JP5759132B2 - Method for producing filamentous fungal peptidase - Google Patents

Description

  The present invention relates to a method for producing a filamentous fungal peptidase, and a gene expression cassette for filamentous fungi, an expression vector for filamentous fungi, and a transformant useful for carrying out the method.

  Peptidase is used in various applications and fields such as detergents for clothes, cosmetics, bath preparations, food modifiers, digestive aids or anti-inflammatory agents, and is the most produced enzyme product. 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 with high nutrient source, Peptidases 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 that are turbid in foods. Peptidases used in the food industry are often derived from microorganisms, and many are from the genus Aspergillus, Bacillus, and Rhizopus. In particular, many peptidases derived from Aspergillus oryzae (Aspergillus oryzae) can cope with a wide range of pH and have various types of molecular species depending on the culture conditions such as liquid culture and solid culture. Since it can provide peptidase enzyme, it is most widely used in the food industry.

  In particular, lactotripeptides such as VPP and IPP derived from milk proteins that have blood pressure lowering activity can be produced advantageously with peptidase enzymes derived from koji molds. Therefore, peptidases derived from koji molds are also very useful for the production of peptides as functional food materials. (Non-patent Document 1).

  However, in the production of functional peptides, the present peptidase enzyme agent derived from Aspergillus oryzae has a weak point. Specifically, such an enzyme agent contains a plurality of peptidases whose characteristics are unknown because it is a culture extract of Aspergillus oryzae. Therefore, when it is allowed to act on food proteins, the target functional peptide is excessive. It is 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 peptidase enzyme agent with a clear molecular species can be provided by filamentous fungi, particularly koji molds, which have been used for the production of brewed foods such as sake and soy sauce since long ago. However, since peptidase has cytotoxicity, it has been considered difficult to introduce a gene so that peptidase is highly expressed in cells, that is, to successfully express a peptidase with a known molecular species in filamentous fungi. Moreover, the substrate specificity of peptidase cannot be inferred from its primary structure, and it is predicted that the cytotoxicity is different for each molecule, and there is a difficulty in devising a high expression system for each molecule.

  Recently, it has been reported that 10 kinds of peptidases derived from Aspergillus were expressed and produced by a self-cloning method using Aspergillus oryzae (Patent Document 1).

JP 2010-4760

British Journal of Nutrition, 94, 84-91 (2005)

  However, Patent Document 1 only describes that 10 types of peptidases were produced using koji molds, and no other peptidase production was disclosed.

  Therefore, the present invention produces a peptidase derived from a filamentous fungus and having a known molecular species other than the peptidase whose production has been reported in Patent Document 1, using a filamentous fungus as a host, particularly by a self-cloning method. It aims to provide a method. Another object of the present invention is to provide materials for use in producing the peptidase, that is, a gene expression cassette for filamentous fungi, an expression vector for filamentous fungi, and a transformant.

  As a result of repeated studies to solve the above-mentioned problems, the present inventors combined a peptidase derived from a filamentous fungus having the amino acid sequence described in any of SEQ ID NOs: 16 to 25 with a specific promoter, thereby forming a filamentous shape. The inventor has found that it can be efficiently produced using a self-cloning method using bacteria as a host. The present invention has been completed based on these findings and has been completed. The present invention provides the following gene expression cassette for filamentous fungi, expression vector for filamentous fungus, filamentous fungus, and peptidase production method.

(I) Gene expression cassette for filamentous fungi
(I-1) A gene for a filamentous fungus having a promoter comprising the following DNA (a) or (b), a peptidase gene comprising the following DNA (c) or (d), and a terminator that functions in the filamentous fungus body Expression cassette:
(A) DNA comprising any one base sequence selected from the group consisting of base sequences described in SEQ ID NOs: 1 to 5
(B) hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to a DNA consisting of any one base sequence selected from the group consisting of the base sequences set forth in SEQ ID NOs: 1 to 5; DNA that functions as a promoter in filamentous fungi
(C) DNA comprising any one base sequence selected from the group consisting of base sequences described in SEQ ID NOs: 6 to 15
(D) hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of any one base sequence selected from the group consisting of base sequences set forth in SEQ ID NOs: 6 to 15; And a DNA encoding a protein having peptidase activity.

(I-2) The promoter is composed of the following DNA (a1) or (b1), and the peptidase gene is composed of the following DNA (c1) or (d1): (I-1) Gene expression cassette for filamentous fungi as described in:
(A1) DNA comprising any one base sequence selected from the group consisting of the base sequences described in SEQ ID NOS: 1 and 2
(B1) hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising any one base sequence selected from the group consisting of the base sequences set forth in SEQ ID NOs: 1 and 2, DNA that functions as a promoter in filamentous fungi
(C1) DNA comprising the base sequence set forth in SEQ ID NO: 6
(D1) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 6 and encodes a protein having peptidase activity.

(I-3) The promoter is composed of the following DNA (a2) or (b2), and the peptidase gene is composed of the following DNA (c2) or (d2), (I-1) Gene expression cassette for filamentous fungi as described in:
(A2) DNA comprising the base sequence set forth in SEQ ID NO: 3
(B2) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 3 and functions as a promoter in filamentous fungi
(C2) DNA comprising any one base sequence selected from the group consisting of the base sequences set forth in SEQ ID NOs: 6, 7, 8, 12, 13 and 14
(D2) DNA and stringent which are complementary to the DNA consisting of any one of the base sequences selected from the group consisting of the base sequences described in SEQ ID NOs: 6, 7, 8, 12, 13 and 14. DNA encoding a protein that hybridizes under mild conditions and has peptidase activity.

(I-4) The promoter is composed of the following DNA (a3) or (b3), and the peptidase gene is composed of the following DNA (c3) or (d3), (I-1) Gene expression cassette for filamentous fungi as described in:
(A3) DNA comprising the base sequence set forth in SEQ ID NO: 4
(B3) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence described in SEQ ID NO: 4 and functions as a promoter in filamentous fungi
(C3) DNA comprising any one base sequence selected from the group consisting of base sequences described in SEQ ID NOs: 6 to 11
(D3) hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of any one base sequence selected from the group consisting of base sequences set forth in SEQ ID NOs: 6 to 11, And a DNA encoding a protein having peptidase activity.

(I-5) The promoter is composed of the following DNA (a4) or (b4), and the peptidase gene is composed of the following DNA (c4) or (d4), (I-1) Gene expression cassette for filamentous fungi as described in:
(A4) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 5
(B4) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 5 and functions as a promoter in filamentous fungi
(C4) DNA comprising the base sequence set forth in SEQ ID NO: 15
(D4) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 15 and encodes a protein having peptidase activity.

(II) Expression vector for filamentous fungi
(II-1) An expression vector for filamentous fungi comprising a selection marker gene that functions in filamentous fungi and the gene expression cassette according to any one of (I-1) to (I-5).

  (II-2) The expression vector for filamentous fungi according to (II-1), which is a self-cloning vector consisting essentially of nucleotides derived from filamentous fungi.

  (II-3) The expression vector for filamentous fungi according to (II-1) or (II-2), which is linear.

  (II-4) The expression vector for filamentous fungi according to any one of (II-1) to (II-3), wherein the filamentous fungus is a filamentous fungus belonging to the genus Aspergillus.

