JP2008263862A - Method for creating transformant of white-rot fungus and transformant obtained by the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a strain highly producing YK-LiPs (lignin peroxidases) exhibiting excellent characteristics of degrading polymer lignins and derived from a Phanerochaete sordida YK-624 strain and to provide a means of highly expressing each transferred gene in UV-#64 strain (a strain derived from the YK-624 strain). <P>SOLUTION: The uracil-requiring mutant strain UV-#64 using the Phanerochaete sordida YK-624 strain as a parent strain is provided. A method for creating a transformant of the YK-624 strain comprising using the UV-#64 strain as a host strain is provided. GPD-YKLiP1 No. 11 and GPD-YKLiP2 No. 3 (strains having each gene transferred thereinto) obtained by the method and highly expressing the YK-LiPs are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a transformant of white rot fungus, and more specifically, a method for producing a transformant of Usukiirokawatake YK-624, and a lignin peroxidase derived from YK-624 produced using the method It relates to a new strain that produces a high amount of.

White rot fungi are the only organisms that highly degrade lignin, a persistent macromolecule. When white rot decomposes lignin, it secretes redox enzymes such as lignin peroxidase and manganese peroxidase to the outside of the cell. Among them, lignin peroxidase, in addition to direct oxidative degradation of polymer lignin, is difficult to decompose such as dioxin. Since it has the ability to decompose aromatic compounds, it is expected to be used in the paper and pulp industry (biopulping and biobleaching) and bioremediation.
Plant biomass is mainly composed of cellulose, hemicellulose, and lignin. By decomposing lignin, the cellulose changes to a state where it is easily degraded by cellulase. Therefore, lignin peroxidase is useful in a bioethanol production process by decomposing cellulose and a digestibility improving process by delignification of feed crops.

A typical white rot fungus is Phanerochaete chrysosporium (no Japanese name, hereinafter referred to as P. chrysosporium ), and many of the past developments in polymer lignin degradation technology have been carried out using this fungus. There are many known bacteria that strongly degrade lignin, such as Kawatake and Oyster mushrooms, but these bacteria have the disadvantage of degrading macromolecular lignin and at the same time degrading cellulose, which is a useful biomass. .
On the other hand, the present inventor screened more than 100 white rot fungi collected from Yakushima, and as a result, Phanerochaete sordida (selective white rot fungus) that selectively decomposes high molecular weight lignin Phanerochaete sordida ( Usukiirokawatake , The following P. sordida ) YK-624 strain was found. When cultured for 28 days various white rot fungi in beech containing a large amount of polymer lignin, P. chrysosporium, and Coriolus versicolor 24% polymeric lignin respectively, whereas the decomposed 27%, P. sordida YK-624 The strain was degraded by 41%. On the other hand, the degradation amount of cellulose of YK-624 strain was about 1/4 of that of P. chrysosporium and Kawaratake.

A typical example of lignin peroxidase is LiPH8 produced by P. chrysosporium , and most lignin peroxidase studies have been conducted using this enzyme. On the other hand, the YK-624 strain is characterized by producing novel lignin peroxidases (YK-LiP1 and YK-LiP2) that are superior to LiPH8 in degrading high molecular weight lignin. The dimeric lignin model compound has the ability to decompose about 95% for YK-LiP1 and about 50% for YK-LiP2, while LiPH8 decomposes only about 30% in 24 hours.
However, since the expression level of YK-LiPs (YK-LiP1,2) in the YK-624 strain is low, the production of a strain that highly expresses YK-LiPs was desired for industrial use of this strain. .

In the meantime, a manganese peroxidase gene that also exhibits lignin-degrading activity has been patented from the YK-624 strain (see, for example, Patent Document 1). In addition, a oyster mushroom-derived manganese peroxidase gene that also exhibits the degradation activity of polymer lignin has been patented (see, for example, Patent Document 2).
In addition, P. chrysosporium has been reported to overexpress LipH8, which is a typical lignin peroxidase, by a gene introduction method (see, for example, Non-Patent Document 1). Furthermore, a gene transfer system developed for the purpose of expressing lignin peroxidase and the like in white rot fungus, Arakawakawatake, has been patented (for example, see Patent Document 3).

  However, since these bacterial species degrade lignin and cellulose at approximately the same rate, it is inevitable that the cellulose content is reduced during delignification treatment, and especially when kraft pulp biopulping is performed, degradation of cellulose is inevitable. Causes deterioration of paper quality. For this reason, it is particularly desirable for delignification treatment of plant biomass to express lignin peroxidase with high polymer lignin degrading ability in bacterial species with high original selective lignin degrading ability such as YK-624 strain. It was thought.

JP 2000-152786 A Japanese Unexamined Patent Publication No. 7-313156 JP 2000-342275 A Appl Environ Microbiol. 65, 1670-1674 (1999)

There are many well-known bacteria that strongly degrade lignin, such as P. chrysosporium , which is a typical white rot fungus, Kawaratake, oyster mushroom , etc., but these bacteria decompose cellulose lignin at the same time as degrading high molecular lignin. There was a disadvantage that the same amount was decomposed.
Therefore, in the present invention, the purpose is to produce a strain that highly produces YK-LiPs by decoding the full-length amino acid sequence of YK-LiPs and newly developing a gene transfer system for the YK-624 strain. It was.

In order to solve the above problems, in the present invention, first, the N-terminal amino acid sequence of YK-LiP that has already been purified as a protein is analyzed, and partial information of the cDNA sequence is obtained by performing degenerate PCR based on that information. I got it. Subsequently, the full-length YK-LiP gene ( YK-LiP1 , YK-LiP2 ) was cloned from the genomic DNA of the YK-624 strain by 3'-RACE and 5'-RACE methods, and the entire nucleotide sequence and deduced amino acid sequence were revealed. (SEQ ID NO: 78, 79).

Next, in the P. sordida species including the YK-624 strain, unlike the white rot fungus P. chrysosporium, which has already established a transformation system, conidia are not formed, and fruit bodies are formed and spores are recovered. Since the conditions were not found and there has been no successful gene transfer system so far, we attempted to construct a gene transfer system in the YK-624 strain and completed a stable gene transfer technique.

In addition, since no gene expression vector that has been confirmed to function in P. sordida has been reported so far, the full length of the constitutive gene GPDH (also referred to as glycerol triphosphate dehydrogenase, GPD ) is newly obtained from the YK-624 strain. And a gene expression vector that strongly expresses the transgene was constructed by using the promoter and terminator region (FIG. 10). This vector had the ability to express the transgene strongly (1.2% of the total soluble protein) (FIG. 12).

  The present inventors introduced YK-LiP1 and YK-LiP2 genes by transforming YK-LiP1 and YK-LiP2 genomic DNA into YK-624 strain using the above gene transfer method and gene expression vector. A number of clones were obtained (FIGS. 13 and 14). The present invention was completed by selecting strains (YK-LiP1 No11, YK-LiP2 No3) having higher lignin peroxidase activity than the parent strain from those clones (FIG. 15).

As described above, the present invention uses the original P. sordida YK-624 strain having high selective lignin-degradability as a producing bacterium, a gene introduction method, and gene expression for the first time in a selective white-rot fungus P. sordida . It is novel in that the vector was constructed, and that YK-LiP1 and YK-LiP2, which are superior in the ability to decompose high molecular lignin compared to the typical lignin peroxidase LiPH8, were highly expressed.

