JP2004329143A - Cyclic nucleotide derived from mold of genus aspergillus, method for producing self-cloning strain of mold of genus aspergillus and self-cloning strain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a self-cloning strain of a mold of the genus Aspergillus, a cyclic nucleotide to be used in the method and a self-cloning strain of a mold of the genus Aspergillus. <P>SOLUTION: The cyclic nucleotide contains a marker gene derived from a mold (A) of the genus Aspergillus and selected from niaD, argB, sC, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene and hygromycin resistance gene and the objective gene expression cassette derived from the mold (A) of the genus Aspergillus. The invention further provides a method for producing a self-cloning strain by the transformation of the mold (A) of the genus Aspergillus with the cyclic nucleotide. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００８２】
表１から、形質転換により目的遺伝子が導入されたセルフクローニング株を得るに当たり、目的遺伝子を線状のままで用いると実質的に形質転換は起こらないが、環状ヌクレオチドの形にすることにより極めて高効率で形質転換が起こることが分かる。また、その効率は目的遺伝子を大腸菌ベクターに組み込んで用いる場合よりはるかに高いことも分かる。
【００８３】
【発明の効果】
本発明によれば、アスペルギルス属糸状菌のセルフクローニング株の効率的な生産方法、この方法に使用する環状ヌクレオチド、及び、アスペルギルス属糸状菌のセルフクローニング株が提供された。
さらにいえば、麹菌アスペルギルスオリザに代表されるアスペルギルス属糸状菌の育種は取得効率の低い変異法、又は、取得効率はよいが食品産業でパブリックアクセプタンスが低い遺伝子組み換え法に依存していた。このことから、食品産業界では、遺伝子組み換え法のような簡便な技術を用いて、異種生物の遺伝子を含まないセルフクローニング株を育種することが長年にわたり渇望されてきた。本発明によれば、目的とする性質を、非常に簡便に、効率的かつ安全な方法でアスペルギルス属糸状菌に導入することができた。
【００８４】
【配列表】

【図面の簡単な説明】
【図１】実施例１において得たプラスミドｐＮＩＡ２の構成を示す図である。
【図２】実施例１において得られた形質転換株の染色体ＤＮＡにｆｌｅＡが組み込まれていることを示す図である。
【図３】実施例１において得られた形質転換株がフコースレクチンを大量に生産していることを示す図である。
【図４】実施例２において得られた形質転換体の染色体ＤＮＡにｇｌａＢが組み込まれていることを示す図である。
【図５】実施例２において得られた形質転換株がグルコアミラーゼを大量に生産していることを示す図である。
【図６】実施例３において得られた形質転換体の染色体ＤＮＡにｆｌｅＡが組み込まれていることを示す図である。
【図７】実施例３において得られた形質転換株がフルコースクチンを大量に生産していることを示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cyclic nucleotide used for preparing a self-cloning strain of Aspergillus filamentous fungus, a method for producing a self-cloning strain of Aspergillus filamentous fungus using this cyclic nucleotide, and a self-cloning strain obtained by this method.
[0002]
[Prior art]
Aspergillus filamentous fungi are used in industries around the world as various enzyme sources and various secondary metabolite sources. In particular, Aspergillus oryzae, a typical strain of koji mold, is a representative food microorganism in Japan, and has been used in the brewing industry for sake, soy sauce, miso, mirin, shochu, and the like for many years.
[0003]
Aspergillus oryzae has attracted attention as a host for heterologous protein production because of its high protein-producing ability and safety as a brewing microorganism. In addition, by performing genetic recombination, it is possible to efficiently obtain bacterial species whose production amounts of various proteins and the like have increased several hundred times.
[0004]
In recent years, industrial enzyme agents have been produced using genetically modified koji molds, utilizing the excellent protein secretory production ability of Aspergillus oryzae aspergillus (Non-Patent Document 1, Patent Document 1). As a method for transforming Aspergillus oryzae, a method using niaD of a nitrate reductase gene has been established (Non-Patent Document 2). In addition, the present inventors have found that in the production of a heterologous protein using Aspergillus oryzae as a host, a higher production amount can be obtained by using a gene derived from a closely related species such as another species of the genus Aspergillus. It is reported that a very large amount of protein is produced when a gene derived from Aspergillus oryzae is expressed under the control of a high expression promoter of Aspergillus oryzae (Patent Document 2).
[0005]
However, since an E. coli vector / E. Coli system is usually used as a host for efficient gene recombination, recombinants obtained by these methods are derived from heterologous organisms such as E. coli vectors and E. coli-derived marker genes. Including the sequence of Aspergillus oryzae is often used in the food industry, but in Japan, the use of genetically modified organisms in food is still shunned by many consumers.
[0006]
For this reason, breeding of Aspergillus is mainly performed by a conventional mutagen or a mutation method using ultraviolet rays. The mutagenesis method is very inefficient as a method for obtaining a bacterial strain that highly expresses a target protein. In addition, protein production is improved only several times.
[0007]
Therefore, in order to commercialize a gene recombinant containing only the DNA derived from Aspergillus aspergillus oryzae and expressing the desired protein at a high level, the breeding method is genetically modified, but the resulting transformant is derived from Aspergillus oryzae. Desirably, it is a self-cloning strain or a natural occurrence strain that contains only DNA. Self-cloning refers to the case where the host, vector and inserted DNA all belong to the same species. A natural occurrence strain refers to a strain in which living cells having a gene constitution equivalent to that of a recombinant exist in the natural world (http://www.mhlw.go.jp/topics/idenshi/anzen/tuchi2.html). .
[0008]
The self-cloning strain is different from the strain obtained by the conventional mutagenesis method or the mutation method using ultraviolet light in that it does not contain vector genes derived from Escherichia coli, antibiotic resistance genes, other proteins derived from heterologous organisms, or low molecular weight compounds. There is no. In addition, the production of the enzyme is equivalent to the production of the genetically modified product, and is a production that is too high to be obtained by the mutation method.
[0009]
Examples of such self-cloning microorganisms include breeding examples using budding yeast and lactic acid bacteria. These are produced by a method of constructing a genetically modified product and then using a susceptibility gene to loop out and remove only unnecessary heterologous genes from the chromosome (Non-Patent Document 3).
