Mutant filamentous fungus and substance production method using same

Abstract

A mutant filamentous fungus according to the present invention lacks at least a part of the GAG biosynthesis cluster and at least one hydrophobin gene, and does not biosynthesize α-1,3-glucan.

Description

Mutant filamentous fungus and method for producing substances using said mutant filamentous fungus

[0001] The present invention relates to a mutant filamentous fungus and a method for producing a substance using the mutant filamentous fungus.

[0002] Filamentous fungi are a general term for fungi composed of tubular cells called hyphae, and are used in the fermentative production of low-molecular-weight compounds such as organic acids, pigments, and chemical products including agricultural chemicals, pharmaceuticals including penicillin and statins, and industrial enzymes such as amylase, cellulase, protease, and lipase.

[0003] For example, Patent Document 1 describes a method for producing cellulase, which includes a step of adding β-glucosidase derived from a thermophilic bacterium to a glucose-containing solution to produce a disaccharide-containing solution by a condensation reaction, and a step of producing cellulase by culturing a filamentous fungus in a medium containing the disaccharide-containing solution.

[0004] Furthermore, Patent Document 2 describes a method for producing phospholipase, which includes a step of processing a fungal peptide to remove a peptide from the C-terminus and / or a peptide from the N-terminus, thereby generating a core peptide having phospholipase activity and consisting of a specific amino acid sequence.

[0005] Furthermore, Patent Documents 3 to 7 describe expression vectors constructed to allow filamentous fungi to function as hosts, with the aim of improving the efficiency of substance production by filamentous fungi, and methods for producing transformants by introducing into filamentous fungi a plasmid in which a gene encoding a homologous or heterologous protein is functionally linked to the expression vector, and further describe how the use of the transformants contributes to increased production of enzymes such as amylase and cellulase, and low-molecular-weight compounds such as penicillin.

[0006] As described above, filamentous fungi have the advantage of being able to produce a wide variety of useful substances. However, in the liquid culture process, filamentous fungi have problems such as the inability to achieve high-density culture due to entanglement of hyphae, which causes fungal bodies to clump together, resulting in a decrease in the amount of useful substances produced, and the complexity of the process for producing useful substances (e.g., Patent Documents 8 and 9).

[0007] Under these circumstances, the present inventors have discovered that by using a mutant filamentous fungus that does not biosynthesize α-1,3-glucan, aggregation of fungal cells during culture is suppressed more than conventional methods, resulting in relatively uniform dispersion of fungal cells in the medium, and that by deleting at least a portion of the galactosaminogalactan (GAG) biosynthesis gene cluster from the mutant that does not biosynthesize α-1,3-glucan, the dispersibility of the fungal cells in the medium is further increased, and have developed a substance production method (Patent Documents 10 and 11). However, even when using these filamentous fungi, there is still a problem of reduced productivity of useful substances due to their biological characteristics, namely, adhesion of fungal cells to the inner walls of the fermenter tank and to tank installations such as the agitator blades and agitator shaft, resulting in a decrease in the liquid component. From the perspective of improving substance production efficiency, there is a need for the development of filamentous fungi that have reduced adhesion to the inner walls of the fermenter tank.

[0008] JP 2010-227032, JP 2010-172343, JP 2001-46078, JP 2005-52116, JP 2009-118783, JP 11-506025, JP 2007-508022, JP 2002-218970, JP 2010-227031, WO 2014 / 073674, WO 2018 / 203566

[0009] Fontaine T. et al. (2011) Galactosaminogalactan, a New Immunosupressive Polysacharide of Aspergillus fumigatus, PLoS Pathogens, 7:e1002372Rappleye C.A. et al. (2004) RNA interference in Histoplasma capsulatum demonstrates a role for α-(1,3)-glucan in virulence. Mol. Microbiol. 53:153-165.Beauvais A. et al. (2005) Two α(1-3) Glucan Synthases with Different Functions in Aspergillus fumigatus. Appl. Environ. Microbiol. 71:1531-1538.Maubon D. et al. (2006) AGS3, an α(1-3)glucan synthase gene family member of Aspergillus fumigatus, modulates mycelium growth in the lung of experimentally infected mice. Fungal Genet. Biol. 43:366-375.Henry C. et al. (2011) α1,3 glucans are dispensable in Aspergillus fumigatus. Eukaryot. Cell 11:26-29Mizutani O. et al. (2008) A defect of LigD(human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fung. Genet. Biol., 45:878-889.Zhang S. et al.(2017) Self-excising Cre / mutant lox marker recycling system for multiple gene integrations and consecutive gene deletion in Aspergillus oryzae. J. Biosci. Bioengin. 123:403-411Gomi K. et al. (1987) Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 51:2549-2555Bamford N. et al.(2015) Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus.J Biol Chem 290, 27438Takahashi T. et al. (2015) Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1. Mol. Microbiol. 96(1):14-27. doi: 10.1111 / mmi.12915Aimanianda V. et al.(2009) Surface hydrophobin prevents immune recognition of airborne fungal spores. Nat. Cell Biol. 2009, 460, 1117-1121. https: / / doi.org / 10.1038 / nature08264Mizutani O. et al.(2016) Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae. Biosci. Biotech. Biochem. 80 (9):1781-1791.

[0010] An object of the present invention is to provide a mutant filamentous fungus that has reduced adhesion to culture vessels compared to conventional filamentous fungi.

[0011] Under these circumstances, the present inventors have conducted extensive research into the various factors possessed by filamentous fungi from the perspective of adhesion of fungal cells to the inner walls of a culture vessel, and as a result have discovered the surfactant protein hydrophobin as a new factor. The present inventors have found that by deleting the function of the GAG ​​biosynthesis gene cluster and further deleting the hydrophobin gene in filamentous fungi that have been modified so as not to biosynthesize α-1,3-glucan or in filamentous fungi that do not originally possess the α-1,3-glucan synthase gene ags, they have surprisingly found that adhesion of fungal cells to the inner walls of a culture vessel is suppressed, the concentration of fungal cells in the culture medium is increased, the stirring efficiency of the culture medium is improved, the oxygen transfer rate within the culture vessel is increased, and the efficiency of substance production is also increased. The present invention is based on this novel finding.

[0012] Accordingly, the present invention provides the following: Item 1. A mutant filamentous fungus that lacks at least a portion of a galactosaminogalactan (GAG) biosynthetic gene cluster and at least one hydrophobin gene, and that does not biosynthesize α-1,3-glucan. Item 2. The filamentous fungus according to Item 1, wherein at least a portion of the GAG ​​biosynthetic gene cluster includes at least one gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3. Item 3. The filamentous fungus according to Item 1 or 2, wherein at least one of the hydrophobin genes includes at least one gene selected from the group consisting of rolA, hypB, hypC, hypD, and hypE. Item 4. The filamentous fungus according to any one of Items 1 to 3, wherein at least one of the hydrophobin genes includes rolA. Item 5. Item 6. The filamentous fungus according to any one of Items 1 to 4, which does not biosynthesize α-1,3-glucan due to a deficiency in at least one of the α-1,3-glucan synthases Ags. Item 7. The filamentous fungus according to any one of Items 1 to 6, which belongs to the genus Aspergillus, Botrytis, Cochliobolus, or Emmarisella. Item 8. The filamentous fungus according to any one of Items 1 to 7, which is Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus fumigatus, Botrytis cinerea, Cochliobolus heterostrophos, or Emmarisella nidulans. Item 9. A method for producing a substance, comprising the step of culturing the filamentous fungus according to any one of Items 1 to 8 and allowing the filamentous fungus to produce a substance. Item 10. Item 10. The method according to Item 9, wherein the substance is a substance other than a constituent component of the cell wall of a filamentous fungus or a hydrolysate thereof.

[0013] According to the present invention, it is possible to provide a mutant filamentous fungus that has reduced adhesion to the inner walls of a culture tank, etc., compared to conventional filamentous fungi. Furthermore, in a typical embodiment, the filamentous fungus of the present invention is useful because it is more likely to increase the fungal cell concentration in a culture medium and more likely to increase the efficiency of substance production than conventional filamentous fungal mutants.

[0014] Photographs of the flask appearance after culture in a 72-hour culture test using chemically defined medium medium are shown. The wall-adhering bacterial cell mass after culture in a 72-hour culture test using chemically defined medium medium is shown. It was 0.47±0.03 for the AG-deficient strain and 0.27±0.08 for the AG-rolA-deficient strain. Recombinant enzyme activity after culture in a culture test using chemically defined medium medium. Bacterial cell concentration after culture in a culture test using chemically defined medium medium. Photographs of the culture vessel after culture in a liquid shaking culture test using YPD medium are shown. Weight of wall-adhering bacterial cells after culture in a liquid shaking culture test using YPD medium. Recombinant enzyme activity after culture in a culture test using chemically defined medium medium of Example 2. Bacterial cell concentration after culture in a culture test using chemically defined medium medium of Example 2. Amylase activity results for a 48-hour culture test using chemically defined medium medium of Example 3 are shown. Bacterial cell concentration results for a 48-hour culture test using chemically defined medium medium of Example 3 are shown. 1 shows the results of amylase activity per cell in a 48-hour culture test using a chemically defined medium in Example 3.

[0015] As used herein, the singular forms (a, an, the, etc.) include both the singular and the plural unless otherwise specified herein or clearly contradictory in context. As used herein, "comprise" is a concept that also encompasses "consist essentially of" and "consist of."

[0016] As used herein, the term "wild-type strain" refers to the original filamentous fungal strain that has been subjected to artificial modification such as gene introduction, and which has not been artificially modified.

[0017] The present invention provides a mutant filamentous fungus that lacks at least a part of the galactosaminogalactan (GAG) biosynthetic gene cluster and at least one hydrophobin gene, and that does not biosynthesize α-1,3-glucan.

[0018] Filamentous Fungal Mutant Strain In the present invention, the term "mutant filamentous fungus that does not biosynthesize α-1,3-glucan" refers to a filamentous fungal mutant strain that does not biosynthesize α-1,3-glucan at all, and also includes mutant strains that do not substantially biosynthesize α-1,3-glucan. More specifically, a mutant strain that does not substantially biosynthesize α-1,3-glucan refers to a mutant strain that only biosynthesizes a small amount of α-1,3-glucan and in which the entanglement of hyphae, resulting in the formation of clumps of fungal cells, is significantly suppressed. For example, the amount of α-1,3-glucan biosynthesis may be 30% or less of that of a wild-type strain, more preferably 10% or less of that of a wild-type strain. The amount of α-1,3-glucan biosynthesis can be evaluated by analyzing the cell wall constituent sugars using the method described in Non-Patent Document 12. Furthermore, the "filamentous fungus that does not biosynthesize α-1,3-glucan" in the present invention also includes filamentous fungi that do not originally biosynthesize α-1,3-glucan, typically filamentous fungi that do not have the α-1,3-glucan synthase gene ags in the wild type. The "filamentous fungus that does not biosynthesize α-1,3-glucan" in the present invention also includes filamentous fungi such as B. cinerea that have the α-1,3-glucan synthase gene ags but do not biosynthesize α-1,3-glucan because the α-1,3-glucan synthase gene ags is not expressed under specific culture conditions, for example, liquid culture conditions.

[0019] Examples of filamentous fungi include the genus Aspergillus, the genus Penicillium (e.g., Penicillium chrysogenum), the genus Trichoderma, the genus Cephalosporium, the genus Acremonium, the genus Neurospora, the genus Botrytis, the genus Cochliobolus, the genus Emericella, the genus Monascus, etc. Of these, the genera Aspergillus, Botrytis, and Cochliobolus are more preferred, with the genus Aspergillus being more preferred. Examples of filamentous fungi of the genus Aspergillus used in the present invention include Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, and Aspergillus fumigatus. Aspergillus oryzae, Aspergillus sojae, or Aspergillus niger are preferred, Aspergillus oryzae and Aspergillus sojae are more preferred, and Aspergillus oryzae is even more preferred. Examples of filamentous fungi of the genus Botrytis used in the present invention include Botrytis cinerea (teleomorph: Botryotinia fuckeliana), Botrytis alli, Botrytis scamosa, Botrytis bysoysia, etc. Examples of filamentous fungi of the genus Cochliobolus used in the present invention include Cochliobolus heterostrophus (anamorph: Bipolaris maydis), Cochliobolus carbonum, Cochliobolus miavianus, Cochliobolus victoria, etc. Examples of filamentous fungi of the genus Emerysella include Emerysella nidulans, etc. Examples of the Monascus genus include Monascus purpureus (red koji mold), Monascus ruber, and Monascus pilosus.

