JP5344857B2 - Method for producing filamentous fungal protease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a filamentous fungal protease, an expression vector for filamentous fungi and a transformant useful for practicing the method. <P>SOLUTION: The expression vector (preferably self-cloning vector) for filamentous fungi comprises a protease gene composed of any one base sequence selected from the group consisting of a selective marker gene sequence functioning in a filamentous fungus cell, a promoter sequence functioning in a filamentous fungus cell and a specific base sequence, and a terminator sequence functioning in a filamentous fungus cell. The filamentous fungi are transformed with the vector. The method for producing protease comprises culturing and recovering the filamentous fungus. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a filamentous fungal protease, and an expression vector and a transformant useful for the implementation of the method.

  Proteases are used in various applications and fields such as detergents for clothes, cosmetics, bath preparations, food modifiers, digestive aids or anti-inflammatory agents, and are the most produced of industrial enzymes. There are many. Also in the food industry, amino acid production as a seasoning from food protein, reduction of bitterness and miscellaneous taste of food, production of peptide to give richness and richness to food, production of peptide with high intestinal absorption and high nutrient source Proteases have a significant role in the production of peptides as functional food materials such as blood pressure lowering activity and the removal of unwanted proteins turbid in food. Many proteases used in the food industry are derived from microorganisms, and many are from the genus Aspergillus, Bacillus, and Rhizopus. In particular, many proteases derived from Aspergillus oryzae can handle a wide range of pH, and provide protease enzyme agents with various types of molecular species depending on the culture conditions such as liquid culture and solid culture. Because it can, it is most widely used in the food industry.

  In particular, lactotripeptides such as VPP and IPP derived from milk proteins with blood pressure lowering activity can be produced advantageously by protease enzyme agents derived from Aspergillus oryzae. Proteases derived from Aspergillus oryzae are also very useful in peptide production as functional food materials. (Non-patent Document 1).

  However, in the production of functional peptides, the present protease enzyme agent derived from Aspergillus has a weak point. More specifically, such an enzyme agent contains a plurality of peptidases whose characteristics are unknown because it is composed of a culture extract of Aspergillus oryzae. Therefore, when acting on food proteins, the target functional peptide Is excessively decomposed and converted to amino acids.

  In order to solve the above problems, the current situation is that the reaction time, reaction pH, reaction temperature, amount ratio of food protein and enzyme agent, combined use of enzyme agent, etc. must be dealt with on a case-by-case basis.

In order to solve such a problem, it is desirable that a protease enzyme agent with a clear molecular species can be provided by filamentous fungi, particularly koji molds, which have been used for the manufacture of brewed foods such as sake and soy sauce since long ago. Currently, no such technology has been provided.
British Journal of Nutrition, 94, 84-91 (2005)

  In order to industrially produce a protease enzyme agent derived from filamentous fungi useful for the production of functional peptides, particularly koji mold, it is necessary to construct a system capable of producing a protease with a known molecular species. Therefore, an object of the present invention is to provide a method for producing a protease derived from a filamentous fungus and having a known molecular species using the filamentous fungus as a host, particularly a method for producing by a self-cloning method. Another object of the present invention is to provide a material for producing the protease, that is, an expression vector for a filamentous fungus and a transformant.

  As a result of repeated researches to solve the above problems, the present inventors have been able to construct the above system for a protease derived from a filamentous fungus having the amino acid sequence described in any of SEQ ID NOs: 11 to 20, It was confirmed that the protease can be efficiently produced using a self-cloning method using a filamentous fungus as a host and outside the cell (medium supernatant), and the present invention has been completed.

That is, the present invention has the following embodiments.
(I) an expression vector for filamentous fungi (I-1) selected from the group consisting of a selectable marker gene sequence that functions in filamentous fungi, a promoter sequence that functions in filamentous fungi, and the base sequences described in SEQ ID NOs: 1 to 10 An expression vector for a filamentous fungus, comprising a protease gene comprising any one of the nucleotide sequences described above and a terminator sequence that functions in the filamentous fungus body.
(I-2) The expression vector for filamentous fungi according to (I-1), which is a self-cloning vector consisting essentially of nucleotides derived from filamentous fungi.
(I-3) The expression vector for filamentous fungi according to (I-1) or (I-2), which is linear.
(I-4) The expression vector for filamentous fungi according to any one of (I-1) to (I-3), wherein the filamentous fungus is a filamentous fungus belonging to the genus Aspergillus.

(II) A filamentous fungus transformed with the expression vector for filamentous fungus according to any one of the transformants (II-1) (I-1) to (I-4).
(II-2) The filamentous fungus according to (II-1), which is a filamentous fungus belonging to the genus Aspergillus.

(III) Protease production method (III-1) The transformed filamentous fungus described in (II-1) or (II-2) is cultured in a medium, and described in SEQ ID NOs: 11 to 20 from the medium. A method for producing a protease, comprising the step of recovering a protease comprising any one amino acid sequence selected from the group consisting of amino acid sequences.

  According to the expression vector for filamentous fungi and the production method of the present invention, a protease derived from a filamentous fungus with a clear molecular species can be efficiently produced using the filamentous fungus as a host. Therefore, according to the method of the present invention, a desired protease having a clear molecular species can be industrially produced.

  In addition, linear filamentous fungal expression vectors can be prepared with fewer steps compared to circular expression vectors, such as (1) one-step PCR from the constructed plasmid, and (2) leucine as a host. When using a required mutant, there are advantages that the mutation can be directly complemented and (3) the transformation efficiency is higher than that of a circular expression vector.

  Examples of the filamentous fungi targeted by the present invention include koji molds and incomplete filamentous fungi to which koji molds belong. Among these, preferred are filamentous fungi belonging to the genus Aspergillus, Mucor, Candida, Trichoderma, and Neurospora, and more preferred are Aspergillus. It belongs to the filamentous fungus. The Aspergillus genus fungus is not particularly limited, but Aspergillus oryzae, Aspergillus niger, Aspergillus kawachii, Aspergillus awamori, saspergillus sperm ), Aspergillus sojae, Aspergillus tamarii, Aspergillus glaucus, Aspergillus fumigatus, Aspergillus flavus, Aspergilter rusus And Aspergillus nidulans. Among them, Aspergillus oryzae is preferable because it is a safe microorganism that has been used in brewed foods such as sake, soy sauce, miso, and mirin for many years and is a frequently used host in Japanese industries. .

