Patent Description:
A filamentous fungus is a collective name for fungi constructed of tubular cells called hyphae, and is used for fermentative production of: low-molecular-weight compounds, for example, chemical products such as an organic acid, a pigment, and an agricultural chemical bulk, and pharmaceutical products such as penicillin and statins; and industrial enzymes such as amylase, cellulase, protease, and lipase.

For example, in <CIT>, there is a description of a method of producing cellulase, including the steps of: producing a disaccharide-containing solution by adding thermophilic fungus-derived β-glucosidase to a glucose-containing solution and subjecting the mixture to a condensation reaction; and producing cellulase by culturing a filamentous fungus using a medium containing the disaccharide-containing solution.

In addition, in <CIT>, there is a description of a method of producing phospholipase, including a step of processing a fungal peptide to truncate a peptide from the C-terminus and/or a peptide from the N-terminus, to thereby produce a core peptide formed of a specific amino acid sequence having phospholipase activity.

In addition, in <CIT>, <CIT>, <CIT>, <CIT>, and <CIT>, with a view to increasing efficiency of substance production using the filamentous fungus, there is a description of an expression vector constructed so that the filamentous fungus functions as a host, there is also a description of a method involving preparing a transformant by introducing, into the filamentous fungus, a plasmid in which a gene encoding a homologous or heterologous protein is functionally linked to the expression vector, and there is also a description that utilization of the transformant contributes to increased production of enzymes such as amylase and cellulase and low-molecular-weight compounds such as penicillin.

As described above, the filamentous fungus has an advantage of being able to produce a wide variety of useful substances. However, the filamentous fungus causes a problem in that the filamentous fungus cannot be cultured at a high density because of entanglement of hyphae and aggregation of cells in its liquid culture step, a problem in that a production amount of a useful substance lowers, and a problem in that a production step of a useful substance becomes complicated (e.g., <CIT> and <CIT>).

Under such circumstances, the inventors of the present invention have found that, when a mutant filamentous fungus lacking expression of α-<NUM>,<NUM>-glucan is used, aggregation of cells during culture is suppressed more than in the related art and the cells are relatively homogeneously dispersed in a medium, and thus have developed a method of producing a substance (<CIT>). However, although the use of the mutant filamentous fungus suppresses the aggregation of the cells more than in the related art, there is a demand for development of a filamentous fungus that is still less liable to form an aggregate.

Non-Patent Literature <NUM> discloses filamentous fungus Aspergillus oryzae having genes encoding α-<NUM>,<NUM>- glucan synthase knocked out has better hyphal dispersion in culture and increased recombinant protein production (see abstract). The introduction explains that the aggregation of hyphae is thought to be related to cell wall polysaccharides and polypeptides. Also explained is the similarity between cell wall of A. oryzae and the human pathogen Aspergillus fumigatus (see p. <NUM>, column <NUM>).

Non-Patent Literature <NUM> discloses A. fumigatus mutants deficient in the production of α-<NUM>,<NUM>-glucan (see p. <NUM>-<NUM>). fumigatus mutants having genes sph3 or uge3 knocked out being defective in the production of galactosaminogalactan (GAG) are review. Gene sph3 is indicated to be also present in A. oryzae (see end of p. <NUM> and first paragraph of p <NUM>). It is explained that GAG and α-<NUM>,<NUM>-glucan play major roles in hyphae aggregation (see p <NUM>, column <NUM>, paragraph <NUM>).

Non-Patent Literature <NUM> discloses overlapping and distinct roles of Aspergillus fumigatus UDP-glucose <NUM>-epimerases in galactose metabolism and the synthesis of galactose-containing cell wall polysaccharides.

Non-Patent Literature <NUM> is directed to sph3, which is a glycoside hydrolase required for the biosynthesis of galactosaminogalactan in Aspergillus fumigatus.

An object of the present invention is to provide a filamentous fungus mutant strain that is still more suppressed in aggregation of cells (hyphae) in a medium than a related-art filamentous fungus.

Under such circumstances, the inventors of the present invention have made extensive investigations on a wide variety of factors in filamentous fungi from the viewpoint of the aggregation of cells, and as a result, have found a galactosaminogalactan (GAG) biosynthetic cluster to be a novel factor. In Non-patent Literature <NUM>, a disruption strain of the GAG biosynthetic cluster is obtained using Aspergillus fumigatus, and is analyzed for evaluating the effect of the cluster disruption. In Non-patent Literature <NUM>, it is reported that GAG is not observed on the cell wall surface of the disruption strain, but that there is no difference in germination or growth between the disruption strain and a wild-type strain. The inventors of the present invention have caused a filamentous fungus modified so as to be deficient in at least one α-<NUM>,<NUM>-glucan synthase gene comprising agsB and to be deficient in at least one galactosaminogalactan biosynthetic gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3 so that the at least one gene responsible for the expression of galactosaminogalactan is knocked out, and as a result, have observed a further decrease in expression of galactosaminogalactan, and besides, have surprisingly found that the aggregation of cells is suppressed and the cells are completely dispersed. The present invention is based on such novel findings.

According to the present invention, the filamentous fungus mutant strain that is still more suppressed in aggregation of cells in a medium than the related-art filamentous fungus can be provided. When cells aggregate during culture, the inside of the aggregate becomes anaerobic to kill the cells. Therefore, the filamentous fungus of the present invention suppressed in aggregation and the method using the same contribute to efficient culture and substance production of the filamentous fungus, and hence are extremely useful.

The present invention provides a mutant filamentous fungus which is deficient in at least one α-<NUM>,<NUM>-glucan synthase gene comprising agsB, and is deficient in at least one galactosaminogalactan biosynthetic gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3 so that the at least one gene responsible for the expression of galactosaminogalactan is knocked out.

Examples of the filamentous fungus 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, and the genus Monascus. Of those, the genus Aspergillus, the genus Botrytis, or the genus Cochliobolus is preferred, and the genus Aspergillus is more preferred. Examples of the filamentous fungi of the genus Aspergillus to be used in the present invention include Aspergillus oryzae, Aspergillus sojae, Aspergillus nidulans/Emericella nidulans, Aspergillus niger, and Aspergillus fumigatus. Of those, Aspergillus oryzae, Aspergillus sojae, Aspergillus nidulans, or Aspergillus niger is preferred, Aspergillus oryzae or Aspergillus sojae is more preferred, and Aspergillus oryzae is still more preferred. Examples of the filamentous fungi of the genus Botrytis to be used in the present invention include Botrytis cinerea (teleomorph: Botryotinia fuckeliana), Botrytis allii, Botrytis squamosa, and Botrytis byssoidea. Examples of the filamentous fungi of the genus Cochliobolus to be used in the present invention include Cochliobolus heterostrophus (anamorph: Bipolaris maydis), Cochliobolus carbonum, Cochliobolus miyabeanus, and Cochliobolus victoriae. Examples of the genus Monascus include Monascus purpureus, Monascus ruber, and Monascus pilosus.