That is, the present invention described in claim 1 is a uracil-requiring mutant UV- # 64 (NITE AP-344) whose parent strain is Usukiirokawatake YK-624.
The present invention described in claim 2 comprises the nucleotide sequence of SEQ ID NO: 69, and makes the UV- # 64 strain non-uracil-requiring by introducing it into the UV- # 64 strain described in claim 1. YK-624 strain-derived orotate phosphoribosyltransferase functioning as a marker gene for gene transfer systems.
The present invention according to claim 3 is a plasmid containing the gene according to claim 2.
The present invention according to claim 4 is a gene expression vector comprising a DNA consisting of the nucleotide sequence of SEQ ID NO: 75 and having the ability to highly express a transgene in Usukiirokawatake.
The present invention described in claim 5 is a method for producing a transformant of YK-624 strain using the UV- # 64 strain described in claim 1 as a host strain.
The present invention according to claim 6 is the production method according to claim 5, wherein the plasmid according to claim 3 is introduced into the host strain together with the foreign gene.
The present invention described in claim 7 is the production method according to claim 5 or 6, wherein a foreign gene is introduced using the gene expression vector according to claim 4.
The present invention described in claim 8 is a new strain GPD-YKLiP1 No11, wherein the UV- # 64 strain described in claim 1 is used as a parent strain and YK-624 strain-derived lignin peroxidase (YK-LiP1) production ability is higher than that of the parent strain. (NITE AP-342).
The present invention according to claim 9 is a new strain GPD-YKLiP2 No3, which has the UV- # 64 strain according to claim 1 as a parent strain and exhibits higher ability to produce lignin peroxidase (YK-LiP2) derived from the YK-624 strain than the parent strain. (NITE AP-343).
The present invention described in claim 10 is a gene introduction kit for the YK-624 strain comprising the plasmid of claim 3 and / or the gene expression vector of claim 4.

According to the present invention, it has become possible to obtain a high-producing strain of YK-LiP1 and YK-LiP2 that are excellent in the ability to decompose high-molecular-weight lignin in the selective white-rot fungus Usukiirokawatake YK-624. For YK-624 strain without gene transfer, YK-LiP1 and YK-LiP2 were forcibly expressed by gene expression vectors pGPD-LiP1 and pGPD-LiP2 , respectively. In GPD-LiP1 No11 and GPD-LiP2 No3, liquid culture This effect was confirmed by the remarkably high enzyme activity in the supernatant.
Examples of conventional techniques include the production of lignin peroxidase in white-rot fungi such as P. chrysosporium , Aragekawa hirsutus , and oyster mushroom ( Pleurotus ostreatus ). The present invention is based on the degree of cellulose degradation associated with lignin degradation. It is advantageous in that it uses YK-624, which is a low selective white rot fungus.
In addition, by using this gene introduction method, any gene other than YK-LiPs enzyme can be expressed in the YK-624 strain.

Hereinafter, the present invention will be described in detail.
The present invention provides a method for producing a transformant of white-rot fungus Usukiirokawatake.

The method for producing a transformant of the present invention is characterized by using a uracil auxotrophic mutant strain having the parent strain Usukiirokawatake YK-624 as a host strain into which a foreign gene is introduced.
The uracil-requiring mutant strain of YK-624 is a medium containing 5-fluoroorotic acid after creating protoplasts from the vegetative mycelium of parent strain YK-624 and irradiating it with ultraviolet light to introduce mutations. Protoplast regeneration can be performed by selecting uracil-requiring clones. Detailed procedures are described in Examples 5 and 6.

The uracil auxotrophic mutant thus obtained had mutations on the uracil synthase orotic acid phosphoribosyltransferase genes URA3 and URA5 , of which 43 base pairs were missing in the coding region of URA5. Those having a mutation were named UV- # 64 strain. This UV- # 64 strain has been deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation, and the deposit number is NITE AP-344.
The mycological properties of the UV- # 64 strain were the same as the parent strain YK-624 except that it was uracil-requiring. In addition, there is no conspicuous difference in the growth ability of the fungus, and there is no difference in the YK-Lips productivity of the fungus.

  In the present invention, transformation is carried out using the above-mentioned UV- # 64 strain as a host strain. The gene introduction method is not particularly limited, and for example, the polyethylene glycol (PEG) method can be used.

In addition, as a marker for confirming that the target gene has entered the UV- # 64 strain at the time of gene introduction, a marker gene to be incorporated together with the transgene is introduced into the UV- # 64 strain. The strain # 64 can be used as long as it can be made uracil non-requiring and can function as a marker gene for the gene transfer system of the present invention. For example, mouse Yellow River bamboo, and Phanerochaete spp such P.Chrysosporium, Coriolus hirsutus, can be used a gene encoding orotate phosphoribosyltransferase URA5 from Pleurotus ostreatus. Of these, from YK-624 strain URA5 (PsURA5) In particular, PsURA5 is preferred (Example 7).
In addition, even when the marker gene described above is introduced into a uracil-requiring mutant strain of Usukiirokawatake other than the UV- # 64 strain, the host strain can be made non-requiring to uracil, and the gene transfer system marker Can function as a gene.

When the marker gene is used in the present invention, a plasmid containing the marker gene is prepared and mixed with a recombinant vector (gene expression vector) containing the target transgene, for example, into a protoplastized vegetative mycelium. What is necessary is just to introduce | transduce into UV- # 64 strain | stump | stock by a conventional method, such as the method of introducing a gene using PEG method (Examples 16 and 17).
Here, the method for preparing the plasmid containing the marker gene is not particularly limited, and the plasmid to be used can be appropriately selected.

The gene expression vector used in the method for producing a transformant of the present invention is not particularly limited as long as the transgene can be expressed in the host strain, and in particular, those having the ability to highly express the transgene are preferable. For example, a material containing a promoter region and a terminator region of a gene that is strongly expressed in a host strain, such as Ukishirokawatake , particularly glycerol 3-phosphate dehydrogenase ( PsGPD ) derived from YK-624 strain, is preferably used. A specific example of such a gene expression vector is pPsGPD - EGFP prepared in Examples 11 and 12.
In addition, the gene expression vectors described above are host strains such as Ukishirokawatake , such as UV- # 64 and YK-624, Phanerochaete, such as P. chrysosporium, and closely related species, such as Aragekawaratake , oyster mushroom, etc. Also when used, it has the ability to highly express the transgene in the host.

  In the present invention, when the desired transgene is incorporated into the UV- # 64 strain using the above gene expression vector, the transgene is sandwiched between the promoter region and the terminator region of the gene expression vector by a conventional method. Combine. Specifically, it can be carried out according to the methods described in Examples 14 and 15.

The foreign gene to be introduced into the UV- # 64 strain in the method for producing the transformant of the present invention is not particularly limited, but lignin peroxidases YK-LiP1 and YK that selectively degrade the high molecular lignin produced by the YK-624 strain in particular. -LiP2 is preferred. A purification method for the YK-LiPs enzyme is already known (FEMS Microbiol Lett 246: 19-24), but the gene was elucidated for the first time in the present invention (Examples 3 and 4).

The new strains obtained by transforming the above - described YK-LiP1 and YK-LiP2 into the UV- # 64 strain by the method of the present invention are each GPD-YKLiP1 No11 (hereinafter sometimes abbreviated as GL1 No11). And GPD-YKLiP2 No3 (hereinafter sometimes abbreviated as GL2 No3) (Example 18). These transformed strains have been deposited with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, with the accession numbers of NITE AP-342 and NITE AP-343, respectively.
Since the above-described transformants GL1 No11 and GL2 No3 of the present invention had significantly higher lignin peroxidase (LiP) activity than the parent strain, as shown in Example 18, YK− higher than that of the parent strain. It was found to have LiP1 and YK-LiP2 enzyme production ability.