(1) Expression vector for filamentous fungi The expression vector for filamentous fungi of the present invention is a filamentous fungal protease having the amino acid sequence described in any one of SEQ ID NOs: 11 to 20 in the host using the filamentous fungus or a mutant thereof as a host. Is a vector capable of expressing and producing Such a vector, in addition to the protease gene having the base sequence described in any one of SEQ ID NOs: 1 to 10 encoding the protease, a promoter sequence that functions in filamentous fungi, a terminator sequence that functions in filamentous fungi, and It has a selectable marker gene sequence that functions in filamentous fungi and is preferably used for selection of transformants.

(1-1) Filamentous protease The protease targeted by the present invention is a protease derived from a filamentous fungus having any one of the amino acid sequences shown in SEQ ID NOs: 11 to 20, particularly a koji mold. Among these proteases, the proteases shown in SEQ ID NOs: 11 to 13 are alkaline proteases, the acidic carboxypeptidase shown in SEQ ID NOs: 14 to 17, the protease shown in SEQ ID NO: 18 is an acidic protease, and the proteases shown in SEQ ID NOs: 19 to 20 are medium. It can be classified into sex proteases.

  The correspondence between the amino acid sequences of these proteases and the base sequences of the genes (protease genes) encoding them is shown in the following table:

(1-2) Selection marker gene The selection marker gene may be any one that functions as a selectable marker in filamentous fungi, particularly Aspergillus sp., That is, can be expressed, but is preferably a filamentous fungus, particularly Aspergillus sp. It is a selectable marker gene derived from bacteria. Examples of such selectable marker genes include niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6, 386-389, (1984)), sC (Gene, 84, 329-334, (1989)), ptrA (Biosci Biotechnol Biochem, 64, 1416-1421, (2000)), pyrG (Biochem Biophys Res Commun, 112, 284-289, (1983)), amdS (Gene, 26, 205-221, (1983)), aureobasidin resistance gene (Mol Gen Genet, 261, 290-296, (1999)), benomyl resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873, (1986)) And a marker gene selected from a hygromycin resistance gene (Gene, 57, 21-26, (1987)).

  These selectable marker genes can be obtained by PCR using primers designed based on the sequences described in the above-mentioned literature and using the chromosome DNA of Aspergillus filamentous fungi as a template, or based on the sequences described in the above-mentioned literature. It can be easily obtained by a method of obtaining from a chromosomal DNA library of Aspergillus filamentous fungi using a designed probe. In addition, when an auxotrophic mutant such as leucine auxotrophy is used as the host filamentous fungus, a wild-type gene that complements the auxotrophy can also be used as a selection marker gene. For example, when the host is a leucine-requiring mutant, a selectable marker gene that can complement the leucine requirement can be used, such as the gene ANleu2 derived from Aspergillus nidulans encoding β-isopropylmalate dehydrogenase and A gene leu2 derived from Aspergillus oryzae can be mentioned. In these cases, it is necessary to use a strain that does not have a functional gene for the selected selection marker as the filamentous fungus used as a host.

(1-3) Promoter The promoter is not particularly limited as long as it functions as a promoter in filamentous fungi, particularly Aspergillus sp., But is preferably a promoter derived from filamentous fungi, particularly Aspergillus sp. Specific examples of such promoters include, for example, α-amylase gene promoter amyB (Biosci Biotechnol Biochem, 56, 1849-1853, (1992)), glucoamylase gene promoter glaA (Gene, 108, 145-150, (1992)), α-glucosidase gene promoter agdA (Curr Genet, 30, 432-438, (1996)), manganese SOD gene promoter sodM (JP 2000-224381), tyrosinase gene promoter melO (JP 2001-046078), Examples include glucoamylase gene promoter glaB (JP 2000-245465 A, JP 11-243965 A). Preferred examples of the high expression promoter specific to liquid culture include sodM and melO. Preferred examples of the high expression promoter specific to solid culture include glaB.

  The position of the promoter in the expression vector is not particularly limited as long as the promoter functions in the filamentous fungus and can express and produce a desired protease, but is usually located upstream of the protease gene sequence.

(1-4) Terminator The terminator is not particularly limited as long as it functions as a terminator in filamentous fungi, particularly Aspergillus sp., But is preferably a terminator derived from a filamentous fungus, particularly Aspergillus sp. Preferred examples include the terminator of the α-amylase gene or the glucoamylase (glaB) terminator (Gene. 207, 127-134, (1998)) derived from the genus Aspergillus, more preferably from Aspergillus oryzae.

(1-5) Expression vector for filamentous fungi The expression vector for filamentous fungi of the present invention may be one in which these selection marker genes, promoters, protease genes, and terminators are directly linked. A nucleotide of about 1 to 2000 bases may be sandwiched between them. However, these nucleotide sequences are preferably derived from filamentous fungi, and preferably from Aspergillus spp. For example, when the target protease is produced as an intracellular protein, a known secreted protein of a filamentous fungus, preferably an Aspergillus spp., Adjacent to the open reading frame of the target protease gene The target protease is expressed and produced as a fusion protein with the secretory protein, and the fusion protein thus produced is secreted outside the cells. Further, as other nucleotides, a restriction enzyme cleavage site or the like usually provided in a vector may be provided.

  The secretory proteins of Aspergillus fungi include α-amylase, glucoamylase, α-glucosidase, endoglucanase, cellobiohydrolase, β-glucosidase, acid protease, neutral protease, alkaline protease, carboxypeptidase, lipase, Phospholipases, xylanases, galactosidases, fructofuranosidases, xylosidases, pectin lyases, phytases and the like are known.

  In addition, the present invention includes both linear filamentous fungus expression vectors and circular filamentous fungus expression vectors. (1) A circular expression vector that can be obtained from the constructed plasmid by one-step PCR. (2) When using a leucine-requiring mutant as a host, the mutation can be directly complemented, and (3) an expression vector with a circular transformation efficiency. From the standpoint of being higher than the above, linear expression vectors for filamentous fungi are preferred.

