JP2012179011A - Filamentous fungus variant, and method for producing protein using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To markedly improve productivity of an extraneous protein encoded by an extraneous gene in a filamentous fungus.SOLUTION: There is disclosed a filamentous fungus variant transferring an extraneous gene encoding the aimed protein in an expressible way and reduced in function of genes except those involved in degradation of autophagic bodies in an vacuole, among genes associated with autophagy.

Description

  The present invention relates to a filamentous fungal mutant capable of highly expressing a gene encoding a desired target protein and a method for producing a protein using the same.

  Aspergillus oryzae is a microorganism that has long been used for brewing sake, miso, and soy sauce, and is classified as a fungus. Aspergillus oryzae has the ability to secrete and produce a large amount of protein outside the cell. For example, it is known that an enzyme protein such as amylase derived from Aspergillus oryzae itself can be secreted to the outside of the microbial cell per liter of the culture solution. On the other hand, the protein secretory production capacity of yeast, which is well studied, is 1/100 to 1000 times lower than that of Aspergillus oryzae. As described above, Aspergillus oryzae is one of the eukaryotic cells having the highest protein secretory production ability, and its safety is guaranteed from the track record of being used for many years of food production. Therefore, Aspergillus oryzae is expected to be used as a host serving as a protein factory for mass production of various heterologous proteins by genetic recombination.

  However, when a protein (foreign protein) encoded by an externally introduced gene (foreign gene) such as a protein derived from a higher organism such as an animal or plant is secreted and produced as a recombinant protein by Aspergillus oryzae, the amount of secretion is 1000 This is a problem in the production of recombinant heterologous proteins. So far, attempts to improve the production capacity of heterologous proteins by Aspergillus oryzae have been successful in developing powerful high-expression promoters and codon optimization.

  The inventors of the present invention have succeeded in producing a recombinant protein in an vacuolar acidic protease gene-disrupted strain as an Aspergillus oryzae mutant strain serving as a host for high production of the recombinant protein. (Patent Document 1).

WO2007 / 099776

  However, it is a technique that can be applied to filamentous fungi including Aspergillus oryzae and other koji molds, and a technique for greatly improving the expression level of foreign genes has not been well established. Techniques for further improving the expression level of foreign genes introduced into filamentous fungi are desired. With the accumulation of these techniques, protein production methods using filamentous fungi as a host have been put to practical use. It is thought that.

  Therefore, in view of the above-described circumstances, an object of the present invention is to provide a filamentous fungal mutant strain that can highly express a gene encoding a desired target protein, and a protein production method using the same.

  As a result of intensive studies by the present inventors in order to achieve the above-described object, the expression level of a foreign gene can be greatly improved by reducing the function of a gene related to autophagy. It has been found that the productivity of the encoded foreign protein can be greatly improved, and the present invention has been completed.

The present invention includes the following.
(1) A foreign gene encoding the target protein was introduced so that it could be expressed, and among the genes related to autophagy, the functions of the genes except for the genes involved in autophagic body decay were reduced in the vacuole. Filamentous fungal mutant.
(2) The filamentous fungus mutant according to (1), wherein the gene whose function is reduced is a gene encoding a factor essential for autophagosome formation.
(3) The filamentous fungal mutation according to (2), wherein the gene encoding the factor essential for the formation of the autophagosome is a gene encoding any one of the following proteins (a) to (c): stock.
(a) Proteins constituting the Atg1 kinase complex
(b) Proteins constituting the ubiquitin-like binding reaction system involving Atg8 or Atg12
(c) Proteins constituting the PtdIns 3-kinase complex (4) The gene encoding the factor essential for the formation of the autophagosome is a gene selected from the group consisting of Atg1, Atg13, Atg8 and Atg4 genes. The filamentous fungus mutant according to (2), characterized in that it exists.
(5) The filamentous fungus mutant according to (1), which is an Aspergillus genus filamentous fungus or a Trichoderma genus filamentous fungus.
(6) A method for producing a protein comprising a step of culturing the filamentous fungus mutant according to any one of (1) to (5) and a step of recovering a target protein.
(7) The method for producing a protein according to (6), wherein the target protein is recovered from the culture supernatant of the filamentous fungus mutant.

  ADVANTAGE OF THE INVENTION According to this invention, the expression level of the foreign gene in the filamentous fungi containing Aspergillus oryzae and other koji molds can be improved significantly. Therefore, according to the present invention, the productivity of a protein encoded by a foreign gene can be greatly improved using a filamentous fungus as a host.