[0020] The filamentous fungal mutant strain of the present invention that does not biosynthesize α-1,3-glucan includes one that is deficient in at least one of the α-1,3-glucan synthase genes ags. Examples of the α-1,3-glucan synthase gene ags include agsA (Genbank accession no. AN5885) and agsB (Genbank accession no. AN3307) from Emerysella nidulans, agsA, agsB, and agsC from Aspergillus oryzae, agsA, agsB, and agsC from Aspergillus sojae, ags1 from Aspergillus fumigatus (Genbank accession no. AFUA_3G00910), agsE from Aspergillus niger (Genbank accession no. ANI_1_360084), and agsB from Penicillium chrysogenum (Genbank accession no. Pc16g06130). Here, the agsA, agsB, and agsC genes of Aspergillus oryzae are registered in the Aspergillus database AspGD (http: / / www.aspergillusgenome.org) under the gene numbers agsA (AOR_1_956014), agsB (AOR_1_2634154), and agsC (AOR_1_1350024). Examples of the amino acid sequence and nucleotide sequence of the α-1,3-glucan synthase gene ags from Aspergillus oryzae and the like are shown in the sequence listing: Deduced amino acid sequence of AgsA from Aspergillus oryzae (SEQ ID NO: 1) Nucleotide sequence of the nucleic acid molecule encoding AgsA from Aspergillus oryzae (SEQ ID NO: 2) Deduced amino acid sequence of AgsB from Aspergillus oryzae (SEQ ID NO: 3) Nucleotide sequence of the nucleic acid molecule encoding AgsB from Aspergillus oryzae (SEQ ID NO: 4) Deduced amino acid sequence of AgsC from Aspergillus oryzae (SEQ ID NO: 5) Nucleotide sequence of the nucleic acid molecule encoding AgsC from Aspergillus oryzae (SEQ ID NO: 6) Deduced amino acid sequence of AgsA from Emmarisella nidulans (SEQ ID NO: 7) Nucleotide sequence of the nucleic acid molecule encoding AgsA from Emmarisella nidulans (SEQ ID NO: 8) Deduced amino acid sequence of AgsB from Emmarisella nidulans (SEQ ID NO: 9) Nucleotide sequence of the nucleic acid molecule encoding AgsB from Emmarisella nidulans (SEQ ID NO: 10)

[0021] Examples of the amino acid sequences of AgsA, AgsB, and AgsC of Aspergillus sojae include amino acid sequences deduced from gene sequences registered in GenBank (Genbank accession nos. DF093557-DF093585) based on homology with Aspergillus oryzae.

[0022] Examples of the amino acid sequence and nucleotide sequence of the α-1,3-glucan synthase gene ags from Aspergillus sojae and the like are shown in the sequence listing: Deduced amino acid sequence of AgsA from Aspergillus sojae (SEQ ID NO: 11) Deduced nucleotide sequence of the nucleic acid molecule encoding the above AgsA from Aspergillus sojae (SEQ ID NO: 12) Deduced amino acid sequence of AgsB from Aspergillus sojae (SEQ ID NO: 13) Deduced nucleotide sequence of the nucleic acid molecule encoding AgsB from Aspergillus sojae (SEQ ID NO: 14) Deduced amino acid sequence of AgsC from Aspergillus sojae (SEQ ID NO: 15) Deduced nucleotide sequence of the nucleic acid molecule encoding AgsC from Aspergillus sojae (SEQ ID NO: 16) Deduced amino acid sequence of AgsE from Aspergillus niger (SEQ ID NO: 17) Deduced nucleotide sequence of the nucleic acid molecule encoding AgsE from Aspergillus niger (SEQ ID NO: 18) Deduced amino acid sequence of Ags1 from Aspergillus fumigatus (SEQ ID NO: 19) Aspergillus Nucleic acid sequence of a nucleic acid molecule encoding Ags1 from Penicillium fumigatus (SEQ ID NO: 20) Deduced amino acid sequence of AgsB from Penicillium chrysogenum (SEQ ID NO: 71) Nucleic acid sequence of a nucleic acid molecule encoding AgsB from Penicillium chrysogenum (SEQ ID NO: 72)

[0023] Mutant filamentous fungi include those lacking one or more of these α-1,3-glucan synthase genes, with the one or more including at least agsB being preferred, and all three being preferred. Mutant filamentous fungi include those that do not express one or more of these α-1,3-glucan synthase genes, with the one or more including at least agsB being preferred, and all three being preferred. In the present invention, the concept of a deficiency in any of the above-mentioned α-1,3-glucan synthase genes also includes a deficiency in an ortholog of that gene. For example, the term "deficiency in agsB" also includes a deficiency in its ortholog, such as a deficiency in ags1 from Aspergillus fumigatus, agsB from Aspergillus sojae, or agsE from Aspergillus niger. The same applies to the concept of not expressing any of the α-1,3-glucan synthase genes.

[0024] In the present invention, a deficiency in the α-1,3-glucan synthase gene ags refers to a deletion of all or part of the coding region of the α-1,3-glucan synthase in the genome, an insertion of another nucleic acid molecule into all or part of the coding region, or a replacement of all or part of the coding region with another nucleic acid molecule. Deficiency in the α-1,3-glucan synthase gene ags also includes a gene deficiency that suppresses expression of the coding region by inserting, deleting, or substituting a specific nucleic acid molecule into a non-coding region in the genome involved in regulating the expression of the coding region. Furthermore, filamentous fungi lacking the α-1,3-glucan synthase gene ags also include filamentous fungi that have a gene deficiency due to a mutation in the gene region encoding the active center of α-1,3-glucan synthase, resulting in the expression of a protein that is still capable of producing the enzyme protein but lacks its function as a synthase (as a result, α-1,3-glucan biosynthesis is no longer possible). Furthermore, in addition to the addition, deletion, and substitution of a specific nucleic acid molecule in the coding region, conditional gene deletions are also included, which are designed so that α-1,3-glucan is expressed only under certain conditions without the addition of a nucleic acid molecule in the coding region. Examples of conditional gene deletions include those using the Cre-LoxP system. By deleting the α-1,3-glucan synthase gene ags, the gene responsible for α-1,3-glucan biosynthesis can be knocked out. Due to the deficiency of any of the above-mentioned α-1,3-glucan synthase genes ags, the α-1,3-glucan synthase protein is not expressed, the expression level as a synthase protein is 30% or less of that of a wild-type strain of filamentous fungus, more preferably 10% or less of that of the wild-type strain, the protein is expressed as a protein that has lost its function as a synthase (enzymatic activity), or the protein is expressed as a protein whose function as a synthase (enzymatic activity) has been reduced to 30% or less of that of a wild-type strain of filamentous fungus, more preferably 10% or less of that of the wild-type strain, resulting in a filamentous fungal mutant strain that does not biosynthesize α-1,3-glucan according to the present invention.

[0025] The filamentous fungal mutant strain of the present invention not only does not biosynthesize α-1,3-glucan, but also lacks at least a portion of the galactosaminogalactan (GAG) biosynthetic gene cluster.

[0026] Galactosaminogalactan is an extracellular polysaccharide identified in Aspergillus fumigatus in 2011, and is composed of galactose (Gal), N-acetylgalactosamine (GalNAc), and galactosamine (GalN) (Non-Patent Document 1). Genes that make up the GAG ​​biosynthetic gene cluster include uge3, sph3, ega3, agd3, and gtb3.

[0027] Therefore, examples of the filamentous fungal mutant strain of the present invention that is deficient in at least a portion of the GAG ​​biosynthetic gene cluster include those that are deficient in at least one gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3. In one embodiment of the present invention, for example, examples of the filamentous fungal mutant strain that is also deficient in at least a portion of the GAG ​​biosynthetic gene cluster include those that are deficient in at least uge3 and sph3 among these genes. In the present invention, the concept of "defective in at least a portion of the GAG ​​biosynthetic gene cluster" also includes the deletion of orthologs of the genes. For example, the term "defective in uge3" also includes the deletion of orthologs such as uge3 from Aspergillus fumigatus, uge3 from Aspergillus sojae, and uge3 from Aspergillus niger. The same applies to the concept of not expressing at least a portion of the GAG ​​biosynthetic gene cluster.

[0028] Examples of these genes constituting the GAG ​​biosynthesis gene cluster include uge3 (Genbank accession no. AOR_1_2588174), sph3 (Genbank accession no. AOR_1_2586174), ega3 (Genbank accession no. AOR_1_2584174), agd3 (Genbank accession no. AOR_1_2582174), and gtb3 (Genbank accession no. AOR_1_2580174) from Aspergillus oryzae; uge3 (Genbank accession no. AN2951), sph3 (Genbank accession no. AN2952), ega3 (Genbank accession no. AN2953), agd3 (Genbank accession no. AN2954), and gtb3 (Genbank accession no. AN2955) from Emerysella nidulans; sojae uge3, sph3, ega3, agd3, and gtb3; Aspergillus niger uge3 (Genbank accession No. ANI_1_1578024), sph3 (Genbank accession No. ANI_1_3046024), ega3 (Genbank accession No. ANI_1_1582024), agd3 (Genbank accession No. ANI_1_3048024), and gtb3 (Genbank accession No. ANI_1_3050024); Aspergillus fumigatus uge3 (Genbank accession No. AFUA_3G07910), sph3 (Genbank accession No. AFUA_3G07900), ega3 (Genbank accession No. AFUA_3G07890), and agd3 (Genbank accession No. AFUA_3G07910). No. AFUA_3G07870) and gtb3 (Genbank accession No. AFUA_3G07860) and Penicillium chrysogenum uge3 (Genbank accession No. Pc20g06140), sph3 (Genbank accession No.Pc20g06130), ega3 (Genbank accession no. Pc20g06110), agd3 (Genbank accession no. Pc20g06090), and gtb3 (Genbank accession no. Pc20g06080), uge3 (Gene ID: COCHEDRAFT_1185586), sph3 (Gene ID: COCHEDRAFT_1023805), ega3 (Gene ID: COCHEDRAFT_1023806), agd3 (Gene ID: COCHEDRAFT_1146217), gtb3 (Gene ID: COCHEDRAFT_1146218), uge3 (Gene ID: COCHEDRAFT_1146219), and uge3 (Gene ID: COCHEDRAFT_1146220) from Botrytis cinerea. Examples include Bcin01p05750.1), sph3 (Gene ID: Bcin01p05740.1), ega3 (Gene ID: Bcin01p05730.1), agd3 (Gene ID: Bcin01p05720.1), and gtb3 (Gene ID: Bcin01p05710.1).

[0029] Examples of amino acid sequences and nucleotide sequences of Aspergillus oryzae and the like are shown in the sequence listing: Amino acid sequence of Uge3 of Aspergillus oryzae (SEQ ID NO:21) Nucleotide sequence of a nucleic acid molecule encoding Uge3 of Aspergillus oryzae (SEQ ID NO:22) Amino acid sequence of Sph3 of Aspergillus oryzae (SEQ ID NO:23) Nucleotide sequence of a nucleic acid molecule encoding Sph3 of Aspergillus oryzae (SEQ ID NO:24) Amino acid sequence of Ega3 of Aspergillus oryzae (SEQ ID NO:25) Nucleotide sequence of a nucleic acid molecule encoding Ega3 of Aspergillus oryzae (SEQ ID NO:26) Amino acid sequence of Agd3 of Aspergillus oryzae (SEQ ID NO:27) Nucleotide sequence of a nucleic acid molecule encoding Agd3 of Aspergillus oryzae (SEQ ID NO:28) Amino acid sequence of Gtb3 of Aspergillus oryzae (SEQ ID NO:29) Nucleotide sequence of a nucleic acid molecule encoding Gtb3 of Aspergillus oryzae (SEQ ID NO:30) Emerysella Amino acid sequence of Uge3 from Emmalisella nidulans (SEQ ID NO: 31) Nucleotide sequence of a nucleic acid molecule encoding Uge3 from Emmalisella nidulans (SEQ ID NO: 32) Amino acid sequence of Sph3 from Emmalisella nidulans (SEQ ID NO: 33) Nucleotide sequence of a nucleic acid molecule encoding Sph3 from Emmalisella nidulans (SEQ ID NO: 34) Amino acid sequence of Ega3 from Emmalisella nidulans (SEQ ID NO: 35) Nucleotide sequence of a nucleic acid molecule encoding Ega3 from Emmalisella nidulans (SEQ ID NO: 36) Amino acid sequence of Agd3 from Emmalisella nidulans (SEQ ID NO: 37) Nucleotide sequence of a nucleic acid molecule encoding Agd3 from Emmalisella nidulans (SEQ ID NO: 38) Amino acid sequence of Gtb3 from Emmalisella nidulans (SEQ ID NO: 39) Nucleotide sequence of a nucleic acid molecule encoding Gtb3 from Emmalisella nidulans (SEQ ID NO: 40).