(III) Transformant
(III-1) A filamentous fungus obtained by transformation with the expression vector for filamentous fungi according to any one of (II-1) to (II-3).

  (III-2) The filamentous fungus according to (III-1), which is a filamentous fungus belonging to the genus Aspergillus.

(IV) Peptidase production method
(IV-1) A method for producing a peptidase, comprising a step of culturing the filamentous fungus according to (III-1) in a medium and recovering the peptidase from the medium.

  According to the gene expression cassette for filamentous fungi and the production method of the present invention, a peptidase derived from a filamentous fungus with an apparent molecular species can be efficiently produced using the filamentous fungus as a host. For this reason, according to the method of the present invention, a desired peptidase having a clear molecular species can be industrially produced.

It is a graph which shows the result of having measured the enzyme activity of the culture supernatant after liquid culture | cultivation for 30 hours at 30 degreeC and MPP for the peptidase 1 production strain. It is a figure which shows the result of having performed the SDS-PAGE of the culture-medium supernatant after liquid culture | cultivation for 30 degreeC and the peptidase 1 production strain for 64 hours. 1: Control, 2 and 3: PsodM, 4 and 5: PamyA, 6: PglaA, 7 and 8: Phly, 1-8: MPY It is a graph which shows the result of having measured the enzyme activity of the culture-medium supernatant after liquid culture | cultivation for 30 hours, GPC or MPY, and peptidase 2 production strain. It is a figure which shows the result of having performed the SDS-PAGE of the culture-medium supernatant after liquid culture | cultivation for 30 degreeC and 64 hours by GPY or MPY for a peptidase 2 production strain. 1: Control, 2: PsodM, 3 and 4: PamyA, 5 and 6: PglaA, 7 and 8: Phly, 1, 2, 7, and 8: GPY, 3-6: MPY It is a graph which shows the result of having measured the enzyme activity of a culture-medium supernatant or a microbial cell disruption liquid after 30-degree C, 40 or 64 hours, and liquid culture of peptidase 3 production strain. It is a figure which shows the result of having performed the SDS-PAGE of a culture-medium supernatant or a cell disruption liquid after liquid culture for 30 degreeC, 40 or 64 hours by GPY or MPY for a peptidase 3 production strain. 1: Marker, 2: PsodM 40 hr, 3: PsodM 64 hr, 4: PamyA 40 hr, 5: PamyA 64 hr, 6: PglaA 40 hr, 7: PglaA 64 hr, 8: Phly 40 hr, 9: Phly 64 hr, 10: control 40 hr, 11: control 64 hr. It is a graph which shows the result of having measured the enzyme activity of a culture-medium supernatant or a microbial cell lysate after liquid culture | cultivation of peptidase 4 production strain by 30 degreeC and 64 hours. It is a figure which shows the result of having performed the SDS-PAGE of the culture-medium supernatant after liquid culture | cultivation of the peptidase 4 production strain by 30 degreeC and 64 hours. 1: Marker, 2: Control, 3, 4: Peptidase 4 production strain After peptidase 5 production strain is cultured in wheat bran culture (30 ° C., 40 hours), GPY liquid culture (3 ml, 30 ° C., 40 hours or 10 days), or culture in a medium supplemented with 1 mM CoCl ₂ , wheat FIG. 5 is a graph showing the results of measurement of enzyme activity in the bran culture only outside the cells, and in the liquid culture in the cell cultures. After peptidase 6 production strain is subjected to wheat bran culture (30 ° C, 40 hours) or GPY liquid culture (3 ml, 30 ° C, 40 hours or 10 days), wheat bran culture is only outside the cells, liquid culture is It is a graph which shows the result of having measured the enzyme activity inside and outside a microbial cell. After peptidase 5 production strain and peptidase 6 production strain are subjected to liquid culture (30 ° C, 150 rpm, 5 or 10 days) with 100 ml GPY (peptidase 5 is +1 ml CoCl ₂ ), SDS- of the culture supernatant It is a figure which shows the result of having performed PAGE. 1: Marker, 2 and 3: Control, 4 and 5: Peptidase 5 producing strain, 6 and 7: Control, 8 and 9: Peptidase 6 producing strain, 2, 4, 6 and 8: 5 days of culture, 3, 5, 7 and 9: 10 days culture It is a graph which shows the result of having measured the enzyme activity of the culture supernatant after liquid culture (3 ml, 30 degreeC, 40, 64, or 88 hours) of peptidase 7 production strains. It is a figure which shows the result of having performed the liquid culture (100 ml, 30 degreeC, 150 rpm, 88 hours) of the peptidase 7 production strain, and performing SDS-PAGE of the culture supernatant. 1: Marker 2: Control 3: Peptidase 7 production strain It is a figure which shows the result of having performed the SDS-PAGE of the culture-medium supernatant, after performing GPY liquid culture (3 ml, 30 degreeC, 200 rpm, 40 hours) of the peptidase 8 production strain and the peptidase 9 production strain. 1: Marker 2: Control 3: Peptidase 8 producing bacteria 4 and 5: Peptidase 9 producing bacteria It is a figure which shows the result of having performed the liquid culture (5 ml, 30 degreeC, 250 rpm, 3 days) of the peptidase 10 production strain by SDS-PAGE of the culture-medium supernatant. M: Marker, 1: Control, 2-8: Peptidase 10 producing bacterium

  Hereinafter, embodiments of the present invention will be described in detail.

  Examples of the filamentous fungi targeted by the present invention include koji molds and incomplete filamentous fungi to which koji molds belong. Among them, filamentous fungi belonging to the genus Aspergillus, Mucor, Candida, Trichoderma, and Neurospora, more preferably 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 Aspergillus nidulans and the like can be mentioned. Of these, Aspergillus oryzae is preferred because it is a safe microorganism that has been used for brewed foods such as sake, soy sauce, miso, and mirin for many years, and is a frequently used host in Japanese industries.

(1) Gene expression cassette for filamentous fungi The gene expression cassette for filamentous fungi of the present invention is a promoter comprising the following DNA (a) or (b), and a peptidase gene comprising the following (c) or (d) DNA: And having a terminator that functions in filamentous fungi:
(A) DNA comprising any one base sequence selected from the group consisting of base sequences described in SEQ ID NOs: 1 to 5
(B) hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to a DNA consisting of any one base sequence selected from the group consisting of the base sequences set forth in SEQ ID NOs: 1 to 5; DNA that functions as a promoter in filamentous fungi
(C) DNA comprising any one base sequence selected from the group consisting of base sequences described in SEQ ID NOs: 6 to 15
(D) hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of any one base sequence selected from the group consisting of base sequences set forth in SEQ ID NOs: 6 to 15; And a DNA encoding a protein having peptidase activity.

  In the present specification, the promoter composed of the DNA of (b) and the peptidase gene composed of the DNA of (d) are sometimes referred to as variants.

(1-1) Filamentous Peptidase The peptidase targeted by the present invention is a peptidase derived from a filamentous fungus having any amino acid sequence described in SEQ ID NOs: 16 to 26, particularly a koji mold or a variant thereof. Among these peptidases, peptidases represented by SEQ ID NOs: 16 to 21 can be classified as exopeptidases, and peptidases represented by SEQ ID NOs: 22 to 25 can be classified as endopeptidases.