1 is a schematic configuration diagram of an Atg1 protein kinase complex. FIG. 1 is a schematic configuration diagram of a PtdIns 3-kinase complex. FIG. It is a characteristic view which shows the result of the multiple alignment analysis regarding the amino acid sequence of Atg1 protein derived from Aspergillus oryzae, Aspergillus niger, and Trichoderma reesei. FIG. 4 is a continuation of FIG. 3 and is a characteristic diagram showing the results of multiple alignment analysis on the amino acid sequence of Atg1 protein derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei. It is a characteristic view which shows the result of the multiple alignment analysis regarding the amino acid sequence of Atg4 protein derived from Aspergillus oryzae, Aspergillus niger, and Trichoderma reesei. It is a characteristic view which shows the result of the multiple alignment analysis regarding the amino acid sequence of Atg8 protein derived from Aspergillus oryzae, Aspergillus niger, and Trichoderma reesei. It is a characteristic view which shows the result of the multiple alignment analysis regarding the amino acid sequence of Atg13 protein derived from Aspergillus oryzae, Aspergillus niger, and Trichoderma reesei. FIG. 8 is a continuation of FIG. 7 and is a characteristic diagram showing the results of multiple alignment analysis on the amino acid sequence of Atg13 protein derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei. It is a characteristic view which shows the result of having measured the expression level of the bovine chymosin gene in various Atg gene disruption strains at the protein level. It is a characteristic view which shows the result of having measured the expression level of the bovine chymosin gene in the Aoatg4 gene conditional expression suppression strain at the protein level.

  Hereinafter, the filamentous fungus mutant according to the present invention and a protein production method using the same will be described in detail.

  The filamentous fungal mutant according to the present invention is a strain in which a foreign gene is introduced so as to be expressed, and the function of a predetermined gene in the filamentous fungus is reduced. Genes that reduce the function are genes that exclude genes involved in autophagic body decay in vacuoles among genes related to autophagy.

  Here, autophagy is synonymous with so-called macro autophagy, and means a mechanism in which a double membrane structure surrounding a cytoplasmic component is fused with a decomposition compartment, and the cytoplasmic component is decomposed in the decomposition compartment. The double membrane structure that surrounds the cytoplasm component is called `` autophagosome '', and the sac-like membrane structure called `` separation membrane '' generated in the cytoplasm in response to the nutrient starvation signal expands in a cup shape, It is formed by surrounding and closing its edges. The degradation compartment means vacuoles and lysosomes, but in filamentous fungi, it means vacuoles. Autophagosomes fuse with degradation compartments in their outer membranes. When the autophagosome fuses with the degradation compartment, the inner membrane structure of the autophagosome (which incorporates cytoplasmic components inside) is released into the degradation compartment. The inner membrane structure released into the degradation compartment is a single membrane structure and is called “autophagic body”. Inside the decomposition compartment, the membrane structure of the autophagic body rapidly collapses and the cytoplasmic components are decomposed by the action of various degrading enzymes.

  Such a series of autophagy mechanisms are conserved in various organisms, and can be broadly divided into the stages of autophagosome formation and the breakdown of autophagic bodies within the degradation compartment. It has been isolated and identified as a fuzzy gene. The types and functions of autophagy-related genes are summarized in Table 1 below. In Table 1, the character string on the right side of the gene name is the accession number of the gene stored in the UniprotKB database. The amino acid sequence information of the protein encoded by each ATG gene can be obtained from the UniprotKB database by the accession number. Among the accession numbers shown in Table 1, Q6JUT9 of the ATG25 gene is the accession number related to the gene derived from Pichia angusta, and Q5IF00 of the ATG28 gene and C4R5T1 of the ATG30 gene are accession numbers related to the gene derived from Pichia pastoris. Accession numbers other than these are all accession numbers for genes derived from Saccharomyces cerevisiae.

  The filamentous fungus mutant according to the present invention reduces the functions of genes other than the genes involved in the decay of the autophagic body in the vacuole among the aforementioned autophagy-related genes. The genes involved in autophagic body decay in the vacuole are, as described above, the ATG15 gene encoding the lipase-like protein involved in autophagic body decay and the vacuolar membrane involved in autophagic body decay. ATG22 gene that encodes a protein. Therefore, in the present invention, the gene whose function is reduced is, for example, a gene other than the ATG15 gene and the ATG22 gene among the autophagy-related genes listed in Table 1.

  In particular, in the filamentous fungus mutant according to the present invention, the gene that reduces the function is preferably a gene encoding a factor essential for the formation of autophagosomes. The formation of autophagosome involves the Atg1 protein kinase complex and the PtdIns3-kinase complex, and the ubiquitin-like binding reaction system of Atg8 and Atg12, which are ubiquitin-like proteins. Therefore, factors essential for the formation of autophagosomes include: (a) proteins that make up the Atg1 protein kinase complex, (b) proteins that participate in ubiquitin-like binding reactions involving Atg8 or Atg12, and (c) PtdIns Mention may be made of proteins constituting a 3-kinase complex.

  In Table 1 described above, autophagy-related genes encoding factors essential for autophagosome formation are circled. That is, in Table 1 described above, autophagy-related genes encoding factors essential for autophagosome formation are the ATG1-10 gene, the ATG12-14 gene, the ATG16-18 gene, the ATG29 gene, and the ATG31 gene. More specifically, the proteins constituting the Atg1 protein kinase complex are Atg1 protein, Atg13 protein, Atg17 protein, Atg29 protein and Atg31 protein as shown in FIG. The proteins involved in the ubiquitin-like binding reaction system involving Atg8 or Atg12 are Atg3 protein, Atg4 protein, Atg5 protein, Atg7 protein, Atg8 protein, Atg10 protein, Atg12 protein and Atg16 protein. As shown in FIG. 2, the proteins constituting the PtdIns 3-kinase complex are Atg6 protein and Atg14 protein.