The present invention also provides for transgenic for YK-624 strain containing the gene expression vector, comprising a nucleotide sequence of p PsGPD-EGFP of the invention the plasmid p PsURA5, and / or described above containing the gene marker PsURA5 of the present invention described above Provide kit.
In addition to the gene marker and gene expression vector, the gene introduction kit may contain reagents, instruments, instruction manuals and the like that are usually used for gene introduction.

  The present invention will be specifically described below with reference to examples.

Example 1 (N-terminal amino acid analysis of YK-LiPs protein)
YK-LiPs protein was purified from the culture filtrate of strain YK-624 according to the previous report (FEMS Microbiol Lett 246: 19-24). Using 2 micrograms of purified YK-LiPs protein, SDS-PAGE analysis was performed according to the method of laemmli (In “Methods in Carbohydrate Chemistry,” Vol. 1 (Academic Press), pp. 338 (1962)). SDS-PAGE was carried out using a mini slab electrophoresis apparatus for 10 minutes polyacrylamide gel and constant current (20 mA) for 90 minutes. After transferring to a PVDF membrane (clear blot, ATTO), staining was performed using MEMCODE STAIN FOR PVDF (Pierce). The region where the YK-LiPs protein was transcribed was cut out with a cutter knife and subjected to N-terminal analysis (request analysis from Apro Science). The obtained N-terminal amino acid sequence of 21 residues was A (A / T) CSNG (A / K) TVS (A / D) ASCCAWF (A / D / N) VL (/ is a plurality of amino acids detected) Means that).

Example 2 (degenerate PCR analysis based on the N-terminal amino acid sequence of YK-LiPs protein (Fig. 1))
Based on the information on the N-terminal amino acid sequence obtained in Example 1, degenerate PCR was performed. Primer 1 (P1: SEQ ID NO: 1) and primer 2 (P2: SEQ ID NO: 2) were used as forward primers, and primer 3 (P3: SEQ ID NO: 3) was used as a reverse primer. In the following, all primers were synthesized by Fasmac DNA Synthesis Division. HotStart Taq (QIAGEN) was used as the PCR enzyme. Hereinafter, unless otherwise indicated, all PCR reactions were performed using this enzyme. As template DNA for PCR reaction, cDNA synthesized from RNA of YK-624 strain cultured for 5 days to produce YK-LiPs protein under the conditions of Example 1 was used. RNA was purified using RNeasy Mini kit (QIAGEN). Hereinafter, all RNA purification was performed using the RNeasy Mini kit. cDNA synthesis was performed using the reverse transcriptase Reverse Transcriptase XL AMV (TAKARA), it was used to 1st strand cDNA synthesis reaction of the reverse transcription reaction oligodT _18. Hereinafter, unless otherwise specified, all reverse transcription reactions were performed using Reverse Transcriptase XL AMV.

  A 60 bp-sized DNA fragment obtained by PCR was TA cloned using pT7Blue T-Vector (Novagen) and DNA Ligation kit ver2.1 (TAKARA), and transformed into competent cell DH5α (TOYOBO). Hereinafter, unless otherwise specified, TA cloning of all DNA fragments obtained by PCR was performed using this method. Emerging E. coli clones were isolated and plasmids were recovered from each using the QIAprep Spin Miniprep Kit (QIAGEN). Using each plasmid as a template, a sequencing reaction was performed using BigDye Terminator ver3.1 (ABI), and the cloned 60 bp PCR product was analyzed using ABI PRISM 310 Genetic Analyzer (ABI). In the following, all DNA sequences were sequenced using BigDye Terminator ver3.1 and ABI PRISM 310 Genetic Analyzer.

As a result of sequencing the recovered plasmid, the PCR product generated from primer set P1-P3 was DNA sequence 1 (DNA1), and the PCR product generated from primer set P2-P3 was DNA sequence 2 (DNA2). -LiPs enzyme was expected to be a mixture of two genes. YK-LIP1 fragment DNA1, was named YK-LIP2 fragment DNA 2.

Example 3 (Cloning of YK-LiP1 full length (Figure 2))
Based on the DNA sequence of the YK-LiP1 fragment, forward primer 4 (P4: SEQ ID NO: 4) and forward primer 5 (P5: SEQ ID NO: 5) were synthesized. From the RNA described in Example 2, a reverse transcription reaction was performed using primer 6 (P6: SEQ ID NO: 6) for the first strand cDNA synthesis reaction of the reverse transcription reaction. 3′-RACE PCR reaction was performed using the obtained cDNA as a template and a set of P4 and P7 (SEQ ID NO: 7). The obtained PCR reaction product was diluted as a template, and nested PCR was performed using a set of P5 and P7. The obtained PCR product of about 1100 bp was subjected to TA cloning and sequencing. As a result, DNA sequence 3 (DNA3) was obtained. Analysis of amino acid sequence 1 deduced from DNA3 using the FASTA DNA analysis program (http://fasta.genome.jp/) showed high homology with known lignin-degrading enzymes. Was suggested to be part of

Next, 5′-RACE PCR was performed to clone the 5 ′ side of YK-LiP1 . Using the RNA described in Example 2, cDNA used for 5′-RACE PCR was synthesized using GeneRacer Kit (Invitorgen). Reverse primer 8 (P8: SEQ ID NO: 8) and reverse primer 9 (P9: SEQ ID NO: 9) were synthesized. First, 5′-RACE PCR reaction was performed with forward primer 10 (P10: SEQ ID NO: 10) and P8 set. It was. The obtained PCR reaction product was diluted as a template, and nested PCR was performed using a set of P10 and P9. The resulting 250 bp PCR product was TA cloned and sequenced. As a result, DNA sequence 4 (DNA4) was obtained.

Based on the information of DNA4 and DNA3, forward primer 11 (P11: SEQ ID NO: 11) and reverse primer 12 (P12: SEQ ID NO: 12) were synthesized, and PCR enzyme (Platinum Taq DNA polymerase, Invitrogen) with few PCR errors was synthesized. PCR reaction was carried out. As templates, the cDNA synthesized in Example 2 and the genomic DNA purified from YK-624 were used. Each of the obtained PCR products was TA cloned, and 3 clones were compared to confirm that there were no base sequence errors associated with the PCR reaction. As a result, DNA sequence 5 (DNA5: SEQ ID NO: 65) and DNA sequence 6 were finally obtained. (DNA6: SEQ ID NO: 66) was obtained and designated as full-length YK-LiP1 cDNA and YK-LiP1 genomic DNA.

Amino acid sequence 2 (SEQ ID NO: 78) deduced from DNA sequence 5 contains the translation initiation methionine, the deduced signal sequence, and the N-terminal 21 amino acids obtained in Example 1, which was designated as the YK-LiP1 amino acid sequence. . YK-LiP1 genomic DNA consists of 8 exons (indicated by the white box in Fig. 2), 7 introns, short 5'UTR (untranslated region) and 3'UTR by comparison with YK-LiP1 cDNA It was 1707 bp long DNA.

Example 4 (Cloning of YK-LiP2 full length (Figure 3))
Based on the DNA sequence of the YK-LiP2 fragment, forward primer 13 (P13: SEQ ID NO: 13) and forward primer 14 (P14: SEQ ID NO: 14) were synthesized. From the RNA described in Example 2, reverse transcription reaction was performed using P6 for the first strand cDNA synthesis reaction of reverse transcription reaction. A 3′-RACE PCR reaction was performed using the obtained cDNA as a template and a set of P13 and P7. The obtained PCR reaction product was diluted as a template, and nested PCR was performed using a set of P14 and P7. The obtained PCR product of about 1100 bp was subjected to TA cloning and sequencing. As a result, DNA sequence 7 (DNA7) was obtained. Analysis of amino acid sequence 3 deduced from DNA7 using the FASTA DNA analysis program showed high homology with known lignin-degrading enzymes, suggesting that DNA7 is a part of lignin-degrading enzymes.

