketetii. The synthetic oligomer shown in SEQ ID NO: 52 below was prepared based on the nucleotide sequence of the above, and the 3 ′ end was labeled with fluorescein using an oligo DNA labeling kit manufactured by Amersham.
  • втори ⁇ clones could be selected from about 100,000 plaques.
  • Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was carried out using the above-mentioned synthetic DNA. As a result, a clone that hybridized to 5.0 kbp as a single DNA band was observed in the fragment obtained by digestion with the restriction enzymes EcoRV and Ncol.
  • the nucleotide sequence was determined using a BigDye Terminator Cycle Sequencing kit manufactured by Applied Biosystems, followed by electrophoresis using a DNA sequencer PRISM310 manufactured by Applied Biosysteras, and the nucleotide sequence was analyzed.
  • the nucleotide sequence is shown in SEQ ID NO: 50.
  • the cellobio hydrolase ⁇ ⁇ ⁇ gene derived from Coriolus hirustas was separated by six introns.
  • the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 51.
  • Example 42 From the plasmid pCHCBHII obtained in Example 42 containing the promoter sequence of the cellobiohydrolase II gene derived from Coriolus' Hilstas, it was amplified by PCR using the two primers shown in SEQ ID NOS: 53 and 54 below. As a result, a promoter region of 3.1 kbp was obtained.
  • Primer 2 5'-CCATGGTGCGGGGGTGGAGAG-3 '(SEQ ID NO: 54)
  • This DNA fragment obtained by PCR were ligated using T4DNA ligase to Smal site of the E. coli cloning vector P UC18, to obtain a plasmid PCHCBHIIP.
  • a plasmid pBSMPOGl (FERM P-14933) containing a manganese peroxidase gene derived from Coriolus hirustas was amplified by PCR using two primers shown in SEQ ID NOS: 55 and 56 below.
  • Primer 3 5 '-CCATGGCTTTCAAGACACTCG-3' (SEQ ID NO: 55)
  • Primer 4 5'-CCATGGGGAAGGACTGGTGAG-3 '(SEQ ID NO: 56)
  • the DNA fragment obtained by this PCR was inserted into a TA cloning vector using a TA cloning kit manufactured by In Vitrogen, to obtain pTAMP.
  • the obtained pTAMP was digested with the restriction enzyme Ncol, about 2.2 kb was cut out, and Ncol of the above plasmid pCHCBHIIP was digested.
  • the site was introduced to obtain a plasmid pCHCBHIIPMP containing the promoter sequence of the cellobio hydrolase II gene derived from Coriolus hirustas and a manganese peroxidase structural gene (Fig. 10).
  • the protoplast solution 100 having a concentration of 10 6 cells / 100 1 ⁇ m of the plasmid pCHCBHIIPMP prepared in Example 33 was added in a cyclic or linear form. Further as selection markers, the plasmid pUCRl for holding Onorenichin force Luba Moi Honoré Trang Sufuweraze gene derived from Coriolus Hirusutasu 0 - 2 mu epsilon was added, and 30 minutes ice cooling. Next, an equal volume of a PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added to the solution, and the mixture was cooled on ice for 30 minutes. Next, the mixture was mixed with a minimal soft agar medium (agar 1%) containing 0.5 M sucrose and leucine and spread on a plate. The plate was cultured at 28 ° C for 4 days to obtain a transformant.
  • a minimal soft agar medium agar 1%) containing 0.5 M sucrose and le
  • DNA was prepared from the above transformant, and it was confirmed by Southern hybridization that the desired manganese peroxidase expression plasmid pCHCBHIIPMP was incorporated.
  • Enzymatic activity 0.5 M Ma port phosphate sodium buffer (pH 5.5) 50 ⁇ 1, thoroughly mixing the enzyme solution 345 ⁇ 1, 10 m hydrogen peroxide 5 ⁇ 1, 1 mM MnS0 4 100 1, resulting from the reaction ⁇ (III) The increase in absorbance at 2 nm of the malonic acid complex at 2 nm was recorded over time.
  • Mn (III) malonic acid complex generated by the enzymatic activity of manganese peroxidase was observed at 6 ⁇ / ⁇ 1 / ⁇ on day 6 of the culture.
  • the enzyme activity unit was defined as the activity to increase the Mn (III) malonic acid complex by 1 ⁇ mol per minute.
  • it does not contain donor DNA cultured under the same conditions
  • the plasmid pCHCBHIIP obtained in Example 43 was digested with a restriction enzyme Ncol (manufactured by Takara Shuzo Co., Ltd.), and then smoothed using Klenow fragment. After that, it was digested with the restriction enzyme EcoRI to obtain a cellohydrohydrase II gene promoter expression vector.
  • 0J-P0G-E1 (BP-2793) was prepared using the primers of SEQ ID NOs: 57 and 58 shown below.