  Therefore, it is preferable that the fungal mutant according to the present invention has a reduced function of at least one gene among autophagy-related genes encoding factors essential for the formation of these autophagosomes. Among them, the ATG1 gene encoding the Atg1 protein constituting the Atg1 protein kinase complex; the ATG13 gene encoding the Atg13 protein constituting the Atg1 protein kinase complex; involved in the ubiquitin-like binding reaction system involving the ubiquitin-like protein Atg8 It is preferable to reduce the function of at least one gene selected from the group consisting of an ATG8 gene encoding an Atg8 protein; and an ATG4 gene encoding an Atg4 protein involved in a ubiquitin-like binding reaction system involving the ubiquitin-like protein Atg8 .

  More specifically, the sequence information (base sequence and amino acid sequence) of autophagy-related genes in typical filamentous fungi such as Aspergillus oryzae, Aspergillus niger and Trichoderma reesei is summarized in the following table. By using the registration numbers shown in Table 2, the base sequence and amino acid sequence of the autophagy-related gene can be obtained in various databases.

  In Table 2, the registration number of Aspergillus oryzae is the DOGAN database (http://www.bio.nite.go.jp/dogan/project/view/) provided by the National Institute of Technology and Evaluation (NITE). AO) and / or Aspergillus oryzae RIB 40 genome DB (http://nribf2.nrib.go.jp/). In Table 2 above, the registration number of Aspergillus niger is the database provided by the National Center for Biotechnology Information, US National Library of Medicine (http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed) Can be used. Furthermore, in Table 2 above, the registration number of Trichoderma reesei is the database (http://genome.jgi-psf.org/Trire2/Trire2.home.html) based on Nat Biotechnol. 26, 553-560 (2008). Can be used.

  Thus, the base sequence and amino acid sequence of various autophagy-related genes derived from various filamentous fungi can be specified and obtained using the database. In particular, the base sequence and amino acid sequence of autophagy-related genes (such as the Atg1, Atg4, Atg8, and Atg13 genes) that encode factors essential for autophagosome formation are also obtained using known databases. it can. As an example, the base sequences of the coding region of Atg1 gene derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei are shown in SEQ ID NOs: 1, 3 and 5, respectively. The amino acid sequences encoded by the nucleotide sequences shown in SEQ ID NOs: 1, 3 and 5 are shown in SEQ ID NOs: 2, 4 and 6, respectively. In addition, the nucleotide sequences of the coding regions of the Atg4 gene derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei are shown in SEQ ID NOs: 7, 9 and 11, respectively. The amino acid sequences encoded by the nucleotide sequences shown in SEQ ID NOs: 7, 9, and 11 are shown in SEQ ID NOs: 8, 10, and 12, respectively. Furthermore, the base sequences of the coding regions of Atg8 gene derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei are shown in SEQ ID NOs: 13, 15 and 17, respectively. The amino acid sequences encoded by the nucleotide sequences shown in SEQ ID NOs: 13, 15 and 17 are shown in SEQ ID NOs: 14, 16 and 18, respectively. Furthermore, the nucleotide sequences of the coding regions of the Atg13 gene derived from Aspergillus oryzae, Aspergillus niger, and Trichoderma reesei are shown in SEQ ID NOs: 19, 21, and 23, respectively. The amino acid sequences encoded by the nucleotide sequences shown in SEQ ID NOs: 19, 21, and 23 are shown in SEQ ID NOs: 20, 22, and 24, respectively.

  3 and 4 show the results of alignment analysis of the amino acid sequences of Atg1 proteins derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei shown in SEQ ID NOs: 2, 4 and 6, using CLUSTAL W (1.81). Similarly, FIG. 5 shows the results of alignment analysis of the amino acid sequences of Atg4 proteins derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei shown in SEQ ID NOs: 8, 10 and 12, using CLUSTAL W (1.81). FIG. 6 shows the result of alignment analysis using CLUSTAL W (1.81) for the amino acid sequence of Atg8 protein derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei shown in SEQ ID NOs: 14, 16 and 18. 7 and 8 show the results of alignment analysis of the amino acid sequence of Atg13 protein derived from Aspergillus oryzae, Aspergillus niger and Trichoderma reesei shown in SEQ ID NOs: 20, 22 and 24 using CLUSTAL W (1.81).

  As shown in FIG. 3 to FIG. 8, in Aspergillus oryzae, Aspergillus niger and Trichoderma reesei, Atg1, Atg4, Atg8 and Atg13 proteins show very high homology. This shows that the Atg1 gene, Atg4 gene, Atg8, and Atg13 gene are very likely to play the same functions and roles in the autophagy mechanism in Aspergillus oryzae, Aspergillus niger, and Trichoderma reesei. That is, for example, the phenomenon (including phenotype) observed when the function of the Atg1 gene in Aspergillus oryzae is reduced when the function of the homologous gene is reduced in other filamentous fungi such as Aspergillus niger and Trichoderma reesei. Similarly, the probability of being observed is high.