The filamentous fungus mutant strain, which is deficient in at least one α-<NUM>,<NUM>-glucan synthase gene, comprises agsB according to the present invention. Examples of the α-<NUM>,<NUM>-glucan synthase gene ags include: agsA (Genbank accession No. AN5885) and agsB (Genbank accession No. AN3307) of Aspergillus nidulans; agsA, agsB, and agsC of Aspergillus oryzae; agsA, agsB, and agsC of Aspergillus sojae; ags1 (Genbank accession No. AFUA_3G00910) of Aspergillus fumigatus; agsE (Genbank accession No. ANI_1_360084) of Aspergillus niger; and agsB (Genbank accession No. Pc16g06130) of Penicillium chrysogenum. In this connection, agsA, agsB, and agsC of Aspergillus oryzae are registered in the Aspergillus database AspGD (http://www. aspergillusgenome. org) with the following gene numbers: agsA (AOR_1_956014), agsB (AOR_1_2634154), and agsC (AOR_1_1350024). The putative amino acid sequence of AgsA of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding AgsA of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. The putative amino acid sequence of AgsB of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding AgsB of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. The putative amino acid sequence of AgsC of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding AgsC of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. The putative amino acid sequence of AgsA of Aspergillus nidulans (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding AgsA of Aspergillus nidulans (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. In addition, the putative amino acid sequence of AgsB of Aspergillus nidulans (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding AgsB of Aspergillus nidulans (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. Examples of the amino acid sequences of AgsA, AgsB, and AgsC of Aspergillus sojae include amino acid sequences estimated from gene sequences registered in GenBank (Genbank accession Nos. DF093557 to DF093585) on the basis of homology with Aspergillus oryzae. The putative amino acid sequence of AgsA of Aspergillus sojae (SEQ ID NO: <NUM>) is shown in <FIG>, and the putative base sequence of a nucleic acid molecule encoding the above-mentioned AgsA of Aspergillus sojae (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. In addition, the putative amino acid sequence of AgsB of Aspergillus sojae (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding AgsB of Aspergillus sojae (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. In addition, the putative amino acid sequence of AgsC of Aspergillus sojae (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding AgsC of Aspergillus sojae (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. The putative amino acid sequence of AgsE of Aspergillus niger (SEQ ID NO: <NUM>) is shown in <FIG>. The base sequence of a nucleic acid molecule encoding AgsE of Aspergillus niger (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. The putative amino acid sequence of Ags1 of Aspergillus fumigatus (SEQ ID NO: <NUM>) is shown in <FIG>. The base sequence of a nucleic acid molecule encoding Ags1 of Aspergillus fumigatus (SEQ ID NO: <NUM>) is shown in <FIG> and <FIG>. The putative amino acid sequence of AgsB of Penicillium chrysogenum (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding AgsB thereof (SEQ ID NO: <NUM>) are shown in <FIG>.

Examples of the mutant filamentous fungus include mutant filamentous fungi each deficient in one or two or more of those α-<NUM>,<NUM>-glucan synthase genes. Of those, a mutant filamentous fungus deficient in all the three genes is preferred.

In the present invention, examples of the deficiency in α-<NUM>,<NUM>-glucan synthase gene ags include: a deletion of the whole or part of the coding region of α-<NUM>,<NUM>-glucan synthase in a genome; an insertion of another nucleic acid molecule into the whole or part of the coding region; and a substitution of the whole or part of the coding region by another nucleic acid molecule. In addition, the deficiency in α-<NUM>,<NUM>-glucan synthase gene ags encompasses an addition, deletion, and substitution of a predetermined nucleic acid molecule to the above-mentioned coding region. Also disclosed is a conditional gene deficiency designed so that α-<NUM>,<NUM>-glucan is expressed only under a certain condition.

The filamentous fungus mutant strain according to the present invention is deficient in at least one galactosaminogalactan biosynthetic gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3 so that the at least one gene responsible for the expression of galactosaminogalactan is knocked out, and the mutant filamentous fungus is deficient in at least one α-<NUM>,<NUM>-glucan synthase gene comprising agsB.

Galactosaminogalactan is an extracellular polysaccharide identified in Aspergillus fumigatus in <NUM>, and is formed of galactose (Gal), N-acetylgalactosamine (GalNAc), and galactosamine (GalN) (Non-patent Literature <NUM>). In addition, genes constituting the GAG biosynthetic cluster include uge3, sph3, ega3, agd3, and gtb3.

Therefore, an example of the filamentous fungus mutant strain deficient also in at least part of the GAG biosynthetic cluster according to the present invention is a filamentous fungus mutant strain 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, an example of the filamentous fungus mutant strain deficient also in at least part of the GAG biosynthetic cluster is a filamentous fungus mutant strain deficient in at least uge3 and sph3 out of those genes.

Examples of those genes constituting the GAG biosynthetic 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) of 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) of Aspergillus nidulans; uge3, sph3, ega3, agd3, and gtb3 of Aspergillus sojae; 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) of Aspergillus niger; uge3 (Genbank accession No. AFUA_3G07910), sph3 (Genbank accession No. AFUA_3G07900), ega3 (Genbank accession No. AFUA_3G07890), agd3 (Genbank accession No. AFUA_3G07870), and gtb3 (Genbank accession No. AFUA_3G07860) of Aspergillus fumigatus; 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) of Penicillium chrysogenum; uge3 (Gene ID: COCHEDRAFT_1185586), sph3 (Gene ID: COCHEDRAFT_1023805), ega3 (Gene ID: COCHEDRAFT_1023806), agd3 (Gene ID: COCHEDRAFT_1146217), and gtb3 (Gene ID: COCHEDRAFT_1146218) of Cochliobolus heterostrophus (anamorph: Bipolaris maydis); and uge3 (Gene ID: Bcin01p05750. <NUM>), sph3 (Gene ID: Bcin01p05740. <NUM>), ega3 (Gene ID: Bcin01p05730. <NUM>), agd3 (Gene ID: Bcin01p05720. <NUM>), and gtb3 (Gene ID: Bcin01p05710. <NUM>) of Botrytis cinerea.