[0030] In the present invention, the amino acid sequences of Uge3, Sph3, Ega3, Agd3, and Gtb3 of Aspergillus sojae include amino acid sequences deduced from the gene sequences of Aspergillus sojae registered in GenBank (Genbank accession nos. DF093557-DF093585) based on their homology with Aspergillus oryzae.

[0031] Examples of amino acid sequences and nucleotide sequences of Aspergillus sojae and the like are shown in the sequence listing: Deduced amino acid sequence of Uge3 from Aspergillus sojae (SEQ ID NO: 41) Deduced nucleotide sequence of a deduced nucleic acid molecule encoding Uge3 from Aspergillus sojae (SEQ ID NO: 42) Deduced amino acid sequence of Sph3 from Aspergillus sojae (SEQ ID NO: 43) Deduced nucleotide sequence of a deduced nucleic acid molecule encoding Sph3 from Aspergillus sojae (SEQ ID NO: 44) Deduced amino acid sequence of Ega3 from Aspergillus sojae (SEQ ID NO: 45) Deduced nucleotide sequence of a deduced nucleic acid molecule encoding Ega3 from Aspergillus sojae (SEQ ID NO: 46) Deduced amino acid sequence of Agd3 from Aspergillus sojae (SEQ ID NO: 47) Deduced nucleotide sequence of a deduced nucleic acid molecule encoding Agd3 from Aspergillus sojae (SEQ ID NO: 48) Deduced amino acid sequence of Gtb3 from Aspergillus sojae (SEQ ID NO: 49) Deduced nucleotide sequence of a deduced nucleic acid molecule encoding Gtb3 from Aspergillus sojae (SEQ ID NO: 50). Deduced amino acid sequence of Aspergillus niger Uge3 (SEQ ID NO:51) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger Uge3 (SEQ ID NO:52) Deduced amino acid sequence of Aspergillus niger Sph3 (SEQ ID NO:53) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger Sph3 (SEQ ID NO:54) Deduced amino acid sequence of Aspergillus niger Ega3 (SEQ ID NO:55) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger Ega3 (SEQ ID NO:56) Deduced amino acid sequence of Aspergillus niger Agd3 (SEQ ID NO:57) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger Agd3 (SEQ ID NO:58) Deduced amino acid sequence of Aspergillus niger Gtb3 (SEQ ID NO:59) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger Gtb3 (SEQ ID NO:60) Deduced amino acid sequence of Aspergillus fumigatus Uge3 (SEQ ID NO:61) Nucleotide sequence of a nucleic acid molecule encoding Uge3 from Aspergillus fumigatus (SEQ ID NO: 62) Deduced amino acid sequence of Sph3 from Aspergillus fumigatus (SEQ ID NO: 63) Nucleotide sequence of a nucleic acid molecule encoding Sph3 from Aspergillus fumigatus (SEQ ID NO: 64) Deduced amino acid sequence of Ega3 from Aspergillus fumigatus (SEQ ID NO: 65)Nucleotide sequence of a nucleic acid molecule encoding Ega3 from Aspergillus fumigatus (SEQ ID NO: 66) Deduced amino acid sequence of Agd3 from Aspergillus fumigatus (SEQ ID NO: 67) Nucleotide sequence of a nucleic acid molecule encoding Agd3 from Aspergillus fumigatus (SEQ ID NO: 68) Deduced amino acid sequence of Gtb3 from Aspergillus fumigatus (SEQ ID NO: 69) Nucleotide sequence of a nucleic acid molecule encoding Gtb3 from Aspergillus fumigatus (SEQ ID NO: 70) Deduced amino acid sequence of Uge3 from Penicillium chrysogenum (SEQ ID NO: 73) Nucleotide sequence of a nucleic acid molecule encoding Uge3 from Penicillium chrysogenum (SEQ ID NO: 74) Deduced amino acid sequence of Sph3 from Penicillium chrysogenum (SEQ ID NO: 75) Nucleotide sequence of a nucleic acid molecule encoding Sph3 from Penicillium chrysogenum (SEQ ID NO: 76) Deduced amino acid sequence of Ega3 from Penicillium chrysogenum (SEQ ID NO: 77) Nucleotide sequence of a nucleic acid molecule encoding Ega3 from Penicillium chrysogenum (SEQ ID NO: 78) Deduced amino acid sequence of Agd3 from Penicillium chrysogenum (SEQ ID NO: 79) Nucleotide sequence of a nucleic acid molecule encoding Agd3 from Penicillium chrysogenum (SEQ ID NO: 80) Deduced amino acid sequence of Gtb3 from Penicillium chrysogenum (SEQ ID NO: 81) Nucleotide sequence of a nucleic acid molecule encoding Gtb3 from Penicillium chrysogenum (SEQ ID NO: 82) Deduced amino acid sequence of Uge3 from Cochliobolus heterostrophos (anamorph: Bipolaris maydis) (SEQ ID NO: 83) Nucleotide sequence of a nucleic acid molecule encoding Uge3 from Cochliobolus heterostrophos (SEQ ID NO: 84) Deduced amino acid sequence of Sph3 from Cochliobolus heterostrophos (SEQ ID NO: 85) Nucleotide sequence of a nucleic acid molecule encoding Sph3 from Cochliobolus heterostrophos (SEQ ID NO: 86) Cochliobolus Deduced amino acid sequence of Ega3 of Cochliobolus heterostrophos (SEQ ID NO: 87) Nucleotide sequence of a nucleic acid molecule encoding Ega3 of Cochliobolus heterostrophos (SEQ ID NO: 88) Deduced amino acid sequence of Agd3 of Cochliobolus heterostrophos (SEQ ID NO: 89) Nucleotide sequence of a nucleic acid molecule encoding Agd3 of Cochliobolus heterostrophos (SEQ ID NO: 90) Deduced amino acid sequence of Gtb3 of Cochliobolus heterostrophos (SEQ ID NO: 91)Nucleotide sequence of a nucleic acid molecule encoding Gtb3 of Cochliobolus heterostrophos (SEQ ID NO: 92) Deduced amino acid sequence of Ags1 of Botrytis cinerea (SEQ ID NO: 93) Nucleotide sequence of a nucleic acid molecule encoding Ags1 of Botrytis cinerea (SEQ ID NO: 94) Deduced amino acid sequence of Uge3 of Botrytis cinerea (SEQ ID NO: 95) Nucleotide sequence of a nucleic acid molecule encoding Uge3 of Botrytis cinerea (SEQ ID NO: 96) Deduced amino acid sequence of Sph3 of Botrytis cinerea (SEQ ID NO: 97) Nucleotide sequence of a nucleic acid molecule encoding Sph3 of Botrytis cinerea (SEQ ID NO: 98) Deduced amino acid sequence of Ega3 of Botrytis cinerea (SEQ ID NO: 99) Nucleotide sequence of a nucleic acid molecule encoding Ega3 of Botrytis cinerea (SEQ ID NO: 100) Deduced amino acid sequence of Agd3 of Botrytis cinerea (SEQ ID NO: 101) Botrytis Nucleotide sequence of a nucleic acid molecule encoding Agd3 from Botrytis cinerea (SEQ ID NO: 102) Deduced amino acid sequence of Gtb3 from Botrytis cinerea (SEQ ID NO: 103) Nucleotide sequence of a nucleic acid molecule encoding Gtb3 from Botrytis cinerea (SEQ ID NO: 104)

[0032] In the present invention, the deletion of at least a portion of a GAG biosynthetic gene cluster includes, for example, deletion of all or a portion of the coding region of the GAG ​​biosynthetic gene cluster in the genome, insertion of another nucleic acid molecule into all or a portion of the coding region, or replacement of all or a portion of the coding region with another nucleic acid molecule. The deletion of at least a portion of a GAG biosynthetic gene cluster also includes a gene defect that suppresses expression of the coding region by inserting, deleting, or substituting a specific nucleic acid molecule into a non-coding region in the genome involved in regulating the expression of the coding region. Furthermore, filamentous fungi lacking at least a portion of a GAG biosynthetic gene cluster may also include filamentous fungi that have a gene defect that causes expression of a gene that produces the enzyme protein but loses its function as a synthase due to a mutation in the gene region encoding the active center of at least one type of GAG synthase (resulting in no GAG biosynthesis). Furthermore, deletion of at least a portion of the GAG ​​biosynthesis gene cluster includes not only addition, deletion, and substitution of a specific nucleic acid molecule in the coding region, but also conditional gene deletions in which there is no addition of a nucleic acid molecule in the coding region but the GAG ​​biosynthesis genes are designed to be expressed only under certain conditions.

[0033] In the present invention, a deficiency in uge3 refers to, for example, a deletion of all or part of the Uge3 coding region in the genome, an insertion of another nucleic acid molecule into all or part of the coding region, or a replacement of all or part of the coding region with another nucleic acid molecule. In the present invention, a deficiency in sph3 refers to, for example, a deletion of all or part of the Sph3 coding region in the genome, an insertion of another nucleic acid molecule into all or part of the coding region, or a replacement of all or part of the coding region with another nucleic acid molecule. In the present invention, a deficiency in ega3 refers to, for example, a deletion of all or part of the Ega3 coding region in the genome, an insertion of another nucleic acid molecule into all or part of the coding region, or a replacement of all or part of the coding region with another nucleic acid molecule. In the present invention, agd3 deficiency refers to, for example, a deletion of all or part of the Agd3 coding region in the genome, a deletion of all or part of the coding region with another nucleic acid molecule, a substitution of all or part of the coding region with another nucleic acid molecule, etc. In the present invention, gtb3 deficiency refers to, for example, a deletion of all or part of the Gtb3 coding region in the genome, a deletion of all or part of the Gtb3 coding region with another nucleic acid molecule, a substitution of all or part of the coding region with another nucleic acid molecule, etc.

[0034] Furthermore, for uge3, sph3, ega3, agd3, and gtb3, deletion of these genes includes not only addition, deletion, and substitution of a predetermined nucleic acid molecule in the coding region, but also gene deletion that suppresses expression of the coding region by inserting, deleting, or substituting a predetermined nucleic acid molecule in a non-coding region in the genome involved in regulating the expression of the coding region, as well as conditional gene deletion designed so that the GAG ​​biosynthesis genes are expressed only under certain conditions.By deleting the GAG ​​biosynthesis gene cluster, the genes responsible for GAG biosynthesis can be knocked out.

[0035] The filamentous fungal mutant strain of the present invention, which is deficient in at least a portion of the GAG ​​biosynthetic gene cluster, preferably includes not only one that does not express any GAG biosynthetic genes at all, but also one that does not substantially express any GAG biosynthetic genes. More specifically, a mutant strain that does not substantially express any GAG biosynthetic genes refers to a mutant strain that expresses only a very small amount of the GAG ​​biosynthetic genes and in which clumping of fungal cells due to entanglement of hyphae is significantly suppressed, and examples thereof include a strain in which the amount of GAG biosynthesis is 30% or less of that of the wild-type strain, more preferably 10% or less of that of the wild-type strain. Due to the deletion of at least a portion of any of the GAG ​​biosynthesis gene clusters described above, in the filamentous fungal mutant strain of the present invention, at least one of the proteins uge3, sph3, ega3, agd3, and gtb3 is not expressed, or the amount of protein expressed is 30% or less of that in a wild-type strain of filamentous fungi, more preferably 10% or less of that in a wild-type strain, or the protein is expressed as a protein that has lost its original function, or the protein is expressed whose original function is reduced to 30% or less of that in a wild-type strain of filamentous fungi, more preferably 10% or less of that in a wild-type strain.