[0010]
However, as for Aspergillus fungi including Aspergillus oryzae, a method of breeding a self-cloning strain effective for all strains has not been reported. If such a self-cloning technique can be developed for Aspergillus oryzae or other Aspergillus filamentous fungi used for industry, it can be applied to the food industry.
[0011]
[Patent Document 1]
JP-A-62-272988
[0012]
[Patent Document 2]
JP-A-11-154271
[0013]
[Non-patent document 1]
Biotechnology, vol 6, p1419 (1988).
[0014]
[Non-patent document 2]
Mol. Gen. Genet. , Vol 218, p99-104 (1989).
[0015]
[Non-Patent Document 3]
Yeast, vol 15, p1-10, (1999)
[0016]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for efficiently producing a self-cloning strain of Aspergillus genus fungi, a cyclic nucleotide used in this method, and a self-cloning strain of Aspergillus genus fungi.
[0017]
[Means for Solving the Problems]
In order to achieve the above object, the present inventor has repeated studies and obtained the following findings.
{Circle around (1)} A marker gene derived from a filamentous Aspergillus (A) species selected from the group consisting of niaD, argB, sC, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene and hygromycin resistance gene, and the like. By transforming a filamentous fungus (A) species with a cyclic nucleotide containing a target gene expression cassette derived from the filamentous fungus (A) species, the marker of the chromosomal DNA of the filamentous fungus (A) species can be very efficiently used. Insertion type homologous recombination occurs at the gene site, and as a result, a transformant having the target gene inserted into the marker gene site of the chromosomal DNA of the filamentous fungus (A) species can be efficiently obtained.
[0018]
In this method, it is important to use any one of the above nine genes as a marker gene and to circularize the DNA containing the marker gene and the target gene expression cassette in order to improve the efficiency of transformation. is there.
{Circle around (2)} By including the high expression enhancer derived from the filamentous fungus (A) species in the target gene expression cassette, this transformant can express the target gene with high expression. In addition, since this transformant does not contain DNA derived from a heterologous organism, it can ensure the same safety as a conventional mutant strain when used in various industries such as the food industry, and can also produce the substance. The sex is expected to be so high that it cannot be obtained with the conventional mutant strain and to be comparable to that of the genetically modified product.
[0019]
The present invention has been completed based on the above findings, and provides the following cyclic nucleotides and the like.
[0020]
Item 1. niaD, argB, sC, ptrA, pyrG, amdS, an aureobasidin resistance gene, a benomyl resistance gene, a hygromycin resistance gene, a marker gene derived from a filamentous Aspergillus (A) species, and a filamentous fungus of the genus Aspergillus (A) a cyclic nucleotide containing a target gene expression cassette derived from a species;
[0021]
Item 2. Item 2. The cyclic nucleotide according to Item 1, wherein the marker gene is niaD.
[0022]
Item 3. Item 3. The cyclic nucleotide according to Item 1 or 2, consisting only of nucleotides derived from the filamentous fungus (A) of the genus Aspergillus.
[0023]
Item 4. Item 4. The cyclic nucleotide according to item 1, 2 or 3, for producing a self-cloning strain of Aspergillus spp.
[0024]
Item 5. Item 5. A method for producing a self-cloning strain of Aspergillus spp., Which transforms Aspergillus spp. Filamentous fungus (A) using the cyclic nucleotide according to any one of Items 1 to 4.
[0025]
Item 6. (I) using the cyclic nucleotide according to any one of items 1 to 4 to transform a mutant strain of Aspergillus spp. (A) in which a marker gene contained in the cyclic nucleotide is mutated;
(Ii) selecting a transformant in which the target gene in the cyclic nucleotide has been introduced using the expression of the marker gene as an index;
A method for producing a self-cloning strain of a filamentous fungus of the genus Aspergillus comprising:
[0026]
Item 7. Item 5. A self-cloning strain of Aspergillus spp. Obtained by transforming Aspergillus spp. Filamentous fungus (A) with the cyclic nucleotide according to any one of Items 1 to 4.
[0027]
Item 8. A state in which a target gene expression cassette derived from a filamentous Aspergillus (A) species is sandwiched between two identical marker genes derived from a filamentous Aspergillus (A) species in the chromosomal DNA of the filamentous Aspergillus (A) species. Wherein the marker gene is selected from the group consisting of niaD, argB, sC, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene and hygromycin resistance gene. stock.
[0028]
Item 9. Item 9. A self-cloning strain of Aspergillus spp. According to Item 8, wherein one of the two marker genes is a mutated marker gene.
[0029]
Hereinafter, the present invention will be described in detail.
(I) Cyclic nucleotide
Basic configuration
The cyclic nucleotide of the present invention is a marker derived from a filamentous Aspergillus (A) species selected from the group consisting of niaD, argB, sC, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene, and hygromycin resistance gene. It is a cyclic nucleotide containing a gene and a gene expression cassette derived from a filamentous fungus (A) of the genus Aspergillus.
Aspergillus fungi (A) seed
Aspergillus spp. Filamentous fungus (A) species is not particularly limited. For example, Aspergillus oryzae (Aspergillus oryzae), Aspergillus niger (Aspergillus niger), Aspergillus awamori (Aspergillus awamori), Aspergillus kawachii (Aspergillus kawachii), Aspergillus Usami (Aspergillus usamii), Aspergillus sojae (Aspergillus sojae), Aspergillus nidulans (Aspergillus nidulans ) Can be used. In particular, Aspergillus oryzae is preferred because it is a safe microorganism that has been used in brewed foods such as sake, soy sauce, miso, and mirin in Japan for many years and is a host that is extremely frequently used in Japanese industry.
Marker gene
The marker gene is a marker gene derived from a filamentous fungus of the genus Aspergillus (A), and includes niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)) and argB (Enzyme Microbiol Technology, 6, 386-386). 389, (1984)), sC (Gene, 84, 329-334, (1989)), ptrA (Biosci Biotechnol Biochem, 64, 1416-14211, (2000)), pyrG (Biochem Biophys Res. 289, (1983)), amdS (Gene, 26, 205-221, (1983)), aureobasidin resistance gene (Mol en Genet, 261, 290-296, (1999)), benomyl resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873, (1986)) and hygromycin resistance gene (Gene, 57, 21-26, (1987)). )), A marker gene selected from the group consisting of:
[0030]
Unlike the transformation of bacteria and budding yeast that only cause homologous recombination, transformation of filamentous fungi of the genus Aspergillus can involve both homologous recombination and non-homologous recombination. When the self-cloning strain is used in, for example, the food industry, it is desirable from the viewpoint of safety that the transformed gene is integrated at a specific position on the host chromosome. By using the specific marker gene described above, insertion type homologous recombination into the marker gene site of the host occurs very efficiently, and as a result, the target gene is inserted into the host chromosomal DNA very efficiently. You. Particularly, niaD is preferable.