The amino acid sequence of Uge3 of Aspergillus oryzae (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Uge3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Sph3 of Aspergillus oryzae (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Ega3 of Aspergillus oryzae (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Ega3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Agd3 of Aspergillus oryzae (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Agd3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Gtb3 of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding Gtb3 of Aspergillus oryzae (SEQ ID NO: <NUM>) is shown in <FIG>.

The amino acid sequence of Uge3 of Aspergillus nidulans (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Uge3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Sph3 of the above-mentioned Aspergillus nidulans (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Ega3 of Aspergillus nidulans (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Ega3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Agd3 of Aspergillus nidulans (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Agd3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the amino acid sequence of Gtb3 of Aspergillus nidulans (SEQ ID NO: <NUM>) is shown in <FIG>, and the base sequence of a nucleic acid molecule encoding Gtb3 of Aspergillus nidulans (SEQ ID NO: <NUM>) is shown in <FIG>.

In the present invention, examples of the amino acid sequences of Uge3, Sph3, Ega3, Agd3, and Gtb3 of Aspergillus sojae include amino acid sequences estimated from gene sequences of Aspergillus sojae registered in GenBank (Genbank accession Nos. DF093557 to DF093585) on the basis of homology with Aspergillus oryzae. The putative amino acid sequence of Uge3 of Aspergillus sojae (SEQ ID NO: <NUM>) and the putative base sequence of a nucleic acid molecule encoding Uge3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Sph3 of Aspergillus sojae (SEQ ID NO: <NUM>) and the putative base sequence of a nucleic acid molecule encoding Sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Ega3 of Aspergillus sojae (SEQ ID NO: <NUM>) and the putative base sequence of a nucleic acid molecule encoding Ega3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the putative amino acid sequence of Agd3 of Aspergillus sojae (SEQ ID NO: <NUM>) and the putative base sequence of a nucleic acid molecule encoding Agd3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. In addition, the putative amino acid sequence of Gtb3 of Aspergillus sojae (SEQ ID NO: <NUM>) is shown in <FIG>, and the putative base sequence of a nucleic acid molecule encoding Gtb3 of Aspergillus sojae (SEQ ID NO: <NUM>) are shown in <FIG>.

The putative amino acid sequence of Uge3 of Aspergillus niger (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Uge3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Sph3 of Aspergillus niger (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Ega3 of Aspergillus niger (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Ega3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Agd3 of Aspergillus niger (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Agd3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Gtb3 of Aspergillus niger (SEQ ID NO: <NUM>) is shown in <FIG>. The base sequence of a nucleic acid molecule encoding Gtb3 of Aspergillus niger (SEQ ID NO: <NUM>) is shown in <FIG>. The putative amino acid sequence of Uge3 of Aspergillus fumigatus (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Uge3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Sph3 of Aspergillus fumigatus (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Ega3 of Aspergillus fumigatus (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Ega3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Agd3 of Aspergillus fumigatus (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Agd3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Gtb3 of Aspergillus fumigatus (SEQ ID NO: <NUM>) is shown in <FIG>. The base sequence of a nucleic acid molecule encoding Gtb3 of Aspergillus fumigatus (SEQ ID NO: <NUM>) is shown in <FIG>. The putative amino acid sequence of Uge3 of Penicillium chrysogenum (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Uge3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Sph3 of Penicillium chrysogenum (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Ega3 of Penicillium chrysogenum (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule encoding Ega3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of Agd3 of Penicillium chrysogenum (SEQ ID NO: <NUM>) is shown in <FIG>. The base sequence of a nucleic acid molecule encoding Agd3 of Penicillium chrysogenum (SEQ ID NO: <NUM>) is shown in <FIG>. The putative amino acid sequence of Gtb3 of Penicillium chrysogenum (SEQ ID NO: <NUM>) is shown in <FIG>. The base sequence of a nucleic acid molecule encoding Gtb3 of Penicillium chrysogenum (SEQ ID NO: <NUM>) is shown in <FIG>.

The putative amino acid sequence of uge3 of Cochliobolus heterostrophus (anamorph: Bipolaris maydis) (SEQ ID NO: <NUM>), the base sequence of a nucleic acid molecule of uge3 (SEQ ID NO: <NUM>), the putative amino acid sequence of sph3 thereof (SEQ ID NO: <NUM>), and the base sequence of a nucleic acid molecule of sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of ega3 of Cochliobolus heterostrophus (anamorph: Bipolaris maydis) (SEQ ID NO: <NUM>), the base sequence of a nucleic acid molecule of ega3 thereof (SEQ ID NO: <NUM>), the putative amino acid sequence of agd3 thereof (SEQ ID NO: <NUM>), and the base sequence of a nucleic acid molecule of agd3 thereof (SEQ ID NO: <NUM>) are shown in <FIG> and <FIG>. The putative amino acid sequence of gtb3 of Cochliobolus heterostrophus (anamorph: Bipolaris maydis) (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule of gtb3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>.

The putative amino acid sequence of ags1 of Botrytis cinerea (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule of ags1 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of uge3 of Botrytis cinerea (SEQ ID NO: <NUM>), the base sequence of a nucleic acid molecule of uge3 thereof (SEQ ID NO: <NUM>), the putative amino acid sequence of sph3 thereof (SEQ ID NO: <NUM>), and the base sequence of a nucleic acid molecule of sph3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>. The putative amino acid sequence of ega3 of Botrytis cinerea (SEQ ID NO: <NUM>), the base sequence of a nucleic acid molecule of ega3 thereof (SEQ ID NO: <NUM>), the putative amino acid sequence of agd3 thereof (SEQ ID NO: <NUM>), and the base sequence of a nucleic acid molecule of agd3 thereof (SEQ ID NO: <NUM>) are shown in <FIG> and <FIG>. The base sequence of a nucleic acid molecule of gtb3 of Botrytis cinerea (SEQ ID NO: <NUM>) and the base sequence of a nucleic acid molecule of gtb3 thereof (SEQ ID NO: <NUM>) are shown in <FIG>.

In the present invention, examples of the deficiency in at least part of the GAG biosynthetic cluster include: a deletion of the whole or part of a coding region out of the GAG biosynthetic cluster in a genome; an insertion of another nucleic acid molecule into the whole or part of the coding region; and a substitution of the whole or part of the coding region by another nucleic acid molecule. In addition, the deficiency in at least part of the GAG biosynthetic cluster encompasses not only an addition, deletion, and substitution of a predetermined nucleic acid molecule to the above-mentioned coding region but also a conditional gene deficiency designed so that GAG is expressed only under a certain condition.