  • Primer 5 5,-CTCGAGGTTCCAGTCTCTG-3 '(SEQ ID NO: 57)
  • Primer 6 5, -GAATTCCCGGGGACGTATACG-3 '(SEQ ID NO: 58)
  • the obtained DNA fragment by PCR was introduced into a TA-cloning vector in the same manner as in Example 3 to obtain a plasmid pTALAC.
  • the plasmid pTALAC was digested with the restriction enzyme Xhol, and then blunt-ended using the modification enzyme klenow fragment. Then, it was digested with the restriction enzyme EcoRI to obtain a laccase structural gene.
  • Example 44 A protoplast solution of Coriolus' Hilstas was obtained by the method described in 4. To the protoplast solution of about 10 6 ZlOO / l, 2 ⁇ g of the plasmid pCHCBHIIPLAC prepared in Example 46 was added in a cyclic or linear form. Further, as a selection method, 0.2 g of a plasmid pUCRl carrying the orbitin force rubamoinoletransferase gene derived from C. holistus was added, and the mixture was cooled on ice for 30 minutes. Next, add an equal volume of PEG solution (50% PE G3400, 20 mM MOPS (pH 6.4)). In addition, the mixture was cooled on ice for 30 minutes.
  • PEG solution 50% PE G3400, 20 mM MOPS (pH 6.4)
  • the plate was mixed with a minimal soft agar medium (agar 1%) containing 0.5M sucrose and leucine, and wound on a plate.
  • the plate was cultured at 28 ° C. for 4 days to obtain a transformant. Further, DNA was prepared from the above transformant, and it was confirmed by Southern hybridization that the desired laccase expression plasmid pCHCBHIIPLAC was incorporated.
  • Enzyme activity was 50 ⁇ l of 1 M sodium acetate buffer (pH 4.0), 50 ⁇ l of 5 mM ABTS (2,2'-azino-bis (3-ethilbenzthiazoline-6-sulfonate), The enzyme solution was added by adding 400 ⁇ l of the enzyme solution, and the ABTS oxide resulting from the reaction was recorded over time at the increase in absorbance at 420 nm.
  • the enzyme activity unit was the amount of enzyme required to oxidize 1 ⁇ ABTS per minute, while culturing under the same conditions. In the culture supernatant of the 0JI-1078 strain which did not contain the donor DNA, only 5 nit / ml of enzyme activity was observed.
  • the plasmid pCHCBHIIP obtained in Example 43 was digested with a restriction enzyme Ncol (manufactured by Takara Shuzo Co., Ltd.) to obtain a cellobiohydrolase II expression vector portion.
  • a plasmid PBSLP0G7 / E. Coli JM109 (FERM P-12683) containing a high temperature inducible lignin peroxidase gene derived from Coriolus hiltus was subjected to PCR using the two primers of SEQ ID NOs: 59 and 60 shown below. Amplified by the method.
  • Primer 7 5'-CCATGGCGTTCAAGGCTCTTC-3 '(SEQ ID NO: 59)
  • Primer 8 5, -CCATGGAAGCTTCGGAAAGAGC-3 '(SEQ ID NO: 60)
  • the obtained PCR fragment was introduced into the TA-cloning vector as in Example 43, and Smid pTALiP was obtained.
  • the plasmid pTALiP was digested with a restriction enzyme Ncol to obtain a lignin peroxidase structural gene portion.
  • the lignin peroxidase structural gene portion was introduced into the Ncol site of the cellohydrohydrase II expression vector pCHCBHIIP obtained in Example 43 to obtain a plasmid pCHCBHIIPLiP (FIG. 12).
  • Example 44 According to the method described in 4, a protoplast solution of Coriolus hirustas was obtained. 2 g of the plasmid pCHCBHIIPLiP prepared in Example 49 was added cyclically or linearly to about 10 6 ⁇ m of the protoplast solution. Further, as a selection marker, 0.2 g of a plasmid pUCRl carrying an orditin force rubamoinoletransferase gene derived from Coriolus hirustas was added, followed by ice-cooling for 30 minutes.
  • DNA was prepared from the above transformant, and it was confirmed by Southern hybridization that the desired lignin peroxidase expression plasmid pCHCBHIIPLiP was incorporated.
  • the enzyme activity was determined by adding 100 ⁇ l of 1 M sodium tartrate buffer (pH 3.0), 25 ⁇ l of 8 mM veratryl alcohol, 350 ⁇ l of enzyme solution, and 25 ⁇ l of 5.4 mM hydrogen peroxide, and the results of the reaction.
  • the resulting veratraldehyde should be recorded over time as the absorbance increase at 310 nm. Was performed.
  • 1.2 units / ml of enzyme activity was observed on the 5th day from the start of the culture.
  • the enzyme activity unit was defined as the amount of enzyme required to produce 1 z niol of beratol aldehyde per minute.
  • no lignin peroxidase activity was observed in the culture supernatant of Coriolus hirustas 0JI-1078 strain (FERM BP-4210) containing no donor DNA cultured under the same conditions.
