Filamentous fungi and their uses

Abstract

This invention provides a filamentous fungus in which wall growth formation is suppressed, and the use of said filamentous fungus. [Solution] Use a filamentous fungus lacking one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group, or a filamentous fungus in which the expression of one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group is suppressed.

Description

Technical Field

[0001] The present invention relates to filamentous fungi and their use.

Background Art

[0002] Conventionally, various filamentous fungi produced by genetic recombination technology have been used to produce desired substances. In such filamentous fungi, various genes have been modified according to the purpose (see, for example, Patent Documents 1 to 3 and Non-Patent Documents 1 to 3).

[0003] In order to produce a large amount of a desired substance, it is necessary to culture filamentous fungi in large quantities. Therefore, conventionally, a technique for liquid-culturing filamentous fungi has been widely used to produce a large amount of a desired substance.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0005] [[ID=4"]]

Non-Patent Document 1

Non-Patent Document 2

[0006] However, the conventional techniques described above have a problem called wall growth, in which filamentous fungi adhere to the surface of culture equipment (for example, the walls of the culture tank), and these fungi proliferate to form large clumps.

[0007] When wall growth occurs, various problems arise, such as (i) the culture of filamentous fungi becomes unstable, (ii) the filamentous fungi forming the wall growth do not produce the desired substance, resulting in wasted nutrients, and (iii) when the filamentous fungi forming the wall growth detach from the surface of the culture equipment, these fungi can clog pipes and other components.

[0008] One aspect of the present invention aims to provide a filamentous fungus in which the formation of wall growth is suppressed, and the use of said filamentous fungus. [Means for solving the problem]

[0009] As a result of diligent research to solve the above-mentioned problems, the inventors of the present invention have found that filamentous fungi lacking one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group have suppressed wall growth formation, and have completed the present invention. One embodiment of the present invention encompasses the following invention.

[0010] [1] One or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group are deficient; or, A filamentous fungus in which the expression of one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group is suppressed.

[0011] [2] The galactosaminogalactan biosynthesis gene group described above is agdZ, ugeZ, sphZ, egaZ, and gtbZ, and their homologs; and / or the chitin biosynthesis gene group described above is chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ, and their homologs, as described in [1].

[0012] [3] The filamentous fungi described above are those belonging to the genera Aspergillus, Penicillium, Trichoderma, Rhizopus, Mucor, Fusarium, Monascus, Neurospora, or Talaromyces, as described in [1] or [2].

[0013] [4] The above filamentous fungi are those described in any one of [1] to [3] into which foreign genes have been introduced.

[0014] 〔5〕A method for culturing filamentous fungi, comprising a step of culturing the filamentous fungus according to any one of 〔1〕to 〔4〕.

[0015] 〔6〕The method for culturing filamentous fungi according to 〔5〕, wherein in the above step, the filamentous fungus is liquid-cultured in a culture tank.

[0016] 〔7〕A method for producing a substance, comprising a step of recovering a desired substance from a culture of filamentous fungi obtained by the culturing method according to 〔5〕or 〔6〕.

Advantages of the Invention

[0017] According to one aspect of the present invention, it is possible to provide filamentous fungi in which the formation of wall growth is suppressed, and the use of such filamentous fungi.

Brief Description of the Drawings

[0018] [Figure 1] It is a diagram showing the structure of the plasmid for gene disruption used in the examples of the present invention. [Figure 2] In the examples of the present invention, it is an image of wall growth adhering to the wall of a flask after culturing a gene disruption strain. [Figure 3] In the examples of the present invention, it is a graph showing the dry weight of wall growth formed by a gene disruption strain. [Figure 4] In the examples of the present invention, it is a graph showing the dry weight of wall growth formed by a gene disruption strain. [Figure 5] In the examples of the present invention, it is a graph showing the dry weight of wall growth formed by a gene disruption strain. [Figure 6] In the examples of the present invention, it is a graph showing the dry weight of wall growth formed by a gene disruption strain. [Figure 7] In the examples of the present invention, it is a graph showing the analysis results of the production ability of heterologous proteins of a gene disruption strain.

Modes for Carrying Out the Invention

[0019] The following describes in detail some examples of embodiments of the present invention, but the present invention is not limited to these. The present invention can be modified in various ways within the scope of the claims. Embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic and patent documents mentioned herein are incorporated herein by reference. Unless otherwise specified herein, "X~Y" representing a numerical range means "X or more and Y or less".