Next, 5′-RACE PCR was performed to clone the 5 ′ side of YK-LiP2 . Using the RNA described in Example 2, cDNA used for 5′-RACE PCR was synthesized using GeneRacer Kit (Invitorgen). Reverse primer 15 (P15: SEQ ID NO: 15) and reverse primer 16 (P16: SEQ ID NO: 16) were synthesized, and first, 5′-RACE PCR reaction was performed with a set of forward primer 10 (P10) and P15. The obtained PCR reaction product was diluted as a template, and nested PCR was performed using a set of P10 and P16. The resulting 250 bp PCR product was TA cloned and sequenced. As a result, DNA sequence 8 (DNA8) was obtained.

Based on the information of DNA8 and DNA7, forward primer 17 (P17: SEQ ID NO: 17) and reverse primer 18 (P18: SEQ ID NO: 18) were synthesized, and PCR enzyme (Platinum Taq DNA polymerase, Invitrogen) with few PCR errors was synthesized. PCR reaction was carried out. As templates, the cDNA synthesized in Example 2 and the genomic DNA purified from YK-624 were used. Each of the obtained PCR products was TA cloned and compared to 3 clones to confirm that there were no base sequence errors associated with the PCR reaction. As a result, DNA sequence 9 (DNA9: SEQ ID NO: 67) and DNA sequence 10 were finally obtained. (DNA10: SEQ ID NO: 68) was obtained and designated as full-length YK-LiP2 cDNA and YK-LiP2 genomic DNA.

Amino acid sequence 4 (SEQ ID NO: 79) deduced from DNA sequence 9 contains the translation initiation methionine, the deduced signal sequence, and the N-terminal 21 amino acids obtained in Example 1, and this was designated as the YK-LiP2 amino acid sequence. Compared with YK-LiP2 cDNA, YK-LiP2 genomic DNA consists of 9 exons (shown in white box in Fig. 3), 8 introns, 1727 bp long DNA consisting of short 5'UTR and 3'UTR. there were.

Example 5 (Protoplast preparation method from YK-624 strain)
YK-624 strain was liquid cultured in a 500 ml Erlenmeyer flask. Medium CYM (Glucose 20g / l, Polypeptone 0.2g / l, Yeast Extract 0.2g / l, KH 2 PO 4 0.46g / l, K 2 HPO 4 1.0g / l, MgSO 4 -7H 2 O 0.5g / l , Thiamine 0.1 mg / l in distilled H ₂ O all Wako Pure Chemicals), 100 ml was added to the Erlenmeyer flask and autoclaved (121 ° C., 15 min). In addition, all operation below was performed using the autoclave sterilizer and the solution. All reagents below were purchased from Wako Pure Chemical unless otherwise specified.

  First, three pieces of YK-624 mycelia cultured on a CYM agar medium (CYM liquid medium-1.5% agar) were inoculated into one Erlenmeyer flask, and liquid culture was performed. In the following, all the bacteria were cultured at 30 ° C. After 7 days of culture, the entire amount of liquid culture mycelium was homogenized with Cellmaster CM-100 (Iuchi), dispensed into 8 Erlenmeyer flasks, and the culture was continued. After 4 days of culture, the entire amount of liquid culture mycelium was homogenized, and the entire amount was dispensed into 16 Erlenmeyer flasks, and the culture was continued. After 1 day of culture, the entire mycelium was aspirated and collected with a Buchner funnel and a glass filter.

The collected cells were suspended in 80 ml of 0.75 M magnesium sulfate buffer (0.75 M MgSO ₄ -7H ₂ O, 20 mM MES, pH 6.3). Cell wall lysing enzymes, Lyzing Enzyme (Sigma) and Cellulase Onozuka RS (Yakult) were added to a final concentration of 3%, respectively, and gently shaken at 29 ° C. for 18 hours. To separate protoplasts generated during shaking from undissolved mycelium, 7.5 ml of lysate is dispensed into a 15 ml centrifuge tube, and 5.5 ml of 1.0 M sorbitol buffer (1.0 M Sorbitol, 10 mM MES, pH 6.3) is overlaid. did. By centrifuging at 4,000 × g for 30 minutes, protoplasts accumulated at the interface of the overlaid solution, and all undissolved mycelium precipitated. Only protoplasts were collected with a Pasteur pipette, and an equal volume of 1.0 M sorbitol buffer was added and centrifuged (2,000 × g, 15 minutes). Since all protoplasts were precipitated, the supernatant was removed, 15 ml of 1.0 M sorbitol buffer was added, and the mixture was centrifuged again (2,000 × g, 10 minutes). The precipitated protoplast was suspended in 1 ml of 1.0 M sorbitol buffer to prepare a YK-624 protoplast preparation. The cell number was counted with a hematometer.

Example 6 (Production of uracil-requiring mutant strain by mutation treatment of YK-624 strain protoplast (FIGS. 4 and 5))
In order to introduce the gene into the YK-624 strain, an auxotrophic (uracil auxotrophic) mutant of the YK-624 strain was constructed (Fig. 4). ^Six YK-624 strain protoplasts 10 prepared according to Example 5 were suspended in 5 ml of 1.0 M sorbitol buffer and transferred to a 6 cm cell culture dish (Iwaki). Without a lid, UV light having a wavelength of 260 nm was irradiated under 5 cm of a UV transilluminator while stirring the protoplast solution using a miniature rotor having a diameter of 1 mm and a length of 1 cm and a magnetic stirrer. The light intensity was 0.75 mW / cm ² and the irradiation time was 30 seconds. After irradiation, in order to prevent DNA repair enzyme from functioning, it was allowed to stand at 4 ° C. in the dark for 18 hours.

  Low-melting point agarose (SeaPlaque Agarose, TAKARA) was added to a CYM liquid medium containing 1.0 M concentration of Sucrose to make 1%, and a medium maintained at 39 ° C. after autoclaving was used as a regeneration medium. To allow only uracil-requiring mutants to grow, 5-FOA (5-fluoroorotic acid, Wako Pure Chemical, final concentration 0.75 mg / ml), and uracil (Wako Pure Chemical, final concentration 0.1 mg / ml) Was added to the above regeneration medium, and the whole amount of the above protoplast suspension was added, and dispensed into 10 petri dishes for 10 cm cell culture. After culturing for 7 days at 30 ° C., about 100 regenerated protoplast-derived clones were isolated, transferred to CYM agar medium (without uracil) containing 0.75 mg / ml of 5-FOA, and cultured for 7 days.

  As a result of inoculating each strain again on CYM agar medium (excluding uracil) and continuing the culture, the strain does not grow on uracil-free CYM agar medium and grows well on CYM agar medium containing uracil 0.1 mg / ml Was identified as UV- # 64 strain (FIG. 5).

Example 6 (Full-length cloning of uracil biosynthetic gene (orotate phosphoribosyltransferase) from YK-624 strain (FIG. 6))
In order to clone orotate phosphoribosyltransferase from YK-624 strain, the amino acid sequence of related basidiomycete orotate phosphoribosyltransferase gene was compared with CLASTAL-DNA analysis program (http://align.genome.jp/clustalw/) And found a highly conserved area. Based on the amino acid sequence, primer 19 (P19: SEQ ID NO: 19) and primer 20 (P20: SEQ ID NO: 20) were designed and degenerate PCR was performed. The cDNA synthesized in Example 2 was used as a template for the PCR reaction. The obtained PCR product was TA cloned and 6 clones were sequenced. As a result, only DNA sequence 11 (DNA11) was obtained. As a result of analyzing the amino acid sequence 5 deduced from DNA11 by FASTA program, it showed high homology with known orotate phosphoribosyltransferase, suggesting that DNA11 is part of orotate phosphoribosyltransferase, This gene was named Ps ( Phanerochaete sordida ) URA5 fragment.