In addition, in the present invention, examples of the deficiency in uge3 include: a deletion of the whole or part of a Uge3 coding region in a genome; an insertion of another nucleic acid molecule into the whole or part of the coding region; and a substitution of the whole or part of the coding region by another nucleic acid molecule. In the present invention, examples of the deficiency in sph3 include: a deletion of the whole or part of an Sph3 coding region in a genome; an insertion of another nucleic acid molecule into the whole or part of the coding region; and a substitution of the whole or part of the coding region by another nucleic acid molecule. In the present invention, examples of the deficiency in ega3 include: a deletion of the whole or part of an Ega3 coding region in a genome; an insertion of another nucleic acid molecule into the whole or part of the coding region; and a substitution of the whole or part of the coding region by another nucleic acid molecule. In the present invention, examples of the deficiency in agd3 include: a deletion of the whole or part of an Agd3 coding region in a genome; an insertion of another nucleic acid molecule into the whole or part of the coding region; and a substitution of the whole or part of the coding region by another nucleic acid molecule. In the present invention, examples of the deficiency in gtb3 include: a deletion of the whole or part of a Gtb3 coding region in a genome; an insertion of another nucleic acid molecule into the whole or part of the coding region; and a substitution of the whole or part of the coding region by another nucleic acid molecule.

In addition, also for uge3, sph3, ega3, agd3, and gtb3, the deficiency in each of these genes encompasses not only an addition, deletion, and substitution of a predetermined nucleic acid molecule to the above-mentioned coding region but also a conditional gene deficiency designed so that GAG is expressed only under a certain condition. According to the invention, the mutant filamentous fungus is deficient in at least one galactosaminogalactan biosynthetic gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3 so that the at least one gene responsible for the expression of galactosaminogalactan is knocked out.

The filamentous fungus mutant strain deficient in at least part of the GAG biosynthetic cluster according to the present disclosure but not part of the present invention encompasses not only a filamentous fungus mutant strain completely lacking expression of GAG but also a filamentous fungus mutant strain substantially lacking expression of GAG. More specifically, the mutant strain substantially lacking expression of GAG refers to a mutant strain that expresses only a small amount of GAG and shows significant suppression of aggregation of cells and an example thereof is a strain having an expression amount of GAG of <NUM>% or less with respect to that of a wild-type strain, more preferably <NUM>% or less with respect to that of the wild-type strain.

A method of the present invention may be used for the production of useful substances, for example, enzymes such as amylase and cellulase and low-molecular-weight compounds such as penicillin that the filamentous fungus originally has abilities to produce. In the method of the present invention, transformation may be performed so as to enhance the expression of the useful substances that the filamentous fungus originally has abilities to produce, or so as to express substances that the filamentous fungus originally has no abilities to produce. As such transformation method, a method known per se (e.g., methods described in <CIT>, <CIT>, <CIT>, <CIT>, and <CIT>) may be used, the method involving utilizing an expression vector constructed so that the filamentous fungus can function as a host, and a plasmid constructed by functionally linking a gene encoding a homologous or heterologous protein to the expression vector.

A method of producing such mutant strain may be performed by subjecting a filamentous fungus to, for example, the following, by appropriately using a method known per se (e.g., methods described in Non-patent Literatures <NUM> to <NUM>): the construction of a disruption cassette for α-<NUM>,<NUM>-glucan gene and the introduction of the cassette into a genome gene; and the construction of a disruption cassette for a gene constituting the GAG biosynthetic cluster and the introduction of the cassette into a genome gene. In the present invention, as the filamentous fungus to be subjected to such genetic manipulation, there may be used, for example, a filamentous fungus having a mutation of gene ligD disruption and/or gene adeA disruption (preferably both thereof) introduced in advance in order to enable gene introduction into a target site at a high probability. Here, ligD is a gene associated with nonhomologous recombination repair in DNA repair, and is preferred because a transformant having the gene introduced into the target site through homologous recombination can be acquired with relatively high efficiency by disrupting the gene. An example of the mutation that disrupts the gene is a ligD::sC mutation obtained by disruption using an sC marker (Non-patent Literature <NUM>). In addition, adeA is an adenine auxotrophic gene, and an example of the mutation that disrupts the gene is adeAΔ::ptrA obtained by disruption with pyrithiamine resistance gene (ptrA) (Non-patent Literature <NUM>). Therefore, examples of the filamentous fungus mutant strain of the present invention also include filamentous fungus mutant strains further having those mutations.

The filamentous fungus mutant strain according to the present invention may be used for the production of a substance, and may be used for, for example, the following method.

The present invention provides a method of producing a substance, including the steps of:.

The useful substances that can be produced by the method of the present invention are not particularly limited as long as the substances can be produced by the filamentous fungus, and examples thereof include: low-molecular-weight compounds such as penicillin, statins, cephalosporin, kojic acid, citric acid, and malic acid; and high-molecular-weight compounds such as amylase, cellulase, protease, lipase, peptidase, esterase, hydrophobin, and oxidase. In addition, examples of the useful substances include chemical products such as an organic acid, a pigment, and an agricultural chemical bulk, and various substances to be used as pharmaceutical products. In addition, the method of the present invention is also applicable to, for example, the production of bioethanol through biomass decomposition (e.g., one using a mold genetically modified so as to highly produce cellulase or the like). By the method of the present invention, a cell wall constituent component or a hydrolysate thereof may be produced, but a substance other than the cell wall constituent component or the hydrolysate thereof may also be produced. Examples of the cell wall constituent component or the hydrolysate thereof include α-<NUM>,<NUM>-glucan, β-<NUM>,<NUM>-glucan, polygalactose, glucose, galactose, glucosamine, amino acid, mannose, N-acetylglucosamine, N-acetylgalactosamine, and chitin. In the present invention, the "substance" that can be produced by the method of the present invention is understood not to encompass: a compound that harms or kills the filamentous fungus; living cells; a substance that can only be obtained by chemical synthesis; and the like.