  • Example 44 Transformants selected for production of manganese peroxidase with high secretion, selected in 4, were cultured at 28 ° C on a potato dextrose agar medium and then stored at 4 ° C. From this plate, 5 sections cut out with a 5 mm-diameter cork borer were placed in a glucose.peptone medium (glucose 2. polypeptone 0.5%, yeast extract 0.2%,
  • KH 2 P0 4, MgS0 4 0. 05% was inoculated into 300ml Erlenmeyer flasks containing p H4. 5 for preparation) in phosphoric acid by 100ml, 28 ° C, and 1 week shaking cultured at LOOrpra. After the culture, the cells were filtered off, and the medium remaining in the cells was washed with sterile water. The cells were pulverized with sterile water and lOsec in a Waring blender, and inoculated so that the dry weight of the cells was 1 Omg in eucalyptus wood with an absolute dry weight of 1 kg. After inoculation, the mixture was thoroughly stirred so that the bacteria could spread throughout. The culture was allowed to stand still for one week while aerating at 28 ° C. Saturated steam was ventilated as needed so that the chip moisture content was 40 to 65%. The air flow rate was set to O. Olvvm per chip.
  • a chip treatment was carried out in the same manner as in Example 52 using radita-tapain material.
  • the treated wood chips are beaten using a laboratory refiner (manufactured by Kumagai Riki Co., Ltd.) to make the Canadian standard fineness 200 ml.
  • physical tests of pulp handmade sheets were performed in accordance with Tappi method T220 om-83.
  • a power meter Hiokidenki model3133
  • an integrator model3141
  • a chip having an absolute dry weight of 400 g was measured, and the liquid ratio was 5 in an autoclave, the degree of sulfide was 30%, and the effective altitude was 17% (Na 2
  • the cooking white liquor was added to the mixture, and the kraft cooking was performed at a cooking temperature of 150 ° C. After the completion of the kraft digestion, the black liquor was separated, the resulting chips were defibrated by a high-concentration defibrillator, and centrifugal dehydration and water washing with a filter cloth were repeated three times. Then, the unpulped product was removed by a screen and centrifugally dewatered to obtain a pulverized unbleached pulp.
  • the pulp obtained above was subjected to a four-stage bleaching process of D-E-P-D as shown below.
  • D chlorine dioxide
  • the pulp concentration was adjusted to 10% by mass, 0.4% by mass of chlorine dioxide was added, and the treatment was performed at 70 ° C for 40 minutes.
  • Ion After washing and dehydration with water exchange, the pulp concentration was adjusted to 10% by mass, caustic soda was added at 1% by mass, and an alkali extraction treatment (E) was performed at 70 ° (:, 90 minutes). After washing and dehydration, the pulp concentration was adjusted to 10% by mass, and 0.5% by mass of hydrogen peroxide and 0.5% by mass of caustic soda were sequentially added.
  • the pulp slurry having a pulp concentration of 4% by mass obtained above was beaten by a refiner so that the freshness became 410 ml (CSF).
  • Controll is a pulp sheet prepared by processing wood chips without microbial treatment.
  • the present invention provides a promoter sequence that functions in basidiomycetes of a host, particularly Coriolus hirustas, and that allows the host to produce a large amount of lignin-degrading enzyme. It is possible to efficiently produce lignin-degrading enzymes such as mangan phenol oxidase, lignin peroxidase, and laccase, which have been considered difficult, and to treat wood chips with host cells that highly produce the enzymes. Can be. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un promoteur qu'on utilise pour produire à haut rendement de la ligninase dans un hôte basidiomycète, un système vecteur-hôte comprenant ce promoteur, un procédé de production de ligninase à haut rendement à l'aide de ce système et un procédé de traitement de copeaux de bois à l'aide de cette enzyme.
PCT/JP2003/002057 2002-02-25 2003-02-25 Fragment d'adn ayant une activite de promoteur WO2003070939A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003211681A AU2003211681A1 (en) 2002-02-25 2003-02-25 Dna fragment having promoter activity
JP2003569832A JP4016948B2 (ja) 2002-02-25 2003-02-25 プロモーター活性を有するｄｎａ断片