  In addition, although the multiple alignment was shown in FIG. 3 thru | or FIG. 8 about the autophagy related gene which codes the factor indispensable for formation of an autophagosome, very high homology is seen similarly about another autophagy related gene. For other autophagy-related genes, homology can be known by obtaining amino acid sequence information from the above-mentioned database and using alignment analysis software such as CLUSTAL W (1.81).

  By the way, the filamentous fungus mutant according to the present invention has a reduced function of a specific autophagy-related gene as described above. Here, “reducing the function of the gene” means reducing the expression level of the gene or reducing the activity of the protein encoded by the gene. Further, “reducing the gene expression level” means reducing the gene transcription level and / or reducing the gene translation level. Although there is no particular limitation on reducing the amount of gene transcription, for example, a method of modifying the expression control region of the target gene or a method of deleting all or part of the target gene is applied. Although it does not specifically limit in order to reduce the translation amount of a gene, For example, the method of utilizing what is called RNA interference and antisense RNA can be mentioned. In other words, in order to reduce the expression level of a predetermined gene, any method known as a method for destroying the gene may be applied.

  “Reducing the activity of the protein encoded by the gene” is not particularly limited. For example, there is a substance that inhibits the activity of the protein encoded by the gene of interest (low molecular compound, high molecular compound such as an antibody). It is sufficient to adopt a technique for making them.

  On the other hand, the filamentous fungal mutant according to the present invention can be prepared by using a conventionally known filamentous fungus as a host without any limitation. Filamentous fungi that can be used for the host are not particularly limited, but include Aspergillus aculeatus, Aspergillus awamori, Aspergillus caesiellus, Aspergillus candidus, Aspergillus carneus, Aspergillus clavatus, Aspergillus deflectus, Aspergillus fischerianus, Aspergillus asperus, Aspergillus sperum, Aspergillus sperum Aspergillus niger, Aspergillus ochraceus, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae, Aspergillus sydowi, Aspergillus tamari, Aspergillus terreus, Aspergillus ustus, Aspergillus usgil and Aspergillus versicolor, etc. As hosts, Trichoderma aggressivum, Trichoderma asperellum, Trichoderma atroviride, Trichoderma aureoviride, Trichoderma austrokoningii, Trichoderma brevicompactum, Trichoderma candidum, Trichoderma caribbaeum var.aequatoriale, Trichoderma , Trichoderma chlorosporum, Trichoderma chromospermum, Trichoderma cinnamomeum, Trichoderma citrinoviride, Trichoderma crassum, Trichoderma cremeum, Trichoderma dingleyeae, Trichoderma dorotheae, Trichoderma effusum, Trichoderma erinaceum, Trichoderma estonicum, Trichoderma fertile, Trichoderma gelatinosus, Trichoderma ghanense, Trichoderma hamatum, Trichoderma harzianum, Trichoderma helicum, Trichoderma intricatum, Trichoderma konilangbra, Trichoderma koningii, Trichoderma koningiopsis, Trichoderma longibrachiatum, Trichoderma longipile, Trichoderma minutisporum, Trichoderma oblong isporum, Trichoderma ovalisporum, Trichoderma petersenii, Trichoderma phyllostahydis, Trichoderma piluliferum, Trichoderma pleuroticola, Trichoderma pleurotum, Trichoderma polysporum, Trichoderma pseudokoningii, Trichoderma pubescens, Trichoderma reesei, Trichoderma rogersonii, Trichoderma rossicum, Trichoderma saturnisporum, Trichoderma sinensis, Trichoderma sinuosum, Trichoderma sp. MA 3642, Trichoderma sp. And Trichoderma genus filamentous fungi. Furthermore, as hosts, Rhizomucor pusillus, Rhizomucor miehei and other Rhizomucor genus fungi, Penicillium notatum, Penicillium chrysogenum and other Penicillium genus fungi, Rhizopus oryzae and other Rhizopus genus fungi, Acremonium cellulolyticus, Humicola griur a Can do. In particular, the host is preferably Aspergillus filamentous fungi, especially Aspergillus oryzae and Trichoderma spp., Especially Trichoderma reesei.

  The filamentous fungus used as the host may be a wild strain of the aforementioned filamentous fungus, or may be a mutant strain into which various mutations are introduced. For example, WO2007 / 099776 discloses an Aspergillus oryzae mutant strain in which the vacuolar acid protease gene is disrupted, but this mutant strain can also be used as a host. In addition, the Aspergillus oryzae mutant strain disclosed in WO2007 / 099776 is known as a mutant strain that produces a recombinant protein at a high level. By further reducing the function of the above-mentioned autophagy-related gene in this Aspergillus oryzae mutant strain, it can be expected that the protein productivity is further improved.

  In these various filamentous fungi, the autophagy-related gene can be identified based on the amino acid sequence or base sequence that can be obtained by the accession numbers shown in Table 1. That is, if the above-described genome sequence information of the filamentous fungus is known, the homologous gene of the gene identified by the accession number shown in Table 1 should be identified using homology search software such as Blast. Can do. When the genome sequence information of the filamentous fungus described above is unknown, as an example, a cDNA library or the like is prepared, and a probe that specifically hybridizes to the gene specified by the accession number shown in Table 1 is used. Thus, homologous genes can be identified from a cDNA library.