[0036] The filamentous fungal mutant strain of the present invention is also deficient in a hydrophobin gene. Hydrophobins are amphipathic proteins secreted by filamentous fungi, and RolA is known to be involved in the degradation of biodegradable polyesters (Non-Patent Document 10). Furthermore, in human-infectious filamentous fungi, the hydrophobin spore coating inhibits recognition by macrophages, contributing to the establishment of infection (Non-Patent Document 11). The filamentous fungal mutant strain of the present invention is preferably deficient in hydrophobin genes, particularly the RolA gene (rolA). In the present invention, the concept of being deficient in the hydrophobin gene also includes the deletion of orthologs of the gene. For example, the term "deficient in rolA" may also include the deletion of orthologs such as Aspergillus fumigatus rodA, Aspergillus sojae rolA, Aspergillus niger rodA, Emerysella nidulans rodA, and Penicillium chrysogenum rodA. The same applies to the concept of not expressing any of the hydrophobin genes.

[0037] Genes encoding hydrophobins include rolA (Gene ID: AO090020000588), hypB (Gene ID: AO090012000143), hypC (Gene ID: AO090012000370), hypD (Gene ID: AO090701000610), and hypE from Aspergillus oryzae, rodA (Gene ID: AN8803), DewA (Gene ID: AN8006), DewB (Gene ID: AN1837), DewC (Gene ID: AN6401), DewD (Gene ID: AN0940), and DewE (Gene ID: AN7539) from Emerysella nidulans, and Aspergillus sojae rolA, hypB, hypC, hypD, and hypE; Aspergillus fumigatus rodA (Gene ID: Afu5g09580), rodB (Gene ID: Afu1g17250), rodC (Gene ID: Afu8g07060), rodD (Gene ID: Afu05g01490), and rodE (Afu08g05890); Aspergillus niger rodA (Gene ID: An04g08500), hyp1 (Gene ID: An07g03340), hfbA (Gene ID: An03g02400), hfbB (Gene ID: An03g02360), hfbD (Gene ID: An08g09880), and hfbE (Gene ID: An12g05020), Penicillium chrysogenum rodA (Gene ID: Pc22g14290), hfbB (Gen ID: Pc21g18350), hfbC (Gene ID: Pc21g23770), hfbD (Gene ID: Pc22g25460), and hfbE (Gene ID: Pc21g16130).

[0038] Examples of amino acid sequences and nucleotide sequences of RolA from Aspergillus oryzae and the like are shown in the sequence listing: Deduced amino acid sequence of Aspergillus oryzae RolA (SEQ ID NO: 105) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus oryzae RolA (SEQ ID NO: 106) Deduced amino acid sequence of Aspergillus oryzae HypB (SEQ ID NO: 107) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus oryzae HypB (SEQ ID NO: 108) Deduced amino acid sequence of Aspergillus oryzae HypC (SEQ ID NO: 109) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus oryzae HypC (SEQ ID NO: 110) Deduced amino acid sequence of Aspergillus oryzae HypD (SEQ ID NO: 111) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus oryzae HypD (SEQ ID NO: 112) Deduced amino acid sequence of Aspergillus oryzae HypE (SEQ ID NO: 113) Aspergillus Nucleotide sequence of a nucleic acid molecule encoding HypE of Emmarisella oryzae (SEQ ID NO:114) Deduced amino acid sequence of RodA of Emmarisella nidulans (SEQ ID NO:115) Nucleotide sequence of a nucleic acid molecule encoding RodA of Emmarisella nidulans (SEQ ID NO:116) Deduced amino acid sequence of DewA of Emmarisella nidulans (SEQ ID NO:117) Nucleotide sequence of a nucleic acid molecule encoding DewA of Emmarisella nidulans (SEQ ID NO:118) Deduced amino acid sequence of DewB of Emmarisella nidulans (SEQ ID NO:119) Nucleotide sequence of a nucleic acid molecule encoding DewB of Emmarisella nidulans (SEQ ID NO:120) Deduced amino acid sequence of DewC of Emmarisella nidulans (SEQ ID NO:121) Nucleotide sequence of a nucleic acid molecule encoding DewC of Emmarisella nidulans (SEQ ID NO:122) Deduced amino acid sequence of DewD of Emmarisella nidulans (SEQ ID NO:123) The base sequence of the nucleic acid molecule encoding DewD of Emmalisella nidulans (SEQ ID NO: 124) The predicted amino acid sequence of DewE of Emmalisella nidulans (SEQ ID NO: 125) The base sequence of the nucleic acid molecule encoding DewE of Emmalisella nidulans (SEQ ID NO: 126)

[0039] Examples of the amino acid sequences of RolA, HypB, HypC, HypD, and HypE of Aspergillus sojae include amino acid sequences deduced from the gene sequences of Aspergillus sojae registered in GenBank (Genbank accession nos. DF093557-DF093585) based on homology with Aspergillus oryzae. Examples of amino acid sequences and nucleotide sequences of RolA from Aspergillus sojae and the like are shown in the sequence listing: Deduced amino acid sequence of RolA from Aspergillus sojae (SEQ ID NO: 127) Nucleotide sequence of a nucleic acid molecule encoding RolA from Aspergillus sojae (SEQ ID NO: 128) Deduced amino acid sequence of HypB from Aspergillus sojae (SEQ ID NO: 129) Nucleotide sequence of a nucleic acid molecule encoding HypB from Aspergillus sojae (SEQ ID NO: 130) Deduced amino acid sequence of HypC from Aspergillus sojae (SEQ ID NO: 131) Nucleotide sequence of a nucleic acid molecule encoding HypC from Aspergillus sojae (SEQ ID NO: 132) Deduced amino acid sequence of HypD from Aspergillus sojae (SEQ ID NO: 133) Nucleotide sequence of a nucleic acid molecule encoding HypD from Aspergillus sojae (SEQ ID NO: 134) Deduced amino acid sequence of HypE from Aspergillus sojae (SEQ ID NO: 135) Aspergillus Nucleotide sequence of a nucleic acid molecule encoding HypE of Aspergillus fumigatus (SEQ ID NO: 136) Deduced amino acid sequence of RodA of Aspergillus fumigatus (SEQ ID NO: 137) Nucleotide sequence of a nucleic acid molecule encoding RodA of Aspergillus fumigatus (SEQ ID NO: 138) Deduced amino acid sequence of RodB of Aspergillus fumigatus (SEQ ID NO: 139) Nucleotide sequence of a nucleic acid molecule encoding RodB of Aspergillus fumigatus (SEQ ID NO: 140) Deduced amino acid sequence of RodC of Aspergillus fumigatus (SEQ ID NO: 141) Nucleotide sequence of a nucleic acid molecule encoding RodC of Aspergillus fumigatus (SEQ ID NO: 142) Deduced amino acid sequence of RodD of Aspergillus fumigatus (SEQ ID NO: 143) Nucleotide sequence of a nucleic acid molecule encoding RodD of Aspergillus fumigatus (SEQ ID NO: 144) Deduced amino acid sequence of RodE of Aspergillus fumigatus (SEQ ID NO: 145) Nucleic acid sequence of Aspergillus fumigatus RodE (SEQ ID NO: 146)Deduced amino acid sequence of Aspergillus niger RodA (SEQ ID NO: 147) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger RodA (SEQ ID NO: 148) Deduced amino acid sequence of Aspergillus niger Hyp1 (SEQ ID NO: 149) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger Hyp1 (SEQ ID NO: 150) Deduced amino acid sequence of Aspergillus niger HfbA (SEQ ID NO: 151) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger HfbA (SEQ ID NO: 152) Deduced amino acid sequence of Aspergillus niger HfbB (SEQ ID NO: 153) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger HfbB (SEQ ID NO: 154) Deduced amino acid sequence of Aspergillus niger HfbD (SEQ ID NO: 155) Nucleotide sequence of a nucleic acid molecule encoding Aspergillus niger HfbD (SEQ ID NO: 156) Aspergillus Deduced amino acid sequence of HfbE from Aspergillus niger (SEQ ID NO:157) Nucleotide sequence of a nucleic acid molecule encoding HfbE from Aspergillus niger (SEQ ID NO:158) Deduced amino acid sequence of RodA from Penicillium chrysogenum (SEQ ID NO:159) Nucleotide sequence of a nucleic acid molecule encoding RodA from Penicillium chrysogenum (SEQ ID NO:160) Deduced amino acid sequence of HfbB from Penicillium chrysogenum (SEQ ID NO:161) Nucleotide sequence of a nucleic acid molecule encoding HfbB from Penicillium chrysogenum (SEQ ID NO:162) Deduced amino acid sequence of HfbC from Penicillium chrysogenum (SEQ ID NO:163) Nucleotide sequence of a nucleic acid molecule encoding HfbC from Penicillium chrysogenum (SEQ ID NO:164) Deduced amino acid sequence of HfbD from Penicillium chrysogenum (SEQ ID NO:165) Penicillium the nucleotide sequence of a nucleic acid molecule encoding HfbD from Penicillium chrysogenum (SEQ ID NO: 166); the deduced amino acid sequence of HfbE from Penicillium chrysogenum (SEQ ID NO: 167); and the nucleotide sequence of a nucleic acid molecule encoding HfbE from Penicillium chrysogenum (SEQ ID NO: 168).

[0040] In a preferred embodiment, the "at least one hydrophobin gene" to be deleted in the filamentous fungus of the present invention includes, for example, rolA, hypB, hypC, etc., preferably rolA, etc. Therefore, it is preferable that the "at least one hydrophobin gene" to be deleted in the filamentous fungus of the present invention includes at least one selected from the group consisting of rolA, hypB, hypC, hypD and hypE, preferably at least rolA.

[0041] In the present invention, a hydrophobin gene deficiency refers to a deletion of all or part of the hydrophobin coding region in the genome, an insertion of another nucleic acid molecule into all or part of the coding region, or a replacement of all or part of the coding region with another nucleic acid molecule. A hydrophobin gene deficiency also includes a gene deficiency resulting in suppression of expression of the coding region due to the insertion, deletion, or substitution of a specific nucleic acid molecule into a non-coding region in the genome involved in regulating the expression of the coding region. A hydrophobin gene deficiency also includes a gene deficiency resulting in the production of a protein that lacks hydrophobin function, despite the protein being produced due to a mutation in the gene region encoding amino acids necessary for the functional expression of hydrophobin. Furthermore, in addition to the addition, deletion, or substitution of a specific nucleic acid molecule into the coding region, a conditional gene deficiency, in which no nucleic acid molecule is added to the coding region but the hydrophobin gene is designed to be expressed only under certain conditions, is also included. An example of a conditional gene deficiency is one that uses the Cre-LoxP system. The hydrophobin gene can be knocked out by deleting the hydrophobin gene. Due to the deletion of any of the hydrophobin genes described above, in the filamentous fungal mutant strain of the present invention, the hydrophobin protein is not expressed, the amount of protein expressed is 30% or less, more preferably 10% or less, of that in a wild-type strain of filamentous fungus, or the protein is expressed as a protein that has lost its original function, or the protein expressed has its original function reduced to 30% or less, more preferably 10% or less, of that in a wild-type strain of filamentous fungus.

[0042] In the present invention, a deficiency in the RolA gene (rolA) refers to a deletion of all or part of the coding region of RolA in the genome, an insertion of another nucleic acid molecule into all or part of the coding region, or a replacement of all or part of the coding region with another nucleic acid molecule. Furthermore, a deficiency in the RolA gene (rolA) also includes a gene deficiency that suppresses expression of the coding region by inserting, deleting, or substituting a specific nucleic acid molecule into a non-coding region in the genome involved in regulating the expression of the coding region. Furthermore, in addition to the addition, deletion, or substitution of a specific nucleic acid molecule into the coding region, the deficiency also includes a conditional gene deficiency that is designed so that rolA is expressed only under certain conditions, even without the addition of a nucleic acid molecule to the coding region. Examples of conditional gene deficiencies include those using the Cre-LoxP system. By deleting the RolA gene (rolA), rolA can be knocked out. Due to any of the above-mentioned rolA deletions, in the filamentous fungal mutant strain of the present invention, the RolA protein is not expressed, the amount of protein expressed is 30% or less, more preferably 10% or less, of that in the wild-type strain of filamentous fungi, or the protein is expressed as a protein that has lost its original function, or the protein expressed has its original function reduced to 30% or less, more preferably 10% or less, of that in the wild-type strain of filamentous fungi.