(2) Transformant The transformant of the present invention is obtained by transforming a filamentous fungus with the aforementioned expression vector for filamentous fungi of the present invention.

  The host may be a filamentous fungus, but in order to express a high promoter activity, a strain of the genus Aspergillus is preferable, and in particular, Aspergillus oryzae, Aspergillus niger, Aspergillus kawachii (Aspergillus kawachii) ), Aspergillus awamori, Aspergillus saitoi, Aspergillus sojae, Aspergillus tamarii, Aspergillus glaucus, Aspergillus glaucus, Aspergillus glaucus, Aspergillus glaucus, Aspergillus glaucus Aspergillus flavus, Aspergillus terrus, Aspergillus nidulans and other Aspergillus nidulans are preferred. Among these, Aspergillus oryzae, Aspergillus niger, Aspergillus kawachi, Aspergillus awamori, Aspergillus saito, Aspergillus sojae, Aspergillus tamari, Aspergillus glaucus are useful bacterial species in the food industry. From the viewpoint of high protein production ability and safety as a brewing microorganism, it is particularly preferable to use Aspergillus oryzae as a host. The filamentous fungi used as the host are not limited to wild strains, and may be mutants thereof as long as they are derived from filamentous fungi. Examples of such mutant strains include strains that are mutated so as not to have a functional gene related to the selection marker described above, and specifically, are responsible for leucine biosynthesis so that leucine cannot be normally biosynthesized. A leucine-requiring mutant in which a gene is mutated or deleted: an arginine-requiring mutant in which a gene responsible for arginine biosynthesis is mutated or deleted so that arginine biosynthesis cannot be normally performed: normal methionine production A methionine-requiring mutant in which the gene responsible for methionine biosynthesis is mutated or deleted so that synthesis cannot be performed: histidine in which the gene responsible for histidine biosynthesis is mutated or deleted so that histidine biosynthesis cannot be performed normally Requirement mutant: niaD mutant with a nitrate-assimilating gene mutated or deleted so that nitrate cannot normally be assimilated: Normal Etc. sC mutants nitrate ions nitrate ions assimilating gene so as not to assimilate are deleted can be exemplified.

  The host transformation method is not particularly limited, and can be performed using a conventionally known method. For example, the method of Cohen et al. (Calcium chloride method) (Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)), protoplast method (Mol. Gen. Genet., 168: 111 (1979)), competent Method (J. Mol. Biol., 56: 209 (1971)), electroporation method and the like.

(3) Protease Production Method The protease production method of the present invention includes a step of culturing the aforementioned filamentous fungus transformant of the present invention and a step of recovering the target protein from the culture.

  The culturing step can be performed by either solid culture using a solid medium or liquid culture using a liquid medium. Liquid culture using a liquid medium is preferred.

  As the solid medium, a known solid medium used for culturing filamentous fungi can be used without limitation. The solid medium refers to a solid medium in which the solid support carrier contains a nutrient source or a nutrient source is added to the solid support carrier, on which filamentous fungi can grow. Such solid media mainly include bran (cereal shells such as wheat), starch powder, raw or steamed rice / wheat / soybean, and membranes and porous artifacts (eg vermiculite used in horticulture) ) And the like to which a nutrient source is added. In particular, bran and steamed rice are preferred.

As the liquid medium, a known liquid medium used for culturing filamentous fungi can be used without limitation. For example, the medium to be used includes those containing carbohydrates such as glucose, fructose, glycerol and starch as a carbon source. Further, inorganic or organic nitrogen sources (for example, ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, bactotryptone, beef extract, etc.) can be mentioned. These carbon sources and nitrogen sources do not need to be used in pure form, and those having low purity are advantageous because they are rich in trace amounts of growth factors and inorganic nutrients. Further, if desired, other nutrient sources [eg, inorganic salts (eg, sodium or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg, vitamin B1) Antibiotics (eg, ampicillin, kanamycin, etc.) may be added to the medium. Specific liquid media include, for example, potato dextrose medium (Nissui), or minimal medium (2% glucose (or starch), 0.3% NaNO ₃ , 0.2% KCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.002% FeSO ₄ , pH 6.0) and the like. These can also be prepared as a solid medium by adding about 1.5% agar.

  The transformant can be cultured usually at pH 5.5 to 8.5, preferably pH 6 to 8; usually 25 to 42 ° C, preferably 30 to 37 ° C. The culture time varies depending on other conditions, but is usually about 2 to 7 days, preferably about 3 to 5 days.

  According to the method of the present invention, the target protease (SEQ ID NO: 1 to 10) is produced outside the cells as shown in the Examples. For this reason, the culture supernatant may be recovered upon obtaining the protease. Moreover, what is necessary is just to collect | recover the agar medium as a culture supernatant also when using a solid medium like an agar medium. Furthermore, the target protease can be isolated and recovered by subjecting these supernatants to various known chromatographic methods such as ion exchange, hydrophobicity, gel filtration, and affinity.

(4) Self-cloning vector and self-cloning strain In the present invention, preferable filamentous fungus expression vectors are derived from a selectable marker gene derived from a filamentous fungus, a promoter derived from a filamentous fungus, a protease gene derived from a filamentous fungus, and a filamentous fungus. And a self-cloning vector consisting essentially of nucleotides derived from filamentous fungi. In the present invention, a preferred transformant is a filamentous fungus obtained by transformation with the above self-cloning vector. Among the filamentous fungi, a koji mold is preferable, and among the koji molds, a koji mold belonging to the genus Aspergillus is preferable.

  In the present invention, the “self-cloning strain” refers to a strain composed substantially of nucleotides derived from the same species as the host filamentous fungus as described above. Moreover, when nucleotides not derived from filamentous fungi are included, it is at most about 1 to 10 bases, particularly about 6 to 10 bases. An insertion sequence of this size may be inserted, for example, to introduce a restriction enzyme site.

  As the host filamentous fungus, a filamentous fungus from which the marker gene, promoter, terminator and protease gene to be expressed are derived, preferably a strain of the same type as the Aspergillus spp. It is preferable to use a mutant strain in which a gene having the same function as or a function of the marker gene to be introduced is deleted or mutated so that a transformant can be easily selected. In this case, after transforming the mutant strain using the filamentous fungus expression vector described above, the target protease gene in the filamentous fungus expression vector is expressed by using the expression of the introduced marker gene as an index. The introduced transformant can be selected.