  By the way, the filamentous fungal mutant strain according to the present invention is a strain into which a foreign gene has been introduced so that it can be expressed. Here, the foreign gene means a gene derived from an organism other than the filamentous fungus that is the origin of the filamentous fungal mutant. For example, when the filamentous fungal mutant according to the present invention is produced from Aspergillus oryzae, the foreign gene means a gene present in an organism other than Aspergillus oryzae. Therefore, if the filamentous fungal mutant according to the present invention is produced from Aspergillus oryzae, genes derived from other Aspergillus spp. Other than Aspergillus oryzae are also included in the foreign gene.

  However, the filamentous fungal mutant according to the present invention is preferably one in which a gene derived from a higher organism such as an animal cell or a plant cell is introduced as a foreign gene. This is because genes derived from higher organisms such as animal cells and plant cells were not highly expressed even when introduced into filamentous fungi such as Aspergillus oryzae. That is, in the filamentous fungal mutant according to the present invention, even a foreign gene derived from these higher organisms can be highly expressed, and a protein encoded by the foreign gene can be produced at high yield.

  Here, the protein to be produced is not limited in any way, and may be any molecular weight, any biological species, any isoelectric point, or any amino acid sequence. That is, as the foreign gene, for example, alkaline protease gene, α-amylase gene, ascorbate oxidase gene, aspartic protease gene, cellobiohydrolase gene, cellulase gene, cutinase gene, endoglucanase gene, glucoamylase, β-glucosidase gene, Glyoxal oxidase gene, laccase gene, lignin oxidase gene, lignin peroxidase gene, lipase gene, manganese peroxidase gene, 1,2-α-mannosidase gene, nuclease gene, pectin lyase gene, pectin methylesterase gene, acid phosphatase gene, polygalactururo Examples include a nuclease gene, a xylanase gene, a β-xylosidase gene, etc. Kill. In particular, the proteins to be produced include lysozyme, chymosin, lectin, interleukin, lactoferrin, taste-modifying protein miraculin, anti-Fas antibody and other antibody drugs, mite allergen, pollen allergen, and cellulolytic enzyme for degradation of woody biomass In addition, cytokines can be exemplified as proteins encoded by genes derived from higher organisms.

  The foreign genes described above can be introduced into filamentous fungi in the form of commonly used expression vectors. Vectors typically have a selectable marker gene, a cloning site, and control regions (promoter and terminator). This vector is well known in the art and is commercially available.

  The promoter contained in the vector may be either a constitutive expression promoter or an inducible promoter as long as it can function in filamentous fungi. That the promoter can function means that the downstream gene can be transcribed in the host filamentous fungus. Examples of the promoter include a promoter of a gene encoding glucoamylase, alpha-amylase, or alpha-glucosidase derived from Aspergillus filamentous fungi such as Aspergillus niger. Also, A. nidulans gpdA gene, oliC gene or trpC gene promoter, Neurospora crassa cbh1 or trp1 gene promoter, A. niger or Rhizomucor miehei aspartic proteinase encoding gene promoter, Trichoderma reesei derived cbh1 gene, cbh2 Promoters of genes encoding genes, egl1 genes, egl2 genes or other cellulases can be used. In particular, when the host filamentous fungus is Trichoderma reesei, it is possible to use a promoter of a gene encoding a cbh1 gene, cbh2 gene, egl1 gene, egl2 gene or other cellulase derived from Trichoderma reesei. In particular, it is more preferable to use the promoter of the cbh1 gene.

  In the present invention, an expression vector into which the above-mentioned foreign gene is incorporated is introduced according to a standard method to produce a protein encoded by the foreign gene. As a method for introducing an expression vector, various conventionally known methods such as transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method and the like can be used.

  This expression vector can be introduced into a filamentous fungus having a reduced function of the aforementioned predetermined autophagy-related gene. Alternatively, after the expression vector is introduced into the filamentous fungus that retains the function of the predetermined autophagy-related gene described above, the function of the autophagy-related gene may be reduced. Furthermore, the introduction of the expression vector and the process for reducing the function of the predetermined autophagy-related gene described above may be performed simultaneously. In any method, it is possible to produce a filamentous fungal mutant strain in which a foreign gene is introduced so that it can be expressed, and the function of a predetermined autophagy-related gene described above is reduced.

  The filamentous fungus mutant prepared as described above has a high expression level of a foreign gene and can produce a protein encoded by the foreign gene in high production. Here, the expression level of the foreign gene is high as compared to the expression level of the foreign gene when the expression vector is introduced into the filamentous fungus that retains the function of the predetermined autophagy-related gene described above. This means that the expression level of the foreign gene in the filamentous fungal mutant according to the present invention is significantly high.