  The correspondence between the amino acid sequence of each of these peptidases and the base sequence of the gene encoding them (peptidase gene) is shown in the following table:

  In addition, the peptidase gene in the present invention is stringent under conditions that are stringent with DNA consisting of a base sequence complementary to DNA consisting of any one base sequence selected from the group consisting of base sequences set forth in SEQ ID NOs: 6-15. It may be composed of DNA that hybridizes underneath and encodes a protein having peptidase activity.

  Here, the above-mentioned stringent conditions are, for example, hybridization at 5 ° C. in a 5 × SSC solution (composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), and It means the condition of washing at 65 ° C. with 0.5 × SSC solution containing 0.1% SDS. Each operation of hybridization under stringent conditions is performed by a conventionally known method such as the method described in "Molecular Cloning (Third Edition)" (J. Sambrook & DW Russell, Cold Spring Harbor Laboratory Press, 2001). Can be done. Generally, the higher the temperature and the lower the salt concentration, the higher the stringency.

  DNA that hybridizes under stringent conditions usually has a certain level of identity with the base sequence of DNA used as a probe, and the identity is, for example, 70% or more, preferably 80% or more, and more preferably Is 90% or more, particularly preferably 95% or more. The identity of the base sequence can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). The identity (%) of the base sequence can be determined using a program commonly used in the art (for example, BLAST, FASTA, etc.) by default.

  Such a modified peptidase gene can be produced according to a conventional method.

(1-3) Promoter The promoter is a promoter derived from a filamentous fungus having any one of the base sequences described in SEQ ID NOs: 1 to 5, particularly an Aspergillus spp. The promoters shown in these SEQ ID NOs: 1 to 5 are SEQ ID NO: 1: α-amylase gene promoter amyA (Patent No. 3005618), SEQ ID NO: 2: glucoamylase gene promoter glaA (Gene, 108, 145-150, (1992). ), SEQ ID NO: 3: Manganese SOD gene promoter sodM (Japanese Patent Laid-Open No. 2000-224381), SEQ ID NO: 4: hly (Japanese Patent Laid-Open No. 2009-296958), SEQ ID NO: 5: Peptidylprolyl cis-trans isomerase gene promoter pdiA ( JP 2002-320477 A).

  In addition, the promoter in the present invention is a stringent condition with a DNA comprising a base sequence complementary to a DNA comprising any one base sequence selected from the group consisting of the base sequences set forth in SEQ ID NOs: 1-5. It may be composed of DNA that hybridizes with and functions as a promoter in filamentous fungi.

  The stringent conditions here are the same as described above. In addition, such a modified promoter can be produced according to a conventional method.

  The position of the promoter in the gene expression cassette is not particularly limited as long as the promoter functions in the filamentous fungus so that a desired peptidase can be expressed and produced, but it is usually located upstream of the peptidase 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) Gene expression cassette for filamentous fungi Preferred combinations of peptidase genes and promoters in the gene expression cassette for filamentous fungi of the present invention are shown below.
-The peptidase gene having the base sequence described in SEQ ID NO: 6 and the modified form thereof are preferably the promoter having the base sequence described in SEQ ID NOs: 1 to 4 and the modified form thereof, and the base sequences described in SEQ ID NOs: 1 and 2 A promoter having a nucleotide sequence set forth in SEQ ID NO: 1 and a variant thereof are particularly preferred.
-The peptidase gene having the base sequence described in SEQ ID NO: 7 and the modified form thereof are preferably the promoter having the base sequence described in SEQ ID NOs: 3 and 4 and the modified form thereof, and the promoter having the base sequence described in SEQ ID NO: 3. And its variants are particularly preferred.
-The peptidase gene having the base sequence described in SEQ ID NO: 8 and the modified form thereof are preferably the promoter having the base sequence described in SEQ ID NOs: 1, 3 and 4 and the modified form thereof, and the bases described in SEQ ID NOs: 3 and 4 A promoter having a sequence and a variant thereof are more preferred, and a promoter having the base sequence described in SEQ ID NO: 3 and a variant thereof are more preferred.
-The peptidase gene having the base sequence described in SEQ ID NOs: 9 to 11 and its variant are preferably the promoter having the base sequence described in SEQ ID NO: 4 and its variant.
-The peptidase gene having the base sequence described in SEQ ID NOs: 12 to 14 and the modified form thereof are preferably the promoter having the base sequence described in SEQ ID NO: 3 and the modified form thereof.
-The peptidase gene having the base sequence described in SEQ ID NO: 15 and its modified form are preferably the promoter having the base sequence described in SEQ ID NO: 5 and its modified form.

  In the gene expression cassette for filamentous fungi of the present invention, these promoter, peptidase gene, and terminator may be directly linked, and a nucleotide of about 1 to 2000 bases is sandwiched between them. May be. However, these nucleotide sequences are preferably derived from filamentous fungi, and preferably from Aspergillus spp. For example, when the target peptidase is produced as an intracellular protein, a known secreted protein of a filamentous fungus, preferably an Aspergillus filamentous fungus, upstream, adjacent to the open reading frame of the gene of the target peptidase Thus, the target peptidase is expressed and produced as a fusion protein with the secretory protein, and the thus produced fusion protein 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.

  In addition, as secretory proteins of Aspergillus filamentous fungi, 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.

(2) Expression vector for filamentous fungi The expression vector for filamentous fungi of the present invention has a selection marker gene that functions in filamentous fungi and the gene expression cassette.

(2-1) Selectable marker gene The selectable marker gene may be any one that functions as a selectable marker in filamentous fungi, particularly Aspergillus spp., That is, can be expressed, but is preferably a filamentous fungus, particularly Aspergillus spp. 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 are obtained by performing PCR using primers designed based on the sequences described in the above-mentioned literature and using chromosomal DNA of Aspergillus filamentous fungi as a template, and on the basis of 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 the 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 capable of complementing the leucine-requiring gene 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.

(2-2) Expression vector for filamentous fungi The expression vector for filamentous fungi of the present invention may be one in which these selectable marker genes and gene expression cassettes are directly linked, with 1 between them. A nucleotide of about ˜2000 bases may be inserted. However, these nucleotide sequences are preferably derived from filamentous fungi, and preferably from Aspergillus spp.

  The present invention includes both linear filamentous fungus expression vectors and circular filamentous fungus expression vectors. (1) A circular expression vector such that it can be obtained from the constructed plasmid by one-step PCR. (2) When a leucine-requiring mutant is used 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.

(3) 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. Aspergillus filamentous fungi such as Soya, Aspergillus tamari, Aspergillus glaucus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus tereus, 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 strain in which a gene responsible for methionine biosynthesis is mutated or deleted so that synthesis cannot be performed: histidine in which a gene responsible for histidine biosynthesis is mutated or deleted so that histidine biosynthesis cannot be performed normally Requirement mutant: niaD mutant, in which the nitrate-assimilating gene is mutated or deleted so that nitrate cannot be assimilated normally: There can be mentioned, such as sC mutants nitrate assimilation genes are deleted so as not to be assimilated.

  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.

(4) Peptidase Production Method The peptidase production method of the present invention includes a step of culturing the above-described filamentous fungus transformant of the present invention and a step of recovering the target protein from the culture.

  The culture 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, maltose, 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 diphosphate, 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), 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). 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 peptidase (SEQ ID NOs: 16 to 25) can also be produced outside the cells as shown in the Examples. For this reason, the culture supernatant may be recovered when obtaining the peptidase. 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 peptidase can be isolated and recovered by subjecting these supernatants to various chromatographic methods such as known protein purification methods such as ion exchange, hydrophobicity, gel filtration, and affinity.