  In addition, Aspergillus filamentous fungi that do not have a deletion or mutation in the same or the same function as the marker gene to be introduced can also be used as a host. In this case, a transformant can be selected by using the expression of the target protease gene or an increase in the expression level as an index. 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, or a microinjection method can be employed without limitation.

  The self-cloning strain thus obtained is present in a state in which the above-described expression vector for filamentous fungi of the present invention is incorporated in the chromosomal DNA of a filamentous fungus, preferably Aspergillus sp. This nucleotide is substantially absent.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in detail, this invention is not limited to these. In addition, the general experiment method followed the experiment book (Sambrook, J., Molecular Cloning: A Laboratory Manual 3rd edition). In the following examples, all PCRs were performed using LA-Taq (Takara Holdings). In the following Examples, OSI 1013 strain was used as Aspergillus oryzae.

  In addition, in the following examples, the AO number described accompanying each protease is GOGAN [microorganism genome database sequenced at Genome Analysis Center of nite (National Institute of Technology and Evaluation) (http: / /www.bio.nite.go.jp/dogan/Top), which means a gene encoding a protein derived from Aspergillus oryzae.

Example 1 Construction of an Expression Vector Cassette for Filamentous Fungi (1) Preparation of Selection Marker Gene (SEQ ID NO: 21) Amplification was performed by PCR under the following conditions using the genomic DNA of Aspergillus oryzae as a template and the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and a selection marker gene fragment was excised using a QIAquick Gel Extraction kit (QIAGEN).

<Primer>
Primer 1: 5'-gcgtggttta ctagctttag tgctaccaaa-3 '(SEQ ID NO: 22)
Primer 2: 5'-ccgtacgcggggagtgtgcttaaggcgatg-3 '(SEQ ID NO: 23)
<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C 5 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(2) Preparation of terminator (SEQ ID NO: 24) Amplification was performed by PCR using Aspergillus oryzae genomic DNA as a template and the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and a terminator fragment was excised using a QIAquick Gel Extraction kit (QIAGEN).

<Primer>
Primer 3: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTC-3 '(SEQ ID NO: 25)
Primer 4: 5'-CTGCAGATCGGCTGAAGTTAGGAGCGGCCATTGTC-3 '(SEQ ID NO: 26)
<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (1 minute) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(3) Preparation of sodM promoter (SEQ ID NO: 27) Amplification was carried out by PCR under the following conditions using the following primers with genomic DNA of Aspergillus oryzae as a template. The obtained PCR amplification product was subjected to agarose gel electrophoresis, the obtained PCR amplification product was subjected to agarose gel electrophoresis, and the sodM promoter fragment was excised using a QIAquick Gel Extraction kit (QIAGEN).

<Primer>
Primer 5: 5'-CTGCAGTTATGTACTCCGTACTCGGTTGAATTAT-3 '(SEQ ID NO: 28)
Primer 6: 5'-TTTGGGTGGTTTGGTTGGTATTCTGGTTGAGGGTTTTGAG-3 '(SEQ ID NO: 29)
<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (1 minute) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(4) Preparation of glaA promoter (SEQ ID NO: 30) Amplified by PCR under the following conditions using aspergillus oryzae genomic DNA as a template and the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, the obtained PCR amplification product was subjected to agarose gel electrophoresis, and the glaA promoter fragment was excised using a QIAquick Gel Extraction kit (QIAGEN).

<Primer>
Primer 7: 5'-GAATTCTGTA GCTGCTCTAT TTCTATTACT-3 '(SEQ ID NO: 31)
Primer 8: 5'-CTTGCTTCGACTTCGTTTGCTGATGTG-3 '(SEQ ID NO: 32)
<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (1 minute) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(5) Preparation of Expression Vector Cassette for Filamentous Fungi An expression vector for Aspergillus was prepared as follows using pUC118 (Takara Bio, Japan) as a basic plasmid.

  The selective marker gene (SEQ ID NO: 21) prepared above was blunt-ended at the SmaI site of pUC118 with Blunting High (Toyobo, Japan) and subcloned with DNA Ligation Kit ver. 1 (Takara Bio, Japan). This plasmid was designated as pUCL1.

  Next, a terminator prepared as described above and blunt-ended by Blunting High (Toyobo, Japan) was subcloned into pUCL1. Specifically, PCR was performed under the following conditions using primer 2 (SEQ ID NO: 23) and primer 9 (5′-ggggatcctctagagtcgacctgca-3 ′ (SEQ ID NO: 33)) using pUCL1 as a template. The amplified product was subjected to agarose gel electrophoresis, and the target fragment was excised using QIAquick Gel Extraction kit (QIAGEN) to prepare a vector.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (7 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

  The obtained vector and the terminator fragment prepared above were subcloned using DNA Ligation Kit ver. 1 (Takara Bio, Japan), and the resulting plasmid was designated as pUCLT1.

  Next, a promoter (sodM promoter, glaA promoter) was subcloned into pUCLT1 using the following method.

(A) Subcloning of sodM promoter
The sodM promoter (SEQ ID NO: 27) prepared above was subcloned into pUCLT1. Specifically, PCR was performed using primer 2 (SEQ ID NO: 23) and primer 3 (SEQ ID NO: 25) using pUCLT1 as a template, and the obtained PCR amplification product was subjected to agarose gel electrophoresis, and QIAquick Gel Extraction The target fragment was excised using a kit (QIAGEN) to prepare a vector.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (8 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

  The above vector and sodM promoter were subcloned with DNA Ligation Kit ver. 1 (Takara Bio, Japan), and the resulting plasmid was designated as pUCLTS1.

(B) Subcloning of glaA promoter
The glaA promoter (SEQ ID NO: 30) prepared above was subcloned into pUCLT1. Specifically, PCR was performed using primer 2 (SEQ ID NO: 23) and primer 3 (SEQ ID NO: 25) using pUCLT1 as a template, and the obtained PCR amplification product was subjected to agarose gel electrophoresis, and QIAquick Gel Extraction The target fragment was excised using a kit (QIAGEN) to prepare a vector.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (8 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

  The above vector and the glaA promoter were subcloned using DNA Ligation Kit ver. 1 (Takara Bio, Japan), and the resulting plasmid was designated as pUCLTA1.