[0020] [1. Filamentous fungi] A filamentous fungus according to one embodiment of the present invention is a filamentous fungus that (i) lacks one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group; or (ii) has suppressed expression of one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group.

[0021] The above-mentioned group of galactosaminogalactan biosynthesis genes may include genes involved in galactosaminogalactan biosynthesis. Examples of such genes include genes involved in the synthesis of galactosaminogalactan or its intermediates, genes involved in the transport of galactosaminogalactan or its intermediates, genes involved in the modification of galactosaminogalactan or its intermediates, and genes involved in the demodification of galactosaminogalactan or its intermediates.

[0022] The above-mentioned group of chitin biosynthesis genes may include genes involved in chitin biosynthesis. Examples of such genes include genes involved in the synthesis of chitin or its intermediates, genes involved in the transport of chitin or its intermediates, genes involved in the modification of chitin or its intermediates, and genes involved in the demodification of chitin or its intermediates.

[0023] The above-mentioned filamentous fungi may be filamentous fungi (or filamentous fungi in which the function is lost under specific conditions) in which (i) the function of one or more genes selected from the galactosaminogalactan biosynthesis gene group and the function of one or more genes selected from the chitin biosynthesis gene group and / or (ii) the function of the protein encoded by one or more genes selected from the galactosaminogalactan biosynthesis gene group and the function of the protein encoded by one or more genes selected from the chitin biosynthesis gene group are lost, and the specific mode of deficiency is not limited.

[0024] Furthermore, the above-mentioned filamentous fungi may be filamentous fungi in which (iii) the expression of one or more genes selected from the galactosaminogalactan biosynthesis gene group, the expression of one or more genes selected from the chitin biosynthesis gene group, and / or (iv) the expression of proteins encoded by one or more genes selected from the galactosaminogalactan biosynthesis gene group, and the expression of proteins encoded by one or more genes selected from the chitin biosynthesis gene group are suppressed (or filamentous fungi whose expression is suppressed under specific conditions), and the specific mode of suppression is not limited.

[0025] For example, the above filamentous fungi may be filamentous fungi in which (i) the function of a particular gene and / or the function of a protein encoded by a particular gene is lost (or lost under certain conditions) due to (a) the deletion of the entire base sequence of a particular gene, (b) the deletion of a part of the base sequence of a particular gene, or (c) the introduction of a mutation (e.g., substitution, deletion, insertion, or addition) within the base sequence or expression regulatory region (e.g., promoter) of a particular gene, or (ii) the expression of a particular gene and / or the expression of a protein encoded by a particular gene is suppressed (or suppressed under certain conditions).

[0026] The number of genes selected from the missing galactosaminogalactan biosynthesis gene group may be one or more, for example, two or more, three or more, four or more, or five or more. There is no upper limit to this number; for example, it may be five, seven, or ten.

[0027] The number of genes selected from the missing chitin biosynthesis gene group mentioned above may be one or more, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. The upper limit of this number is not limited and may be, for example, 10, 15, or 20.

[0028] The above galactosaminogalactan biosynthesis gene group is preferably agdZ, ugeZ, sphZ, egaZ, and gtbZ, and their homologs; and / or, more preferably, the above chitin biosynthesis gene group is preferably chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ, and their homologs. With this configuration, filamentous fungi in which wall growth is suppressed can be efficiently obtained. In this specification, the term "homolog" includes "ortholog" and "paralog" insofar as they have the same function.

[0029] The above agdZ, ugeZ, sphZ, egaZ, gtbZ, chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ are the names of genes found in Aspergillus oryzae.

[0030] The nucleotide sequences and other information for the above-mentioned agdZ, ugeZ, sphZ, egaZ, gtbZ, chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ are publicly known. For information regarding these nucleotide sequences, please refer to Table 5, which will be described later.

[0031] The homologs mentioned above may be orthologues or paralogs of these genes (agdZ, ugeZ, sphZ, egaZ, gtbZ, chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ) present in filamentous fungi other than Aspergillus oryzae. Information on these homologs, orthologues, and paralogs (e.g., gene names, nucleotide sequences, and amino acid sequences) is registered in public databases (for example, see the GenBank database of the National Center for Biotechnology Information (NCBI)), and these databases are referenced herein. Examples of information on these homologs are shown in Tables 1-4 below.