The method of the present invention includes a step of culturing a mutant filamentous fungus, which is deficient in at least one galactosaminogalactan biosynthetic gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3 so that the at least one gene responsible for the expression of galactosaminogalactan is knocked out, and the mutant filamentous fungus is deficient in at least one α-<NUM>,<NUM>-glucan synthase gene comprising agsB to allow the filamentous fungus to produce a substance. A medium to be used in the step is not particularly limited, and there may be used a wide range of media that may be used for the culture of a filamentous fungus. Examples thereof include CD minimal medium, YPD medium, TSB medium, malt medium, and PDA medium. To the medium, glucose, starch, soluble starch, or the like may be added as a carbon source. The addition amount of such carbon source is not particularly limited and may be appropriately set within a range of, for example, from <NUM>% to <NUM>%, more preferably from <NUM>% to <NUM>%. A culture temperature is not particularly limited and may be appropriately set within a range of from <NUM> to <NUM>, more preferably from <NUM> to <NUM>. A culture time is also not particularly limited and may be appropriately set within a range of, for example, from <NUM> hours to <NUM> hours, more preferably from <NUM> hours to <NUM> hours. In addition, as described above, the filamentous fungus mutant strain according to the disclosure but not part of the present invention relates to a filamentous fungus mutant strain having a conditional gene deficiency designed so that α-<NUM>,<NUM>-glucan and/or GAG is expressed only under a certain condition. Therefore, the method of the present disclosure but not part of the present invention also relates to a method including a step of culturing a mutant strain having the conditional gene deficiency under such a condition that α-<NUM>,<NUM>-glucan and GAG are not expressed (or their expressions are suppressed).

A method of collecting the useful substance from the culture medium is not particularly limited, and there may be appropriately used a method known per se (e.g., centrifugation, recrystallization, a distillation method, a solvent extraction method, or chromatography). The method of the present invention is a method of collecting a useful substance. Therefore, a method of decomposing and detecting constituent components of a filamentous fungus mutant for the purpose of analyzing constituent components of cells of a filamentous fungus itself or the like for research thereof is essentially different from the method of the present invention.

Now, an embodiment of the present invention is more specifically described by way of Examples, and the action and effect of the present invention are demonstrated.

In this study, a modified strain of an NS4 strain (genotype; niaD-,sC-) was used as a wild-type strain of a filamentous fungus A. The NS4 modified strain used in this study is a strain having introduced therein a ligDΔ::sC mutation capable of gene introduction into a target site at a high probability and an adeAΔ::ptrA mutation having adenine auxotrophy (ligDΔ::sC,adeAΔ::ptrA). In addition, a strain deficient in three kinds of α-<NUM>,<NUM>-glucan synthase genes (agsAΔagsBΔagsCΔ) was used as an AG-deficient strain. Other produced gene mutant strains and genotypes thereof are as shown in Table <NUM>.

In this study, Czapek-Dox (CD) medium was used as a selection minimal medium for A. In addition, YPD medium was used as a nutrient-rich medium. Herein, CD medium having added thereto <NUM> sodium glutamate in place of sodium nitrate as a nitrogen source was used in the culture of a niaD- strain (this medium is hereinafter referred to as CDE medium). In addition, in the culture of an adeA- strain, adenine sulfate was added at a final concentration of <NUM>% (this medium is hereinafter referred to as CDEA medium). The compositions of media and culture solutions are as shown in Table <NUM>. In the case of using a medium as an agar plate medium, agar was added to the medium at a final concentration of <NUM>% (w/v).

In this study, the culture of A. oryzae was performed at <NUM>, unless otherwise stated. In the case of agar plate culture, a plate was left to stand still, and in the case of liquid culture, rotary shaking culture was performed at <NUM> rpm.

Conidia of A. oryzae were inoculated into agar plate medium of CD medium satisfying the auxotrophy of each kind of mutant strain, and were cultured at <NUM> for about <NUM> days until conidia were sufficiently formed. Further, the conidia were subcultured in malt medium at <NUM> for about <NUM> days until conidia were sufficiently formed. <NUM> per plate of a sterilized conidial suspended solution (<NUM> NaCl, <NUM>% Tween <NUM>, <NUM> phosphate buffer (pH <NUM>)) was poured on agar plate medium, and the conidia were scraped with a cell spreader to be suspended. For the purpose of removing hyphae mixed in the suspension, the suspension was filtered using a sterilized cell strainer (pore size: <NUM>) or sterilized MIRACLOTH (Calbiochem), and only the conidia were collected in a Falcon tube having a volume of <NUM> or a volume of <NUM> to prepare a conidial suspension. The number of the conidia was measured using a Thoma hemocytometer.

The uge3 and sph3 genes of A. oryzae are adjacent to each other. Therefore, a disruption cassette for disrupting both the genes at once was produced. First, a uge3 downstream (<NUM>' side) region (amplicon <NUM>) and an sph3 downstream (<NUM>' side) region (amplicon <NUM>) were amplified by PCR using genomic DNA of A. oryzae as a template. In addition, AnadeA gene (amplicon <NUM>) was amplified from plasmid TOPO-<NUM>-adeA by PCR (1st round of PCR). PCR amplification was performed using: primers Sph3+Uge3-LU and Sph3+Uge3-LL+Ade for amplicon <NUM>; primers Sph3+Uge3-RU+Ade and Sph3+Uge3-RL for amplicon <NUM>; and primers Sph3+Uge3-AU and Sph3+Uge3-AL for amplicon <NUM> (Table <NUM>). The primers Sph3+Uge3-LL+Ade, Sph3+Uge3-AU, Sph3+Uge3-RU+Ade, and Sph3+Uge3-AL each contain, on the <NUM>' side, a homologous sequence to a complementary strand, for linking by fusion PCR. The PCR product was gel-extracted, and subjected to PCR using the primers Sph3+Uge3-LU and Sph3+Uge3-RL to link those three fragments (2nd round of PCR). A main band of the PCR product was gel-extracted, and used as a uge3/sph3 gene disruption cassette.