[0031]
These marker genes can be obtained by a method of performing PCR using chromosomal DNA of Aspergillus spp. Filamentous fungus (A) as a template using primers designed based on the sequence described in the above-mentioned document, or a method based on the sequence described in the above-mentioned document. It can be easily obtained from the chromosomal DNA library of Aspergillus spp. (A) using the designed probe by a method such as hybridization.
[0032]
In the present invention, “derived from a filamentous fungus of the genus Aspergillus (A)” means that the sequence is present in a specific species (A) of the filamentous bacterium of the genus Aspergillus.
Target gene expression cassette
The target gene is not particularly limited, and may be a gene derived from a filamentous fungus (A) of the genus Aspergillus, as long as it can impart the desired properties to the filamentous fungus (A) serving as a host. Typical examples of such a gene include a gene encoding a protein (including a protein that eventually becomes a glycoprotein). A self-cloning strain is prepared using a cyclic nucleotide containing such a gene by the method described below. This will allow the protein to be produced in large quantities by self-cloning strains.
[0033]
The target gene expression cassette of a gene encoding a protein usually includes an enhancer (including a promoter sequence), a structural gene (open reading frame) of the protein, and a terminator.
[0034]
The gene expression cassette of interest may be used as it is in the sequence present in the chromosomal DNA of Aspergillus spp. (A), or may be present in the chromosomal DNA of Aspergillus spp. Promoter, open reading frame, enhancer, terminator, etc., may be separately isolated and then fused. In any case, it is sufficient that the target gene expression cassette is composed of a sequence derived from the filamentous fungus (A) of the genus Aspergillus.
[0035]
Examples of the structural gene include, but are not limited to, phytase, lyase, pectin degrading enzymes such as pectinase, amylase, glucoamylase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, mannosidase, isomerase, invertase. , Transferase, ribonuclease, deoxyribonuclease, chitinase, catalase, laccase, phenol oxidase, oxidase, oxidoreductase, cellulase, xylanase, peroxidase, lipase, hydrolase, esterase, cutinase, protease such as protease, aminopeptidase or carboxypeptidase, etc. And the structural gene of the protein.
These structural genes can be easily obtained by searching the koji mold EST database (http://www.nrib.go.jp/ken/EST/db/blast.html). In addition, the present inventor has proposed a glucoamylase gene glaB (Gene, 207, 127-134, (1998) and glA (Gene, 108, 145-150, (1992)) derived from Aspergillus oryzae aspergillus and a fucose lectin gene fleA (JP 2002-112786) and the like.
[0036]
For controlling the expression of a gene derived from a filamentous fungus, an enhancer to which a transcription control factor binds and a promoter to which a basic transcription factor such as RNA polymerase binds are indispensable. Although enhancer sequences are diverse, there is no consensus sequence common to all genes, but promoter sequences are generally TATA motifs, which are often located 50-150 bp upstream of the start codon (Crit Rev Biochem and Mol). Biol, 25, 185-221 (1990)). For example, in the case of the glaB enhancer, tataaa of base numbers 269 to 274 of SEQ ID NO: 4, in the sodM enhancer, tattat of base numbers 901 to 906 of SEQ ID NO: 15, and in the melO enhancer, the tatttaa of base numbers 1037 to 1043 of SEQ ID NO: 22 correspond to the promoter sequence. Guessed. Therefore, both the enhancer and promoter sequences are essential for the expression of the target gene in self-cloning.
The terminator can be used without limitation as long as it is a terminator gene derived from a filamentous fungus of the genus Aspergillus (A). For example, glucoamylase glaB terminator (SEQ ID NO: 6) can be used.
[0037]
The enhancer is not particularly limited as long as it is derived from a filamentous fungus (A) of the genus Aspergillus. For example, α-amylase gene enhancer amyB (Biosci Biotechnol Biochem, 56, 1849-1853, (1992)), glucoamylase gene enhancer glaA (Gene, 108, 145-150, (1992)), α-glucosidase gene enhancer agdA (Cur). Genet, 30, 432-438, (1996)), manganese SOD gene enhancer sodM (JP-A-2001-224381), tyrosinase gene enhancer melO (JP-A-2001-0446078), glucoamylase gene enhancer glaB (JP-A-2000-245465, JP-A-11-243965) and the like. In particular, sodM or melO is preferable in that it is a liquid culture-specific high expression enhancer, and glaB is preferable in that it is a solid culture-specific high expression enhancer.
[0038]
The position of the enhancer is not particularly limited, and may be located upstream or downstream of the unit comprising the structural gene and the terminator. The structural gene, the terminator and the enhancer may be directly linked, or a nucleotide of about 1 to 2000 bases may be interposed between them. However, the sequence of these nucleotides is preferably a sequence derived from a filamentous fungus (A) of the genus Aspergillus. For example, when the target protein is to be produced as an intracellular protein, a known secretory protein gene of a filamentous fungus (A) species is placed upstream adjacent to the open reading frame of the target gene, thereby obtaining the target protein. It can be secreted outside the cells as a fusion protein of the protein and its secretory protein.
[0039]
In addition, examples of the target gene expression cassette include a gene silencing cassette such as antisense, cosuppression, or RNAi (complementary double-stranded ribonucleotide). These nucleotides also use those derived from the filamentous fungus (A) of the genus Aspergillus. Although gene disruption of Aspergillus fungi is extremely difficult, transformation of Aspergillus species (A) using a cyclic nucleotide containing a gene silencing cassette as a target gene expression cassette is industrially undesirable. Expression of genes such as enzymes can be suppressed. For example, the expression of a tyrosinase gene involved in browning of brewed food, a gene of a useful enzyme, or a gene of an enzyme involved in decomposition of a useful secondary metabolite can be suppressed.