[0032] [Table 1]

[0033] [Table 2]

[0034] [Table 3]

[0035] [Table 4]

[0036] For example, whether a particular gene A is a homolog (e.g., ortholog or paralog) of gene B (agdZ, ugeZ, sphZ, egaZ, gtbZ, chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ) can be determined based on the following criteria.

[0037] For example, (i) the sequence identity between the base sequence of a specific gene A and the base sequence of gene B is 40% or more, preferably 45% or more, more preferably 50% or more, more preferably 55% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and more preferably 95% or more, and the physiological activity of the protein encoded by the specific gene A and the physiological activity of the protein encoded by gene B (e.g., galactosaminogalactan biosynthesis activity or chitin biosynthesis activity) are substantially the same, or (ii) the amino acids of the protein encoded by the specific gene A If the sequence identity between the sequence and the amino acid sequence of the protein encoded by gene B is 40% or more, preferably 45% or more, more preferably 50% or more, more preferably 55% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and more preferably 95% or more, and if the physiological activity of the protein encoded by a specific gene A is substantially the same as the physiological activity of the protein encoded by gene B (for example, galactosaminogalactan biosynthesis activity or chitin biosynthesis activity), then it can be determined that a specific gene A is a homolog of gene B. The above-described determination method is merely an example, and the present invention is not limited to this determination method.

[0038] Examples of gene B mentioned above include agdZ, ugeZ, sphZ, egaZ, gtbZ, chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ. However, gene B is not limited to these genes and may be homologs (e.g., orthologs or paralogs) of these genes. In this case, gene A can be determined based on the homologs of these genes.

[0039] By determining whether gene A is a homolog of gene B as described above, it is possible to identify genes belonging to the galactosaminogalactan biosynthesis gene group and genes belonging to the chitin biosynthesis gene group in filamentous fungi whose genomic information is known (for example, Aspergillus sojae).

[0040] The relationship between the base sequence of a specific gene A and the base sequence of gene B is preferably such that, when gene A is searched using NCBI's Blast search based on the base sequence of gene B, the base sequence of gene A has a score of 200 or higher. With this configuration, it is possible to more accurately determine whether a specific gene A is a homolog of gene B.

[0041] The above-mentioned physiological activity (for example, galactosaminogalactan biosynthesis activity or chitin biosynthesis activity) can be measured by known methods. For example, after mixing the protein encoded by gene A with the necessary substrate, it can be measured by checking whether (i) galactosaminogalactan, chitin, or its intermediates are synthesized, (ii) galactosaminogalactan, chitin, or its intermediates are transported, (iii) galactosaminogalactan, chitin, or its intermediates are modified, or (iv) galactosaminogalactan, chitin, or its intermediates are demodified. If the above synthesis, transport, modification, or demodification is confirmed, it can be determined that the protein encoded by gene A has the above-mentioned physiological activity.

[0042] The identity of two arrays can be determined, for example, using a mathematical algorithm. Such mathematical algorithms are not limited to, and include, for example, the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithm of Smith et al (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and the improved algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0043] The above-mentioned filamentous fungi are not limited to those belonging to the genera Aspergillus, Penicillium, Trichoderma, Rhizopus, Mucor, Fusarium, Monascus, Neurospora, or Talaromyces.

[0044] The filamentous fungi of the genus Aspergillus mentioned above are not limited to, and may include, for example, Aspergillus oryzae, Aspergillus sojae, Aspergillus nidulans (Emericella nidulans), Aspergillus niger, Aspergillus fumigatus, Aspergillus aculeatus, Aspergillus luchuensis, Aspergillus usamii, Aspergillus terreus, or Aspergillus phoenicis.

[0045] The filamentous fungi of the genus Penicillium mentioned above are not limited to, but may include, for example, Penicillium camemberti, Penicillium chrysogenum, Penicillium multicolor, Penicillium purpurogenum, or Penicillium roqueforti.

[0046] The filamentous fungi of the genus Trichoderma mentioned above are not limited to those listed above, and may include, for example, Trichoderma reesei or Trichoderma viride.

[0047] The filamentous fungi of the genus Rhizopus mentioned above are not limited to those listed above and may include, for example, Rhizopus oryzae or Rhizopus japonicus.