Transformation of A. oryzae was performed using a protoplast-PEG method (Non-patent Literature <NUM>). The wild-type strain and the AG-deficient strain (agsAΔagsBΔagsCΔ) were each used as a host strain. <NUM>×<NUM><NUM> conidia of the host strain were inoculated into <NUM> of YPD liquid medium in an Erlenmeyer flask having a volume of <NUM>, and were subjected to rotary shaking culture at <NUM> for <NUM> hours. The cells were filtered through sterilized MIRACLOTH (Calbiochem) to collect the cells. The cells were washed with distilled water, and the cells were dehydrated by being pressed with a sterilized spatula. The collected cells were placed in a Falcon tube having a volume of <NUM>, and suspended by adding <NUM> of a protoplast forming solution [<NUM>/mL Lysing Enzymes (Sigma), <NUM>/mL Cellulase Onozuka (Yakult Pharmaceutical Ind. , Ltd), <NUM>/mL Yatalase (TaKaRa), Lysing enzyme buffer (Table <NUM>)] that had been filtered through a filter DISMIC-25CS (ADVANTEC) having a pore size of <NUM>. The suspension was shaken at <NUM> and <NUM> rpm for <NUM> hours to digest cell walls, to thereby prepare protoplasts. After the reaction, undigested cells were filtered through sterilized MIRACLOTH, and the filtrate was centrifuged at <NUM> and <NUM>,<NUM>×g for <NUM> minutes to collect the protoplasts. The collected protoplasts were washed with <NUM> NaCl, and centrifuged at <NUM> and <NUM>,<NUM>×g for <NUM> minutes to precipitate and collect the protoplasts. The protoplasts were added to Sol. I (Table <NUM>) at <NUM>×<NUM><NUM> protoplasts/mL and suspended therein. After that, a <NUM>/<NUM> amount of Sol. II (Table <NUM>) was added, and the contents were mixed well. <NUM>µL of the protoplast liquid was dispensed in a Falcon tube having a volume of <NUM>, a DNA solution was added in an appropriate amount (from about <NUM>µg to about <NUM>µg), and the contents were mixed well and left to stand in ice for <NUM> minutes. Next, <NUM> of Sol. II (Table <NUM>) was added, and the contents were mixed well and then left to stand at room temperature for <NUM> minutes. <NUM> of Sol. I was added, and the contents were mixed well and then centrifuged at room temperature and <NUM>,<NUM>×g for <NUM> minutes. The supernatant was removed, and <NUM>µL of Sol. I was added. The protoplasts were homogeneously suspended, and seeded into CD selection medium (Table <NUM>) containing <NUM> NaCl. After that, <NUM> of soft agar medium of the same composition [<NUM>% (w/v) Agar] that had been warmed to <NUM> was poured from the periphery and overlaid so as to quickly and homogeneously suspend the protoplasts. After that, the protoplasts were cultured at <NUM> until colonies were formed.

In order to confirm whether the genomic DNA of the resulting transformed strain candidate had been transformed as intended, the genomic DNA was simply extracted from the conidia of the strain, and the transformed strain was selected by PCR using designed primers. <NUM>µL of YPD liquid medium was taken in a <NUM> Eppendorf tube, and the conidia of the transformed strain candidate were poked with a sterilized toothpick and inoculated, followed by culture at <NUM> until cells were grown. After centrifugation, the medium was removed. Glass beads in an amount equal to that of the cells and <NUM>µL of Nuclei Lysis Sol. (Promega) were added, and the cells were pulverized with Micro Smash™ MS-100R (TOMY) at <NUM>,<NUM> rpm for <NUM> minutes. The resultant was left to stand at <NUM> for <NUM> minutes, <NUM>µL of Protein Prep. (Promega) was added, and the contents were mixed well. The mixture was left to stand at room temperature for <NUM> minutes and centrifuged at <NUM> and <NUM>,<NUM> rpm for <NUM> minutes, and then the supernatant was transferred to another <NUM> tube. A <NUM>/<NUM> amount of <NUM> sodium acetate and a <NUM>-fold amount of ethanol were added, and the contents were mixed. After centrifugation at <NUM> and <NUM>,<NUM> rpm for <NUM> minutes, the pellets were washed with <NUM> of <NUM>% ethanol and dissolved in <NUM>µL of RNase-containing TE. The resulting solution was defined as a genomic DNA solution, and stored at <NUM> until being used as a template for PCR.

The transformed strain candidate of interest was grown on minimum agar plate medium, and the collected conidial suspension was passed through a mononucleation filter (ISOPORE TM MEMBRANE FILTERS, <NUM> TMTP, Millipore) that had been sterilized by autoclave treatment in advance, to thereby collect mononucleate conidia. The conidial suspension subjected to the mononucleation treatment was appropriately diluted, and grown on minimum agar plate medium. The resulting strain candidate was confirmed again by PCR. Thus, the transformed strain of interest was purified.

The wild-type strain, the AG-deficient strain, or the AG-GAG-deficient strain was used and subjected to liquid shaking culture in YPD medium for <NUM> hours. The temperature was set to <NUM>, the number of revolutions was set to <NUM> rpm, the scale of the medium was set to a <NUM>/<NUM> Erlenmeyer flask (without baffles), and conidia were inoculated at <NUM>×<NUM><NUM> conidia/mL.

The produced AG-GAG-deficient strain highly expressing cutL1 was subjected to a PBSA decomposition ability test in order to confirm the introduction of the plasmid for highly expressing cutL1. CDE (<NUM>% maltose) medium containing <NUM>% PBSA was used for the test. Conidia were inoculated at the center of the medium and subjected to static culture at <NUM> for <NUM> days, and formed halos were observed. As controls, a wild-type strain highly expressing cutL1 and an AG-GAG-deficient strain not highly expressing cutL1 were used.

Proteins in <NUM>µL of the culture supernatant were purified by TCA precipitation, appropriately diluted, and subjected to SDS-PAGE (buffer compositions were as shown in Table <NUM>). As a standard, <NUM>µg to <NUM> ug of purified α-amylase (derived from A. oryzae, Sigma-Aldrich) or <NUM> ng to <NUM> ng of purified CutL1 quantified by a BCA method was used. An image of a gel detected by SDS-PAGE was taken into ImageJ, and a band of interest was converted into a pixel value. A calibration curve was prepared from the standard to quantify an endogenous amylase or CutL1 secretion amount.

The wild-type strain highly expressing cutL1, the AG-deficient strain highly expressing cutL1, or the AG-GAG-deficient strain highly expressing cutL1 was used and subjected to liquid shaking culture in YPM medium (Table <NUM>) for <NUM> hours. The temperature was set to <NUM>, the number of revolutions was set to <NUM> rpm, the scale of the medium was set to a <NUM>/<NUM> Erlenmeyer flask, and conidia were inoculated at <NUM>×<NUM><NUM> conidia/mL.

On the basis of five gene cluster sequences considered to be responsible for GAG biosynthesis in A. fumigatus, a database (AspGD) was searched as to whether or not the same gene cluster was also present in A. As a result, it was suggested that the cluster gene sequences were also present in A. oryzae, and the ORFs of gtb3 (AOR_1_2580174), agd3 (AOR_1_2582174), ega3 (AOR_1_2584174), sph3 (AOR_1_2586174), and uge3 (AOR_1_2588174) were GAG biosynthetic genes in A. oryzae (<FIG>).

The wild-type strain and the AG-deficient strain were each used as a parental strain, and the uge3/sph3 gene disruption cassette was introduced into the genome thereof by a protoplast-PEG method. The selection of transformants was performed with adeA-free CDE agar plate medium. The resulting transformants were subjected to nucleus purification, and confirmed by PCR amplification using primers Sph3+Uge3-LU and Sph3+Uge3-RL.