(5) Self-cloning vector and self-cloning strain In the present invention, preferable filamentous fungal expression vectors are derived from a selection marker gene derived from a filamentous fungus, a promoter derived from a filamentous fungus, a peptidase 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 peptidase 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 or a function as 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 expression vector for filamentous fungi described above, the target peptidase gene in the expression vector for filamentous fungus is obtained 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 peptidase 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 PrimeSTAR (registered trademark) HS DNA Polymerase (Takara Bio Inc.). In the following examples, OSI 1013 strain was used as Aspergillus oryzae.

  In the examples, ten types of peptidases (peptidases 1 to 10) shown in the following table were used.

Method for Measuring Exopeptidase Activity A solution having the following composition was reacted at 37 ° C., and the absorbance at 405 nm was measured every reaction time (5 minutes interval up to 20 minutes). The enzyme activity that liberates 1 μmol of pNA (p-nitroanilide) per minute was defined as 1 unit.

20 mM substrate / sH ₂ O 5 μl (final concentration 0.5 mM)
10 μl of 1M buffer (final concentration 50 mM)
Enzyme ^* + sH ₂ O 185 μl
200 μl total
^* Culture supernatant or cell disruption solution

The substrate and the buffer shown in the following table were used according to the type of enzyme to be measured.

Endopeptidase Activity Measurement Method A solution having the following composition was reacted at 37 ° C. for 1 hour, 1 ml of 10% (w / v) TCA was added, vortexed, centrifuged, and the absorbance of the supernatant at 325 nm was measured. The enzyme activity that increases the absorbance by 1 per minute was defined as 1 unit.

2% Azocasein / sH ₂ O 250μl (final concentration 1%)
1M buffer 50μl (final concentration 100mM)
Enzyme ^* + sH ₂ O 200 μl
500 μl total
^* Culture supernatant

The buffers shown in the following table were used according to the type of enzyme to be measured.

Example 1 Construction of Expression Vector Cassette for Filamentous Fungus and Transformant Plasmid vector IE-232 described in Patent Document 1 (JP 2010-4760) was used to construct the following plasmid vector.

(1) Construction of peptidase 1 expression plasmid
(A) Construction of peptidase 1 expression plasmid under the control of sodM promoter (SEQ ID NO: 3)
Using the plasmid DNA of IE-232 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 1: 5'-TTTTGGGTGGTTTGGTTGGTATTCTGGTTGA-3 '(SEQ ID NO: 26)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 1 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 3: 5'-ACCAAACCACCCAAAATGAAGGTACGTCAATTCCACTGAT-3 '(SEQ ID NO: 28)
Primer 4: 5'-CACTGGAAAGTACATCTACTCCTCCAAGTCCTTCTTAGTC-3 '(SEQ ID NO: 29)
The 5 'terminal 15 base of primer 3 is a sequence homologous to the 3' terminal of the sodM promoter (SEQ ID NO: 3), and the 5 'terminal 15 base of primer 4 is a sequence homologous to the 5' terminal of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (2 minutes and 30 seconds) The above two PCR amplification products are converted into In-Fusion ^™ Advantage PCR via a homologous region of 15 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated IE-300.

(B) Construction of peptidase 1 expression plasmid under the control of amyA promoter (SEQ ID NO: 1)
Using the plasmid DNA of IE-300 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 6: 5′-ATGAAGGTACGTCAATTCCACTGAT -3 ′ (SEQ ID NO: 32)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes 30 seconds) amyA by PCR under the following conditions using Aspergillus oryzae genomic DNA as a template and the following primers: The promoter (SEQ ID NO: 1) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 7: 5′-ACACTCCCCGCGTACGGTACGCCTCCGGGTAGTAG -3 ′ (SEQ ID NO: 33)
Primer 8: 5′-AATTGACGTACCTTCATAAATGCCTTCTGTGGGGT-3 ′ (SEQ ID NO: 34)
The 5 ′ terminal 17 base of primer 7 is a sequence homologous to the 3 ′ terminal of the leuA marker (SEQ ID NO: 35), and the 5 ′ terminal 17 base of primer 8 is a sequence homologous to the 5 ′ terminal of the peptidase 1 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBaAiP014.

(C) Construction of peptidase 1 expression plasmid under the control of glaA promoter (SEQ ID NO: 2)
Using the plasmid DNA of IE-300 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 6: 5′-ATGAAGGTACGTCAATTCCACTGAT -3 ′ (SEQ ID NO: 32)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes and 30 seconds) glaA by PCR using the genomic DNA of Aspergillus oryzae as a template and the following primers: The promoter (SEQ ID NO: 2) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 9: 5′-ACACTCCCCGCGTACGGGAATTCTGTAGCTGCTCT-3 ′ (SEQ ID NO: 36)
Primer 10: 5'-TTGACGTACCTTCATCTTGCTTCGACTTCGTTTGCTGATG-3 '(SEQ ID NO: 37)
The 5 'terminal 17 base of primer 9 is a sequence homologous to the 3' terminal of the leuA marker (SEQ ID NO: 35), and the 5 'terminal 15 base of primer 10 is a sequence homologous to the 5' terminal of the peptidase 1 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are passed through the homologous region at each end, and the In-Fusion ^™ Advantage PCR Cloning Kit (Clontech) was used for fusion, and the resulting plasmid was designated IE-324.

(D) Construction of peptidase 1 expression plasmid under the control of hly promoter (SEQ ID NO: 4)
Using the plasmid DNA of IE-300 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 6: 5′-ATGAAGGTACGTCAATTCCACTGAT -3 ′ (SEQ ID NO: 32)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes and 30 seconds) Using the genomic DNA of Aspergillus oryzae as a template and the following primers, The promoter (SEQ ID NO: 4) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 11: 5′-ACACTCCCCGCGTACGGCTACAGCATGGTCTGGAT-3 ′ (SEQ ID NO: 38)
Primer 12: 5′-AATTGACGTACCTTCATGGTGTTGTGGTGTGAAGG-3 ′ (SEQ ID NO: 39)
The 5 ′ terminal 17 base of primer 11 is a sequence homologous to the 3 ′ terminal of the leuA marker (SEQ ID NO: 35), and the 5 ′ terminal 17 base of primer 12 is a sequence homologous to the 5 ′ terminal of the peptidase 1 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBhiP014.

(2) Construction of peptidase 2 expression plasmid
(A) Construction of peptidase 2 expression plasmid under the control of sodM promoter (SEQ ID NO: 3)
Using the plasmid DNA of IE-232 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 1: 5'-TTTTGGGTGGTTTGGTTGGTATTCTGGTTGA-3 '(SEQ ID NO: 26)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 2 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 13: 5'-TACCAACCAAACCACCCAAAATGAAGTCGGTACTCAACAT-3 '(SEQ ID NO: 40)
Primer 14: 5′-ACACGCACTGGAAAGTACATTCACGGGTTATAATCCACAA-3 ′ (SEQ ID NO: 41)
The 5 'end 20 bases of primer 13 are homologous to the 3' end of the sodM promoter (SEQ ID NO: 3), and the 5 'end 20 bases of primer 14 are homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (3 minutes) The above two PCR amplification products are passed through a homologous region of 20 bases at each end, and the In-Fusion ^™ Advantage PCR Cloning Kit Fusion was performed using (Clontech), and the resulting plasmid was designated as pBsP002.