Example 2 Preparation of an expression vector for filamentous fungi and expression of alkaline protease by self-cloning Self-cloning using Aspergillus oryzae alkaline protease (AO090003001036) (for convenience, this is referred to as “protease 1”) It was expressed and produced by the method.

(1) Preparation of alkaline protease gene The amino acid sequence of alkaline protease is shown in SEQ ID NO: 11, and the base sequence of the alkaline protease gene encoding it is shown in SEQ ID NO: 1. This alkaline protease gene was prepared by the following method.

  PCR was carried out using Aspergillus oryzae genomic DNA as a template using the following primers, and the resulting PCR amplification product was subjected to agarose gel electrophoresis, and using QIAquick Gel Extraction kit (QIAGEN), SEQ ID NO: 1 A fragment consisting of the base sequence described in (1) was excised to prepare an alkaline protease gene.

<Primer>
Primer 10: 5′-ATGCAGTCCATCAAGCGTACCTTGCTCCTCCTCGG-3 ′ (SEQ ID NO: 34)
Primer 11: 5'-TTAAGCGTTACCGTTGTAGGCAAGCAGGTT-3 '(SEQ ID NO: 35)
<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (2 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(2) Preparation of Expression Vector for Filamentous Fungi (2-1) Plasmid Vector First, PCR was performed using the above pUCLT1 as a template and using primer 3 (SEQ ID NO: 25) and primer 6 (SEQ ID NO: 29), and the resulting PCR The amplification product was subjected to agarose gel electrophoresis, the target fragment was excised using QIAquick Gel Extraction kit (QIAGEN), and this was used as a vector.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (9 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

  The obtained vector and the alkaline protease gene prepared above were subcloned with DNA Ligation Kit ver. 1 (Takara Bio, Japan). The plasmid was designated as a plasmid vector (IE-232).

(2-2) Self-cloning vector Using the plasmid vector (IE-232) obtained above as a template, PCR was performed using primer 1 (SEQ ID NO: 22) and primer 4 (SEQ ID NO: 26), and the resulting PCR The amplification product was purified using QIAquick Gel Extraction kit (QIAGEN), and this was used as a self-cloning vector.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (9 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(3) Preparation of transformant The plasmid vector (IE-232) or self-cloning vector (IE-) prepared above according to the standard method PEG-calcium method (Mol Gen Genet, 218, 99-104, (1989)) 232) were used to transform leucine-requiring mutant strains. The leucine-requiring mutant gonococcal strain is a strain obtained by mutating a gene encoding leucine of Aspergillus oryzae so that normal leucine cannot be synthesized, and a selection marker gene that complements leucine-requiring requirement is incorporated. Otherwise, it usually cannot grow on media without leucine. The leucine-requiring mutant strain is deposited as Aspergillus oryzae leu-5 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, which has an address at Tsukuba Center Center 6-1, Tsukuba City, Ibaraki Prefecture. (Deposit number FERM P-20079).

  The transformed strain was then transformed into a Czapek-Dox medium (2% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate, 0.001) using nitric acid as the single nitrogen source. Multiple transformants for each plasmid vector (IE-232) and self-cloning vector (IE-232) by selecting strains that can grow on this medium. It was.

(3-1) Transformation efficiency The transformation efficiency of plasmid vector (IE-232) was 1 ± 0.5 / μg-DNA, while that of self-cloning vector was 8.6 ± 3.4. / Μg-DNA. From this, it was confirmed that when a self-cloning vector (IE-232) was used, the transformation efficiency was increased by about 8 times compared to a vector that was not.

(3-2) Presence or absence of gene transfer Presence or absence of gene transfer was examined by Southern analysis.

  Genomic DNA of each transformant using a self-cloning vector (IE-232) and a plasmid vector (IE-232) was prepared by a conventional method, digested with EcoRI, subjected to agarose electrophoresis, and Hybond N + membrane (Amersham Pharmacia). ). Next, Southern analysis was performed with Gene Image kit (Amersham Pharmacia) using the selection marker gene (SEQ ID NO: 21) as a probe (leuA probe).

  The results are shown in FIG. As can be seen from FIG. 1, 1 copy of the transformant of the plasmid vector (IE-232) and 1 to 3 copies of the transformant of the self-cloning vector (IE-232) compared to the host strain (Host strain). Gene transfer was observed. From this, it was confirmed that a gene can be introduced into Aspergillus using a self-cloning vector consisting only of a gene derived from Aspergillus oryzae.

(3-3) Presence / absence of E. coli-derived sequences Next, Southern analysis was used to confirm whether the obtained transformants had E. coli-derived sequences.

  First, genomic DNA of each transformant using a self-cloning vector (IE-232) and a plasmid vector (IE-232) was digested with EcoRI, subjected to agarose electrophoresis, and transferred to a Hybond N + membrane (Amersham Pharmacia). Next, Southern analysis was performed with Gene Image kit (Amersham Pharmacia) using the pUC118 gene (Takara Bio, Japan) as a probe (pUC probe).

  The results are shown in FIG. As can be seen from FIG. 2, the plasmid vector (IE-232) transformant showed E. coli-derived sequences, whereas the self-cloning vector (IE-232) transformant showed E. coli-derived sequences. I was not able to admit. From this, it was confirmed that the said transformant is a self-cloning strain | stump | stock by which a koji mold is transformed only with the gene derived from a koji mold.

(4) Production of alkaline protease Each of the transformants prepared above was sporulated in a potato dextrose medium, and at 30 ° C. for 3 days, a GPY liquid medium (2% glucose, 1% peptone, 0.5% yeast extract, 0.1% The cells were cultured in 1 potassium dihydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate, 0.001% iron sulfate, and 0.3% sodium nitrate). Next, 100 μl of the culture supernatant was concentrated and dried and subjected to SDS-PAGE. In addition, as a control test, the plasmid pUCLTS1 prepared in Example 1 was used and cultured in the same manner using a strain obtained by transforming a Neisseria gonorrhoeae leucine-requiring mutant, and subjected to SDS-PAGE.