In order to clone the full length of PsURA5, forward primer 21 (P21: SEQ ID NO: 21) and forward primer 22 (P22: SEQ ID NO: 22) were synthesized based on DNA11. Using the cDNA synthesized in Example 3 as a template, 3′-RACE PCR reaction was performed using P21 and P7. The obtained PCR reaction product was diluted as a template, and nested PCR was performed using P22 and P7. The obtained PCR product of about 520 bp was subjected to TA cloning and sequencing. As a result, DNA sequence 12 (DNA12) was obtained. Analysis of amino acid sequence 6 deduced from DNA12 using the FASTA DNA analysis program showed high homology with known orotate phosphoribosyltransferase, suggesting that PsURA5 is part of orotate phosphoribosyltransferase It was done.

Next, TAIL-PCR was performed to clone the 5 ′ side of PsURA5 . TAIL-PCR was performed according to a previous report (Microbiology 148 2797-809 (2002)). Reverse primer 23, 24, 25 (P23: SEQ ID NO: 23, P24: SEQ ID NO: 24, P25: SEQ ID NO: 25) was synthesized, and YK-624 genomic DNA was prepared against TAIL primer NGTCGASWGANAWGAA (P26: SEQ ID NO: 26). Three TAIL-PCRs were performed as a template. The obtained PCR product was TA cloned to obtain DNA sequence 13 (DNA13).

Similarly, TAIL-PCR was also performed to clone the 3 ′ side of PsURA5 . Forward primer 27 (P27: SEQ ID NO: 27) was synthesized, and three TAIL-PCRs were sequentially performed on TAIL primer CANGCNWSGTNTSCAA (P28: SEQ ID NO: 28) using primers P21, P22, and P27. The obtained PCR product was TA cloned to obtain DNA sequence 14 (DNA14).

Based on DNA13 and DNA14, primer 29 and primer 30 (P29: SEQ ID NO: 29, P30: SEQ ID NO: 30) were synthesized, and using a highly accurate PCR enzyme (PrimeSTAR HS DNA Polymerase, TAKARA), YK-624 The full length PsURA5 was amplified from the genomic DNA of the strain. The obtained 5 kbp long PCR product was cloned into the cloning plasmid pCR4Blunt-TOPO using Zero Blunt TOPO Cloning Kit for sequencing (Invitrogen). As a result of sequencing the sequences obtained from the 4 clones, a clone having no mutation associated with PCR was obtained, and its DNA sequence 15 (DNA15: SEQ ID NO: 69) was designated as the full length PsURA5 . Further, the p PsURA5 plasmids containing PsURA5 entire length, and a transgenic marker plasmid in Example 9.

In addition, primers 31, 32 (P31: SEQ ID NO: 31, P32: SEQ ID NO: 32) were synthesized based on the sequence information of DNA15, and RT-PCR was performed using the cDNA synthesized in Example 2 as a template. 16 (DNA16: SEQ ID NO: 70) was obtained and designated as PsURA5 cDNA. Amino acid sequence 7 (SEQ ID NO: 80) deduced from PsURA5 cDNA consists of 232 amino acids including translation initiation methionine and stop codon, and this was designated as PsURA5 amino acid sequence. PsURA5 genomic DNA (DNA15) was found to contain 2 exons (indicated by the white box in FIG. 6) and 1 intron by comparison with PsURA5 cDNA.

Example 8 ( Ploning of PsURA5 gene in uracil-requiring UV- # 64 strain (FIG. 6))
In order to identify the gene responsible for the phenotype (uracil requirement) in the UV- # 64 strain prepared in Example 6, RNA was recovered from the UV- # 64 strain cultured on CYM agar medium containing 0.1 mg / ml uracil. Then, cDNA was synthesized according to the method of Example 2. Next, the cDNA of PsURA5 was PCR amplified using primers P31 and P32. As a result of TA cloning of the amplified PCR product and sequencing, DNA sequence 17 (DNA17: SEQ ID NO: 71) was obtained. As a result of comparing DNA17 with DNA16, it was confirmed that there was a 43 bp deletion mutation in the translation region. Therefore, the uracil requirement in the UV- # 64 strain was confirmed to be due to the loss of PsURA5 function.

Example 9 (Generation system construction in uracil-requiring UV- # 64 strain (FIGS. 7 and 8))
It was shown by Example 8 that the phenotype (uracil requirement) of the UV- # 64 strain produced in Example 6 was caused by loss of function of PsURA5 . Therefore, the p PsURA5 containing full-length PsURA5 (DNA15) obtained in Example 7 was introduced into UV-# 64 strain as a marker gene, a UV-# 64 was constructed gene transfer system to non-uracil-requiring (Figure 7).

First, according to Example 5, protoplasts were prepared from the UV- # 64 strain. In the liquid culture process, uracil was added to the CYM liquid medium (final concentration 0.1 mg / ml). The marker gene pPsURA5 was added to the obtained protoplasts (2 × 10 ⁷ cells) and incubated with CaCl ₂ on ice for 10 minutes. The composition of the solution is shown below (protoplast 2 × 10 ⁷ cells, p PsURA5 1.25-20 μg, CaCl ₂ 40 mM, 1.0 M Sorbitol, 10 mM MES, pH 6.2).

  Next, an equal volume of polyethylene glycol solution (PEG4000 50%, 1.0 M Sorbitol, 10 mM MES, pH 6.2) was added, gently mixed and then incubated on ice. Ten minutes later, the entire amount was added to the regeneration medium (without uracil) described in Example 6, and after mixing well, was dispensed into 15 dishes for 10 cm cell culture. After culturing at 30 ° C. for 7 days, the regenerated protoplast-derived clones were isolated, transferred to CYM agar medium (without uracil), and cultured. As a result, many clones showing uracil non-requirement were obtained (FIG. 8). .

Example 10 (Cloning of GPD (glycerol 3-phosphate dehydrogenase) gene derived from YK-624 strain (FIG. 9))
To construct a gene expression vector that strongly expresses the transgene using the promoter and terminator region of the GPD (glycerol triphosphate dehydrogenase) gene, which is known to be strongly and constitutively expressed in many organisms. GPD cloning from YK-624 strain genomic DNA was performed.

In order to clone GPD from the YK-624 strain, the GPD amino acid sequences of closely related basidiomycetes were compared using a CLASTAL DNA analysis program, and a highly conserved region was found. Based on the amino acid sequence, primer 33 (P33: SEQ ID NO: 33) and primer 34 (P34: SEQ ID NO: 34) were designed and degenerate PCR was performed. The cDNA synthesized in Example 2 was used as a template for the PCR reaction. The obtained PCR product was TA cloned and 6 clones were sequenced. As a result, only DNA sequence 18 (DNA18) was obtained. The amino acid sequence 8 deduced from DNA18 was analyzed by FASTA DNA analysis program, since it shows a known GPD high homology, suggesting that DNA sequence 18 is a GPD gene fragment, the present gene Ps ( Phanerochaete sordida ) It was named GPD .