(B) Construction of peptidase 2 expression plasmid under the control of amyA promoter (SEQ ID NO: 1)
Using the plasmid DNA of pBsP002 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 15: 5′-ATGAAGTCGGTACTCAACATGGGAG-3 ′ (SEQ ID NO: 42)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes 30 seconds) amyA by PCR under the following conditions using Aspergillus oryzae genomic DNA as a template and the following primers: The promoter (SEQ ID NO: 1) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 7: 5'-ACACTCCCCGCGTACGGTACGCCTCCGGGTAGTAG-3 '(SEQ ID NO: 33)
Primer 16: 5'-TTGAGTACCGACTTCATAAATGCCTTCTGTGGGGT-3 '(SEQ ID NO: 43)
The 5 'terminal 17 base of primer 7 is a sequence homologous to the 3' terminal of the leuA marker (SEQ ID NO: 35), and the 5 'terminal 17 base of primer 16 is a sequence homologous to the 5' terminal of the peptidase 2 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBaAP002.

(C) Construction of peptidase 2 expression plasmid under the control of glaA promoter (SEQ ID NO: 2)
Using the plasmid DNA of pBsP002 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 15: 5′-ATGAAGTCGGTACTCAACATGGGAG-3 ′ (SEQ ID NO: 42)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes and 30 seconds) glaA by PCR using the genomic DNA of Aspergillus oryzae as a template and the following primers: The promoter (SEQ ID NO: 2) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 9: 5'-ACACTCCCCGCGTACGGGAATTCTGTAGCTGCTCT-3 '(SEQ ID NO: 36)
Primer 17: 5'-TTGAGTACCGACTTCATCTTGCTTCGACTTCGTTT-3 '(SEQ ID NO: 44)
The 5 ′ terminal 17 bases of primer 9 are homologous to the 3 ′ terminal of the leuA marker (SEQ ID NO: 35), and the 5 ′ terminal 17 bases of primer 17 are homologous to the 5 ′ terminal of the peptidase 2 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBgAP002.

(D) Construction of peptidase 2 expression plasmid under the control of hly promoter (SEQ ID NO: 4)
Using the plasmid DNA of pBsP002 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 15: 5′-ATGAAGTCGGTACTCAACATGGGAG-3 ′ (SEQ ID NO: 42)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes and 30 seconds) Using the genomic DNA of Aspergillus oryzae as a template and the following primers, The promoter (SEQ ID NO: 4) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 11: 5′-ACACTCCCCGCGTACGGCTACAGCATGGTCTGGAT-3 ′ (SEQ ID NO: 38)
Primer 18: 5'-TTGAGTACCGACTTCATGGTGTTGTGGTGTGAAGG-3 '(SEQ ID NO: 45)
The 5 ′ terminal 17 bases of primer 11 are homologous to the 3 ′ terminal of the leuA marker (SEQ ID NO: 35), and the 5 ′ terminal 17 bases of primer 18 are homologous to the 5 ′ terminal of the peptidase 2 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBhP002.

(3) Construction of peptidase 3 expression plasmid
(A) Construction of peptidase 3 expression plasmid under the control of sodM promoter (SEQ ID NO: 3)
Using the plasmid DNA of IE-232 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 1: 5'-TTTTGGGTGGTTTGGTTGGTATTCTGGTTGA-3 '(SEQ ID NO: 26)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C (10 seconds), 58 ° C (5 seconds), 72 ° C (9 minutes) Peptidase 3 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 19: 5′-TACCAACCAAACCACCCAAAATGACCATTCGTCCAATGAA-3 ′ (SEQ ID NO: 46)
Primer 20: 5'-ACACGCACTGGAAAGTACATTTATAGAGACATATTAGCCA-3 '(SEQ ID NO: 47)
The 5 'end 20 bases of primer 19 is a sequence homologous to the 3' end of the sodM promoter (SEQ ID NO: 3), and the 5 'end 20 bases of primer 20 is a sequence homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (2 minutes 30 seconds) In-Fusion ^™ Advantage PCR Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBsP003.

(B) Construction of peptidase 3 expression plasmid under the control of amyA promoter (SEQ ID NO: 1)
Using the plasmid DNA of pBsP003 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 21: 5′-ATGACCATTCGTCCAATGAAGTTCA-3 ′ (SEQ ID NO: 48)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes 30 seconds) amyA by PCR under the following conditions using Aspergillus oryzae genomic DNA as a template and the following primers: The promoter (SEQ ID NO: 1) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 7: 5′-ACACTCCCCGCGTACGGTACGCCTCCGGGTAGTAG -3 ′ (SEQ ID NO: 33)
Primer 22: 5′-ATTGGACGAATGGTCATAAATGCCTTCTGTGGGGT-3 ′ (SEQ ID NO: 49)
The 5 ′ terminal 17 bases of primer 7 are homologous to the 3 ′ terminal of the leuA marker (SEQ ID NO: 35), and the 5 ′ terminal 17 bases of primer 22 are homologous to the 5 ′ terminal of the peptidase 3 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBaAP003.

(C) Construction of peptidase 3 expression plasmid under the control of glaA promoter (SEQ ID NO: 2)
Using the plasmid DNA of pBsP003 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5'-CCGTACGCGGGGAGTGTGCTTAAGG-3 '(SEQ ID NO: 31)
Primer 21: 5′-ATGACCATTCGTCCAATGAAGTTCA-3 ′ (SEQ ID NO: 48)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes and 30 seconds) glaA by PCR using the genomic DNA of Aspergillus oryzae as a template and the following primers: The promoter (SEQ ID NO: 2) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 9: 5′-ACACTCCCCGCGTACGGGAATTCTGTAGCTGCTCT-3 ′ (SEQ ID NO: 36)
Primer 23: 5'-ATTGGACGAATGGTCATCTTGCTTCGACTTCGTTT-3 '(SEQ ID NO: 50)
The 5 ′ terminal 17 bases of primer 9 are homologous to the 3 ′ terminal of the leuA marker (SEQ ID NO: 35), and the 5 ′ terminal 17 bases of primer 23 are homologous to the 5 ′ terminal of the peptidase 3 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBgAP003.

(D) Construction of peptidase 3 expression plasmid under the control of hly promoter (SEQ ID NO: 4)
Using the plasmid DNA of pBsP003 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 5: 5′-CCGTACGCGGGGAGTGTGCTTAAGG-3 ′ (SEQ ID NO: 31)
Primer 21: 5′-ATGACCATTCGTCCAATGAAGTTCA-3 ′ (SEQ ID NO: 48)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (10 minutes and 30 seconds) Using the genomic DNA of Aspergillus oryzae as a template and the following primers, The promoter (SEQ ID NO: 4) was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 11: 5′-ACACTCCCCGCGTACGGCTACAGCATGGTCTGGAT-3 ′ (SEQ ID NO: 38)
Primer 24: 5′-ATTGGACGAATGGTCATGGTGTTGTGGTGTGAAGG-3 ′ (SEQ ID NO: 51)
The 5 ′ terminal 17 bases of primer 11 are homologous to the 3 ′ terminal of the leuA marker (SEQ ID NO: 35), and the 5 ′ terminal 17 bases of primer 24 are homologous to the 5 ′ terminal of the peptidase 3 gene.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 17 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBhP003.