Other
The cyclic nucleotide may include the marker gene and the target gene expression cassette. Any other sequence may be included, but it is preferable that a sequence not derived from the filamentous fungus (A) of Aspergillus species (a sequence derived from an organism of another species or an artificial sequence) is not included. As a result, the filamentous fungus obtained by the transformation has extremely high safety.
[0040]
The size of the cyclic nucleotide is usually about 3,000 to 20,000 bases, particularly preferably about 5,000 to 10,000 bases. Within the above range, a cyclic nucleotide can be stably formed by intramolecular bonding, and an enhancer, a terminator, a promoter and a target gene can be selected from a wide range.
Method for producing cyclic nucleotide
The cyclic nucleotide of the present invention can be produced, for example, by the following method.
[0041]
First, an Escherichia coli plasmid (I) is constructed by incorporating a marker gene (for example, niaD) of Aspergillus spp. Separately, an enhancer (including a promoter) derived from a filamentous fungus (A) species, an open reading frame of a target gene derived from a filamentous fungus (A) species, and a terminator gene derived from a filamentous fungus (A) species are fused by combination PCR. A target gene expression cassette as a gene is obtained. Next, the target gene expression cassette is incorporated into the Escherichia coli plasmid (I) so that the marker gene and the target gene expression cassette in the Escherichia coli plasmid (I) are adjacent to each other to obtain the Escherichia coli plasmid (II).
[0042]
Further, using Escherichia coli plasmid (II) as a template, a unit consisting of a marker gene and a target gene expression cassette is amplified by PCR, the ends are phosphorylated and blunt-ended, and then cyclic nucleotides are obtained by intramolecular ligation (self-ligation). .
(II) Method for producing self-cloning strain of Aspergillus filamentous fungus
The method for producing the self-cloning strain of Aspergillus filamentous fungus of the present invention is a method of transforming Aspergillus species (A) using the cyclic nucleotide of the present invention described above.
[0043]
In the present invention, the “self-cloning strain” refers to a strain substantially composed of nucleotides derived from the same organism as the species of the host filamentous fungus (A). Even when a sequence not derived from the filamentous fungus (A) species is contained, it is at most about 1 to 10 bases, particularly about 6 to 10 bases. Insertion sequences of this size may be inserted, for example, to introduce restriction enzyme sites.
[0044]
As the host filamentous fungus, a strain of Aspergillus species filamentous fungus (A) from which the marker gene and the target gene are derived is used. It is preferable to use a mutant in which a marker gene to be introduced is mutated so that a transformant can be easily selected. In this case, (i) using the above-described cyclic nucleotide to transform a mutant of Aspergillus sp. (A) in which the marker gene contained in the cyclic nucleotide is mutated, and then (ii) using the marker gene Transformants in which the target gene in the cyclic nucleotide has been introduced may be selected by using the expression of the as an index.
Alternatively, a filamentous fungus (A) of the genus Aspergillus in which the marker gene is not mutated can also be used as a host. do it.
The transformation method is not particularly limited, and a known method such as a PEG-calcium method, a calcium phosphate method, a DEAE dextran method, an electroporation method, a lipofection method, and a microinjection method can be adopted without limitation.
(III) Self-cloning strain
The self-cloning strain of the present invention thus obtained is a self-cloning strain of the present invention, wherein the expression gene cassette of the Aspergillus spp. (A) is contained in the chromosomal DNA of the Aspergillus spp. Exists between two identical marker genes derived from a filamentous fungus (A) species, and the marker genes are niaD, argB, sC, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene, and hygromycin It is selected from the group consisting of resistance genes. The marker gene is preferably niaD.
In the above-described method for producing a self-cloning strain, when a host in which the marker gene contained in the circular DNA used is mutated is used, one of the two marker genes is mutated. For example, a self-cloning strain obtained by transforming a host in which niaD has been mutated using a circular DNA containing niaD and a glaB expression cassette has a mutant niaD and a wild-type niaD in its chromosomal DNA. It has a sequence in which the glaB expression cassette is interposed.
In addition, nucleotides derived from a heterologous organism such as an E. coli vector are not substantially present in the chromosomal DNA. In addition, when the self-cloning strain is transformed using a cyclic nucleotide containing an enhancer, the expression level of the target gene is improved to about 3 to 1000 times that of a wild-type strain, depending on the type of the enhancer. Become.
[0045]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
Example 1 (Breeding of fucose lectin-producing self-cloning strain under the control of glaB enhancer)
<Incorporation of marker gene into E. coli vector>
The nitrate reductase gene niaD (SEQ ID NO: 1) derived from Aspergillus oryzae Aspergillus oryzae was converted into a PstI-HindIII fragment by using primers A (5′-aactgcagaacagccccaaattcaattataattgca-3 ′: SEQ ID NO: 2) and primer B (5′-cccagattctactcctactg). : SEQ ID NO: 3) and amplified by PCR under the following conditions using LA-Taq (Takara Holdings) using koji mold genomic DNA as a template.
PCR conditions
・ One cycle of 96 ° C (5 minutes)
・ 30 cycles of 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (5 minutes)
・ One cycle of 72 ° C (7 minutes)
The obtained PCR amplification product was treated at 37 ° C. with a restriction enzyme PstI-HindIII, and then cut out by agarose gel electrophoresis. For cutting out, a QIAquick Gel Extraction kit (QIAGEN) was used. Escherichia coli plasmid pUC119 (Takara Holdings) was ligated with DNA Ligation kit ver. 1 (Takara Holdings) and transformed into Escherichia coli (E. coli) strain JM109. As a result, a plasmid pNIA2 in which the niaD marker was subcloned was obtained. The construction of plasmid pNIA2 is shown in FIG.
<Preparation of fucose lectin gene expression cassette>
The glucoamylase gene glaB enhancer (glaB enhancer) 0.36-kb (SEQ ID NO: 4), the fucose lectin gene (fleA) open reading frame 1.1-kb (SEQ ID NO: 5) and the glucose amylase gene glaB terminator (glaB terminator) ) An attempt was made to construct a fusion gene expression cassette consisting of 1.0-kb (SEQ ID NO: 6).