[0043] In one embodiment, the filamentous fungus of the present invention has a spore count of 5.0 x 10 ５When 100 mL of a culture medium containing the filamentous fungus added so that the concentration of conidia is 1 / mL is cultured in a 500 mL Sakaguchi flask at 30°C with an amplitude of 50 mm and swirling at 125 rpm from the start of culture until 12 hours later, and then at 125 rpm from 12 to 72 hours after the start of culture for 72 hours, the weight (dry weight) of fungal cells adhering to the wall of the culture vessel is preferably 0.40 g or less (preferably 0.35 g or less). In one embodiment, the filamentous fungus of the present invention is preferably one that, when cultured under the above conditions, produces a significantly reduced weight (dry weight) of fungal cells adhering to the wall of the culture vessel (preferably 0.80-fold or less, more preferably 0.70-fold or less) compared to a control mutant strain that lacks at least a portion of the GAG ​​biosynthetic cluster and does not express α-1,3-glucan but does not lack the hydrophobin gene. Furthermore, in one embodiment, the filamentous fungus of the present invention is preferably one that, when cultured under the above conditions, significantly increases the cell concentration in the medium (e.g., increases by 5.0% by mass or more) compared to a control mutant strain that lacks at least a portion of the GAG ​​biosynthetic cluster and does not express α-1,3-glucan but does not lack the hydrophobin gene. Furthermore, in one embodiment, the filamentous fungus of the present invention is preferably one that, when cultured under the above conditions, significantly increases the substance production per unit volume of culture solution (e.g., increases by 5.0% by mass or more) compared to a control mutant strain that lacks at least a portion of the GAG ​​biosynthetic cluster and does not express α-1,3-glucan but does not lack the hydrophobin gene. The substance production per unit volume of culture solution can be evaluated, for example, by the method described in the Examples of the present application. Furthermore, the filamentous fungus of the present invention reduces the amount of adherence to the culture vessel, thereby increasing the volume of the culture solution minus the volume of the adhered cells, i.e., the volume of the culture solution that can be effectively used for cultivation (sometimes referred to as the effective volume herein). Therefore, when the filamentous fungus of the present invention is used, not only can the cell concentration and substance production amount per unit volume be increased, but also the cell mass and substance production amount per culture vessel can be significantly increased.In one embodiment, the filamentous fungus of the present invention is preferably one that, when cultured under the above conditions, results in a significant increase in [cell concentration] x [effective volume] (e.g., an increase of 5.0% by mass or more, preferably 10% by mass or more) compared to a control mutant strain that is defective in at least a portion of the GAG ​​biosynthetic cluster and does not express α-1,3-glucan but is not defective in the hydrophobin gene. In one embodiment, the filamentous fungus of the present invention is preferably one that, when cultured under the above conditions, results in a significant increase in [substance production per unit volume of culture medium] x [effective volume] (e.g., an increase of 5.0% by mass or more, preferably 10% by mass or more, more preferably 15% by mass or more) compared to a control mutant strain that is defective in at least a portion of the GAG ​​biosynthetic cluster and does not express α-1,3-glucan but is not defective in the hydrophobin gene. In these embodiments, the culture conditions (e.g., medium composition) described in the Examples below can be used.

[0044] The filamentous fungus of the present invention may be used to produce useful substances, such as enzymes such as amylase and cellulase that the filamentous fungus is naturally capable of producing, or low-molecular-weight compounds such as penicillin. In this case, the filamentous fungus of the present invention may be transformed to enhance expression of the useful substance that the filamentous fungus is naturally capable of producing. Alternatively, the filamentous fungus of the present invention may be transformed to express a substance that the filamentous fungus is not naturally capable of producing, and the transformant may be used to produce the substance that the filamentous fungus is not naturally capable of producing. These transformation methods can be performed using known methods (e.g., methods described in JP-A Nos. 2001-46078, 2005-52116, 2009-118783, JP-A Nos. 11-506025, and 2007-508022) that utilize an expression vector constructed to allow the filamentous fungus to serve as a host, and a plasmid constructed by functionally linking a gene encoding a homologous or heterologous protein to the expression vector.

[0045] The filamentous fungal mutant strain of the present invention can be produced by appropriately using known methods (e.g., the methods described in Non-Patent Documents 2 to 5) on filamentous fungi, such as constructing a disruption cassette for an α-1,3-glucan gene and introducing the cassette into a genomic gene, or constructing a disruption cassette for a gene constituting a GAG biosynthetic cluster and introducing the cassette into a genomic gene. In the present invention, the filamentous fungus subjected to these genetic manipulations may be one into which a mutation has been introduced in advance, such as a disruption of the ligD gene and / or a disruption of the adeA gene (preferably both), to enable high probability of gene introduction into the target site. Here, ligD is a gene involved in non-homologous recombination repair in DNA repair, and disruption of this gene is preferred because it enables relatively high efficiency in obtaining transformants in which the gene has been introduced into the target site by homologous recombination. Examples of mutations that disrupt this gene include the ligD::sC mutation (Non-Patent Document 6), which is disrupted using the sC marker. Furthermore, adeA is an adenine-requiring gene, and examples of mutations that disrupt this gene include adeAΔ::ptrA, which is disrupted by a pyrithiamine resistance gene (ptrA) (Non-Patent Document 7). Therefore, filamentous fungal mutant strains of the present invention also include those that further have these mutations.

[0046] The filamentous fungal mutant strain of the present invention can be used for substance production, for example, in the following methods.

[0047] Substance Production Method The present invention provides a substance production method comprising the steps of culturing the aforementioned filamentous fungal mutant and causing the filamentous fungus to produce a substance.

[0048] Useful substances that can be produced by the method of the present invention are not particularly limited as long as they can be produced by filamentous fungi, and examples include low-molecular-weight compounds such as penicillin, statins, cephalosporins, kojic acid, citric acid, and malic acid; and high-molecular-weight compounds such as amylase, cellulase, protease, lipase, peptidase, esterase, hydrophobin, xylanase, and oxidase. Other useful substances include organic acids, pigments, and chemical products such as agricultural chemicals, as well as various substances used in pharmaceuticals. The method of the present invention can also be applied to the production of bioethanol by biomass decomposition (e.g., using fungi genetically modified to produce high levels of cellulase). While the method of the present invention can produce cell wall components or hydrolysates thereof, substances other than cell wall components or hydrolysates thereof can also be produced. Examples of cell wall components or hydrolysates thereof include α-1,3-glucan, β-1,3-glucan, galactomannan, glucose, galactose, glucosamine, amino acids, mannose, N-acetylglucosamine, N-acetylgalactosamine, chitin, etc. In the present invention, the "substance" that can be produced by the method of the present invention is not considered to include compounds that kill filamentous fungi, living cells, substances that can only be obtained by chemical synthesis, etc.

[0049] Culturing Step: The method of the present invention includes a step of culturing a mutant filamentous fungus that lacks at least a portion of the GAG ​​biosynthetic cluster and a hydrophobin gene and does not express α-1,3-glucan, and allowing the filamentous fungus to produce a substance. The medium used in this step is not particularly limited, and a wide variety of mediums that can be used to culture filamentous fungi can be used. Examples include CD minimal medium, YPD medium, TSB medium, malt medium, and PDA medium. The above medium may also contain a carbon source such as glucose, starch, or soluble starch. The amount of carbon source added is not particularly limited, but can be appropriately set within a range of, for example, 0.5 to 10%, more preferably 1 to 4%. The culture temperature is not particularly limited, but can be appropriately set within a range of 20 to 45°C, more preferably 25 to 37°C. The culture time is also not particularly limited, but can be appropriately set within a range of, for example, 12 to 72 hours, more preferably 24 to 48 hours. As described above, the filamentous fungal mutant strains of the present invention also include those having a conditional gene deletion designed so that at least one selected from the group consisting of α-1,3-glucan, GAG, and hydrophobin is expressed only under certain conditions. Therefore, the methods of the present invention also include a method comprising the step of culturing the conditionally gene-deleted mutant strain under conditions in which α-1,3-glucan, GAG, and hydrophobin are not expressed (or their expression is suppressed). This step also includes culturing a mutant filamentous fungus (e.g., a B. cinerea mutant) having the α-1,3-glucan synthase gene ags under conditions in which α-1,3-glucan is not expressed.

[0050] Recovery Step In one embodiment, the method of the present invention may further comprise a step of recovering the substance obtained in the culturing step. The method for recovering the useful substance from the culture medium is not particularly limited, and any known method (centrifugation, recrystallization, distillation, solvent extraction, chromatography, etc.) can be used as appropriate. The method of the present invention is a method for recovering a useful substance. Therefore, methods for decomposing and detecting components of filamentous fungal mutants for the purpose of analyzing the components of the fungal body for research on the filamentous fungi themselves are essentially different from the method of the present invention.

[0051] The following examples are provided to further explain the embodiments of the present invention and to illustrate the effects of the present invention. These examples are provided for illustrative and descriptive purposes, and the present invention is not limited to these examples.

[0052] Example 1 Materials and Methods Strains In this example, the NS4 strain (genotype: niaD) was used as a wild-type strain of the filamentous fungus Aspergillus oryzae. － , sC － ) modified strain was used. The NS4 modified strain used in this example is a strain (ligDΔ::sC, adeAΔ::ptrA) into which the ligDΔ::sC mutation, which enables gene introduction into the target site with high probability, and the adeAΔ::ptrA mutation, which causes adenine auxotrophy, were introduced. In addition, a strain lacking three types of α-1,3-glucan synthase genes (agsAΔagsBΔagsCΔ) was used as the AG-deficient strain. Other gene mutant strains prepared and their genotypes are shown in Table 1.

[0053]

[0054] Culture medium In this example, the following liquid medium was used unless otherwise specified: <Chemically defined medium (modified from Diano et al., (2009) BB, 103(5)> 3% glucose, 1% K2HPO4 [pH 7.8], 0.3% NaNO3, 0.035% (NH4)2SO4, 0.1% MgSO4・7H2O, 0.1% NaCl, 0.1% Tween 80, 0.05% Antifoam, 0.1% Trace metal solution*, 0.005% chloramphenicol [*Trace metal solution] 7.2 mg / L ZnSO4・7H2O, 1.3 mg / L CuSO4・5H2O, 0.3 mg / L NiCl2・6H2O, 3.5 mg / L MnCl2, 6.9 mg / L FeSO4・7H2O <YPD medium> 6% glucose, 6% peptone, 1% Yeast extract, 20mM Succinate buffer[pH 7.0], 0.05% Antifoam

[0055] Cultivation In this example, Aspergillus oryzae was cultivated at 30°C unless otherwise specified.

[0056] Spore suspension: Conidia of Aspergillus oryzae were inoculated onto agar plates of CD medium that met the nutritional requirements of each mutant strain, and cultured at 30°C for approximately 7 days until sufficient conidia were formed. The plates were then subcultured onto Malt medium and cultured at 30°C for approximately 4 days until sufficient conidia were formed. Ten mL of sterilized conidial suspension solution (150 mM NaCl, 0.1% Tween 20, 10 mM phosphate buffer (pH 7.2)) was poured onto each agar plate, and the conidia were scraped off and suspended with a conical stick. To remove any mycelia contaminating the suspension, the suspension was filtered using a sterilized cell strainer (70 μm pore size) or sterilized Miracloth (Calbiochem), and only the conidia were collected in a 50 mL or 15 mL Falcon tube to prepare a conidial suspension. The number of conidia was counted using a Thoma hemocytometer.

[0057] Because the GAG ​​biosynthetic gene clusters uge3 and sph3 genes of Aspergillus oryzae are adjacent, a disruption cassette was constructed to disrupt both genes simultaneously. First, the downstream (5') region of uge3 (amplicon 1) and the downstream (3') region of sph3 (amplicon 2) were amplified by PCR using Aspergillus oryzae genomic DNA as a template. Furthermore, the Ana deA gene (amplicon 3) was amplified by PCR from the TOPO-2.1-adeA plasmid (first round of PCR). Primers Sph3+Uge3-LU and Sph3+Uge3-LL+Ade were used for PCR amplification in Amplicon 1, primers Sph3+Uge3-RU+Ade and Sph3+Uge3-RL for Amplicon 2, and primers Sph3+Uge3-AU and Sph3+Uge3-AL for Amplicon 3 (Table 2). The 5' ends of primers Sph3+Uge3-LL+Ade, Sph3+Uge3-AU, Sph3+Uge3-RU+Ade, and Sph3+Uge3-AL contain homologous sequences with the complementary strand for fusion PCR. The PCR product was gel extracted, and PCR was performed using primers Sph3+Uge3-LU and Sph3+Uge3-RL to ligate these three fragments (second round of PCR). The main band of the PCR product was gel extracted and identified as the uge3, sph3 gene disruption cassette.