(4) Construction of peptidase 7 expression plasmid
Using the plasmid DNA of IE-232 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 1: 5'-TTTTGGGTGGTTTGGTTGGTATTCTGGTTGA-3 '(SEQ ID NO: 26)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 7 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 25: 5'-TACCAACCAAACCACCCAAAATGCATATGCTCTTATTTAT-3 '(SEQ ID NO: 52)
Primer 26: 5'-ACACGCACTGGAAAGTACATTTAGATCTTCTTCTGGCACA-3 '(SEQ ID NO: 53)
The 5 'end 20 bases of primer 25 is a sequence homologous to the 3' end of the sodM promoter (SEQ ID NO: 3), and the 5 'end 20 bases of primer 26 is a sequence homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (2 minutes 30 seconds) In-Fusion ^™ Advantage PCR Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBsP004.

(5) Construction of peptidase 9 expression plasmid
Using the plasmid DNA of IE-232 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 1: 5'-TTTTGGGTGGTTTGGTTGGTATTCTGGTTGA-3 '(SEQ ID NO: 26)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 9 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 27: 5'-TACCAACCAAACCACCCAAAATGCGGGGCGCATCTCTCCT-3 '(SEQ ID NO: 54)
Primer 28: 5′-ACACGCACTGGAAAGTACATTTACAAAGCAAGAAGAAGAC-3 ′ (SEQ ID NO: 55)
The 5 'end 20 bases of primer 27 is a sequence homologous to the 3' end of the sodM promoter (SEQ ID NO: 3), and the 5 'end 20 bases of primer 28 is a sequence homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
98 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (2 minutes) 30 cycles of the above two PCR amplification products via a homologous region of 20 bases at each end, In-Fusion ^™ Advantage PCR Cloning Kit Fusion was performed using (Clontech), and the resulting plasmid was designated as pBsiP020.

(6) Construction of peptidase 8 expression plasmid
Using the plasmid DNA of IE-232 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 1: 5'-TTTTGGGTGGTTTGGTTGGTATTCTGGTTGA-3 '(SEQ ID NO: 26)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 8 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 29: 5'-TACCAACCAAACCACCCAAAATGAGATTTCTTCTGTCTTT-3 '(SEQ ID NO: 56)
Primer 30: 5'-ACACGCACTGGAAAGTACATCTATTTGGGGGATGCAACTC-3 '(SEQ ID NO: 57)
The 5 'end 20 bases of primer 29 is a sequence homologous to the 3' end of the sodM promoter (SEQ ID NO: 3), and the 5 'end 20 bases of primer 30 is a sequence homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 20 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBsP005.

(7) Construction of peptidase 4 expression plasmid
Using the plasmid DNA of pBhiP014 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 31: 5'-GGTGTTGTGGTGTGAAGGGTGATTGATGTG-3 '(SEQ ID NO: 58)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 4 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 32: 5'-ACCCTTCACACCACAACACCATGCGTTTCCTCCCCTGCAT-3 '(SEQ ID NO: 59)
Primer 33: 5'-ACACGCACTGGAAAGTACATTTACAGCGAATCTGCGAAGG-3 '(SEQ ID NO: 60)
The 5 'end 20 bases of primer 32 is a sequence homologous to the 3' end of the hly promoter (SEQ ID NO: 4), and the 5 'end 20 bases of primer 33 is a sequence homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (1 minute 30 seconds) The above two PCR amplification products are subjected to In-Fusion ^™ Advantage PCR via a homologous region of 20 bases at each end. Fusion was performed using Cloning Kit (Clontech), and the resulting plasmid was designated as pBhP006.

(8) Construction of peptidase 6 expression plasmid
Using the plasmid DNA of pBhiP014 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 31: 5'-GGTGTTGTGGTGTGAAGGGTGATTGATGTG-3 '(SEQ ID NO: 58)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 6 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 34: 5′-ACCCTTCACACCACAACACCATGGCTGCCAAACTAGTAGA-3 ′ (SEQ ID NO: 61)
Primer 35: 5′-ACACGCACTGGAAAGTACATCTAATCAATAGAGTCGTCCC-3 ′ (SEQ ID NO: 62)
The 5 'end 20 bases of primer 34 is a sequence homologous to the 3' end of the hly promoter (SEQ ID NO: 4), and the 5 'end 20 bases of primer 35 is a sequence homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
98 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (2 minutes) 30 cycles of the above two PCR amplification products via a homologous region of 20 bases at each end, In-Fusion ^™ Advantage PCR Cloning Kit Fusion was performed using (Clontech), and the resulting plasmid was designated as pBhiP021.

(9) Construction of peptidase 5 expression plasmid
Using the plasmid DNA of pBhiP014 as a template, the vector side was amplified by PCR under the following conditions using the following primers. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 31: 5'-GGTGTTGTGGTGTGAAGGGTGATTGATGTG-3 '(SEQ ID NO: 58)
Primer 2: 5'-ATGTACTTTCCAGTGCGTGTAGTCTACTCT-3 '(SEQ ID NO: 27)
<PCR conditions>
30 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (9 minutes) Peptidase 5 gene by PCR using Aspergillus oryzae genomic DNA as a template and the following primers: Was amplified. The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was excised and purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
<Primer>
Primer 36: 5'-ACCCTTCACACCACAACACCATGACTTCGAAAATCGCCCA-3 '(SEQ ID NO: 63)
Primer 37: 5'-ACACGCACTGGAAAGTACATTCAGTCAACAAAGATTGTCT-3 '(SEQ ID NO: 64)
The 5 'end 20 bases of primer 36 is a sequence homologous to the 3' end of the hly promoter (SEQ ID NO: 4), and the 5 'end 20 bases of primer 37 is a sequence homologous to the 5' end of the glaB terminator (SEQ ID NO: 30). It is.
<PCR conditions>
98 cycles of 98 ° C. (10 seconds), 58 ° C. (5 seconds), 72 ° C. (2 minutes) 30 cycles of the above two PCR amplification products via a homologous region of 20 bases at each end, In-Fusion ^™ Advantage PCR Cloning Kit Fusion was performed using (Clontech), and the resulting plasmid was designated as pBhiP007.

(10) Acquisition of various peptidase high-producing transformants Using each plasmid DNA constructed above as a template, DNA fragments to be introduced into koji molds were amplified by PCR under the following conditions using the following primers. As a control, the leuA marker was amplified by PCR under the following conditions using primer DNA 38 and primer 5 using IE-232 plasmid DNA as a template. The obtained PCR amplification product was purified and ethanol precipitated using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega). Aspergillus was transformed with the obtained DNA fragment.
<Primer>
Primer 38: 5′-GCGTGGTTTACTAGCTTTAGTGCTA-3 ′ (SEQ ID NO: 65)
Primer 39: 5′-ATCGGCTGAAGTTAGGAGCGGCCATTG-3 ′ (SEQ ID NO: 66)
<PCR conditions>
30 cycles of 98 ° C (10 seconds), 58 ° C (5 seconds), 72 ° C (8 minutes 30 seconds)

(11) According to the PEG-calcium method (Mol Gen Genet, 218, 99-104, (1989)), which is a standard method for preparing transformants , each of the leucine-requiring mutant strains using the self-cloning vectors prepared above. Was transformed. The leucine-requiring mutant strain is a strain that mutates a gene encoding leucine of Aspergillus oryzae so that normal leucine cannot be synthesized. If not incorporated, it usually cannot grow on media without leucine. The leucine-requiring mutant strain was 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 Central 6th, 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. % Ferrous sulfate, 0.3% sodium nitrate), and a transformant was obtained for each self-cloning vector by selecting a strain capable of growing on the medium.