  The results are shown in FIG.

  As shown in FIG. 3, in the self-cloning strain (IE-232), a large amount of alkaline protease bands (indicated by → in the figure) that were not observed in the control strain were detected. Some of the self-cloning strains exceeded the production of alkaline protease by the plasmid vector transformant.

  Next, protease activity was measured for the culture supernatant.

  The protease activity was measured by mixing 400 μl of a 1.25% azocasein solution dissolved in 50 mM Tris buffer (pH 8.0) and 100 μl of culture supernatant, reacting at 37 ° C. for 1 hour, and then adding 1 ml of 10% trichloroacetic acid. After vigorous mixing, the mixture was centrifuged at 19000 × g for 5 minutes, and the absorbance (335 nm) of the supernatant was measured. As a blank value, add 1 ml of 10% trichloroacetic acid to 100 μl of culture supernatant, add 400 μl of 1.25% azocasein solution dissolved in 50 mM Tris buffer (pH 8.0), mix vigorously and then centrifuge at 19000 × g for 5 minutes. After separation, the value obtained by measuring the absorbance (335 nm) of the supernatant was used.

  The absorbance of the control strain minus the blank value (OD335) was not detected, whereas the absorbance of the self-cloning strain minus the blank value (OD335) was 2.87 at maximum. From this, it was confirmed that the self-cloning strain (IE-232) is a strain that expresses and produces alkaline protease and secretes it outside the cell body (medium supernatant).

Example 3 Preparation of an expression vector for filamentous fungi using sodM promoter and self-cloning expression of various protease genes Six types of proteases (proteases 2 to 7) derived from koji molds were expressed by a self-cloning method using koji molds. Produced (using sodM promoter).

(1) Preparation of protease genes Six types of protease genes shown in Table 2 were amplified under the following PCR conditions using Aspergillus oryzae genomic DNA as a template and two primers shown in the table. Prepared. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target fragment was excised using a QIAquick Gel Extraction kit (QIAGEN) to obtain a protease gene.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (2 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(2) Preparation of an expression vector for filamentous fungi (2-1) Plasmid vector First, pUCLTS1 was used as a template, and PCR was performed using primer 3 (SEQ ID NO: 25) and primer 6 (SEQ ID NO: 29) under the following conditions. The PCR amplification product was subjected to agarose gel electrophoresis, the target fragment was excised using a QIAquick Gel Extraction kit (QIAGEN), and this was used as a vector.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (9 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

  The vector thus prepared and each of the protease genes prepared above were subcloned using DNA Ligation Kit ver. 1 (Takara Bio, Japan) to prepare six types of plasmids (plasmid vectors) incorporating the respective protease genes. Hereinafter, these plasmid vectors are referred to as follows corresponding to each protease.

(2-2) Self-cloning vector Using the above six types of plasmids (IE233, IE292, IE338, IE339, IE340, IE341) as templates, respectively, using primer 1 (SEQ ID NO: 22) and primer 4 (SEQ ID NO: 26), PCR was performed under the following conditions to amplify only the gonococcal gene portion. The obtained PCR amplification product was purified using QIAquick Gel Extraction kit (QIAGEN), and this was used as a self-cloning vector (IE233, IE292, IE338, IE339, IE340, IE341).

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (9 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(3) Preparation of transformant According to the standard method of PEG-calcium, leucine-requiring mutant strains (Aspergillus oryzae leu-5 (deposit number: FERM P-20079)) using each of the self-cloning vectors prepared above Was transformed. This is cultured in a Czapek-Dox medium containing nitric acid as a single nitrogen source, and 10 transformants are obtained for each self-cloning vector by selecting strains that can grow on this medium. did. All these transformants were subjected to Southern analysis, and all were confirmed to be self-cloning strains that do not contain E. coli-derived genes.

(4) Protease production Each of the transformants prepared above was sporulated in a potato dextrose medium, and at 30 ° C. for 3 days, a GPY liquid medium (2% glucose, 1% peptone, 0.5% yeast extract, 0.1% phosphorus) 1 potassium hydrogen acid, 0.05% potassium chloride, 0.05% magnesium sulfate, 0.001% iron sulfate, 0.3% sodium nitrate). Next, 100 μl of the culture supernatant was concentrated and dried and subjected to SDS-PAGE. As a control test, pUCLTS1 prepared in Example 1 was used to culture in the same manner using a strain obtained by transforming a gonococcal leucine-requiring mutant, 100 μl of the culture supernatant was concentrated and dried, and then subjected to SDS-PAGE. Provided (control strain). As a result, no protease was confirmed outside the cell (culture supernatant) in the control strain, whereas protease was produced outside the cell (culture supernatant) in any of the transformants (self-cloning strain). I was able to confirm clearly.

  The results of measuring the protease production and protease activity of each self-cloning strain (IE233, IE292, IE338, IE339, IE340, IE341) are as follows.

(4-1) Self-cloning strain (IE-233)
(a) Protease Production FIG. 4 shows the result of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE together with the result of the control strain. As shown in FIG. 4, in the self-cloning strain (IE-233), a large amount of protease bands not found in the control strain were detected.

(b) Measurement of protease activity Protease activity was measured for the culture supernatant. The measurement was performed by mixing 400 μl of a 1.25% azocasein solution dissolved in 50 mM Tris buffer (pH 7.0) and 100 μl of culture supernatant, reacting at 37 ° C. for 1 hour, and then adding 1 ml of 10% trichloroacetic acid. Mixed. Subsequently, centrifugation was performed at 19000 × g for 5 minutes, and the absorbance (335 nm) of the supernatant was measured. As a blank value, add 1 ml of 10% trichloroacetic acid to 100 μl of culture supernatant, add 400 μl of 1.25% azocasein solution dissolved in 50 mM Tris buffer (pH 7.0), mix vigorously, and then centrifuge at 19000 × g for 5 minutes. After separation, the value obtained by measuring the absorbance (335 nm) of the supernatant was used. The absorbance (OD335) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance (OD335) of the culture supernatant of the self-cloning strain minus the blank value was 3.42 at the maximum. Met.