[0048] The filamentous fungi of the genus Mucor mentioned above are not limited to any one species, and could include, for example, Mucor sp.

[0049] The filamentous fungi of the genus Fusarium mentioned above are not limited to any one species, and could include, for example, Fusarium oxysporum.

[0050] The filamentous fungi of the genus Monascus mentioned above are not limited to any one species, and could include, for example, Monascus purpureus.

[0051] The filamentous fungi of the genus Neurospora mentioned above are not limited to those listed above, and could include, for example, Neurospora crassa.

[0052] The filamentous fungi of the genus Talaromyces mentioned above are not limited to this species and could include, for example, Talaromyces cellulolyticus.

[0053] The filamentous fungi described above may have foreign genes introduced into them. This configuration allows for the conferring of desired functions to the filamentous fungi (e.g., the ability to produce heterologous and / or homologous proteins, low molecular weight compounds, organic acids, and / or nucleic acids). The foreign genes may be genes derived from organisms of a different species from the filamentous fungi (heterologous genes), or genes derived from organisms of the same species as the filamentous fungi (homologous genes). Proteins encoded by heterologous genes are called heterologous proteins, and proteins encoded by homologous genes are called homologous proteins.

[0054] The above-mentioned foreign genes are not limited to, but may include, for example, polynucleotides (e.g., expression vectors) for expressing heterologous and / or homologous proteins, polynucleotides (e.g., expression vectors) for expressing synthases of compounds (e.g., small molecules and / or organic acids), and / or polynucleotides (e.g., promoter sequences, terminator sequences, and / or non-coding RNAs, etc.) for regulating the expression of specific genes.

[0055] The polynucleotides included in the above polynucleotide (e.g., expression vector) are not limited and may be appropriately selected depending on heterogeneous proteins, homogeneous proteins, and compounds. The polynucleotides included in the above polynucleotide (e.g., expression vector) may be, for example, polynucleotides encoding heterogeneous proteins and / or homogeneous proteins, or polynucleotides encoding enzymes involved in compound synthesis, or polynucleotides that can function as promoters and / or terminators.

[0056] The number of foreign genes introduced into the filamentous fungus is not limited; for example, it may be one or two or more. A number and type of foreign genes capable of conferring a desired function to the filamentous fungus should be introduced into the fungus.

[0057] [2. Method for culturing filamentous fungi] A method for culturing filamentous fungi according to one embodiment of the present invention comprises a step of culturing filamentous fungi according to one embodiment of the present invention. According to this configuration, it is possible to cultivate filamentous fungi while suppressing the formation of wall growth that occurs during cultivation.

[0058] The filamentous fungi cultured in the above process may be filamentous fungi without introduced foreign genes, or filamentous fungi with introduced foreign genes. If filamentous fungi without introduced foreign genes are cultured in the above process, the culture may contain substances that the filamentous fungi can originally produce. On the other hand, if filamentous fungi with introduced foreign genes are cultured in the above process, the culture may contain substances that the filamentous fungi can originally produce, as well as substances that become artificially produced by the foreign genes. These substances may be substances to be recovered in the method for producing substances according to one embodiment of the present invention, which will be described later.

[0059] The substances that the above-mentioned filamentous fungi can originally produce are not limited to those mentioned above, but include, for example, α-amylase, glucoamylase, protease, lipase, nuclease, kojic acid, deferriferricrysin, and biotin.

[0060] The substances that can be artificially produced by the above-mentioned foreign genes are not limited to those mentioned above, but include, for example, chymosin, human lysozyme, cellulase, oxidase, miraculin, and neocrine.

[0061] The method for culturing filamentous fungi in the above process is not limited; any general method used for culturing filamentous fungi should be adopted.

[0062] The culture medium used in the above process is not limited to, and may include, for example, DPY medium, modified DPY medium, CD minimal medium, YPD medium, TSB medium, malt medium, and PDA medium. These media may also be supplemented with a carbon source, such as glucose, dextrin, starch, and / or soluble starch.

[0063] The time for culturing the filamentous fungi in the above process is not limited and could be, for example, 12 hours to 7 days, 12 hours to 5 days, 12 hours to 4 days, or 12 hours to 3 days. This time should be set appropriately depending on the substance to be produced.