[0046]
The glaB enhancer was prepared by using primer C (5'-aactgcagcgaggtagactagaagaatgcgggacacgg-3 ': SEQ ID NO: 7) and primer D (5'-gccaggagtagagacatatgatggtgctgaagaqAqaac-Aqaac-Aqaac-Aqaac-Aqaac-Aqaac-Aqaac-Aqaac-Aqaac-qaac-qaac-qaac-qaac-qaac-aac-qaac-aacAqaac-Aqaac-qaacqaacAqaacAqaacAqaqaacAqaacAqaacAqaacAqaacAqaacAqaacAqaacAqaacAcaAqaacAqaacAqaacAqaacAcaAqaacAqaacAqaacAqaacAqaacAacAacAacAacAacAacAacAacAacAacAacAga with a DNA sequence of a bacterium and a germ line with a DNA sequence as a genomic sequence. Holdings). fleA open reading frame, primers E (5'- gaagtcaccaccatcatgtctactcctggcgcccaagaagttcttttcc -3 'SEQ ID NO: 9) and primer F (5'- gcactggaaagtacatctactcagccggagggagagcccggcgaccc-3': LA-Taq (Takara the Aspergillus genomic DNA as a template using the SEQ ID NO: 10) Holdings). The glaB terminator was prepared by using primer G (5′-cccctcggtagtagattacttttcccagtgcgtgtagtagcc-3 ′: SEQ ID NO: 11) and primer H (5′-acgcgtcgacgcgaacagctcatacctTacactAq using the template as a genomic strain, and using a KojigaagcctaccacctTacactAq as a sequence for the Koji Lactase gene. ) Was used for PCR amplification. The PCR conditions were as follows.
[0047]
・ One cycle of 96 ° C (5 minutes)
・ 30 cycles of 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (5 minutes)
・ One cycle of 72 ° C (7 minutes)
The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target fragment was cut out using QIAquick Gel Extraction kit (QIAGEN).
[0048]
Next, as a second stage PCR, these three fragments were mixed, and PCR amplification by LA-Taq (Takara Holdings) was performed using primer C and primer H. The PCR conditions were as follows.
[0049]
・ One cycle of 96 ° C (5 minutes)
・ 30 cycles of 96 ° C (20 seconds) and 68 ° C (7 minutes)
・ One cycle of 68 ° C (7 minutes)
As a result of the PCR, an approximately 2.5-kb fusion gene product was amplified. The obtained PCR amplification product was treated with a restriction enzyme PstI-SalI at 37 ° C., then subjected to agarose gel electrophoresis, and a target DNA fragment was cut out using a QIAquick Gel Extraction kit (QIAGEN).
<Preparation of fusion gene of marker gene / target gene>
This DNA fragment was transferred to pNIA2 using DNA Ligation kit ver. By ligation using No. 1 (Takara Holdings), a plasmid pNBF into which a fusion gene of the marker gene and the target gene expression cassette was subcloned was obtained. This plasmid is used for E. coli E. coli. coli JM109 strain was transformed.
[0050]
Next, PCR for obtaining the cyclic nucleotide of the present invention was performed. Using plasmid pNBF as a template, primer I (5'-gcgaacagctcatacctctcatacctttctggacgaa-3 ': SEQ ID NO: 13) and primer J (5'-actctttggattttcctactctctcaataccaqatAq-Aq with PCRAqaq-AqAq-qqqqqqqqqAqAq-qqqAqqqqqqqqqAqqAqqqqqAqqqqqAqqqqqqqqq8a9) went. The PCR conditions were as follows.
[0051]
・ One cycle at 96 ° C (5 minutes)
・ 30 cycles at 96 ° C (20 seconds) and 68 ° C (10 minutes)
・ One cycle at 68 ° C (7 minutes)
As a result, an approximately 7.6-kb fusion gene product (marker gene / target gene expression cassette) was amplified. The obtained PCR amplification product was cut out by agarose gel electrophoresis, and extracted with QIAquick Gel Extraction kit (QIAGEN).
[0052]
This gene was treated with T4 DNA polynucleotide kinase (Takara Holdings) for 1 hour at 37 ° C., followed by treatment with T4 DNA polynucleotide kinase (Takara Holdings) for 1 hour at 37 ° C., followed by phenol chloroform extraction and ethanol precipitation. This was treated with T4 DNA ligase at 16 ° C. for 20 hours to allow self-ligation, followed by ethanol precipitation. Thereby, the cyclic nucleotide of the present invention was obtained.
<Preparation of self-cloning strain>
Using the above-mentioned cyclic nucleotide, a niaD mutant of A. oryzae (Patent Depositary of the National Institute of Advanced Industrial Science and Technology, AIST) was obtained by the conventional PEG-calcium method (Mol Gen Genet, 218, 99-104, (1989)). (Deposited as FERM P-13034) in a Czapek-Dox medium (2% glucose, 0.1% dipotassium hydrogen phosphate, 0.1% phosphate, 0.05% nitrate). % Of potassium chloride, 0.05% magnesium sulfate, 0.001% iron sulfate, 0.3% sodium nitrate) to obtain a plurality of transformants retaining fleA.
<Chromosome of self-cloning strain DNA Analysis>
The genomic DNA of these transformants was extracted by a conventional method, treated with SalI, subjected to agarose gel electrophoresis, alkali-transferred from the agarose gel to Hybond N + membrane, and further subjected to Southern blotting using Gene Image kit (Amersham Bioscience). Analyzed
FIG. 2 shows the results of Southern blotting. When niaD was used as a probe, a single band of about 16-kb was detected, indicating that one copy was transformed at the niaD site. When the E. coli vector pUC119 was used as a probe, no band could be confirmed. This indicates that a self-cloning strain was obtained in which no gene derived from Escherichia coli was present and only one gene derived from Aspergillus oryzae was introduced into the niaD site by one copy.
<Confirmation of expression level of target gene in self-cloning strain>
The fucose lectin productivity of this self-cloning strain was examined as follows. Since the glaB enhancer is an enhancer that is specifically expressed in solid culture (JP-A-2000-245465, JP-A-11-243965), the self-cloning strain is cultured in solid culture using rice koji (30-35 ° C.) with 70% polished rice. For 3 days). 10 g of the obtained rice koji was extracted and filtered with 50 ml of water at room temperature for 3 hours, and 50 μl of the supernatant was concentrated to dryness and subjected to SDS-PAGE.
[0053]
FIG. 3 shows the results of SDS-PAGE. As is apparent from FIG. 3, a band having a molecular weight of 35,000, which was not observed in the wild strain used as a control, was detected. This indicates that in the self-cloning strain, fucose lectin (molecular weight: 35,000) (Biosci. Biotechnol. Biochem. 66, 1002-1008 (20002)) is secreted and produced in large amounts outside the cells.