[0058]

[0059] A disruption cassette for disrupting the hydrophobin gene rolA of Aspergillus oryzae was constructed. First, the downstream (5') region of rolA (amplicon 1) and downstream (3') region of rolA (amplicon 2) were amplified by PCR using Aspergillus oryzae genomic DNA as a template. The AdenA gene (amplicon 3) was also amplified by PCR from the TOPO-2.1-adeA plasmid (first round of PCR). Primers RolA-LU and RolA-LL + Ade were used for PCR amplification of Amplicon 1, primers RolA-RU + Ade and RolA-RL for PCR amplification of Amplicon 2, and primers RolA-AU and RolA-AL for PCR amplification of Amplicon 3 (Table 3). The 5' ends of primers RolA-LL+Ade and RolA-AU, and RolA-RU+Ade and RolA-AL contain sequences homologous to the complementary strands for fusion PCR. The PCR product was gel extracted, and PCR was performed using primers RolA-LU and RolA-RL to ligate these three fragments (second round of PCR). The main band of the PCR product was gel extracted and identified as the rolA gene disruption cassette.

[0060]

[0061] Transformation of Aspergillus oryzae by the protoplast-PEG method Aspergillus oryzae was transformed using the protoplast-PEG method (Non-Patent Document 8). A wild-type strain and an AG-deficient strain (agsAΔagsBΔagsCΔ) were used as host strains. 2 × 10 conidia of the host strain were ８The cells were inoculated into 200 mL of YPD liquid medium in a 500 mL Erlenmeyer flask, and cultured at 30°C for 20 hours with rotary shaking. The cells were then filtered through sterilized Miracloth (Calbiochem), washed with distilled water, and pressed with a sterilized spatula to dehydrate them. The collected cells were placed in a 50 mL Falcon tube and suspended in 25 mL of protoplasting solution [10 mg / mL Lysing Enzymes (Sigma), 5 mg / mL Cellulase Onozuka (Yakuult Pharmaceutical Ind. Co., Ltd.), 2.5 mg / mL Yatalase (TaKaRa), Lysing enzyme buffer (Table 4)] filtered through a 0.20 μm pore size filter DISMIC-25CS (ADVANTEC). The suspension was shaken at 30 ° C. and 83 rpm for 3 hours to digest the cell walls, preparing protoplasts. After the reaction, undigested cells were filtered through sterilized Miracloth, and the filtrate was centrifuged at 2,000 × g at 4 ° C for 5 minutes to recover protoplasts. The recovered protoplasts were washed with 0.8 M NaCl and centrifuged at 2,000 × g at 4 ° C for 5 minutes to precipitate and recover the protoplasts. The number of protoplasts was 2 × 10 ８ Sol. I (Table 4) was added to the protoplasts to a concentration of 1000 cells / mL, and the suspension was followed by the addition of 1 / 5 volume of Sol. II (Table 4) and thorough mixing. 240 μL of the protoplast solution was dispensed into a 15 mL Falcon tube, and an appropriate amount of DNA solution (approximately 1-10 μg) was added, mixed thoroughly, and left on ice for 25 minutes. Next, 1 mL of Sol. II (Table 4) was added, mixed thoroughly, and then left at room temperature for 20 minutes. 10 mL of Sol. I was added, mixed thoroughly, and then centrifuged at room temperature at 2,000 × g for 5 minutes. The supernatant was removed, and 300 μL of Sol. I was added. The protoplasts were uniformly suspended and plated on CD selective medium containing 0.8 M NaCl. Five mL of soft agar medium of the same composition (0.6% (w / v) Agar) preheated to 55°C was poured over the suspension, and the protoplasts were quickly and uniformly suspended. The suspension was then cultured at 30°C until colonies formed.

[0062]

[0063] Selection of Transformation Candidate Strains To confirm whether the desired transformation had occurred using the genomic DNA of the resulting transformation candidate strains, genomic DNA was simply extracted from the conidia of the strain, and transformants were selected by PCR using designed primers. 500 μL of YPD liquid medium was placed in a 1.5 mL Eppendorf tube, and conidia of the transformation candidate strain were inoculated by poking with a sterilized toothpick. The culture was then cultured at 30°C until the cells grew. After centrifugation, the medium was removed, and an equal amount of glass beads and 150 μL of Nuclei Lysis Sol. (Promega) were added. The cells were then pulverized using a Micro Smash® MS-100R (TOMY) for 2 minutes at 4,500 rpm. The mixture was left at 65°C for 15 minutes, and 100 μL of Protein Prep Sol. (Promega) was added and mixed well. The mixture was left at room temperature for 5 minutes and centrifuged at 15,000 rpm at 4°C for 5 minutes. The supernatant was transferred to a separate 1.5 mL tube, and 1 / 10 volume of 3 M sodium acetate and 2.5 volumes of ethanol were added and mixed. After centrifugation at 15,000 rpm at 4°C for 20 minutes, the mixture was washed with 1 mL of 70% ethanol, and the pellet was dissolved in 50 μL of TE buffer containing RNase. This solution was used as the genomic DNA solution and stored at 4°C until use as a PCR template.

[0064] Nucleus Purification The desired transformant candidate strain was grown on a minimal nutrient agar plate medium, and the collected conidial suspension was passed through a mononucleation filter (ISOPORE (registered trademark) MEMBRANE FILTERS, 5.0 μm TMTP, Millipore) that had been pre-sterilized by autoclaving to collect mononuclear conidia. The mononucleated conidial suspension was diluted appropriately and grown on a minimal nutrient agar plate medium. The obtained candidate strain was confirmed again by PCR, and the desired transformant strain was purified.

[0065] Liquid Shaking Culture in Chemically Defined Medium The wild-type strain, the AG-deficient strain (AG + GAG-deficient), and the AG-rolA-deficient strain (AG + GAG + rolA-deficient) were cultured in liquid using the aforementioned chemically defined medium. The culture conditions are as shown in Table 5:

[0066]

[0067] Measurement of Xylanase Activity The culture broth was filtered, and the resulting filtrate was used as the culture supernatant. This was then appropriately diluted with ultrapure water to prepare a sample solution. 200 μL of xylan substrate solution (1% xylan (Serva), 0.02 M citric acid buffer (pH 6.0)) was incubated at 58°C for 5 minutes, and 50 μL of the sample solution was quickly added. The reaction was then allowed to proceed at 58°C for 10 minutes. The reaction was stopped by incubation at 100°C for 5 minutes and then rapidly cooled on ice. 100 μL of the reaction solution was diluted with 300 μL of DNS reagent (33 mM 3,5-Dinitrosalicylic acid, 766 mM Potassium sodium (+)-tartrate tetrahydrate, 59 mM phenol, NaOH, NaHCO ). ３ The mixture was mixed with a 100°C buffer and reacted for 5 minutes in the dark at 100°C, then rapidly cooled in ice. 400 μL of the DNS reaction solution was mixed with 2.5 mL of ultrapure water, and the absorbance at 500 nM was measured. A calibration curve was created from the absorbance when xylose of known concentrations was reacted with the DNS reagent in the same manner, and the enzyme activity (U / mL) of the sample was calculated from the calibration curve.

[0068] Measurement of bacterial cell concentration The culture medium was collected, and 5 mL of it was filtered through a pre-weighed Miracloth (Calbiochem). The filter residue was washed twice with distilled water, and the bacteria and Miracloth were wrapped in aluminum foil and placed in a dryer (Advantec) set to 60°C to dry until the weight remained constant. After the bacteria were dried, they were weighed, and the weight of the Miracloth was subtracted to determine the bacterial cell weight. The bacterial cell concentration (g / L) was calculated by dividing the weight by the volume of the filtered culture medium.

[0069] After the cultivation was completed, a silicone tube was inserted through the opening of the cultivation vessel, taking care not to drop the bacterial cells adhering to the inner wall of the cultivation vessel. The entire volume of the culture medium was aspirated and collected using a syringe (Terumo Corporation) attached to the silicone tube or a vacuum pump V-700 (Buchi), and the total volume of the culture medium was measured using a measuring cylinder.

[0070] Liquid shaking culture in YPD medium The wild-type strain, the AG-deficient strain (AG + GAG-deficient), and the AG-rolA-deficient strain (AG + GAG + rolA-deficient) were cultured in YPD medium with liquid shaking. The culture vessel used was a 5 L microbial culture device BMS-05KP3 manufactured by Biot. The conditions were as follows: YPDS medium 2.5 L, 30°C, 800 rpm, 60 hours, spore count at the start of culture 1.0 x 10 ５ / mL

[0071] Measurement of the power required for agitation The power required for agitation during liquid shaking culture (P ｇＶ / m ３ ) was measured using a mixing torque meter ST-300II (manufactured by Satake Multimix Co., Ltd.).

[0072] Measurement of oxygen transfer rate Oxygen transfer rate k in the culture tank during liquid shaking culture Ｌ a ( / h) was measured using an exhaust gas analyzer EG-910 (manufactured by Satake Multimix Co., Ltd.).

[0073] Experimental Results Results of Liquid Shaking Cultivation in Chemically Defined Medium Medium Figure 1A shows the appearance of the flask after 72 hours of culture in chemically defined medium medium. The amount of bacterial cells attached to the wall after 72 hours of culture is shown in the graph in Figure 1B. The effective volume after 72 hours of culture is shown in Table 6. As is clear from Figures 1A and 1B, the filamentous fungus that does not express α-1,3-glucan, is defective in the GAG ​​biosynthesis cluster, and is also defective in the hydrophobin gene, showed a significantly reduced amount of cells attached to the culture vessel compared to the strain that did not lack the hydrophobin gene, and therefore showed an increased effective volume.

[0074]

[0075] The results of measuring the recombinant enzyme activity after culture in a chemically defined medium medium are shown in Figure 2A and Table 7 (average values ​​for N = 3). The results of measuring the bacterial cell concentration after culture in a chemically defined medium medium are shown in Figure 2B and Table 8 (average values ​​for N = 3). As is clear from these results, the filamentous fungus that does not express α-1,3-glucan, is defective in the GAG ​​biosynthetic cluster, and is also defective in the hydrophobin gene had increased enzyme activity per unit volume and bacterial cell concentration compared to the strain that did not lack the hydrophobin gene.

[0076]

[0077]

[0078] The total activity (10 3 The amount of the culture medium (μg) and the total amount of the cells (g) are shown in Table 9.

[0079]

[0080] Results of cultivation in a jar fermenter using YPD medium Figure 3A shows a photograph of the culture vessel after cultivation in a jar fermenter using YPD medium. The weight of the bacterial cells adhering to the wall is shown in the graph of Figure 3B. The volume change of the culture solution components is shown in Table 10. The oxygen transfer rate k in the culture tank Ｌ The results of the measurements of the agitation power required for mixing the culture medium are shown in Table 11. As is clear from these results, the filamentous fungus that does not express α-1,3-glucan, lacks the GAG ​​biosynthesis cluster, and also lacks the hydrophobin gene rolA, showed a significant decrease in the amount of adhesion to the culture vessel compared to the strain that does not lack rolA, and therefore the effective volume increased. ｇＶ decreases, and the stirring efficiency under the same stirring conditions increases, resulting in an increase in the oxygen transfer rate k Ｌ a has risen.

[0081]

[0082]

[0083] Example 2 Materials and Methods Strains In this example, the NS4 strain (genotype: niaD) was used as a wild-type strain of the filamentous fungus Aspergillus oryzae. － , sC － ) modified strain was used. The NS4 modified strain used in this example is a strain (ligDΔ::sC, adeAΔ::ptrA) into which the ligDΔ::sC mutation, which enables gene introduction into the target site with a high probability, and the adeAΔ::ptrA mutation, which causes adenine auxotrophy, were introduced. In addition, a strain lacking three types of α-1,3-glucan synthase genes (agsAΔagsBΔagsCΔ) was used as the AG-deficient strain. Other gene mutant strains prepared and their genotypes are as shown in Table 12.