  In addition, since the protein encoded by the foreign gene is secreted and produced outside the cells, it can be recovered according to a conventional method without performing a treatment such as destroying the cells. The protein can be measured by directly analyzing a supernatant sample of the medium by dodecyl sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) known in the art. The protein of interest produced during cell culture is secreted into the medium and purified or isolated, for example, by removing unwanted components from the cell medium. For purification of target protein, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resin such as DEAE, chromatofocusing, SDS -PAGE, ammonium sulfate precipitation, gel filtration, etc. can be used alone or in combination.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, a mutant strain in which the ATG gene (hereinafter referred to as Aoatg gene) in A. oryzae was disrupted was prepared, and the change in the expression level of the bovine chymosin gene introduced as a foreign gene was measured. Specifically, Aoatg1 gene disruption strain, Aoatg4 gene disruption strain, Aoatg8 gene disruption strain, Aoatg13 gene disruption strain, Aoatg15 gene disruption strain, and Aoatg4 gene conditional expression suppression strain were prepared as mutant strains.

Bacterial strains used, culture medium and transformation The strains used in this example are shown in Table 3 below.

Aspergillus oryzae wild strain RIB40 was used as a DNA source, and NSRKu70-1-1 and NSPlD1 strains were used as transformation hosts. For the growth of strains, DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH ₂ PO ₄ and 0.05% MgSO ₄ .7H ₂ O [pH 5.5]) and PDA medium (Nissui Pharmaceutical) Made). Minimal medium containing 0.15% methionine [0.2% NH ₄ Cl, 0.1% (NH ₄ ) ₂ SO ₄ , 0.05% KCl, 0.05% NaCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ · 7H ₂ O, 0.002 % FeSO ₄ · 7H ₂ O and 2% glucose (pH 5.5)] (M + Met) are used to obtain each Aoatg gene (Aoatg1, Aoatg4, Aoatg13, Aoatg8, Aoatg15) disrupted strain, Aoatg4 conditional expression suppression strain Used as a selective medium. 5 × DPY (pH 5.5) (10% dextrin, 5% polypeptone, 2.5% yeast extract, 0.5% KH ₂ PO ₄ and 0.05% MgSO ₄ .7H ₂ O [pH 5.5]) is a production medium for bovine chymosin (CHY) Using. Czapek-Dox containing 0.0015% of the methionine (CD) medium _{(0.3% NaNO 3, 0.2%} KCl, 0.1% KH 2 PO 4, 0.05% MgSO 4 · 7H 2 O, 0.002% FeSO 4 · 7H 2 O and 2% glucose [pH 5.5]) was used as a selective medium for the niaD marker of the CHY expression plasmid. Escherichia coli DH5α [supE44 ΔlacU169 (φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was used as a host for DNA manipulation. Transformation of A. oryzae was performed based on the method of Kitamoto (2002) Adv Appl Microbiol 51: 129-153.

Production of Aoatg gene disruption strain
1. Preparation of Aoatg disruption plasmid Using the primer set shown in Table 4 below, the upstream and downstream regions of each Aoatg gene of the Aoatg1, Aoatg4, Aoatg8, Aoatg13, and Aoatg15 genes were extracted from the RIB40 strain genome. Amplified by PCR. In addition, using the primer set shown in Table 4 below, the adeA gene was amplified by PCR using pgEaA (Jin et al (2007) Appl. Microbiol. Biotechnol. 76: 1059-1068) as a template.

  Aoatg gene disruption fragments were prepared for the Aoatg1, Aoatg4, Aoatg8, Aoatg13, and Aoatg15 genes by fusion PCR in which the upstream, downstream, and adeA gene fragments of each Aoatg gene obtained by PCR were mixed. By introducing these fragments into pCR 4Blunt-TOPO, Aoatg1 destruction plasmid pgΔAoatg1, Aoatg4 destruction plasmid pgΔAoatg4, Aoatg8 destruction plasmid pgΔAoatg8, Aoatg13 destruction plasmid pgΔAoatg13, and Aoatg15 destruction plasmid pgΔAoatg15 were prepared.

2. Acquisition of Aoatg gene disruption strain Aoatg disruption by PCR using the obtained Aoatg1 disruption plasmid to Aoatg15 disruption plasmid as a template and using the upstream primer and the downstream primer shown in Table 4 above. The fragment was amplified. The obtained Aoatg disruption fragment was used to transform NSRKu70-1-1 strain using the adeA gene as a marker. As a result of recovering the genome from the transformant and confirming by Southern blot analysis (not shown), Aoatg1 gene disruption strain, Aoatg4 gene disruption strain, Aoatg8 gene disruption strain, Aoatg13 gene disruption strain and Aoatg15 gene disruption strain are obtained. It was.

Preparation of Aoatg4 gene conditional expression suppression strain Aoatg4 gene conditional expression suppression strain having the characteristics that the Aoatg4 gene is disrupted under predetermined conditions was prepared. Specifically, a thiA promoter (PthiA), which is a promoter whose expression is suppressed in the presence of thiamine, was used. Aoatg4 gene conditional expression suppression strains were created by Development of Aspergillus oryzae thiA promoter as a tool for molecular biological studies.Shoji JY, Maruyama J, Arioka M, Kitamoto K. FEMS Microbiol Lett. 2005 Mar 1; 244 (1): 41 Based on -6.