[0054]
As a result, although the bred self-cloning strain contained only DNA derived from Aspergillus oryzae aspergillus, the production of fucose lectin was significantly increased as compared with the wild strain. Since fucose lectin has uses as a research reagent and a clinical reagent, the obtained self-cloning strain is a strain that can be industrially effectively used as a safe non-genetically modified product.
Example 2 (Breeding of glucoamylase-producing self-cloning strain under the control of sodM enhancer)
<Preparation of glucoamylase gene expression cassette>
Manganese SOD gene sodM enhancer (sodM enhancer) 1.0-kb (SEQ ID NO: 15), glucoamylase gene glaB open reading frame 1.6-kb (SEQ ID NO: 16) and glucoamylase gene glaB terminator 1.0-kb An attempt was made to construct a fusion gene expression cassette consisting of (SEQ ID NO: 6).
[0055]
The sodM enhancer is composed of primer K (5'-aactgcagttatgtactccgtactcggttgaattattataatcgggg-3 ': SEQ ID NO: 17) and primer L (5'-gaaggttgttctggtTgtgtgtgtgtgttgtgtgtgttgtgtgtgttgttgttgt). (Takara Holdings). The glaB open reading frame was prepared using primer M (5′-ccaccaccacaccaccaaaatgcggaacacctttctttttttccc-3 ′: SEQ ID NO: 19), primer N (5′-gcactggaagagttactatgccDNActgctAggtggDNA sequence using Kg. -PCR was amplified using Taq (Takara Holdings). The glaB terminator was obtained by PCR using LA-Taq (Takara Holdings) using primer O (5′-ctgttgggtcgtggttagattgtacttcccagtgcgtgttagtctact-3 ′: SEQ ID NO: 21) and primer H (SEQ ID NO: 12) with Koji mold genomic DNA as a template. . The PCR conditions were as follows.
[0056]
・ One cycle at 96 ° C (5 minutes)
・ 30 cycles at 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (5 minutes)
・ One cycle at 72 ° C (7 minutes)
The obtained PCR amplification product was subjected to agarose gel electrophoresis, and a target fragment was cut out using a QIAquick Gel Extraction kit (QIAGEN).
[0057]
Next, as a second stage PCR, these three fragments were mixed, and PCR amplification by LA-Taq (Takara Holdings) was performed using primer K and primer H. The PCR conditions were as follows.
[0058]
・ One cycle at 96 ° C (5 minutes)
・ 30 cycles at 96 ° C (20 seconds) and 68 ° C (7 minutes)
・ One cycle at 68 ° C (7 minutes)
As a result of the PCR, a fusion gene product of about 3.7-kb was amplified. The obtained PCR amplification product was treated with a restriction enzyme PstI-SalI at 37 ° C., subjected to agarose gel electrophoresis, and a target DNA fragment was cut out using a QIAquick Gel Extraction kit (QIAGEN).
<Preparation of fusion gene of marker gene / target gene>
This DNA fragment was added to pNIA2 obtained in Example 1 using DNA Ligation kit ver. By ligation using No. 1 (Takara Holdings), a plasmid pNMB in which a fusion gene of the marker gene and the target gene expression cassette was subcloned was obtained. This plasmid is used for E. coli E. coli. coli JM109 strain was transformed.
[0059]
Next, PCR for obtaining the cyclic nucleotide of the present invention was performed. PCR amplification by LA-Taq (Takara Holdings) was performed using primer I and primer J with plasmid pNMB as a template. The PCR conditions were as follows.
[0060]
・ One cycle at 96 ° C (5 minutes)
30 cycles at 96 ° C (20 seconds) and 68 ° C (10 minutes)
・ One cycle at 68 ° C (7 minutes)
As a result, a fusion gene product (marker gene / target gene expression cassette) of about 8.8-kb was amplified. The obtained PCR amplification product was cut out by agarose gel electrophoresis, and extracted with QIAquick Gel Extraction kit (QIAGEN).
[0061]
This gene was treated with T4 DNA polynucleotide kinase (Takara Holdings) for 1 hour at 37 ° C., and treated with T4 DNA polynucleotide kinase (Takara Holdings) for 1 hour at 37 ° C., followed by phenol chloroform extraction and ethanol precipitation. This was treated with T4 DNA ligase at 16 ° C. for 20 hours to allow self-ligation, followed by ethanol precipitation. Thereby, the cyclic nucleotide of the present invention was obtained.
<Preparation of self-cloning strain>
The PEG-calcium method was used to obtain A. oryzae niaD mutant strain FERM P-13034 was transformed. By selecting a strain capable of growing in a medium using nitric acid as the sole nitrogen source as in Example 1, a plurality of transformants carrying the glaB gene were obtained.
<Chromosome of self-cloning strain DNA Analysis>
Genomic DNAs of these transformants were obtained by a conventional method, treated with SalI, subjected to agarose gel electrophoresis, alkali-transferred from the agarose gel to Hybond N + membrane, and further subjected to Southern blot using Gene Image kit (Amersham Bioscience). Analysis was performed.
[0062]
FIG. 4 shows the results of Southern blotting. When niaD was used as a probe, a single band of about 17-kb was detected, indicating that one copy was transformed at the niaD site. When the E. coli vector pUC119 was used as a probe, no band could be confirmed. This indicates that a self-cloning strain in which one copy of the gene derived from Aspergillus aliza was introduced into the niaD site without the gene derived from Escherichia coli was obtained.
<Confirmation of expression level of target gene in self-cloning strain>
Glucoamylase productivity of this self-cloning strain was examined as follows. Since the sodM enhancer is an enhancer that is specifically expressed in liquid culture (Japanese Patent Application Laid-Open No. 2001-224381), the present self-cloning strain is transformed into a liquid medium (dextrin 6%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.5%). 1%, potassium chloride 0.05%, ammonium sulfate 0.2%, iron sulfate 0.00001M, pH 6.3) and cultured at 37 ° C. for 3 days.
[0063]
Glucoamylase activity was measured using a saccharification power measurement kit manufactured by Kikkoman Corporation, and glucoamylase having a specific activity of 0.36 U / mg was used as a standard glucoamylase for the measurement. As a result, the extracellular glucoamylase production was 1.2 g / 1 L-broth.