[0084]

[0085] Culture medium In this example, the following liquid medium was used unless otherwise specified: <Chemically defined medium (modified from Diano et al., (2009) BB, 103(5)> 3% glucose, 1% K2HPO4 [pH 7.8], 0.3% NaNO3, 0.035% (NH4)2SO4, 0.1% MgSO4・7H2O, 0.1% NaCl, 0.1% Tween 80, 0.05% Antifoam, 0.1% Trace metal solution*, 0.005% chloramphenicol [*Trace metal solution] 7.2 mg / L ZnSO4・7H2O, 1.3 mg / L CuSO4・5H2O, 0.3 mg / L NiCl2・6H2O, 3.5 mg / L MnCl2, 6.9 mg / L FeSO4・7H2O <YPD medium> 6% glucose, 6% peptone, 1% Yeast extract, 20mM Succinate buffer[pH 7.0], 0.05% Antifoam

[0086] Cultivation In this example, Aspergillus oryzae was cultivated at 30°C unless otherwise specified.

[0087] Spore suspension: Conidia of Aspergillus oryzae were inoculated onto agar plates of CD medium that met the nutritional requirements of each mutant strain, and cultured at 30°C for approximately 7 days until sufficient conidia were formed. The plates were then subcultured onto Malt medium and cultured at 30°C for approximately 4 days until sufficient conidia were formed. Ten mL of sterilized conidial suspension solution (150 mM NaCl, 0.1% Tween 20, 10 mM phosphate buffer (pH 7.2)) was poured onto each agar plate, and the conidia were scraped off and suspended with a conical stick. To remove any mycelia contaminating the suspension, the suspension was filtered using a sterilized cell strainer (70 μm pore size) or sterilized Miracloth (Calbiochem), and only the conidia were collected in a 50 mL or 15 mL Falcon tube to prepare a conidial suspension. The number of conidia was counted using a Thoma hemocytometer.

[0088] Because the GAG ​​biosynthetic gene clusters uge3 and sph3 genes of Aspergillus oryzae are adjacent, a disruption cassette was constructed to disrupt both genes simultaneously. First, the downstream (5') region of uge3 (amplicon 1) and the downstream (3') region of sph3 (amplicon 2) were amplified by PCR using Aspergillus oryzae genomic DNA as a template. Furthermore, the Ana deA gene (amplicon 3) was amplified by PCR from the TOPO-2.1-adeA plasmid (first round of PCR). Primers Sph3+Uge3-LU and Sph3+Uge3-LL+Ade were used for PCR amplification in Amplicon 1, primers Sph3+Uge3-RU+Ade and Sph3+Uge3-RL were used for PCR amplification in Amplicon 2, and primers Sph3+Uge3-AU and Sph3+Uge3-AL were used for PCR amplification in Amplicon 3 (Table 13). The 5' ends of primers Sph3+Uge3-LL+Ade and Sph3+Uge3-AU, Sph3+Uge3-RU+Ade and Sph3+Uge3-AL contain homologous sequences with the complementary strand for fusion PCR ligation. The PCR product was gel extracted, and PCR was performed using primers Sph3+Uge3-LU and Sph3+Uge3-RL to ligate these three fragments (second round of PCR). The main band of the PCR product was gel extracted and identified as the uge3, sph3 gene disruption cassette.

[0089]

[0090] A disruption cassette was constructed to disrupt the hydrophobin gene hypB of Aspergillus oryzae. First, the hypB downstream (5'-side) region (amplicon 1) and the hypB downstream (3'-side) region (amplicon 2) were amplified by PCR using Aspergillus oryzae genomic DNA as a template. Furthermore, the Ana deA gene (amplicon 3) was amplified by PCR from the TOPO-2.1-adeA plasmid (1st round of PCR). Primers HypB-LU and HypB-LL+Ade were used for PCR amplification in Amplicon 1, primers HypB-RU+Ade and HypB-RL for Amplicon 2, and primers HypB-AU and HypB-AL for Amplicon 3 (Table 14). The 5' ends of primers HypB-LL+Ade and HypB-AU, and HypB-RU+Ade and HypB-AL contain homologous sequences with the complementary strand for fusion PCR ligation. The PCR product was gel extracted, and PCR was performed using primers HypB-cLU and HypB-cRL to ligate these three fragments (second round of PCR). The main band of the PCR product was gel extracted and identified as the hypB gene disruption cassette.

[0091]

[0092] Transformation of Aspergillus oryzae by the protoplast-PEG method Aspergillus oryzae was transformed using the protoplast-PEG method (Non-Patent Document 8). A wild-type strain and an AG-deficient strain (agsAΔagsBΔagsCΔ) were used as host strains. 2 × 10 conidia of the host strain were ８The cells were inoculated into 200 mL of YPD liquid medium in a 500 mL Erlenmeyer flask, and cultured at 30°C for 20 hours with rotary shaking. The cells were then filtered through sterilized Miracloth (Calbiochem), washed with distilled water, and pressed with a sterilized spatula to dehydrate them. The collected cells were placed in a 50 mL Falcon tube and suspended in 25 mL of protoplasting solution [10 mg / mL Lysing Enzymes (Sigma), 5 mg / mL Cellulase Onozuka (Yakuult Pharmaceutical Ind. Co., Ltd.), 2.5 mg / mL Yatalase (TaKaRa), Lysing enzyme buffer (Table 15)] filtered through a 0.20 μm pore size filter DISMIC-25CS (ADVANTEC). The suspension was shaken at 30 ° C. and 83 rpm for 3 hours to digest the cell walls, preparing protoplasts. After the reaction, undigested cells were filtered through sterilized Miracloth, and the filtrate was centrifuged at 2,000 × g at 4 ° C for 5 minutes to recover protoplasts. The recovered protoplasts were washed with 0.8 M NaCl and centrifuged at 2,000 × g at 4 ° C for 5 minutes to precipitate and recover the protoplasts. The number of protoplasts was 2 × 10 ８ Sol. I (Table 15) was added to the protoplasts to a concentration of 1000 cells / mL, and the suspension was followed by the addition of 1 / 5 volume of Sol. II (Table 15) and thorough mixing. 240 μL of the protoplast solution was dispensed into a 15 mL Falcon tube, and an appropriate amount of DNA solution (approximately 1-10 μg) was added, mixed thoroughly, and left on ice for 25 minutes. Next, 1 mL of Sol. II (Table 15) was added, mixed thoroughly, and then left at room temperature for 20 minutes. 10 mL of Sol. I was added, mixed thoroughly, and then centrifuged at room temperature at 2,000 × g for 5 minutes. The supernatant was removed, and 300 μL of Sol. I was added. The protoplasts were uniformly suspended and plated on CD selective medium containing 0.8 M NaCl. Five mL of soft agar medium of the same composition (0.6% (w / v) Agar) preheated to 55°C was poured over the suspension, and the protoplasts were quickly and uniformly suspended. The suspension was then cultured at 30°C until colonies formed.

[0093]

[0094] Selection of Transformation Candidate Strains To confirm whether the desired transformation had occurred using the genomic DNA of the resulting transformation candidate strains, genomic DNA was simply extracted from the conidia of the strain, and transformants were selected by PCR using designed primers. 500 μL of YPD liquid medium was placed in a 1.5 mL Eppendorf tube, and conidia of the transformation candidate strain were inoculated by poking with a sterilized toothpick. The culture was then cultured at 30°C until the cells grew. After centrifugation, the medium was removed, and an equal amount of glass beads and 150 μL of Nuclei Lysis Sol. (Promega) were added. The cells were then pulverized using a Micro Smash® MS-100R (TOMY) for 2 minutes at 4,500 rpm. The mixture was left at 65°C for 15 minutes, and 100 μL of Protein Prep Sol. (Promega) was added and mixed well. The mixture was left at room temperature for 5 minutes and centrifuged at 15,000 rpm at 4°C for 5 minutes. The supernatant was transferred to a separate 1.5 mL tube, and 1 / 10 volume of 3 M sodium acetate and 2.5 volumes of ethanol were added and mixed. After centrifugation at 15,000 rpm at 4°C for 20 minutes, the mixture was washed with 1 mL of 70% ethanol, and the pellet was dissolved in 50 μL of TE buffer containing RNase. This solution was used as the genomic DNA solution and stored at 4°C until use as a PCR template.

[0095] Nucleus Purification: The desired transformant candidate strain was grown on a minimal nutrient agar plate, and the collected conidial suspension was passed through a mononucleation filter (ISOPORE MEMBRANE FILTERS, 5.0 μm TMTP, Millipore) that had been pre-sterilized by autoclaving to collect mononuclear conidia. The mononucleated conidial suspension was appropriately diluted and grown on a minimal nutrient agar plate. The obtained candidate strain was again confirmed by PCR, and the desired transformant was purified.

[0096] Liquid Shaking Culture in Chemically Defined Medium The wild-type strain, the AG-deficient strain (AG + GAG-deficient), and the AG-hypB-deficient strain (AG + GAG + hypB-deficient) were cultured in the above-mentioned chemically defined medium under the culture conditions shown in Table 16:

[0097]

[0098] Measurement of Xylanase Activity The culture broth was filtered, and the resulting filtrate was used as the culture supernatant. This was then appropriately diluted with ultrapure water to prepare a sample solution. 200 μL of xylan substrate solution (1% xylan (Serva), 0.02 M citric acid buffer (pH 6.0)) was incubated at 58°C for 5 minutes, and 50 μL of the sample solution was quickly added. The reaction was then allowed to proceed at 58°C for 10 minutes. The reaction was stopped by incubation at 100°C for 5 minutes and then rapidly cooled on ice. 100 μL of the reaction solution was diluted with 300 μL of DNS reagent (33 mM 3,5-Dinitrosalicylic acid, 766 mM Potassium sodium (+)-tartrate tetrahydrate, 59 mM phenol, NaOH, NaHCO ). ３ The mixture was mixed with a 100°C buffer and reacted for 5 minutes in the dark at 100°C, then rapidly cooled in ice. 400 μL of the DNS reaction solution was mixed with 2.5 mL of ultrapure water, and the absorbance at 500 nM was measured. A calibration curve was created from the absorbance when xylose of known concentrations was reacted with the DNS reagent in the same manner, and the enzyme activity (U / mL) of the sample was calculated from the calibration curve.

[0099] Measurement of bacterial cell concentration The culture medium was collected, and 5 mL of it was filtered through a pre-weighed Miracloth (Calbiochem). The filter residue was washed twice with distilled water, and the bacteria and Miracloth were wrapped in aluminum foil and placed in a dryer (Advantec) set to 60°C to dry until the weight remained constant. After the bacteria were dried, they were weighed, and the weight of the Miracloth was subtracted to determine the bacterial cell weight. The bacterial cell concentration (g / L) was calculated by dividing the weight by the volume of the filtered culture medium.

[0100] Experimental Results: Figure 4A shows the results of measuring the recombinant enzyme activity after 72 hours of culture in a chemically defined medium medium. Figure 4B shows the results of measuring the bacterial cell concentration after culture in a chemically defined medium medium. As is clear from Figures 4A and 4B, even in the case of a filamentous fungus that does not express α-1,3-glucan, is defective in the GAG ​​biosynthetic cluster, and is defective in the hydrophobin gene HypB, both the enzyme activity and the bacterial cell concentration increased compared to the strain that was not defective in HypB.

[0101] Example 3 Materials and Methods Strains In this example, a modified strain of the ABPU1 strain (genotype: biA1, pyrG89, wA3, argB2, pyroA4) was used as a wild-type strain of the filamentous fungus Emerysella nidulans. The modified ABPU1 strain used in this example was a strain (ligDΔ::ptrA, AoargB::argB) that contained a ligDΔ::ptrA mutation, which allows for high-probability gene transfer to the target site, and an AoargB::argB mutation, which confers arginine auxotrophy. In addition, a strain lacking two α-1,3-glucan synthase genes (agsAΔagsBΔ) was used as the AG-deficient strain. Other gene mutant strains and their genotypes that were prepared are shown in Table 17.

[0102]

[0103] Culture medium In this example, the following liquid medium was used unless otherwise specified: <Chemically defined medium (modified from Diano et al., (2009) BB, 103(5)> 3% glucose, 1% K2HPO4 [pH 7.8], 0.3% NaNO3, 0.035% (NH4)2SO4, 0.1% MgSO4・7H2O, 0.1% NaCl, 0.1% Tween 80, 0.05% Antifoam, 0.1% Trace metal solution*, 0.005% chloramphenicol, 0.02 mg / L biotin, 0.5 g / L pyridoxine [*Trace metal solution], 7.2 mg / L ZnSO4・7H2O, 1.3 mg / L CuSO4・5H2O, 0.3 mg / L NiCl2・6H2O, 3.5 mg / L MnCl2, 6.9 mg / L FeSO4・7H2O

[0104] Cultivation In this example, Emerysella nidulans was cultivated at 37°C unless otherwise specified.