The strain deficient only in GAG formed a large hyphal aggregate as compared to the wild-type strain. In addition, the hyphae aggregated to form a hyphal aggregate in each of the wild-type strain and the AG-deficient strain, whereas the AG-GAG-deficient strain did not show aggregation of hyphae and was observed to be in a state in which the hyphae were completely dispersed in liquid medium. Hitherto, a mutant strain of A. oryzae showing culture properties of being completely dispersed as described above has not been known. In addition, on agar plate medium, the AG-GAG-deficient strain showed growth comparable to that of the wild-type strain.

The AG-GAG-deficient strain was used as a host strain and transformed with pNGA-gla-cut (Takahashi et al. , <NUM>), a plasmid for highly expressing cutL1. For the selection of a strain highly expressing cutL1, CD medium containing <NUM> NaCl was used, and a transformed strain candidate showing nitric acid autotrophy was acquired. The resulting transformant was subjected to nucleus purification, and confirmed by PCR amplification using primers niaD-tail-Fw and cutL1-RT-F.

Further, a halo formation test was performed using CDE (<NUM>% maltose) medium containing <NUM>% PBSA. As a result, in the AG-GAG-deficient strain highly expressing cutL1, halo formation comparable to that of the wild-type strain highly expressing cutL1 used as a control was observed. This suggested that the plasmid for highly expressing cutL1 had been properly introduced.

As a result of culture in YPM medium, the dry cell weight at <NUM> hours of culture had increased in the order of the wild-type strain, the AG-deficient strain, and the AG-GAG-deficient strain. In particular, the dry cell weight of the AG-GAG-deficient strain had significantly increased to be about <NUM> times that of the wild-type strain (<FIG>). In addition, the CutL1 production amount had also increased in the order of the wild-type strain, the AG-deficient strain, and the AG-GAG-deficient strain, and the CutL1 production amounts of the AG-deficient strain and the AG-GAG-deficient strain had significantly increased to be about <NUM> times that of the wild-type strain and about <NUM> times that of the wild-type strain, respectively (<FIG>). The results suggested that the AG-GAG-deficient strain showing complete dispersibility had properties suitable for high production of a substance in high-density culture.

<NUM>µL of a conidial suspension of the WT strain, the AGΔ strain, or the AG-GAGΔ strain prepared at <NUM>×<NUM><NUM>/uL was spotted (a total of <NUM>×<NUM><NUM> conidia/plate) at the center of CD agar medium containing Congo red (CR) at <NUM> ug/mL, <NUM> ug/mL, <NUM> ug/mL, <NUM> ug/mL, <NUM> ug/mL, or <NUM> ug/mL, and incubated at <NUM> for <NUM> days. A colony diameter after the <NUM> days was measured, and a growth rate on CR-containing medium was calculated with reference to a colony diameter in the case of no CR (<FIG>). The results were as follows: the AG-GAGΔ strain had lower growth rates than the wild-type strain and the AGΔ strain at all concentrations. This suggested that not only AG but also GAG was associated with CR sensitivity.

The cell wall components of the wild-type (WT) strain, AGΔ strain, AG-GAGΔ strain, and GAGΔ strain of an Aspergillus were analyzed by fractionating the polysaccharide components of cells through the use of a hot water/alkali extraction method, and quantifying monosaccharide components contained in a sulfuric acid hydrolysate of each fraction. First, conidia of each strain were inoculated into <NUM> of YPD medium (<NUM>% peptone, <NUM>% yeast extract, <NUM>% glucose) at a final concentration of <NUM>×<NUM><NUM>/mL, and subjected to shaking culture at <NUM> and <NUM> rpm for <NUM> hours. After the culture, the culture solution was filtered through MIRACLOTH. The resulting cells were washed with water. The cells were lyophilized, and then pulverized with a mixer mill. Next, <NUM> of dry cell powder of each strain was fractionated into a hot-water-soluble (HW) fraction, an alkali-soluble/water-soluble (AS1) fraction, an alkali-soluble (AS2) fraction, and an alkaliinsoluble (AI) fraction in accordance with the method of Yoshimi et al. (<NPL>) (Table <NUM>).

It is known from the report of Yoshimi et al. that α-<NUM>,<NUM>-glucan is mainly contained in the AS2 fraction, and β-<NUM>,<NUM>-glucan and chitin are mainly contained in the AI fraction (<NPL>). <NUM> of each of those four fractions was used and heated in the presence of <NUM> N H<NUM>SO<NUM> at <NUM> for <NUM> hours to decompose the polysaccharide components in the fraction into monosaccharides. Each of the hydrolyzed fractions was neutralized with barium carbonate, and centrifuged to provide a supernatant. The monosaccharide components contained in the hydrolysate of each fraction were separated using an anion-exchange column Carbo PAC PA-<NUM> (<NUM>×<NUM>, DIONEX) and a guard column Carbo PAC under the conditions of a flow rate of <NUM>/min, a column temperature of <NUM>, a compartment temperature of <NUM>, and an eluent of <NUM> NaOH, and were detected using a pulsed amperometric detector. The monosaccharide components were quantified using <NUM> ug/mL to <NUM> ug/mL galactosamine, glucosamine, galactose, glucose, and mannose as standards. In addition, <NUM> ug/mL fucose was used as an internal standard. As a result, no significant difference was found among the monosaccharide components of the HW, AS1, and AI fractions of each strain (<FIG>). In contrast, in the AS2 fraction, the AGΔ strain and the AG-GAGΔ strain were remarkably reduced in glucose amount as compared to the wild-type strain (<FIG>). This suggested that the cell walls of the AGΔ strain and the AG-GAGΔ strain hardly contained α-<NUM>,<NUM>-glucan.

In addition, <NUM>µg of the HW fraction of each strain was subjected to sulfuric acid hydrolysis and monosaccharide component analysis by the same methods as described above. The results were as follows: the wild-type strain and the AGΔ strain contained <NUM> and <NUM> of galactosamine per <NUM> of grown cells, respectively, whereas no galactosamine was detected in the AG-GAGΔ strain (<FIG>). No previous study has reported a polysaccharide containing galactosamine other than GAG in the cell wall of a filamentous fungus of the genus Aspergillus. This revealed that the cell wall of the AG-GAGΔ strain contained no GAG.