[0064] In the above process, it is preferable to culture the filamentous fungi in liquid culture in a culture tank. With this configuration, it is possible to culture a large amount of filamentous fungi while suppressing the formation of wall growth, which frequently occurs during liquid culture.

[0065] The above-mentioned culture vessels are not limited to test tubes, and may include, for example, test tubes, flasks (e.g., Erlenmeyer flasks, Sakaguchi flasks), jar fermenters, or large culture vessels.

[0066] The capacity of the culture vessel described above is not limited and may be, for example, 1 mL or more, 10 mL or more, 100 mL or more, 500 mL or more, 1 L or more, 5 L or more, 10 L or more, or 100 L or more. The upper limit of the capacity of the culture vessel described above is not limited and may be, for example, 10,000 L, 3,000 L, 1,000 L, 500 L, or 100 L.

[0067] [3. Methods of producing substances] A method for producing a substance according to one embodiment of the present invention includes a step of recovering a desired substance from a culture of filamentous fungi obtained by a culture method according to one embodiment of the present invention.

[0068] The method for recovering the desired substance in the above process is not limited and can be appropriately selected depending on the substance to be produced. For example, (i) the desired substance may be recovered by recovering a culture of filamentous fungi (e.g., culture medium, cells), and / or (ii) the desired substance may be recovered by subjecting the recovered culture of filamentous fungi (e.g., culture medium, cells) to a separation process (e.g., centrifugation, filtration, recrystallization, distillation, chromatography). The separation process is not limited and any known separation process can be employed.

[0069] According to one embodiment of the present invention, it is possible to provide filamentous fungi in which the formation of wall growth is suppressed, and to provide a means for utilizing said filamentous fungi. Therefore, one embodiment of the present invention may also contribute to achieving United Nations Sustainable Development Goal (SDG) 9, "Build resilient infrastructure, promote inclusive and sustainable industrialization and foster innovation," etc. [Examples]

[0070] The following are specific examples of the present invention, but the present invention is not limited thereto.

[0071] <1. Analysis of the ability to inhibit wall growth formation> <1-1. Strains> In this study, the wild-type strain of the filamentous fungus Aspergillus oryzae was identified as strain NS4 (genotype: sC). - niaD - A modified NS4 strain was used. This modified NS4 strain is a strain into which the ΔligD mutation, which improves the efficiency of gene transfer to the target site, has been introduced.

[0072] <1-2. Selection of genes to disrupt> In this study, the genes selected to be disrupted were those belonging to the galactosaminogalactan biosynthesis gene group and those belonging to the chitin biosynthesis gene group, as shown in Table 5 below.

[0073] The nucleotide sequence information for these genes, as well as the nucleotide sequences before and after these genes, was obtained from the CAoGD (Comprehensive Aspergillus oryzae genome database).

[0074] [Table 5]

[0075] <1-3. Preparation of plasmids for gene disruption> For each gene (target gene for disruption) listed in Table 5, sequence information A (1000-3000 base pairs upstream of the open reading frame (ORF)) and sequence information B (1000-3000 base pairs downstream of the open reading frame) were obtained from CAoGD.

[0076] Based on the acquired sequence information A, primers were designed to amplify 1000-3000 bases upstream of the open reading frame. Using these primers and Aspergillus oryzae genomic DNA as a template, PCR was performed to amplify 1000-3000 bases upstream of the open reading frame, obtaining amplified fragment A.

[0077] Based on the acquired sequence information B, primers were designed to amplify 1000-3000 bases downstream of the open reading frame. Using these primers and Aspergillus oryzae genomic DNA as a template, PCR was performed to amplify 1000-3000 bases downstream of the open reading frame, yielding amplified fragment B.

[0078] As a selection marker for transformants, the sC gene (nidsC) of Aspergillus nidrans was amplified by PCR using primers specific to the sC gene and Aspergillus nidrans genomic DNA as a template to obtain nidsC amplified fragments.

[0079] Furthermore, linearized pUC118 plasmids were obtained by cleaving the pUC118 plasmid (manufactured by Takara Bio Inc.) with appropriate restriction enzymes such as EcoRI.

[0080] Each of the aforementioned amplification fragments A, B, nidsC, and linearized pUC118 plasmid was separated by agarose gel electrophoresis, and regions of the agarose gel containing amplification fragment A, B, nidsC, or linearized pUC118 plasmid were excised. Using a commercially available kit, amplification fragment A, B, nidsC, and linearized pUC118 plasmid were purified from each of the excised agarose gels.