[0105] Spore suspension: Conidia of Emerisella nidulans were inoculated onto agar plates of CD medium that met the nutritional requirements of each mutant strain, and cultured at 37°C for approximately 4 days until sufficient conidia were formed. Ten milliliters of sterilized conidial suspension solution (150 mM NaCl, 0.1% Tween 20, 10 mM phosphate buffer (pH 7.2)) was poured onto each agar plate, and the conidia were scraped and suspended with a conical rod. To remove any contaminating hyphae from the suspension, the suspension was filtered using a sterilized cell strainer (70 μm pore size) or sterilized Miracloth (Calbiochem), and only the conidia were collected in a 50 mL or 15 mL Falcon tube to prepare a conidial suspension. The number of conidia was counted using a Thoma hemocytometer.

[0106] Because the GAG ​​biosynthetic gene clusters uge3 and sph3 genes of Emmarisella nidulans are adjacent, a disruption cassette was created to disrupt both genes simultaneously. First, the downstream (5') region of uge3 (amplicon 1) and the downstream (3') region of sph3 (amplicon 2) were amplified by PCR using Emmarisella nidulans genomic DNA as a template. Furthermore, the AopyrG gene (amplicon 3) was amplified by PCR from the genomic DNA of a wild-type strain of Aspergillus oryzae (first round of PCR). Primers Sph3+Uge3-LU and Sph3+Uge3-PL were used for PCR amplification in Amplicon 1, primers Sph3+Uge3-PU and Sph3+Uge3-LL were used for PCR amplification in Amplicon 2, and primers Sph3+Uge3-RU and Sph3+Uge3-RL were used for PCR amplification in Amplicon 3 (Table 18). The 5' ends of primers Sph3+Uge3-PL and Sph3+Uge3-RU, Sph3+Uge3-PU and Sph3+Uge3-RL contain sequences homologous to the complementary strand for fusion PCR ligation. The PCR product was gel extracted, and PCR was performed using primers Sph3+Uge3-LU and Sph3+Uge3-RL to ligate these three fragments (second round of PCR). The main band of the PCR product was gel extracted and identified as the uge3, sph3 gene disruption cassette.

[0107]

[0108] Preparation of rodA gene disruption cassette A disruption cassette was prepared to disrupt the hydrophobin gene rodA of Emmarisella nidulans. First, the rodA downstream (5' end) region (amplicon 1) and the rodA downstream (3' end) region (amplicon 2) were amplified by PCR using Emmarisella nidulans genomic DNA as a template. The pyroA gene (amplicon 3) was also amplified by PCR from the genomic DNA of a wild-type strain of Emmarisella nidulans (1st round of PCR). Primers RodA-LU and RodA-PL were used for PCR amplification in Amplicon 1, primers RolA-PU and RodA-RL for Amplicon 2, and primers RodA-AU and RodA-AL for Amplicon 3 (Table 19). The 5' ends of primers RodA-PL and RodA-PU contain sequences homologous to their complementary strands for fusion PCR. The PCR product was gel extracted, and PCR was performed using primers RodA-cLU and RodA-cRL to ligate these three fragments (second round of PCR). The main band of the PCR product was gel extracted and identified as the RodA gene disruption cassette.

[0109]

[0110] Transformation of Emmaricella nidulans by the protoplast-PEG method Transformation of Emmaricella nidulans was carried out using the protoplast-PEG method (Non-Patent Document 8). A wild-type strain and an AG-deficient strain (agsAΔagsB) were used as host strains. 2 × 10 conidia of the host strain were ８The cells were inoculated into 200 mL of YPD liquid medium in a 500 mL Erlenmeyer flask, and cultured at 30°C for 20 hours with rotary shaking. The cells were then filtered through sterilized Miracloth (Calbiochem), washed with distilled water, and pressed with a sterilized spatula to dehydrate them. The collected cells were placed in a 50 mL Falcon tube and suspended in 25 mL of protoplasting solution [10 mg / mL Lysing Enzymes (Sigma), 5 mg / mL Cellulase Onozuka (Yakuult Pharmaceutical Ind. Co., Ltd.), 2.5 mg / mL Yatalase (TaKaRa), Lysing enzyme buffer (Table 20)] filtered through a 0.20 μm pore size filter DISMIC-25CS (ADVANTEC). The tube was shaken at 30 ° C. and 83 rpm for 3 hours to digest the cell walls, preparing protoplasts. After the reaction, undigested cells were filtered through sterilized Miracloth, and the filtrate was centrifuged at 2,000 × g at 4 ° C for 5 minutes to recover protoplasts. The recovered protoplasts were washed with 0.8 M NaCl and centrifuged at 2,000 × g at 4 ° C for 5 minutes to precipitate and recover the protoplasts. The number of protoplasts was 2 × 10 ８ Sol. I (Table 20) was added to a concentration of 1000 cells / mL, and the suspension was followed by the addition of 1 / 5 volume of Sol. II (Table 20) and thorough mixing. 240 μL of the protoplast solution was dispensed into a 15 mL Falcon tube, and an appropriate amount of DNA solution (approximately 1-10 μg) was added, mixed thoroughly, and left on ice for 25 minutes. Next, 1 mL of Sol. II (Table 20) was added, mixed thoroughly, and then left at room temperature for 20 minutes. 10 mL of Sol. I was added, mixed thoroughly, and then centrifuged at room temperature at 2,000 × g for 5 minutes. The supernatant was removed, and 300 μL of Sol. I was added. The protoplasts were uniformly suspended and plated on CD selective medium containing 0.8 M NaCl. Five mL of soft agar medium of the same composition (0.6% (w / v) Agar) preheated to 55°C was poured over the suspension, and the protoplasts were quickly and uniformly suspended. They were then cultured at 37°C until colonies formed.

[0111]

[0112] Selection of Transformation Candidate Strains To confirm whether the desired transformation had occurred using the genomic DNA of the resulting transformation candidate strains, genomic DNA was simply extracted from the conidia of the strain, and transformants were selected by PCR using designed primers. 500 μL of YPD liquid medium was placed in a 1.5 mL Eppendorf tube, and conidia of the transformation candidate strain were inoculated by poking with a sterilized toothpick. The culture was then cultured at 30°C until the cells grew. After centrifugation, the medium was removed, and an equal amount of glass beads and 150 μL of Nuclei Lysis Sol. (Promega) were added. The cells were then pulverized using a Micro Smash MS-100R (TOMY) for 2 minutes at 4,500 rpm. The mixture was left at 65°C for 15 minutes, and 100 μL of Protein Prep Sol. (Promega) was added and mixed well. The mixture was left at room temperature for 5 minutes and centrifuged at 15,000 rpm at 4°C for 5 minutes. The supernatant was transferred to a separate 1.5 mL tube, and 1 / 10 volume of 3 M sodium acetate and 2.5 volumes of ethanol were added and mixed. After centrifugation at 15,000 rpm at 4°C for 20 minutes, the mixture was washed with 1 mL of 70% ethanol, and the pellet was dissolved in 50 μL of TE buffer containing RNase. This solution was used as the genomic DNA solution and stored at 4°C until use as a PCR template.

[0113] Nucleus Purification: The desired transformant candidate strain was grown on a minimal nutrient agar plate, and the collected conidial suspension was passed through a mononucleation filter (ISOPORE MEMBRANE FILTERS, 5.0 μm TMTP, Millipore) that had been pre-sterilized by autoclaving to collect mononuclear conidia. The mononucleated conidial suspension was diluted appropriately and grown on a minimal nutrient agar plate. The resulting candidate strain was again confirmed by PCR, and the desired transformant was purified.

[0114] Liquid Shaking Culture in Chemically Defined Medium The wild-type strain, the AG-deficient strain (AG + GAG-deficient), and the AG-rodA-deficient strain (AG + GAG + rodA-deficient) were cultured in the aforementioned chemically defined medium under the culture conditions shown in Table 21.

[0115]

[0116] Measurement of amylase activity: The cells were filtered from the culture medium, and the amylase activity of the culture supernatant was measured. Specifically, the cells were filtered through Miracloth 48 hours after the start of cultivation, and the amylase activity of the culture filtrate was measured using an α-amylase measurement kit (Kikkoman Biochemifa Corporation). The measurement method was according to the attached instruction manual, and the amylase activity in the culture supernatant was evaluated as a titer of 1 U = 1 μmol of CNP released from N3-G5-β-CNP per minute.

[0117] Measurement of bacterial cell concentration The culture medium was collected, and 5 mL of it was filtered through a pre-weighed Miracloth (Calbiochem). The filter residue was washed twice with distilled water, and the bacteria and Miracloth were wrapped in aluminum foil and placed in a dryer (Advantec) set to 60°C to dry until the weight remained constant. After the bacteria were dried, they were weighed, and the weight of the Miracloth was subtracted to determine the bacterial cell weight. The bacterial cell concentration (g / L) was calculated by dividing the weight by the volume of the filtered culture medium.

[0118] Experimental Results: The results of measuring amylase activity after 48 hours of culture in a chemically defined medium medium are shown in Figure 5. The results of measuring the cell concentration after culture in a chemically defined medium medium are shown in Figure 6. Furthermore, the amylase activity per cell mass is shown in Figure 7. As is clear from Figure 7, the filamentous fungus that does not express α-1,3-glucan, is defective in the GAG ​​biosynthesis cluster, and is also defective in the hydrophobin gene rodA, had increased enzyme activity per unit volume and cell concentration compared to the strain that was not defective in rodA.

[0119] Based on the above, by deleting the hydrophobin gene in a mutant filamentous fungus that is deficient in GAG and does not biosynthesize α-1,3-glucan, adhesion to the inner wall of the culture vessel during filamentous fungal culture can be reduced, resulting in an increase in the amount of fungal cells retained in the culture medium, thereby improving the yield of the target enzyme and the fungal cells themselves. In Example 1 above, a uge3 and sph3 gene disruption strain was used as the GAG-deficient strain. However, it is known that the deletion of just one gene in the GAG ​​biosynthetic gene cluster can result in the loss of GAG expression (Non-Patent Document 9). Therefore, deleting the hydrophobin gene in a mutant filamentous fungus that is deficient in one gene in the GAG ​​biosynthetic gene cluster and does not biosynthesize α-1,3-glucan is also expected to reduce adhesion to the inner wall of the culture vessel during filamentous fungal culture, compared to a strain without these deletions.

[0120] According to the present invention, in a method for producing a substance using a filamentous fungus, the production amount of a useful substance can be dramatically increased. Furthermore, the useful substances that can be produced by the method of the present invention are not particularly limited and are diverse, making them extremely useful industrially.

Claims

1. A mutant filamentous fungus that lacks at least a part of the galactosaminogalactan (GAG) biosynthetic gene cluster and at least one hydrophobin gene and does not biosynthesize α-1,3-glucan.

2. The filamentous fungus according to claim 1, wherein at least a portion of the GAG ​​biosynthetic gene cluster comprises at least one selected from the group consisting of uge3, sph3, ega3, agd3 and gtb3.

3. The filamentous fungus according to claim 1, wherein at least one of the hydrophobin genes comprises at least one selected from the group consisting of rolA, hypB, hypC, hypD and hypE.

4. The filamentous fungus of claim 3, wherein at least one of the hydrophobin genes comprises rolA.

5. The filamentous fungus according to claim 1, which does not biosynthesize α-1,3-glucan due to a deficiency in at least one of the α-1,3-glucan synthases Ags.

6. The filamentous fungus according to claim 5, wherein at least one of the α-1,3-glucan synthases Ags includes AgsB.

7. The filamentous fungus according to claim 1, which belongs to the genus Aspergillus, Botrytis, Cochliobolus, or Emerysella.

8. The filamentous fungus according to claim 7, which is Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus fumigatus, Botrytis cinerea, Cochliobolus heterostrophos, or Emmarisella nidulans.

9. A method for producing a substance, comprising the step of culturing the filamentous fungus according to any one of claims 1 to 8 and causing the filamentous fungus to produce the substance.

Images