In order to clone the full length of PsGPD , forward primer 35 (P35: SEQ ID NO: 35) and forward primer 36 (P36: SEQ ID NO: 36) were synthesized based on DNA sequence 18. Using the cDNA synthesized in Example 3 as a template, 3′-RACE PCR reaction was performed using P35 and P7. The obtained PCR reaction product was diluted as a template, and nested PCR was performed using P36 and P7. The obtained PCR product of about 670 bp was subjected to TA cloning and sequencing. As a result, DNA sequence 19 (DNA19) was obtained. As a result of analyzing the amino acid sequence 9 deduced from DNA19 by the FASTA DNA analysis program, it showed high homology with known GPD, suggesting that DNA19 is a part of the glycerol 3-phosphate dehydrogenase gene.

Next, TAIL-PCR was performed to clone the 5 ′ side of PsGPD . TAIL-PCR was performed according to Example 7. Reverse primers 37, 38 and 39 (P37: SEQ ID NO: 37, P38: SEQ ID NO: 38, P39: SEQ ID NO: 39) were synthesized, and TAIL-PCR was performed three times against TAIL primer CANGCNWSGTNTSCAA (P40: SEQ ID NO: 40). went. YK-624 genomic DNA was used as a template. The obtained PCR product was TA cloned to obtain DNA sequence 20 (DNA20).

Similarly, TAIL-PCR was also performed to clone the 3 ′ side of PsGPD . Forward primer 41 (P41: SEQ ID NO: 41) was synthesized, and three TAIL-PCRs were sequentially performed on TAIL primer GTNCGASWCANAWGTT (P42: SEQ ID NO: 42) using P35, P36, and P41. YK-624 genomic DNA was used as a template. The obtained PCR product was TA cloned to obtain DNA sequence 21 (DNA21).

Based on DNA20 and DNA21, primers 43 and 44 (P43: SEQ ID NO: 43, P44: SEQ ID NO: 44) were synthesized, and using a highly accurate PCR enzyme (PrimeSTAR HS DNA Polymerase, TAKARA), YK-624 strain The full length PsGPD was amplified from genomic DNA. The obtained 3.7 kbp long PCR product was cloned into the cloning plasmid pCR4Blunt-TOPO using Zero Blunt TOPO Cloning Kit for sequencing (Invitrogen). As a result of sequencing the sequences obtained from the 4 clones, a clone having no mutation associated with PCR was obtained, and its DNA sequence 22 (DNA22: SEQ ID NO: 72) was defined as the full length PsGPD . The plasmid containing DNA22 was designated as pPsGPD .

In addition, primers P45 and P46 (P45: SEQ ID NO: 45, P46: SEQ ID NO: 46) were synthesized based on the sequence information of DNA22, and RT-PCR was performed using the cDNA synthesized in Example 2 as a template. Sequence 23 (DNA23: SEQ ID NO: 73) was obtained and designated as PsGPD cDNA. Amino acid sequence 10 (SEQ ID NO: 81) deduced from PsGPD cDNA consists of 337 amino acids including a translation initiation methionine and a stop codon, and this was defined as the PsGPD amino acid sequence. PsGPD genomic DNA ( DNA23 ) was found to contain 7 exons (indicated by the white box in FIG. 9) and 6 introns by comparison with PsGPD cDNA.

Example 11 (Construction 1 of a gene transfer vector derived from pPsGPD 1 (FIG. 10))
p To construct a gene transfer vector using PsGPD promoter and terminator, and to demonstrate that the gene transfer vector has the ability to strongly express the transgene in the UV- # 64 strain, EGFP , a fluorescent gene A pPsGPD- derived vector that expresses the protein was constructed.
PrimeSTAR HS DNA Polymerase was used with a set of primer 47 (P47: SEQ ID NO: 47) -48 (P48: SEQ ID NO: 48) and primer 49 (P49: SEQ ID NO: 49) -50 (P50: SEQ ID NO: 50). PCR was performed using pPsGPD as a template. Both obtained PCR products (DNA24, DNA25) were mixed, and PCR was performed again using PrimeSTAR HS DNA Polymerase with a set of primers P47-P50 (procedure 1 in FIG. 10). The PCR product (DNA26) was cloned into the cloning plasmid pCR4Blunt-TOPO and sequenced using Zero Blunt TOPO Cloning Kit for sequencing. As a result, it was confirmed that just before the restriction enzyme Asc I site of the translation termination codon of PsGPD has been introduced. Then, the restriction enzymes Sac I site of two places originally present in the DNA26, was Sac I degraded DNA insert DNA26. Similarly, pPsGPD was digested with Sac I to form a DNA arm and dephosphorylated with shrimp alkaline phosphatase (Boehringer Mannheim). Result (step 2 in FIG. 10) ligating the DNA arms and DNA insert using DNA ligase ver2.1, p PsGPD the restriction enzyme Asc I site immediately before the stop codon of GPD coding region was introduced (+ Asc I ) A plasmid (DNA sequence 27: SEQ ID NO: 74) could be constructed.

Example 12 (Construction of gene transfer vector derived from pPsGPD 2 (FIG. 10))
In order to introduce an amino acid linker on the N-terminal side of the EGFP gene (Clontec), PCR amplification was performed with PrimeSTAR HS DNA Polymerase using primers P51 (SEQ ID NO: 51) -P52 (SEQ ID NO: 52). The obtained PCR product (DNA28) was again subjected to PCR using primers P53 (SEQ ID NO: 53) -P52. As a result, modified EGFP (L-EGFP) having a long amino acid linker GSGGGSGGG was obtained (DNA29). Furthermore, DNA29 was amplified with a set of primers 54-55 (P54: SEQ ID NO: 54, P55: SEQ ID NO: 55). As a result, Asc I site was introduced at both ends of the EGFP (DNA30). By cutting the cloning plasmid pCR4Blunt-TOPO containing DNA30 with Asc I, we were prepared EGFP fragment (DNA inserts) with Asc I site on both ends. Next, the pPsGPD (+ Asc I) plasmid prepared in Example 11 was similarly digested with Asc I, and the resulting DNA fragment was dephosphorylated using shrimp alkaline phosphatase to prepare a DNA arm.

The above DNA arm and DNA insert were ligated using DNA ligase ver2.1 (procedure 3 in FIG. 10). As a result, a new plasmid p PsGPD-EGFP (DNA31: SEQ ID NO: 75) was obtained. p PsGPD-EGFP is translated sequence the entire length of PsGPD is a fusion gene linked to EGFP via an amino acid linker sequence GSGGGSGGG, and has a design that is expressed in the cell as a fusion protein results PsGPD and EGFP transgenic.

Example 13 ( pGPD - EGFP gene introduction into UV- # 64 strain and confirmation of EGFP expression (FIGS. 11 and 12))
In order to confirm that the gene transfer vector (p PsGPD-EGFP ) constructed in Example 11 and Example 12 has the ability to strongly express the transgene ( EGFP ) by the operation of the GPD promoter and GPD terminator, UV- # Gene transfer (cotransformation) into 64 strains was performed. The gene introduction method was carried out according to Example 9, and 20 μg of pGPD -EGFP was added together with 5 μg of the marker gene pPsURA5, and the transformation was performed. The obtained mycelia of 24 clones of uracil non-requiring strain were collected and repeatedly freeze-thawed three times in TE-Triton X100 solution (Tris-HCl 10 mM, EDTA 1 mM, Triton-X100 0.05% pH 8.0). Using the solution as a template, PCR was performed with a set of primer sets P56 (SEQ ID NO: 56) and P57 (SEQ ID NO: 57) (FIG. 10) that amplify a partial sequence of the EGFP gene. Since PCR amplification (DNA32) of EGFP sequence was confirmed from 12 clones out of 24 clones, it was confirmed that EGFP gene was introduced into these clones, and names up to E1-E12 were given. Next, 6 clone clones (C1-C6) transformed by introducing only the marker gene were separately prepared, and 200 mg (wet weight) of mycelia pieces of each strain were collected.