[0081] The purified amplification fragments A, B, and nidsC amplification fragment, along with the linearized pUC118 plasmid, were ligated together using seamless cloning to create a plasmid for gene disruption. A schematic diagram of the gene disruption plasmid structure is shown in Figure 1.

[0082] When this gene disruption plasmid is introduced into a filamentous fungus, amplification fragments A and B within the plasmid undergo homologous recombination with regions in the fungal genome that have the same base sequence as amplification fragments A and B. As a result, the nidsC amplification fragment (nidsC gene) from the gene disruption plasmid is inserted into the region in the fungal genome where the target gene exists, and the target gene in the fungal genome is disrupted.

[0083] <1-4. Creation of gene knockout strains> Using the gene disruption vectors for each gene listed in Table 5, prepared by the method described above, NS4 modified strains with the ΔligD mutation were transformed using the protoplast-PEG method.

[0084] Transformants were selected based on the complementation of nutritional requirements by the nidsC gene contained within the gene disruption plasmid.

[0085] Genomic DNA was extracted from the obtained transformants. Real-time PCR was performed using this genomic DNA and primers specific to the target gene. Transformants in which the copy number of the target gene was estimated to be 0 were determined to be transformants in which the target gene had been disrupted.

[0086] When further gene disruption was to be performed on transformants in which these target genes had been disrupted, marker recycling was carried out to enable further gene disruption by removing the nidsC gene inserted into the genome of the transformant by homologous recombination, and then selecting strains from which the nidsC gene had been removed in a selenic acid-containing medium (see Mizutani, O., Masaki, K., Gomi, K. and Iefuji, H.: Modified cre-loxp recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue, Appl. Environ. Microbiol., 78, 4126-4133 (2012)).

[0087] A list of gene knockout strains produced by the above method is shown in Table 6 below.

[0088] [Table 6]

[0089] <1-5. Culture of gene knockout strains and analysis of their ability to suppress wall growth - 1> The gene knockout strains listed in Table 6 were cultured for 4 days at 30°C in modified DPY medium (4% dextrin, 2% polypeptone, 2% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O).

[0090] Figure 2 shows an example of the culture vessel after culturing each gene knockout strain. More specifically, Figure 2 shows the NS4 modified strain (ΔligD) used as the wild-type Aspergillus oryzae, the single gene knockout strains (ΔligD, ΔagdZ) in which only agdZ ​​of the NS4 modified strain was disrupted, the single gene knockout strains (ΔligD, ΔchsB) in which only chsB of the NS4 modified strain was disrupted, the double gene knockout strains (ΔligD, ΔagdZ, ΔchsB) in which both agdZ ​​and chsB of the NS4 modified strain were disrupted, the triple gene knockout strains (ΔligD, ΔagdZ, ΔchsB, ΔchsA2) in which agdZ, chsB and chsA2 of the NS4 modified strain were disrupted, and the triple gene knockout strains (ΔligD, ΔagdZ) in which agdZ, chsB and chsC2 of the NS4 modified strain were disrupted. The images show the following: the state of ΔchsB, ΔchsC2), the state of triple gene knockout strains (ΔligD, ΔagdZ, ΔchsB, ΔchsD) in which agdZ, chsB, and chsD of the NS4 modified strain are disrupted, the state of triple gene knockout strains (ΔligD, ΔagdZ, ΔchsB, ΔchsD2) in which agdZ, chsB, and chsD2 of the NS4 modified strain are disrupted, the state of triple gene knockout strains (ΔligD, ΔagdZ, ΔchsB, ΔchsY) in which agdZ, chsB, and chsY of the NS4 modified strain are disrupted, and the state of triple gene knockout strains (ΔligD, ΔagdZ, ΔchsB, ΔchsZ) in which agdZ, chsB, and chsZ of the NS4 modified strain are disrupted.

[0091] As is clear from Figure 2, it was revealed that in filamentous fungi lacking one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group, wall growth formation is suppressed.

[0092] <1-6. Culture of gene knockout strains and analysis of their ability to suppress wall growth - 2> The gene knockout strains listed in Table 6 were cultured for 4 days at 30°C in modified DPY medium (4% dextrin, 2% polypeptone, 2% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O).