[0064]
The obtained culture supernatant equivalent to 50 μl was concentrated to dryness and subjected to SDS-PAGE. FIG. 5 shows the results of SDA-PAGE. As is clear from FIG. 5, a band having a molecular weight of 50,000 to 100,000, which is not observed in the wild strain used as a control, was detected. This indicates that glucoamylase (molecular weight (50,000-100,000) (J. Ferment. Bioeng., 84, 532-537, 1997)) is secreted and produced in large amounts outside the cells.
[0065]
As a result, although the bred self-cloning strain contained only the gene derived from Aspergillus oryzae aspergillus, the production of glucoamylase was significantly increased as compared with the wild-type strain. Since glucoamylase has uses as an industrial enzyme agent, a research reagent, and a clinical reagent, the obtained self-cloning strain is a strain that can be industrially effectively used as a safe non-genetically modified product.
Example 3(Breeding of fucose lectin-producing self-cloning strain under the control of melO enhancer)
<Preparation of fucose lectin expression cassette>
From the tyrosinase gene melO enhancer 1.2-kb (SEQ ID NO: 22), the open reading frame 1.1-kb of the fucose lectin gene fleA (SEQ ID NO: 5) and the glucoamylase gene glaB terminator 1.0-kb (SEQ ID NO: 6) We tried to construct a fusion gene expression cassette.
[0066]
The melO enhancer is composed of primer P (5'-aactgcagttatgctactccgtactcggttgaatgcttgcctttggtccaatcgttcatg-3 ': SEQ ID NO: 23) and primer Q (5'-cgccaggagtaggag Holdings). The fleA open reading frame was PCR-amplified using primer R (5'-gattacttgtgttcacaattgtctactcctggcgcccagaagagtc-3 ': SEQ ID NO: 25) and primer F and LA-Taq (Takara Holdings) using koji mold genomic DNA as a template. The glaB terminator was PCR-amplified using Primer G and Primer H and LA-Taq (Takara Holdings) using Aspergillus genomic DNA as a template. The PCR conditions were as follows.
[0067]
・ One cycle at 96 ° C (5 minutes)
・ 30 cycles at 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (5 minutes)
・ One cycle at 72 ° C (7 minutes)
The obtained PCR amplification product was subjected to agarose gel electrophoresis, and cut out using a QIAquick Gel Extraction kit (QIAGEN).
[0068]
Next, as a second-stage PCR, these three fragments were mixed, and PCR amplification using LA-Taq (Takara Holdings) was performed using primers P and H. The PCR conditions were as follows.
[0069]
・ One cycle at 96 ° C (5 minutes)
・ 30 cycles at 96 ° C (20 seconds) and 68 ° C (7 minutes)
・ One cycle at 68 ° C (7 minutes)
As a result of the PCR, a fusion gene product of about 3.3-kb was amplified. The obtained PCR amplification product was treated with a restriction enzyme PstI-SalI at 37 ° C., subjected to agarose gel electrophoresis, and a target DNA fragment was cut out using a QIAquick Gel Extraction kit (QIAGEN).
<Preparation of fusion gene of marker gene / target gene expression cassette>
This DNA fragment was added to pNIA2 obtained in Example 1 using DNA Ligation kit ver. By ligation using No. 1 (Takara Holdings), a plasmid pNEF in which a fusion gene of the marker gene and the target gene expression cassette was subcloned was obtained. This plasmid is used for E. coli E. coli. coli JM109 strain was transformed.
[0070]
Next, PCR for obtaining the cyclic nucleotide of the present invention was performed. PCR amplification by LA-Taq (Takara Holdings) was performed using primer p and primer J with plasmid pNEF as a template. The PCR conditions were as follows.
[0071]
・ One cycle at 96 ° C (5 minutes)
30 cycles at 96 ° C (20 seconds) and 68 ° C (10 minutes)
・ One cycle at 68 ° C (7 minutes)
As a result, an approximately 8.4-kb fusion gene product (marker gene / target gene expression cassette) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and extracted with QIAquick Gel Extraction kit (QIAGEN).
[0072]
This gene was treated with T4 DNA polynucleotide kinase (Takara Holdings) for 1 hour at 37 ° C., and treated with T4 DNA polynucleotide kinase (Takara Holdings) for 1 hour at 37 ° C., followed by phenol chloroform extraction and ethanol precipitation. This was treated with T4 DNA ligase at 16 ° C. for 20 hours to allow self-ligation, followed by ethanol precipitation. Thereby, the cyclic nucleotide of the present invention was obtained.
<Preparation of self-cloning strain>
The PEG-calcium method was used to obtain A. oryzae niaD mutant strain FERM P-13034 was transformed. By selecting a strain capable of growing on a medium using nitric acid as a single nitrogen source, a plurality of transformants carrying the fleA gene were obtained.
<Chromosome of self-cloning strain DNA Analysis>
The genomic DNAs of these transformants were obtained by a conventional method, treated with SalI, subjected to agarose gel electrophoresis, alkali-transferred the gel to Hybond N + membrane, and further subjected to Southern using Gene Image kit (Amersham Bioscience). Blot analysis was performed.
[0073]
FIG. 6 shows the results of Southern blotting. When niaD was used as a probe, a single band of about 17-kb was detected, indicating that one copy was transformed at the niaD site. When the E. coli vector pUC119 was used as a probe, no band could be confirmed. This shows that a self-cloning strain in which one copy of the gene derived from Aspergillus oryzae was introduced into the niaD site without the gene derived from Escherichia coli was obtained.
<Confirmation of expression level of target gene in self-cloning strain>
The fucose lectin productivity of this self-cloning strain was examined as follows. Since the melO enhancer is an enhancer that is expressed specifically in liquid culture (Japanese Patent Application Laid-Open No. 2001-046078), this self-cloning strain is transformed into a liquid medium (glucose 3%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.5%). 1%, potassium chloride 0.05%, ammonium sulfate 0.2%, iron sulfate 0.00001M, pH 6.3) and cultured at 37 ° C. for 3 days. The recovered koji culture cells were crushed with sea sand, extracted with PBS, and centrifuged. 20 μg of the obtained crude enzyme cell protein was subjected to SDS-PAGE.