  Specifically, a plasmid vector for homologous recombination was prepared using the Gateway system in order to prepare an Aoatg4 gene conditional expression suppression strain under the control of PthiA.

  First, to create a center entry clone, a pair of primers f-thiA_F (5-tctcgggccacggtaaatacactatcacacaccgaac-3: sequence) using pg5'pt plasmid (Mabashi et al. (2006) Biosci Biotechnol Biochem 70: 1882-1889) as a template No. 47) and pgthiA_R (5-GGGGACCACTTTGTACAAGAAAGCTGGGTtttcaagttgcaatgactatcatctgttagcc-3: SEQ ID NO: 48) were used to amplify PthiA. Then, using pgEpg plasmid (Maruyama and Kitamoto (2008) Biotechnol Lett 30: 1811-1817) as a template, a pair of primers pgPyrG_F (5-GGGGACAAGTTTGTACAAAAAAGCAGGCTggtaatgtgccccaggcttg-3: SEQ ID NO: 49) and f-pyrG_R (5-gtatttaccgtggattccccgggg No. 50) was used to amplify pyrG. Furthermore, these were connected by fusion PCR and introduced into a center entry clone to prepare pgEpGPt plasmid.

  Next, to create a 5 'entry clone, the 5' flanking region (about 1 kb) of the Aoatg4 gene was paired with a pair of primers aB4-5a4_F (5-GGGGACAACTTTGTATAGAAAAGTTGtcatcttcactagagctgtcacg-3: SEQ ID NO: 51) and aB1r-5a4_R (5-GGGGACTGCTTTggtgaggta -3: amplified by PCR using SEQ ID NO: 52) and introduced into 5 'entry clone to prepare pg5'a4 plasmid.

  Next, to create a 3 'entry clone, the ORF region (about 1 kb) starting from ATG of the Aoatg4 gene was paired with a pair of primers aB2r-a4ORF_F (5-GGGGACAGCTTTCTTGTACAAAGTGGatgaacagtgtagacatagggcg-3: SEQ ID NO: 53) and aB3-a4ORF_R (5- Amplification was performed by PCR using GGGGACAACTTTGTATAATAAAGTTGcccttctgccttggttgtaagta: SEQ ID NO: 54), and the product was introduced into a 3 ′ entry clone to prepare pg3′a4ORF plasmid.

  Then, LR reaction was carried out with the center entry clone, 3 'entry clone and 5' entry clone prepared, respectively, to prepare LR plasmids for transformation. Thereafter, the LR plasmid for transformation was transformed into NSPlD1 strain, and NSlD-Ta4 strain (thiA promoter control strain), which is an Aoatg4 gene conditional expression suppression strain, was prepared.

Production of bovine chymosin (CHY) expression strains Aoatg1 gene disruption strain, Aoatg4 gene disruption strain, Aoatg8 gene disruption strain, Aoatg13 gene disruption strain and Aoatg15 gene disruption strain prepared as described above were measured using bovine chymosin. A strain expressing (CHY) was produced. Bovine chymosin expression plasmid pgAKCN (Nemoto et al (2009) Appl Microbiol Biotechnol 82: 1105-1114), control strain (NSKu70-AA strain), Aoatg1 gene disruption strain, Aoatg4 gene disruption strain, Aoatg8 gene disruption strain, Aoatg13 gene disruption strain and Aoatg15 gene disruption strain was transformed. The transformant was selected using the niaD gene as a selection marker, and a strain integrated as a single copy with the niaD gene locus in the A. oryzae genome was selected by Southern blot analysis (not shown).

  Southern blot analysis was in accordance with the method described in Nemoto et al (2009), supra. The strain obtained by introducing bovine chymosin gene into the control strain obtained as described above is referred to as SKu70-AA-AKC1 strain. Aoatg1 gene-disrupted strain, Aoatg4 gene-disrupted strain, Aoatg8 gene-disrupted strain, Aoatg13 gene-disrupted strain and Aoatg15 gene-disrupted strain introduced with bovine chymosin gene, SKu70-ΔAoatg1-AKC2 strain, SKu70-ΔAoatg4-AKC1 strain, They are referred to as ΔAoatg8-AKC1 strain, SKu70-ΔAoatg13-AKC1 strain and SKu70-ΔAoatg15-AKC2 strain.

  Similarly, the bovine chymosin expression plasmid pgAKCN was introduced into the Aoatg4 gene conditional expression-suppressed strain to produce an Aoatg4 gene conditional expression-suppressed strain SlD-Ta4-AKC1 that expresses the bovine chymosin gene.

CHY quantitative results
SKu70-AA-AKC1 strain, SKu70-ΔAoatg1-AKC2 strain, SKu70-ΔAoatg4-AKC1 strain, SKu70-ΔAoatg8-AKC1 strain, SKu70-ΔAoatg13-AKC1 strain and SKu70-ΔAoatg15-AKC2 strain conidia minute to about 2 × 10 ⁵ The cells were collected so as to become individual, cultured in 30 ml of 20 ml of 5 × DPY (pH 5.5) medium for 3 to 6 days, and the CHY activity in the culture supernatant was measured. For the SlD-Ta4-AKC1 strain, the same culture was performed in 5 × DPY (pH5.5) medium and 5 × DPY (pH5.5) medium containing 10 μM thiamine, and the CHY activity in the culture supernatant was measured. .