Each mycelium piece was frozen in liquid nitrogen and crushed in a mortar, and the whole powder was suspended in an EGFP fluorescence intensity measurement buffer. The buffer composition is shown below (0.15 M phosphate buffer, 1 mM EDTA, 400-fold diluted protease inhibitor cocktail (Sigma), pH 7.0, 4 ° C.). The mycelium powder suspension was centrifuged (10,000 × g, 5 minutes), and the supernatant was collected to obtain a cell lysate. The EGFP fluorescence intensity in the cell lysate was measured using Mithras LB940 (Berthold). In order to quantify the amount of EGFP protein in each cell lysate from the fluorescence intensity, a calibration curve was prepared using an EGFP protein preparation (Clontech). In addition, the total amount of protein in each cell lysate was quantified (Dc protein assay kit, Bio-Rad). As a result of calculating the amount of EGFP protein in the solubilized total protein, 1.2% of the solubilized total protein was occupied by EGFP in clone E2, which expresses EGFP most strongly (FIG. 11). When clone E2 growing on CYM agar was irradiated with EGFP excitation light (485 nm), strong green fluorescence was observed (FIG. 12). From the above results, it was shown that the GPD promoter and GPD terminator regions in the pPsGPD-EGFP plasmid have the ability to strongly express the transgene in the UV- # 64 strain.

Example 14 (Preparation of YK-LiP1 gene expression vector (p GPD-YKLiP1 ) (FIG. 13))
In order to confer the property of highly expressing YK-LiP1 to the strain derived from the YK-624 strain (UV- # 64), the pPsGPD-EGFP plasmid confirmed to function in UV- # 64 in Example 13 was used. A gene expression vector that highly expresses YK-LiP1 was prepared. p PsGPD-EGFP (DNA31) was used as a template, and PrimeSTAR HS DNA Polymerase was used, and a part of the GPD promoter was PCR-amplified by primer set P58 (SEQ ID NO: 58) -P59 (SEQ ID NO: 59) (DNA33). Separately, PCR amplification was performed using PrimeSTAR HS DNA Polymerase with YK-LiP1 genomic DNA ( DNA6 ) as a template and primer set P60 (SEQ ID NO: 60) -P61 (SEQ ID NO: 61) (DNA34). Both obtained PCR products (DNA33, DNA34) were mixed, and PCR was performed again using PrimeSTAR HS DNA Polymerase with the set of primers P58-P61 (procedure 1 in FIG. 13). The PCR product was cloned into the cloning plasmid pCR4Blunt-TOPO using the Zero Blunt TOPO Cloning Kit for sequencing and sequenced. As a result, it was confirmed that a partial sequence of the GPD promoter and YK-LiP1 genomic DNA were fused in frame, and a restriction enzyme Asc I site was introduced immediately after the YK-LiP1 stop codon (DNA35).

Next, DNA35 was digested with Nde I- Asc I using the restriction enzyme Nde I site originally present in the GPD promoter and the newly introduced restriction enzyme Asc I site to obtain a DNA insert. Similarly, pPsGPD-EGFP was digested with Nde I- Asc I to form a DNA arm and dephosphorylated with shrimp alkaline phosphatase. As a result of ligation of the above DNA arm and DNA insert using DNA ligase ver2.1 (step 2 in FIG. 13), a DNA sequence containing YK-LiP1 genomic DNA bound to GPD promoter and GPD terminator (DNA36: SEQ ID NO: 76) This led to the construction of the pCR4Blunt-TOPO plasmid with This plasmid was named pGPD-YKLiP1 .

Example 15 ( Production of YK-LiP2 gene expression vector (p GPD-YKLiP2 ) (FIG. 14))
In order to confer the property of highly expressing YK-LiP2 to the strain derived from the YK-624 strain (UV- # 64), the pPsGPD-EGFP plasmid confirmed to function in UV- # 64 in Example 13 was used. A gene expression vector that highly expresses YK-LiP2 was prepared. p PsGPD-EGFP (DNA31) was used as a template and PrimeSTAR HS DNA Polymerase was used, and a part of the GPD promoter was PCR-amplified by using primer set P58-P62 (P62: SEQ ID NO: 62) (DNA37). Separately, PCR amplification was performed using PrimeSTAR HS DNA Polymerase using YK-LiP2 genomic DNA (DNA10) as a template and primer set P63 (SEQ ID NO: 63) -P64 (SEQ ID NO: 64) (DNA38). Both obtained PCR products (DNA37, DNA38) were mixed, and PCR was performed again using PrimeSTAR HS DNA Polymerase with a set of primers P58-P64 (Procedure 1 in FIG. 14). The PCR product was cloned into the cloning plasmid pCR4Blunt-TOPO using the Zero Blunt TOPO Cloning Kit for sequencing and sequenced. As a result, it was confirmed that a partial sequence of the GPD promoter and YK-LiP2 genomic DNA were fused in frame, and a restriction enzyme Asc I site was introduced immediately after the YK-LiP2 stop codon (DNA39).

Next, DNA39 was digested with Nde I- Asc I using the restriction enzyme Nde I site originally present in the GPD promoter and the newly introduced restriction enzyme Asc I site to obtain a DNA insert. Similarly, pPsGPD-EGFP was digested with Nde I- Asc I to form a DNA arm and dephosphorylated with shrimp alkaline phosphatase. As a result of ligation of the above DNA arm and DNA insert using DNA ligase ver2.1 (step 2 in FIG. 14), a DNA sequence containing YK-LiP2 genomic DNA bound to GPD promoter and GPD terminator (DNA40: SEQ ID NO: 77) This led to the construction of the pCR4Blunt-TOPO plasmid with This plasmid was named pGPD-YKLiP2 .

Example 16 (Preparation of UV- # 64 strain introduced with YK-LiP1 gene expression vector)
The pGPD -YKLiP1 vector developed in Example 14 was introduced to give the YK-624 strain-derived strain (UV- # 64) the property of highly expressing YK-LiP1 . The gene introduction method was carried out according to Example 9, and the transformation was performed by adding 20 μg of the pGPD-YKLiP1 vector together with 5 μg of the marker gene pPsURA5 . The resulting mycelia of 25 clones not requiring uracil were collected and repeatedly freeze-thawed three times in TE-Triton X100 solution (Tris-HCl 10 mM, EDTA 1 mM, Triton-X100 0.05% pH 8.0). Using the solution as a template, PCR was performed with a set of primers (P9, P45) that amplify a partial sequence of the pGPD-YKLiP1 vector. Since PCR amplification was confirmed from 11 clones out of 25 clones (DNA41), it was confirmed that the pGPD-YKLiP1 vector was introduced into these clones.

Example 17 (Preparation of UV- # 64 strain introduced with YK-LiP2 gene expression vector)
In order to give the YK-624 strain-derived strain (UV- # 64) the property of highly expressing YK-LiP2 , the pGPD-YKLiP2 vector developed in Example 15 was introduced. The gene introduction method was carried out according to Example 9, and the transformation was carried out by adding 20 μg of the pGPD-YKLiP2 vector together with 5 μg of the marker gene pPsURA5 . The mycelia of 52 clones of the uracil non-requiring strain obtained were collected, and freeze-thaw was repeated three times in TE-Triton X100 solution (Tris-HCl 10 mM, EDTA 1 mM, Triton-X100 0.05% pH 8.0). Using the solution as a template, PCR was performed with a primer set (P16, P45) that amplifies a partial sequence of the pGPD-YKLiP2 vector. Since PCR amplification was confirmed from 32 out of 52 clones (DNA42), it was confirmed that the pGPD-YKLiP2 vector was introduced into these clones.