[0074]
FIG. 7 shows the results of SDS-PAGE. As is clear from FIG. 7, a band having a molecular weight of 35,000, which was not observed in the wild strain used as a control, was detected. This indicates that in the self-cloning strain, fucose lectin (molecular weight 35,000) (Biosci. Biotechnol. Biochem. 66, 1002-1008 (2002)) is produced in large amounts in the cells.
[0075]
As a result, although the bred self-cloning strain contained only the gene derived from Aspergillus oryzae, the production of fucose lectin was significantly increased as compared with the wild strain. Since fucose lectin has uses as a research reagent and a clinical reagent, the obtained self-cloning strain is a strain that can be industrially effectively used as a safe non-genetically modified product.
[0076]
<Comparison of transformation efficiency>
Transform Aspergillus oryzae using a fusion gene of a marker gene and a target gene expression cassette, and determine the transformation efficiency using a cyclic nucleotide as in Examples 1 to 3, a method using a linear nucleotide, and Escherichia coli. Comparisons were made between methods using the plasmid integrated into the vector.
[0077]
Transformation test 1
In Example 1, a cyclic nucleotide obtained by self-ligating DNA amplified by PCR using a plasmid pNBF obtained by subcloning niaD and fleA expression cassette into an Escherichia coli vector as a template, before the self-ligation in Example 1 Using the linear DNA and the E. coli plasmid pNBF prepared in Example 1, an Aspergillus oryzaea niaD mutant (FERMP-13034) was transformed under the same conditions as in Example 1.
[0078]
Transformation test 2
In Example 2, a cyclic nucleotide obtained by self-ligating DNA amplified by PCR using plasmid pNBF obtained by subcloning niaD and glaB expression cassette into an Escherichia coli vector as a template, before the self-ligation in Example 2 Using the linear DNA and the E. coli plasmid pNBF prepared in Example 2, an Aspergillus oryzaea niaD mutant (FERMP-13034) was transformed under the same conditions as in Example 2.
[0079]
Transformation test 3
In Example 3, a cyclic nucleotide obtained by self-ligating DNA amplified by PCR using a plasmid pNBF obtained by subcloning niaD and fleA expression cassette into an Escherichia coli vector as a template, before the self-ligation in Example 3 Using the linear DNA and the E. coli plasmid pNBF prepared in Example 3, an Aspergillus oryzae niaD mutant (FERMP-13034) was transformed under the same conditions as in Example 3.
[0080]
In the above transformation tests 1 to 3, the number of transformants obtained per 1 μg of the DNA subjected to transformation was defined as the transformation efficiency. The results are shown in Table 1 below.
[0081]
[Table 1]

[0082]
From Table 1, it can be seen that in obtaining a self-cloning strain into which a target gene has been introduced by transformation, if the target gene is used in a linear form, substantially no transformation will take place, but by using a cyclic nucleotide form, it will be extremely high. It can be seen that transformation occurs with efficiency. It is also found that the efficiency is much higher than when the target gene is incorporated into an E. coli vector.
[0083]
【The invention's effect】
According to the present invention, a method for efficiently producing a self-cloning strain of Aspergillus genus fungi, a cyclic nucleotide used in the method, and a self-cloning strain of Aspergillus genus fungi are provided.
Furthermore, breeding of Aspergillus genus fungi typified by Aspergillus oryzae depends on a mutation method with low acquisition efficiency or a genetic recombination method with good acquisition efficiency but low public acceptance in the food industry. For this reason, there has been a long-felt desire in the food industry for breeding self-cloning strains that do not contain genes from heterologous organisms using simple techniques such as genetic recombination. ADVANTAGE OF THE INVENTION According to this invention, the target property was able to be introduce | transduced into Aspergillus filamentous fungi very simply, efficiently and safely.
[0084]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing the structure of a plasmid pNIA2 obtained in Example 1.
FIG. 2 is a diagram showing that fleA has been integrated into chromosomal DNA of the transformant obtained in Example 1.
FIG. 3 is a diagram showing that the transformant obtained in Example 1 produces a large amount of fucose lectin.
FIG. 4 is a diagram showing that glaB is integrated into chromosomal DNA of the transformant obtained in Example 2.
FIG. 5 is a view showing that the transformant obtained in Example 2 produces glucoamylase in large amounts.
FIG. 6 is a diagram showing that fleA has been integrated into chromosomal DNA of the transformant obtained in Example 3.
FIG. 7 is a view showing that the transformant obtained in Example 3 produces a large amount of flucosctin.

Claims (9)

niaD, argB, sC, ptrA, pyrG, amdS, an aureobasidin resistance gene, a benomyl resistance gene, a hygromycin resistance gene, a marker gene derived from a filamentous Aspergillus (A) species, and a filamentous fungus of the genus Aspergillus (A) a cyclic nucleotide containing a target gene expression cassette derived from a species; The cyclic nucleotide according to claim 1, wherein the marker gene is niaD. The cyclic nucleotide according to claim 1 or 2, comprising only a nucleotide derived from a filamentous fungus of the genus Aspergillus (A). The cyclic nucleotide according to claim 1, 2 or 3, for producing a self-cloning strain of the filamentous fungus (A) of the genus Aspergillus. A method for producing a self-cloning strain of Aspergillus spp., Wherein the Aspergillus spp. Filamentous fungus (A) is transformed using the cyclic nucleotide according to any one of claims 1 to 4. (I) using the cyclic nucleotide according to any one of claims 1 to 4 to transform a mutant of Aspergillus spp. (A) in which a marker gene contained in the cyclic nucleotide is mutated;
(Ii) selecting a transformant in which the target gene in the cyclic nucleotide has been introduced, using the expression of the marker gene as an index, to produce a self-cloning strain of a filamentous fungus of the genus Aspergillus. A self-cloning strain of a filamentous fungus of the genus Aspergillus obtained by transforming a species of the filamentous fungus of the genus Aspergillus (A) using the cyclic nucleotide according to any one of claims 1 to 4. A state in which a target gene expression cassette derived from a filamentous Aspergillus (A) species is sandwiched between two identical marker genes derived from a filamentous Aspergillus (A) species in the chromosomal DNA of the filamentous Aspergillus (A) species. Wherein the marker gene is selected from the group consisting of niaD, argB, sC, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene and hygromycin resistance gene. stock. 9. The self-cloning strain of Aspergillus spp. According to claim 8, wherein one of the two marker genes is a mutated marker gene.

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