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
JP2002-48674 2002-02-25
JP2002048674 2002-02-25
JP2002-206762 2002-07-16
JP2002206762 2002-07-16
JP2002-252055 2002-08-29
JP2002251923 2002-08-29
JP2002252055 2002-08-29
JP2002-251720 2002-08-29
JP2002-251923 2002-08-29
JP2002251720 2002-08-29

Publications (1)

Publication Number Publication Date
WO2003070939A1 true WO2003070939A1 (fr) 2003-08-28

Family

ID=27761616

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/002057 WO2003070939A1 (fr) 2002-02-25 2003-02-25 Fragment d'adn ayant une activite de promoteur

Country Status (3)

Country Link
JP (1) JP4016948B2 (fr)
AU (1) AU2003211681A1 (fr)
WO (1) WO2003070939A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007518407A (ja) * 2004-01-15 2007-07-12 アンスティテュ ナシオナル ドゥ ラ ルシェルシュ アグロノミック シュタケ（Ｐ．ｃｉｎｎａｂａｒｉｎｕｓ）の一核株によって所定の組換えタンパク質を過剰生成する方法
JPWO2006016596A1 (ja) * 2004-08-12 2008-07-31 王子製紙株式会社 リグノセルロース材料からの繊維成分の製造およびその利用
WO2010005553A1 (fr) * 2008-07-07 2010-01-14 Mascoma Corporation Isolement et caractérisation de cellobiohydrolase i (cbh 1) de schizochytrium aggregatum
JP2011160770A (ja) * 2010-02-15 2011-08-25 Tokyo Univ Of Agriculture & Technology リグニンペルオキシダーゼ遺伝子およびマンガンペルオキシダーゼ遺伝子を検出するためのオリゴヌクレオチド並びにこれを用いたリグニンペルオキシダーゼ遺伝子およびマンガンペルオキシダーゼ遺伝子のスクリーニング方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1029922A2 (fr) * 1999-02-15 2000-08-23 Oji Paper Co., Ltd. Fragments d'ADN de basidiomycètes avec activité de promoteur et leurs utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1029922A2 (fr) * 1999-02-15 2000-08-23 Oji Paper Co., Ltd. Fragments d'ADN de basidiomycètes avec activité de promoteur et leurs utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DUMONCEAUX T.J. ET AL.: "Cloning and sequencing of a gene encoding cellobiose dehydrogenase from trametes versicolor", GENE, vol. 210, no. 2, 1998, pages 211 - 219, XP004118173 *
MOUKHA S.M. ET AL.: "Cloning and analysis of pycnoporus cinnabarinus cellobiose dehydrogenase", GENE, vol. 234, no. 1, 1999, pages 23 - 33, XP004172157 *
SIMS P. ET AL.: "The identification, molecular cloning and characterisation of a gene from phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from trichoderma reesei", GENE, vol. 74, no. 2, 1988, pages 411 - 422, XP002962147 *
YAGUE E. ET AL.: "Correlation of exons with functional domains and folding regions in a cellulase from agaricus bisporus", CURR. GENET., vol. 30, no. 1, 1996, pages 56 - 61, XP001052774 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007518407A (ja) * 2004-01-15 2007-07-12 アンスティテュ ナシオナル ドゥ ラ ルシェルシュ アグロノミック シュタケ（Ｐ．ｃｉｎｎａｂａｒｉｎｕｓ）の一核株によって所定の組換えタンパク質を過剰生成する方法
JP4719690B2 (ja) * 2004-01-15 2011-07-06 アンスティテュ ナシオナル ドゥ ラ ルシェルシュ アグロノミック シュタケ（Ｐ．ｃｉｎｎａｂａｒｉｎｕｓ）の一核株によって所定の組換えタンパク質を過剰生成する方法
JPWO2006016596A1 (ja) * 2004-08-12 2008-07-31 王子製紙株式会社 リグノセルロース材料からの繊維成分の製造およびその利用
JP4682982B2 (ja) * 2004-08-12 2011-05-11 王子製紙株式会社 リグノセルロース材料からの繊維成分の製造およびその利用
WO2010005553A1 (fr) * 2008-07-07 2010-01-14 Mascoma Corporation Isolement et caractérisation de cellobiohydrolase i (cbh 1) de schizochytrium aggregatum
US8470592B2 (en) 2008-07-07 2013-06-25 Mascoma Corporation Isolation and characterization of Schizochytrium aggregatum cellobiohydrolase I (Cbh 1)
JP2011160770A (ja) * 2010-02-15 2011-08-25 Tokyo Univ Of Agriculture & Technology リグニンペルオキシダーゼ遺伝子およびマンガンペルオキシダーゼ遺伝子を検出するためのオリゴヌクレオチド並びにこれを用いたリグニンペルオキシダーゼ遺伝子およびマンガンペルオキシダーゼ遺伝子のスクリーニング方法