Cochliobolus heterostrophus (anamorph: Bipolaris maydis) has in its genome a gene cluster homologous to the galactosaminogalactan biosynthetic gene cluster (sequence information, <FIG>). This cluster includes five genes, and a construct for substituting regions corresponding to sph3 and uge3 out of those genes by hygromycin resistance gene was produced (<FIG>). First, a HITO7711 strain serving as a wild-type strain of Cochliobolus heterostrophus was subjected to shaking culture (<NUM> rpm) in complete medium (CM: <NUM> Ca(NO<NUM>)<NUM>·<NUM><NUM>O, <NUM> MgSO<NUM>·<NUM><NUM>O, <NUM> KCl, <NUM> KH<NUM>PO<NUM>, <NUM> K<NUM>HPO<NUM>, <NUM> glucose, <NUM> tryptone, and <NUM> yeast extract per liter) at <NUM> for <NUM> hours, and the cells were collected by filtration through MIRACLOTH. Next, the cells were treated with a protoplast forming liquid (<NUM>/mL lysing enzyme, <NUM>/mL Cellulase onozuka, <NUM>/mL Yatalase in <NUM> Na phosphate buffer, pH <NUM>) to produce protoplasts. The produced protoplasts were used and transformed by a protoplast-PEG method in accordance with the method of Yoshimi et al. Genomic DNA was extracted from one gene disruption strain candidate thus obtained, and gene disruption was confirmed by PCR. As a result, bands of about <NUM> kb and about <NUM> kb to be amplified only in the case of successful gene disruption were found (<FIG>), and thus the strain was confirmed to be a GAG disruption strain having the regions corresponding to sph3 and uge3 deleted as designed.

Next, the GAG disruption strain thus obtained was subjected to shaking culture in YPM medium using maltose as a carbon source (<NUM>% peptone, <NUM>% yeast extract, <NUM>% maltose) (<NUM>, <NUM> rpm, <NUM> hour), and culture properties were observed. The results were as follows: the wild-type strain grew while forming aggregates of hyphae (<FIG>), whereas the hyphae of the GAG disruption strain tended to be dispersed (<FIG>). This characteristic is similar to that of the AG-GAG-deficient strain of A. oryzae, and hence it is suggested that GAG deficiency provides a culture characteristic suitable for high-density culture also in C. heterostrophus.

Botryotinia fuckeliana (anamorph: Botrytis cinerea) has in its genome a gene cluster homologous to the galactosaminogalactan biosynthetic gene cluster (sequence information, <FIG>). This cluster includes five genes, and a construct for substituting regions corresponding to sph3 and uge3 out of those genes by hygromycin resistance gene was produced (<FIG>). First, an AG disruption strain of Botryotinia fuckeliana was subjected to shaking culture (<NUM> rpm) in complete medium (CM: <NUM> Ca(NO<NUM>)<NUM>·<NUM><NUM>O, <NUM> MgSO<NUM>·<NUM><NUM>O, <NUM> KCl, <NUM> KH<NUM>PO<NUM>, <NUM> K<NUM>HPO<NUM>, <NUM> glucose, <NUM> tryptone, and <NUM> yeast extract per liter) at <NUM> for <NUM> hours, and the cells were collected by filtration through MIRACLOTH. Next, the cells were treated with the protoplast forming liquid described above to produce protoplasts. The produced protoplasts were used and transformed by a protoplast-PEG method in the same manner. Genomic DNA was extracted from <NUM> gene disruption strain candidates thus obtained, and gene disruption was confirmed by PCR. As a result, bands of about <NUM> kb and about <NUM> kb to be amplified only in the case of successful gene disruption were found (<FIG>) in two strains, and thus these strains were each confirmed to be a GAG disruption strain having the regions corresponding to sph3 and uge3 deleted as designed.

Next, the GAG disruption strain thus obtained was subjected to shaking culture in YPM medium using maltose as a carbon source (<NUM>% peptone, <NUM>% yeast extract, <NUM>% maltose) (<NUM>, <NUM> rpm, <NUM> hour), and culture properties were observed. As described later, when AG expression on the cell surface of the GAG disruption strain of B. cinerera was detected with an α-<NUM>,<NUM>-glucanase-glucan binding domain-GFP fusion under the above-mentioned culture conditions (<FIG>), it was found that no expression of AG was detected, which was substantially comparable to that of the AG-GAG disruption strain of A. Accordingly, under the above-mentioned culture conditions, although the GAG disruption strain of B. cinerera is deficient only in GAG, whereas the wild-type strain grew while forming aggregates of hyphae (<FIG>), the hyphae of the GAG disruption strain of B. cinerera tended to be more dispersed than those of the wild-type strain (<FIG>). This property is similar to that of the AG-GAG disruption strain of A. oryzae, suggesting the possibility that Botryotinia fuckeliana can be made suitable for high-density culture merely by GAG disruption.

Cells (WT or AG-GAGΔ of Aspergillus oryzae; WT or GAGΔ of Botrytis cinerea; or WT or GAGΔ of Cochliobolus heterostrophus) were mounted on a slide glass, and fixed by being incubated at <NUM> for <NUM> minutes. The fixed cells were immersed in <NUM>µL of an AGBD-GFP solution (<NUM> ug/mL in <NUM> potassium phosphate buffer) and incubated at <NUM> for <NUM> hours. AGBD-GFP is a recombinant protein obtained by fusing the α-<NUM>,<NUM>-glucan-binding site of α-<NUM>,<NUM>-glucanase and GFP, and can specifically stain α-<NUM>,<NUM>-glucan (<NPL>. After the <NUM> hours of reaction, the cells were washed with <NUM> potassium phosphate buffer <NUM> times, and observed with a fluorescence microscope. As a result, definite fluorescence derived from α-<NUM>,<NUM>-glucan was observed in the cells of A. oryzae serving as an Aspergillus, whereas no fluorescence was observed in the AG-GAGΔ strain (<FIG>). In addition, also in the case of B. cinerea, no fluorescence was observed in each of the wild-type strain and the GAGΔ strain (<FIG>). Therefore, it was suggested that B. cinerea, though having α-<NUM>,<NUM>-glucan synthase, did not express α-<NUM>,<NUM>-glucan under the culture conditions in question.

In addition, as a matter of course, no fluorescence derived from α-<NUM>,<NUM>-glucan was observed in C. heterostrophus having no α-<NUM>,<NUM>-glucan synthase (<FIG>).

Claim 1:
A mutant filamentous fungus, which fulfils both of the following requirements (<NUM>) and (<NUM>):
(<NUM>) the mutant filamentous fungus is deficient in at least one galactosaminogalactan biosynthetic gene selected from the group consisting of uge3, sph3, ega3, agd3, and gtb3 so that the at least one gene responsible for the expression of galactosaminogalactan is knocked out, and
(<NUM>) the mutant filamentous fungus is deficient in at least one α-<NUM>,<NUM>-glucan synthase gene comprising agsB.