Example 2 Production of Peptidase 1 by Self-Cloning Peptidase 1 (assumed molecular weight 86.9 kDa) derived from Aspergillus oryzae was expressed and produced by a self-cloning method using Aspergillus.

  Each transformant prepared above was cultured in MPY liquid medium (2% maltose, 1% peptone, 0.5% yeast extract) at 30 ° C. for 64 hours. After the culture, enzyme activity measurement and SDS-PAGE of the culture supernatant were performed. The result of enzyme activity measurement is shown in FIG. 1, and the result of SDS-PAGE is shown in FIG. Expression was observed with the sodM, amyA, glaA, and hly promoters, and the most secreted production was confirmed, particularly when the amyA promoter was used.

Example 3 Production of Peptidase 2 by Self-Cloning Peptidase 2 (assumed molecular weight 81.1 kDa) derived from Aspergillus oryzae was expressed and produced by a self-cloning method using Aspergillus.

  Each transformant prepared above is cultured in GPY liquid medium (2% glucose, 1% peptone, 0.5% yeast extract) (PsodM, Phly) or MPY liquid medium (PamyA, PglaA) at 30 ° C. for 64 hours. did. After the culture, enzyme activity measurement and SDS-PAGE of the culture supernatant were performed. The results of enzyme activity measurement are shown in FIG. 3, and the results of SDS-PAGE are shown in FIG. Expression was confirmed with the sodM and hly promoters, and the most secretory production was confirmed with the use of the sodM promoter.

Example 4 Production of Peptidase 3 by Self-Cloning Peptidase 3 (assumed molecular weight 78.9 kDa) derived from Aspergillus oryzae was expressed and produced by a self-cloning method using Aspergillus.

  Each transformant prepared above was cultured in GPY liquid medium or MPY liquid medium at 30 ° C. for 40 or 64 hours. After the culture, enzyme activity measurement and SDS-PAGE of the culture supernatant and the cell disruption solution were performed. The results of enzyme activity measurement are shown in FIG. 5, and the results of SDS-PAGE are shown in FIG. By using sodM and hly promoters, the production of peptidase 3 in the microbial cells can also be confirmed by culturing for 64 hours, and the production of peptidase 3 outside the microbial cells can be confirmed, especially when the sodM promoter is used. Production was confirmed.

Example 5 Self-cloning expression of various peptidase genes using hly promoter Three types of peptidases (peptidase 4, peptidase 5 and peptidase 6) derived from Aspergillus oryzae were expressed by a self-cloning method using Aspergillus oryzae. Produced (using hly promoter).

(1) Self-cloning strain (peptidase 4)
The peptidase 4 production strain prepared above was cultured in GPY liquid medium at 30 ° C. for 64 hours. After the culture, enzyme activity measurement and SDS-PAGE of the culture supernatant were performed. The result of enzyme activity measurement is shown in FIG. 7, and the result of SDS-PAGE is shown in FIG. By using the hly promoter, peptidase 4 expression could be confirmed outside and inside the cells, and in particular, a significant amount of productivity could be confirmed inside the cells.

(2) Self-cloning strain (peptidase 5)
The peptidase 5 production strain prepared above is cultured in wheat bran culture (30 ° C., 40 hours), GPY liquid culture (3 ml, 30 ° C., 40 hours or 10 days), or a medium supplemented with 1 mM CoCl _2. Was cultured. After cultivation, enzyme activity was measured only outside the cells in wheat bran culture and outside the cells in liquid culture. The results of enzyme activity measurement are shown in FIG. In the liquid medium using GPY, a significant amount of production was confirmed in the microbial cells, and the addition of cobalt chloride to the medium increased the production in the microbial cells 3-4 times. In addition, we succeeded in the secretion expression outside the cells by solid culture of wheat bran and liquid culture for 10 days.

(3) Self-cloning strain (peptidase 6)
Peptidase 6 production prepared above was subjected to wheat bran culture (30 ° C., 40 hours) or GPY liquid culture (3 ml, 30 ° C., 40 hours or 10 days). After cultivation, enzyme activity was measured only outside the cells in wheat bran culture and outside the cells in liquid culture. The results of enzyme activity measurement are shown in FIG. In a liquid medium using GPY, a significant amount of production can be confirmed in the fungus body, and further, by increasing the culture time from 40 hours to 10 days, the production amount in the fungus body increases by about 30%, Secretory production outside the cells was also confirmed. In addition, the solid expression of wheat bran succeeded in the expression of secretion outside the cells.

(4) Self-cloning strain (100 ml culture of peptidase 5 and peptidase 6)
Conidia were scraped with a microplate or the like from the plates of the peptidase 5 production strain and the peptidase 6 production strain prepared above and inoculated into a 3 ml GPY medium. After pre-culture at 30 ° C, 200 rpm for 24 hours, transfer the culture to a 100 ml GPY (peptidase 5 is +1 ml CoCl ₂ ) / 300 ml Erlenmeyer flask and incubate at 30 ° C, 150 rpm for 5 or 10 days. went. The culture supernatant was filtered with a 0.45 μm filter, and enzyme activity measurement and 150 μl concentration were performed, followed by SDS-PAGE. The results of enzyme activity measurement are shown in Tables 5 and 6, and the results of SDS-PAGE are shown in FIG.

  As can be seen from Table 5, regarding peptidase 5, the secretory production amount to the outside of the cells increased about 4-fold by changing the culture time from 5 days to 10 days. Furthermore, from Table 6, regarding peptidase 6, the secretory production amount outside the cells increased about 6 times by changing the culture time from 5 days to 10 days. Further, from FIG. 11, it has been clarified that the production amount in both the peptidase 5 and the peptidase 6 is significantly increased by changing the culture time from 5 days to 10 days. From this result, it was confirmed that peptidase 5 and peptidase 6 could be produced outside the cells in 10 days of culture even when the culture was scaled up from 3 ml to 100 ml.

Example 6 Self-cloning expression of various peptidase genes using sodM promoter Three types of peptidases derived from Aspergillus oryzae (peptidase 7, peptidase 8 and peptidase 9) were expressed by a self-cloning method using Aspergillus oryzae. Produced (using sodM-promoter).

(1) Self-cloning strain (peptidase 7)
The peptidase 7 production strain prepared above was subjected to GPY liquid culture (3 ml, 30 ° C., 40, 64, or 88 hours). After the culture, the enzyme activity of the culture supernatant was measured. The result of enzyme activity measurement is shown in FIG. As a result, a significant amount of secretory production was confirmed outside the cells by using the sodM promoter. Furthermore, by increasing the culture time to 88 hours, the production volume was increased more than 20 times compared to 24 hours.