Also Published As

Publication number Publication date
JP4016948B2 (ja) 2007-12-05
JPWO2003070939A1 (ja) 2005-06-09
AU2003211681A1 (en) 2003-09-09

Similar Documents

Publication Publication Date Title
JP4385766B2 (ja) セルロース分解酵素遺伝子及び該遺伝子の利用
US9512409B2 (en) Thermostable catalase
JP4922524B2 (ja) 糸状菌の分野における新規発現調節配列および発現産物
KR20150113170A (ko) 탄수화물 분해 폴리펩티드 및 이의 용도
WO2012068236A2 (fr) Nouvelles oxydoréductases fongiques
Yu et al. Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher basidiomycetes)
JP4682982B2 (ja) リグノセルロース材料からの繊維成分の製造およびその利用
JPH0767637A (ja) バチラス種由来のキシラナーゼ、その発現ベクター及び宿主生物
JP4472787B2 (ja) 微生物キシログルカン エンドトランスグリコシラーゼ（ｘｅｔ）
US5426043A (en) Cloning and expression of acetyl xylan esterases from fungal origin
JPH08500727A (ja) 新規な酵素製剤及びその製造方法
WO2003070939A1 (fr) Fragment d'adn ayant une activite de promoteur
JP3585840B2 (ja) タンパク質の製造のための発現系
US7001751B1 (en) PyrF gene and the utilization thereof
CN115052477A (zh) 在多肽生产中具有提高的生产力的丝状真菌细胞的突变体
JP2009124995A (ja) リグノセルロース分解酵素遺伝子およびその利用
EP1029922B1 (fr) Fragments d'ADN de basidiomycètes avec activité de promoteur et leurs utilisation
JP2007104937A (ja) 薬剤耐性遺伝子の利用
EP4012018A1 (fr) Procédé de production d'une composition d'enzyme technique à faible viscosité produite par un champignon filamenteux
JP2008263862A (ja) 白色腐朽菌の形質転換体の作出方法及び該方法により得られた形質転換体
WO2022214679A1 (fr) Procédé de production d'une composition enzymatique technique à faible viscosité produite par un champignon filamenteux
JP2003189855A (ja) 新規薬剤耐性遺伝子
JP2000342275A (ja) 担子菌由来プロモーター活性を有するｄｎａ断片および該プロモーター活性の調節下での外来遺伝子の発現
CN116601302A (zh) 用于产生具有低粘度的丝状真菌全肉汤酶组合物的方法
JP2007300825A (ja) デルタ−９−脂肪酸デサチュラーゼ遺伝子および該遺伝子を導入したアラゲカワラタケ形質転換体

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2003569832

Country of ref document: JP

122 Ep: pct application non-entry in european phase