(4-2) Self-cloning strain (IE-292)
(a) Protease production FIG. 5 shows the result of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE together with the result of the control strain. As shown in FIG. 5, in the self-cloning strain (IE-292), a large amount of protease bands not found in the control strain were detected.

(b) Measurement of protease activity Protease activity was measured for the culture supernatant. Measurement was performed in the same manner as the self-cloning strain (IE-233) except that 50 mM acetate buffer (pH 5.0) was used instead of 50 mM Tris buffer (pH 7.0). As a result, the absorbance (OD335) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance (OD335) of the culture supernatant of the self-cloning strain minus the blank value was The maximum was 1.22.

(4-3) Self-cloning strain (IE-338)
(a) Protease production FIG. 6 shows the results of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE. As shown in FIG. 6, a large amount of bands were detected in the self-cloning strain (IE-338). On the other hand, no corresponding band was detected in the control strain (results not shown).

(b) Measurement of protease activity Protease activity was measured for the culture supernatant. The measurement was performed using an “acid carboxypeptidase measurement kit” which is a Kikkoman brewing analysis kit. This principle yields L-alanine when the substrate carbobenzoxy-L-tyrosyl L-alanine is degraded by acid carboxypeptidase. The produced L-alanine is specifically decomposed by adding alanine dehydrogenase in the presence of NAD + to produce NADH. Therefore, the generated NADH is colored with a tetrazolium salt, PMS, and quantified by absorbance (460 nm).

  Protease activity was measured on 100 μl of culture supernatant. As a blank value, a value measured in the same manner after adding a reaction stop solution before the reaction with the substrate carbobenzoxy-L-tyrosyl L-alanine was used. The absorbance (OD460) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance (OD460) of the self-cloning strain (IE-338) minus the blank value was the maximum. It was 0.69.

(4-4) Self-cloning strain (IE-339)
(a) Protease production FIG. 6 shows the results of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE. As shown in FIG. 6, a large amount of bands were detected in the self-cloning strain (IE-339). On the other hand, no corresponding band was detected in the control strain (results not shown).

(b) Measurement of protease activity Protease activity was measured on 100 μl of culture supernatant. The measurement was performed in the same manner as the self-cloning strain (IE-338) using an “acid carboxypeptidase measurement kit” which is a Kikkoman brewing analysis kit. The absorbance (OD460) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance (OD460) of the self-cloning strain (IE-339) minus the blank value was the maximum. 0.89.

(4-5) Self-cloning strain (IE-340)
(a) Protease production FIG. 6 shows the results of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE. As shown in FIG. 6, a large amount of bands were detected in the self-cloning strain (IE-340). On the other hand, no corresponding band was detected in the control strain (results not shown).

(b) Measurement of protease activity Protease activity was measured on 100 μl of culture supernatant. The measurement was performed in the same manner as the self-cloning strain (IE-338) using an “acid carboxypeptidase measurement kit” which is a Kikkoman brewing analysis kit. The absorbance (OD460) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance (OD460) of the self-cloning strain (IE340) minus the blank value was 0.89 at maximum. there were.

(4-6) Self-cloning strain (IE-341)
(a) Protease production FIG. 6 shows the results of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE. As shown in FIG. 6, a large amount of bands were detected in the self-cloning strain (IE-341). On the other hand, no corresponding band was detected in the control strain (results not shown).

(b) Measurement of protease activity Protease activity was measured on 100 μl of culture supernatant. The measurement was performed in the same manner as the self-cloning strain (IE-338) using an “acid carboxypeptidase measurement kit” which is a Kikkoman brewing analysis kit. The absorbance (OD460) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance (OD460) of the self-cloning strain (IE341) minus the blank value was 0.76 at maximum. there were.

Example 4 Preparation of an expression vector for filamentous fungi using glaA promoter and self-cloning expression of various protease genes Three types of proteases derived from Aspergillus (proteases 8 to 10) were expressed by a self-cloning method using Aspergillus. Produced (using glaA promoter (SEQ ID NO: 30)).

(1) Preparation of protease gene The three types of protease genes shown in Table 4 were amplified under the following PCR conditions using Aspergillus oryzae genomic DNA as a template and two primers shown in the table. Prepared. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target fragment was excised using a QIAquick Gel Extraction kit (QIAGEN) to obtain a protease gene.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (2 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(2) Preparation of an expression vector for filamentous fungi (2-1) Plasmid vector First, pUCLTA1 was used as a template, and PCR was performed using primer 3 (SEQ ID NO: 25) and primer 8 (SEQ ID NO: 32) under the following conditions. The PCR amplification product was subjected to agarose gel electrophoresis, the target fragment was excised using a QIAquick Gel Extraction kit (QIAGEN), and this was used as a vector.

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (9 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

  The vector thus prepared and each of the protease genes prepared above were subcloned using DNA Ligation Kit ver. 1 (Takara Bio, Japan) to prepare three types of plasmids (plasmid vectors) incorporating the respective protease genes. Hereinafter, these plasmid vectors are referred to as follows corresponding to each protease.

(2-2) Self-cloning vector Using the above three types of plasmids (IE326, IE327, IE328) as templates, PCR was performed using primer 1 (SEQ ID NO: 22) and primer 4 (primer 26) under the following conditions. Only the gene part of Aspergillus was amplified. The obtained PCR amplification product was purified using QIAquick Gel Extraction kit (QIAGEN), and this was used as a self-cloning vector (IE-326, IE-327, IE-328).

<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 60 ° C (30 seconds), 72 ° C (9 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle.

(3) Preparation of transformant According to the standard method of PEG-calcium, leucine-requiring mutant strains (Aspergillus oryzae leu-5 (deposit number: FERM P-20079)) using each of the self-cloning vectors prepared above Was transformed. This is cultured in a Czapek-Dox medium containing nitric acid as a single nitrogen source, and 10 transformants are obtained for each self-cloning vector by selecting strains that can grow on this medium. did. All these transformants were subjected to Southern analysis, and all were confirmed to be self-cloning strains that do not contain E. coli-derived genes.