Example 18 (YK-LiP1, and YK-LIP2 gene has been introduced UV-# 64 strain LiP activity measurement (Fig. 15))
The LiP activity of the transformed strains GPD-YKLiP1 No11 strain ( YK-LiP1 gene introduced) and GPD-YKLiP2 No3 strain ( YK-LiP2 gene introduced) obtained in Examples 16 and 17 was measured. After culturing the above strain on a potato dextrose agar medium at 30 ° C. for 3 days, the obtained bacterial cells were punched out with a cork borer, and two bacterial cell disks were added to an Mn-deficient Kirk liquid medium (1% glucose, 1.2 mM ammonium tartrate). 10 ml was homogenized with a Waring blender, and this was statically cultured in a 100 ml Erlenmeyer flask at 30 ° C. for 2 days. The obtained culture broth was concentrated 10 times by ultraconcentration, and then this concentrate was subjected to LiP activity measurement.

LiP activity was measured (1 ml of the reaction system), 1 mM veratryl alcohol (VA) and enzyme solution were added to 20 mM succinate buffer (pH 3.0), pre-incubated for 5 minutes, and then 0.2 mM hydrogen peroxide. The reaction was started and oxidation of VA to veratraldehyde was determined by increasing the absorbance at 310 nm (ε _{310 nm} = 9.3 mM ⁻¹ cm ⁻¹ ). As a result, LiP activity was not detected at all in the wild strain and in the control strain in which the YK-LiPs gene was not introduced, whereas in the GL1 No11 strain, 5.5 pkat / ml, and in the GL2 No3 strain, 2.2 pkat / ml LiP activity was detected. Activity was detected.

  The present invention provides an improved strain of YK-624 that has an excellent selective lignin-degrading ability, and provides a novel strain imparted with a property of producing a high yield of YK-LiPs enzyme having a high ability to degrade macromolecular lignin. It can be applied to pulp bleaching process and soil purification treatment. The present invention also enables mass production of useful substances in the YK-624 strain by providing a transformation system for the YK-624 strain.

It is a figure which shows the degenerate PCR analysis based on the N terminal amino acid sequence of YK-LiPs protein (Example 2). It is a figure which shows the cloning of YK-LiP1 full length (Example 3). In the figure, white box indicates an exon. The straight line between exons indicates an intron. 5′-UTR and 3′-UTR indicate 5 ′ and 3 ′ untranslated regions, respectively. poly-A site indicates a site where polyadenylation occurs to mRNA. (Example 4) which is a figure which shows the cloning of YK-LiP2 full length. In the figure, white box indicates an exon. The straight line between exons indicates an intron. 5′-UTR and 3′-UTR indicate 5 ′ and 3 ′ untranslated regions, respectively. poly-A site indicates a site where polyadenylation occurs to mRNA. (Example 6) which is a figure which shows the preparation method of the uracil requirement mutant by the mutation process of YK-624 stock | strain protoplast. It is a figure which shows the result of the growth test of a uracil requirement mutant (Example 6). A state in which YK-624 (parent strain) and a uracil-requiring mutant strain (UV- # 64) were inoculated on uracil-containing agar medium and non-agar medium in three places and cultured for 3 days. (Example 7) which is a figure which shows the full-length cloning of the uracil biosynthesis gene (orotic acid phosphoribosyltransferase) from a YK-624 strain. In the figure, white box indicates an exon. The straight line between exons indicates an intron. The shaded box indicates a 43 bp DNA region that is missing in the UV- # 64 strain. It is a figure which shows the construction method of the gene introduction system in a uracil requirement UV- # 64 strain | stump | stock (Example 9). (Example 9) which shows the result of gene introduction system construction in uracil requirement UV- # 64 strain. The marker plasmid indicates the amount of pPsURA5 used for transformation. The transformation efficiency indicates the ratio (%) of the number of protoplasts used in the transformation experiment to the number of regenerated clones. FIG. 10 shows cloning of GPD (glycerol 3-phosphate dehydrogenase) gene derived from the YK-624 strain (Example 10). In the figure, white box indicates an exon. The straight line between exons indicates an intron. 3′-UTR indicates a 3 ′ untranslated region. poly-A site indicates a site where polyadenylation occurs to mRNA. FIG. 11 shows the construction of a gene transfer vector derived from p PsGPD (Examples 11 and 12). In the figure, white box indicates an exon. The straight line between exons indicates an intron. 3′-UTR indicates a 3 ′ untranslated region. poly-A site indicates a site where polyadenylation occurs to mRNA. Sac I and Asc I are sites recognized and cleaved by the restriction enzymes Sac I and Asc I, respectively. FIG. 13 is a view showing the results of transgene ( EGFP ) expression using a pPsGPD-EGFP gene-introduced plasmid (Example 13). In the figure, each white circle represents the expression level of EGFP in the pPsGPD-EGFP- introduced strain (12 strains, E1-E12) and the control strain (C1-C6) in which only the marker gene has been introduced (weight of EGFP protein in soluble protein / total solubility) Protein weight (%)). It is a figure which shows visualization of EGFP expression in E2 strain (Example 13). p PsGPD-EGFP- introduced strain (E2) and control strain (C1) into which only the marker gene was introduced were irradiated with EGFP excitation light (485 nm), and the resulting green fluorescence was photographed. (Example 14) which is a figure which shows the preparation methods of a YK-LiP1 gene expression vector ( pGPD-YKLiP1 ). In the figure, Nde I represents a site recognized and cleaved by the restriction enzyme Nde I. (Example 15) which is a figure which shows the preparation methods of a YK-LiP2 gene expression vector ( pGPD-YKLiP2 ). In the figure, Nde I represents a site recognized and cleaved by the restriction enzyme Nde I. YK-LIP1, and a diagram showing the LiP activity measurement results of the YK-LIP2 gene has been introduced UV-# 64 strain (Example 18). Each test is performed in triplicate and is expressed as mean ± standard deviation.

Claims (10)

  Uracil auxotrophic mutant UV- # 64 (NITE AP-344) whose parent strain is Usukiirokawatake YK-624.   It consists of the nucleotide sequence of SEQ ID NO: 69, and can be made non-requiring to uracil by introducing it into the UV- # 64 strain according to claim 1, and a marker gene for gene transfer system A gene encoding orotate phosphoribosyltransferase derived from the YK-624 strain.   A plasmid comprising the gene according to claim 2. A gene expression vector comprising DNA consisting of the nucleotide sequence of SEQ ID NO: 75 and having the ability to highly express a transgene in Usukiirokawatake.   A method for producing a transformant of the YK-624 strain using the UV- # 64 strain according to claim 1 as a host strain.   The production method according to claim 5, wherein the plasmid according to claim 3 is introduced into the host strain together with the foreign gene.   The production method according to claim 5 or 6, wherein a foreign gene is introduced using the gene expression vector according to claim 4.   A new strain GPD-YKLiP1 No11 (NITE AP-342), which uses the UV- # 64 strain of claim 1 as a parent strain and exhibits higher lignin peroxidase (YK-LiP1) -producing ability than the parent strain.   A new strain GPD-YKLiP2 No3 (NITE AP-343), which has the UV- # 64 strain of claim 1 as a parent strain and exhibits higher lignin peroxidase (YK-LiP2) production ability than the parent strain.   A gene transfer kit for the YK-624 strain, comprising the plasmid according to claim 3 and / or the gene expression vector according to claim 4.