[0093] The bacterial cells (wall growth) adhering to the walls of the culture tank after cultivation were removed with tweezers, dehydrated, and then dried in an oven set to 100°C for 2 hours. The weight of the dried bacterial cells was then measured.

[0094] The dry weights of each gene knockout strain are shown in Figures 3-6. As is clear from Figures 3-6, compared to NS4 modified strains and single-gene knockout strains, filamentous fungi lacking one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group showed significantly suppressed wall growth formation.

[0095] <2. Analysis of heterologous protein production capacity in gene knockout strains> <2-1. Creation of heterologous protein expression strains using gene knockout strains as hosts> We evaluated the ability of gene knockout strains to produce heterologous proteins using lipase B (CALB) derived from Candida antarctica as a reporter protein.

[0096] Using a CALB expression vector (see Patent No. 7540709, Example 1), NS4 modified strains with the ΔligD mutation introduced (ΔligD), single-gene knockout strains of the NS4 modified strain with disrupted chsB (ΔchsB), and double-gene knockout strains of the NS4 modified strain with disrupted agdZ ​​and chsB (ΔagdZ, ΔchsB) were transformed by protoplast-PEG.

[0097] For each strain, transformants in which only one copy of the CALB expression vector was introduced into the chromosome were selected using real-time PCR.

[0098] Each selected transformant was cultured for 3 days in DPY medium (2% dextrin, 1% polypeptone, 1% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O), and the culture supernatant was collected as crude enzyme solution.

[0099] <2-2. Measurement of CALB activity> 5 μL of p-nitrophenyl butyrate (PNPB) was added to 250 μL of 100% ethanol, and the mixture was suspended using a pipette. The suspension was then diluted to 50 mL with distilled water to obtain a PNPB solution.

[0100] Next, a substrate solution was obtained by mixing 150 μL of PNPB solution with 50 μL of 20 mM phosphate buffer (pH 7.0).

[0101] To the obtained substrate solution, 2 μL of crude enzyme solution, diluted as appropriate, was added to initiate the reaction. The reaction temperature was 37°C and the reaction time was 3 minutes. CALB activity was measured by measuring the increase in absorbance at 400 nm.

[0102] Figure 7 shows the results of CALB activity measurement. In Figure 7, the CALB activity of single-gene knockout strains (ΔligD, ΔchsB) and double-gene knockout strains (ΔligD, ΔagdZ, ΔchsB) is shown as relative activity to the CALB activity of the NS4 modified strain (ΔligD).

[0103] As is clear from Figure 7, the heterologous protein production capacity of the NS4 modified strain, the single-gene knockout strain, and the double-gene knockout strain was almost identical. This indicates that filamentous fungi lacking one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group possess a sufficiently high heterologous protein production capacity for practical use. [Industrial applicability]

[0104] The present invention can be used in the production of substances (e.g., proteins, small molecule compounds).

Claims

1. One or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group are missing; or, A filamentous fungus in which the expression of one or more genes selected from the galactosaminogalactan biosynthesis gene group and one or more genes selected from the chitin biosynthesis gene group is suppressed.

2. The above-mentioned galactosaminogalactan biosynthesis gene group consists of agdZ, ugeZ, sphZ, egaZ, and gtbZ, and their homologs; and / or The filamentous fungus according to claim 1, wherein the above-mentioned group of chitin biosynthesis genes are chsA, chsA2, chsB, chsC, chsC2, chsD, chsD2, chsY, and chsZ, and their homologs.

3. The filamentous fungus according to claim 1, wherein the filamentous fungus is a filamentous fungus belonging to the genera Aspergillus, Penicillium, Trichoderma, Rhizopus, Mucor, Fusarium, Monascus, Neurospora, or Talaromyces.

4. The filamentous fungus described above is the filamentous fungus according to claim 1, wherein an exogenous gene has been introduced.

5. A method for culturing filamentous fungi, comprising the step of culturing the filamentous fungi described in any one of claims 1 to 4.

6. The method for culturing filamentous fungi according to claim 5, wherein the above step involves liquid culture of the filamentous fungi in a culture tank.

7. A method for producing a substance, comprising the step of recovering a desired substance from a culture of filamentous fungi obtained by the culture method described in claim 5.

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