(4) Protease production Each of the transformants prepared above was sporulated in a potato dextrose medium, and at 30 ° C. for 3 days, a GPY liquid medium (2% glucose, 1% peptone, 0.5% yeast extract, 0.1% phosphorus) 1 potassium hydrogen acid, 0.05% potassium chloride, 0.05% magnesium sulfate, 0.001% iron sulfate, 0.3% sodium nitrate). Next, 100 μl of the culture supernatant was concentrated and dried and subjected to SDS-PAGE. As a control test, pUCLTS1 prepared in Example 1 was used to culture in the same manner using a strain obtained by transforming a gonococcal leucine-requiring mutant, 100 μl of the culture supernatant was concentrated and dried, and then subjected to SDS-PAGE. Provided (control strain). As a result, no protease was confirmed outside the cell (culture supernatant) in the control strain, whereas protease was produced outside the cell (culture supernatant) in any of the transformants (self-cloning strain). I was able to confirm clearly.

  The results of measuring protease production and protease activity of each self-cloning strain (IE-326, IE-327, IE-328) are as follows.

(4-1) Self-cloning strain (IE-326)
(a) Protease Production FIG. 7 shows the result of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE together with the result of the control strain. As shown in FIG. 7, in the self-cloning strain (IE-326), a large amount of protease bands not found in the control strain were detected.

(b) Measurement of protease activity Protease activity was measured for the culture supernatant. The measurement was performed by mixing 400 μl of a 1.25% azocasein solution dissolved in 50 mM acetate buffer (pH 3.0) and 100 μl of culture supernatant, reacting at 37 ° C. for 1 hour, and then adding 1 ml of 10% trichloroacetic acid. Mixed. Subsequently, centrifugation was performed at 19000 × g for 5 minutes, and the absorbance (335 nm) of the supernatant was measured. As blank values, add 1 ml of 10% trichloroacetic acid to 100 μl of culture supernatant, add 400 μl of 1.25% azocasein solution dissolved in 50 mM acetate buffer (pH 3.0), mix vigorously, and then centrifuge at 19000 × g for 5 minutes. After separation, the value obtained by measuring the absorbance (335 nm) of the supernatant was used. The absorbance (OD335) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance of the culture supernatant of the self-cloning strain (IE-326) minus the blank value (OD335) The maximum was 0.84.

(4-2) Self-cloning strain (IE-327)
(a) Protease Production FIG. 7 shows the result of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE together with the result of the control strain. As shown in FIG. 7, in the self-cloning strain (IE-327), a large amount of protease bands not found in the control strain were detected.

(b) Measurement of protease activity Protease activity was measured for the culture supernatant. The measurement was performed in the same manner as the above self-cloning strain (IE-326) except that 50 mM Tris buffer (pH 7.0) was used instead of 50 mM acetate buffer (pH 3.0). As a result, the absorbance (OD335) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance of the culture supernatant of the self-cloning strain (IE-327) minus the blank value. (OD335) was 0.45 at the maximum.

(4-3) Self-cloning strain (IE-328)
(a) Protease production FIG. 7 shows the results of concentrating and drying 100 μl of the culture supernatant obtained by culturing by the above method and subjecting it to SDS-PAGE. As shown in FIG. 7, in the self-cloning strain (IE-328), a large amount of protease bands not found in the control strain were detected.

(b) Measurement of protease activity Protease activity was measured for the culture supernatant. Similar to the self-cloning strain (IE-327), the measurement was performed using the self-cloning strain (IE-327) except that 50 mM Tris buffer (pH 7.0) was used instead of 50 mM acetate buffer (pH 3.0). 326). As a result, the absorbance (OD335) of the culture supernatant of the control strain minus the blank value was not detected, whereas the absorbance of the culture supernatant of the self-cloning strain (IE-328) minus the blank value. (OD335) was 0.63 at the maximum.

For the EcoRI digest of genomic DNA of each transformant using a self-cloning vector (IE-232) and a plasmid vector (IE-232), Southern analysis was performed using the selectable marker gene (SEQ ID NO: 21) as a probe (leuA probe). The results are shown (Example 2). The result of Southern analysis using the pUC probe for the EcoRI digest of the genomic DNA of each transformant using the self-cloning vector (IE-232) and plasmid vector (IE-232) is shown. (Example 2). The results of subjecting each transformant of the self-cloning vector (IE-232) and plasmid vector (IE-232) and the culture supernatant of the control strain to SDS-PAGE are shown (Example 2). The result of having applied the transformant by a self-cloning vector (IE-233) and the culture supernatant of a control strain to SDS-PAGE is shown (Example 3). The results of subjecting the transformant by the self-cloning vector (IE-292) and the culture supernatant of the control strain to SDS-PAGE are shown (Example 3). The result of having applied the culture supernatant of the transformant by a self-cloning vector (IE-338, IE-339, IE-340, IE-341) to SDS-PAGE is shown (Example 3). The result of having applied the culture supernatant of the transformant by a self-cloning vector (IE-326, IE-327, IE-328) and a control strain to SDS-PAGE is shown (Example 4).

SEQ ID NOs: 22, 23, 25, 26, 28, 29, and 31 to 53 show the nucleotide sequences of the primers 1 to 29 used for PCR amplification in the examples.
SEQ ID NO: 24 shows the base sequence of the terminator, SEQ ID NO: 27 shows the base sequence of the sodM promoter, and SEQ ID NO: 30 shows the base sequence of the glaA promoter.

Claims (3)

A selectable marker gene sequence derived from Aspergillus oryzae , (1) any one selected from the group consisting of a promoter sequence consisting of the base sequence set forth in SEQ ID NO: 27 and a base sequence set forth in SEQ ID NOS: 1-7 A protease gene consisting of a base sequence or (2) consisting of any one base sequence selected from the group consisting of a promoter sequence consisting of the base sequence set forth in SEQ ID NO: 30 and a base sequence set forth in SEQ ID NOs: 8-10 An expression vector for a filamentous fungus having a protease gene and a terminator sequence derived from Aspergillus oryzae ,
An expression vector for filamentous fungi, which is a self-cloning vector consisting essentially of nucleotides derived from filamentous fungi and is linear. A filamentous fungus transformed with the expression vector for filamentous fungi according to claim 1. A transformed filamentous fungus according to claim 2 is cultured in a medium, and a protease comprising any one amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 11 to 20 from the medium is obtained. A method for producing a protease, comprising a step of collecting.

Images