(2) Self-cloning strain (100 ml culture of peptidase 7)
Conidia were scraped off from the plate of the peptidase 7 production strain prepared above with a microplate or the like and inoculated into 3 ml GPY medium. After preculture at 30 ° C., 200 rpm for 24 hours, the culture solution was transferred to a 100 ml GPY / 300 ml Erlenmeyer flask and cultured at 30 ° C., 150 rpm for 88 hours. The culture supernatant was filtered with a 0.45 μm filter, and enzyme activity measurement and 150 μl concentration were performed, followed by SDS-PAGE. The results of enzyme activity measurement are shown in Table 7, and the results of SDS-PAGE are shown in FIG.

  As a result, it was confirmed that peptidase 7 could be produced outside the cells even when the culture was scaled up from 3 ml to 100 ml.

(3) Self-cloning strains (peptidase 8 and peptidase 9)
The conidia was scraped from the plate of the peptidase 8 production strain and the peptidase 9 production strain prepared above with a microplate or the like, inoculated into 3 ml GPY medium, and cultured at 30 ° C., 200 rpm for 40 hours. The culture supernatant was filtered with a 0.45 μm filter, and enzyme activity measurement and 150 μl concentration were performed, followed by SDS-PAGE. The results of enzyme activity measurement are shown in Tables 8 and 9, and the results of SDS-PAGE are shown in FIG.

  As a result, by using the sodM promoter, significant secretion production of peptidase 8 and peptidase 9 was confirmed outside the cells.

Example 7 Preparation of an expression vector for filamentous fungi and production of peptidase 10 by self-cloning The peptidase 10 gene derived from Aspergillus oryzae was expressed and produced by a self-cloning method using Aspergillus (trans isomerase ( pdiA) Promoter use).

(1) Preparation of expression vector for filamentous fungi
(1-1) Plasmid vector
(A) Preparation of fusion gene of pdiA promoter and peptidase 10 gene
As for the pdiA promoter, KOD using Koji mold genomic DNA as a template and using primer 40 (5'-cctttttgaccttggtggttgatagaacgg-3 '(SEQ ID NO: 67)) and primer 41 (5'-agggtcgcattttgagatgctgaatcggataa-3' (SEQ ID NO: 68)) Amplification was carried out under the following PCR conditions using a plus DNA polymerase (Toyobo, Japan).
<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.

For peptidase 10 gene, Neisseria gonorrhoeae genomic DNA was used as a template and primer 42 (5'-gattcagcatctcaaaatgcgacccttgtctcatctttc-3 '(SEQ ID NO: 69)) and primer 43 (5'-ctaaggcagatcaagccacaacttcaacatc-3' (SEQ ID NO: 70)) Amplification was performed with KOD plus DNA polymerase (Toyobo, Japan) under the following PCR conditions.
<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.

The pdiA promoter PCR amplification product and the peptidase 10 gene PCR amplification product are mixed, and under the following PCR conditions, a primer 40 phosphorylated at the 5′-end and a primer 43 phosphorylated at the 5′-end are used. PCR was performed with KOD plus DNA polymerase (Toyobo, Japan).
<PCR conditions>
• 96 ° C (5 minutes) for 1 cycle • 96 ° C (20 seconds), 68 ° C (3 minutes) for 30 cycles • 72 ° C (7 minutes) for 1 cycle

  The obtained PCR amplification product was subjected to agarose gel electrophoresis, and the target fragment was excised using QIAquick Gel Extraction kit (QIAGEN) to prepare an insert.

(B) Subcloning of the fusion gene The fusion gene prepared above was subcloned into pUCLT1 described in JP 2010-4760 A ([0055]). Specifically, using the above-mentioned pUCLT1 as a template, primer 44 (5′-ccgtacgcgg ggagtgtgct taaggcgatg-3 ′ (SEQ ID NO: 71)) and primer 45 (5′-atgtactttc cagtgcgtgt agtctactc-3 ′ (SEQ ID NO: 72)) are used. PCR was performed using KOD plus DNA polymerase (Toyobo, Japan), the obtained PCR amplification product was subjected to agarose gel electrophoresis, the target fragment was excised using QIAquick Gel Extraction kit (QIAGEN), and a vector was prepared.
<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 vector and the fusion gene were subcloned using DNA Ligation Kit ver. 1 (Takara Bio, Japan), and the resulting plasmid was designated as pUCLTPA1.

(1-2) Self-cloning vector Using the plasmid vector pUCLTPA1 obtained above as a template, primer 46 (5'-gcgtggttta ctagctttag tgctaccaaa-3 '(SEQ ID NO: 73)) and primer 47 (5'-ctgcagatcg gctgaagtta ggagcggcca ttgtc- PCR was performed using 3 ′ (SEQ ID NO: 74)), and the obtained PCR amplification product was purified using QIAquick Gel Extraction kit (QIAGEN), and this was used as self-cloning vector PUCLTPA1.
<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.

(2) In accordance with the PEG-calcium method (Mol Gen Genet, 218, 99-104, (1989)), which is a standard method for preparing transformants , the leucine-requiring mutant strain using the self-cloning vector PUCLTPA1 prepared above. (Aspergillus oryzae leu-5 (deposit number: FERM P-20079)) was transformed.

  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. % Ferrous sulfate, 0.3% sodium nitrate), and a transformant was obtained for the self-cloning vector PUCLTPA1 by selecting a strain capable of growing in such a medium.

(3) Production of peptidase 10 The peptidase 10-producing bacteria prepared above were inoculated into 5 ml of GPY liquid medium, and then cultured in a disposable test tube at 30 ° C. and 250 rpm for 3 days. After culturing, 100 μl of the culture supernatant was centrifuged and concentrated, suspended in 20 μl of sample buffer, boiled at 100 ° C. for 3 minutes, 20 μl was applied, and SDS-PAGE was performed. As a control, a strain obtained by transforming an OSI1013Leu-requiring mutant with the plasmid pUCLT1 described in JP 2010-4760 ([0055]) was used. The results of SDS-PAGE are shown in FIG. As a result, by using the pdiA promoter, a significant amount of peptidase 10 having a g / L-broth level was secreted and produced outside the cells as a broad band to which an N-linked sugar chain was added.

Claims (6)

Promoter consisting of the DNA of the following (a) or (b), the following (c) or peptidase gene comprising the DNA of (d), and gene expression Aspergillus fungi having a terminator that function in Aspergillus fungi body cassette:
(A) nucleotide sequence or Ranaru DNA set forth in SEQ ID NO: 1
(B) DNA having 90% or more identity with the base sequence described in SEQ ID NO: 1 and functioning as a promoter in Aspergillus filamentous fungi
(C) nucleotide sequence or Ranaru DNA described in SEQ ID NO: 6
(D) DNA encoding a protein having 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 6 and having peptidase activity. An expression vector for Aspergillus filamentous fungi comprising a selectable marker gene that functions in Aspergillus filamentous fungi and the gene expression cassette according to claim 1 . That is substantially self-cloning vector comprising only nucleotides from Aspergillus filamentous fungi, Aspergillus filamentous fungi expression vector of claim 2. The expression vector for Aspergillus filamentous fungi according to claim 2 or 3 , wherein the expression vector is linear. It claims 2-4 or transformed Aspergillus fungi comprising in Aspergillus filamentous fungi expression vectors according to one of. A method for producing a peptidase, comprising culturing the Aspergillus filamentous fungus according to claim 5 in a medium and recovering the peptidase from the medium.

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