CHY activity is based on the Foltmann method (OLTMANN, B. "Methods in Enzymology," Vol. XIX, ed. By PERLMANN, GE and LORAND, L., p. 421-436, Academic Press Inc., New York, NY, 1970) Based on. That is, about 100 μl of the culture supernatant was collected every 24 hours and reacted with 1 ml of 12% skim milk (containing 10 mM CaCl ₂ ). The CHY production amount was calculated along a standard curve using a commercially available CHY standard (manufactured by Sigma).

result
CHY activity of SKu70-AA-AKC1 strain, SKu70-ΔAoatg1-AKC2 strain, SKu70-ΔAoatg4-AKC1 strain, SKu70-ΔAoatg8-AKC1 strain, SKu70-ΔAoatg13-AKC1 strain and SKu70-ΔAoatg15-AKC2 strain was measured. 9 shows. As shown in FIG. 9, the production amount of the heterologous protein showed the maximum value on the fourth day of culture. The maximum production of each Aoatg gene disruption strain on the 4th day of culture is 59.1 mg / L for SKu70-ΔAoatg1-AKC2, 80.3 mg / L for SKu70-ΔAoatg4-AKC1, 64.4 mg / L for SKu70-ΔAoatg8-AKC1 The SKu70-ΔAoatg13-AKC1 strain was 37.2 mg / L. On the other hand, the maximum production amount of the control strain SKu70-AA-AKC1 was 26 mg / L. In addition, the maximum production of SKu70-ΔAoatg15-AKC2 strain was 24.1 mg / L.

  From the results of the SKu70-ΔAoatg15-AKC2 strain, the productivity of heterologous proteins is significantly higher than that of the control strain even if the functions of autophagy-related genes and genes involved in autophagic body decay are reduced. It turns out that it does not improve. In contrast, when the functions of autophagy-related genes other than those involved in autophagic body disruption are reduced, the productivity of heterologous proteins is significantly improved compared to control strains. I understood.

  In particular, the maximum production of the SKu70-ΔAoatg4-AKC1 strain was revealed to be about 3 times higher than that of the control strain. It can also be seen that the maximum production amount of the SKu70-ΔAoatg8-AKC1 strain is very high compared to other disrupted strains. From this result, it was found that it is preferable to reduce the function of a gene encoding a protein involved in a ubiquitin-like binding reaction system involving Atg8 or Atg12 as an autophagy-related gene.

  On the other hand, FIG. 10 shows the results of measuring the CHY activity of the SlD-Ta4-AKC1 strain, which is an Aoatg4 gene conditional expression suppression strain. FIG. 10 also shows the results of measuring the CHY activity of the control strains SlD-AKC1 and SlDa4-AKC3. SlD-AKC1 is a strain in which pgAKCN plasmid is introduced into the niaD locus of NSlD. In addition, the SlDa4-AKC3 strain is a strain in which pgAKCN plasmid is introduced into the niaD locus after disrupting the Aoatg4 gene from the NSPlD1 strain using the pyrG marker.

  As shown in FIG. 10, the Aoatg4 gene conditional expression suppression strain produced the same production results as the Aoatg4 gene disruption on the fourth day of culture. That is, in the presence of thiamine such as 5 × DPY (pH5.5) medium and 5 × DPY (pH5.5) medium containing 10 μM thiamine, the Aoatg4 gene of the Aoatg4 gene condition expression-suppressed strain was disrupted and the production of heterologous proteins Was found to be significantly improved compared to the control strain.

Claims (7)

  Filamentous fungal mutation that introduces a foreign gene encoding the target protein in an expressible manner and reduces the function of genes other than those involved in autophagic body disruption in the vacuole among genes related to autophagy stock.   2. The filamentous fungus mutant according to claim 1, wherein the gene whose function is reduced is a gene encoding a factor essential for autophagosome formation. The filamentous fungus mutant according to claim 2, wherein the gene encoding the factor essential for the formation of the autophagosome is a gene encoding any one of the following proteins (a) to (c).
(a) Proteins constituting the Atg1 kinase complex
(b) Proteins constituting the ubiquitin-like binding reaction system involving Atg8 or Atg12
(c) Protein constituting the PtdIns 3-kinase complex   The filamentous fungus mutant according to claim 2, wherein the gene encoding a factor essential for the formation of the autophagosome is a gene selected from the group consisting of Atg1, Atg13, Atg8 and Atg4 genes.   The filamentous fungus mutant according to claim 1, which is an Aspergillus fungus or Trichoderma fungus.   A method for producing a protein comprising a step of culturing the filamentous fungus mutant according to any one of claims 1 to 5 and a step of recovering a target protein.   The method for producing a protein according to claim 6, wherein the target protein is recovered from the culture supernatant of the filamentous fungus mutant.

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