Mutant filamentous fungi and method for producing protein using the same

Abstract

The present invention provides a method for producing a mutant filamentous fungus. The method includes the steps of changing the expression of XYR1 and ACE3 of a parent filamentous fungus. The change of XYR1 is a substitution, deletion, insertion or addition of at least one amino acid residue in the region corresponding to positions 810 to 833 of sequence number 1 for a polypeptide consisting of sequence number 1 or an amino acid sequence at least 90% identical to the sequence thereof and functioning as a transcriptional activator of cellulase and hemicellulase, and the change in ACE3 expression is an enhanced expression of a partial polypeptide of ACE3.

Description

Technical Field

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

[0002] Filamentous fungi produce a variety of cellulases and hemicellulases and therefore have attracted attention as plant polysaccharide degrading bacteria. Among them, Trichoderma can produce cellulases and hemicellulases simultaneously and in large quantities and therefore has attracted attention as a microorganism for producing cellulase-based biomass degrading enzymes.

[0003] It is hoped that the carbon source used for microbial cultivation in industry is cheap and soluble. At present, glucose is generally used as the carbon source for microbial cultivation. However, in the presence of glucose, due to the control mechanism known as catabolite repression, the material productivity of the microorganism is decreased or saturated. It has been reported that wide-area control transcription factors CreA, CreB, CreC, CreD, etc. are involved in the catabolite repression of filamentous fungi such as Aspergillus (Aspergillus) (patent document 1). There is the possibility of regulating the catabolite repression of Aspergillus by controlling these transcription factors, but sufficient results have not been obtained. Regarding Trichoderma, the mechanism of catabolite repression has also been analyzed (patent document 2, non-patent document 1). However, the mechanism of catabolite repression of Trichoderma still has many unclear places, and the avoidance of repression has not been achieved.

[0004] In the production of proteins such as enzymes using microorganisms, inducing substances are sometimes needed. For example, in Trichoderma, the expression of the main cellulase genes cbh1, cbh2, egl1 and egl2 is induced by cellulose, cellobiose, etc., and inducing substances are necessary in the production of cellulase (Non-patent Document 2). Usually, microcrystalline cellulose, i.e., Avicel, etc., are inducing substances used for cellulase production. However, cellulose substrates are expensive and mostly insoluble, so the production process increases the load, and from the perspective of cost and equipment, it is not easy to use for industrial purposes.

[0005] Disclosed are a method for producing cellulase using soluble lactose instead of cellulose as an inducing substance (Patent Document 3), and a method for synthesizing inducible sugars such as sophorose and gentiobiose from glucose by reacting cellulase (including β-glucosidase, endoglucanase, cellobiohydrolase) from Trichoderma with glucose at high temperature (Patent Document 4). However, the production of cellulase using inducible sugars also has disadvantages from the perspective of cost and process load.

[0006] In order to make a microorganism that can express cellulase and xylanase even without using inducing substances by changing the transcription control factor, the analysis of the cellulase expression mechanism was carried out. As the positive transcription factors involved in the induction expression of cellulase in Trichoderma, XYR1, ACE2, ACE3, HAP2 / 3 / 5, etc. (non-patent literature 3) were reported. It was reported that by shortening the C-terminal 140 amino acids of XYR1, the cellulase productivity of Trichoderma was reduced, and in Trichoderma with A824V mutation of XYR1, the control of xylanase was released, and the production of cellulase increased (non-patent literature 3, 4). In non-patent literature 5, it was reported that in Trichoderma strains, by combining the enhanced expression of ACE2 with the V821F mutation of XYR1, in a culture medium with glucose or sucrose as a carbon source, protein productivity was improved. In addition, it is currently believed that in the gene sequence of XYR1, there is an intron of a 20 amino acid region upstream of the coding region of the filamentous fungus-specific transcription factor domain (non-patent document 6), so A824 and V821 of XYR1 disclosed in the above document are temporarily revised to A804 and V801, respectively. However, on the other hand, there have been reports that the 20 amino acid region is not an intron but is translated into amino acids. Recently, the latter view is considered to be correct (for example, non-patent documents 5 and 7), and in this case, A824 and V821 disclosed in the above document are considered to represent the correct amino acid numbering on the sequence of XYR1.

[0007] In non-patent document 8, it is suggested that ACE3 interacts with XYR1 and regulates the expression of cellulase genes in Trichoderma reesei. Patent document 5 discloses a method for increasing and decreasing the productivity of cellulase and the like in Trichoderma reesei by increasing and decreasing the expression of the tre77513 (ACE3) gene. Patent document 6 and non-patent document 9 report that the filamentous fungus that enhances the expression of mutant ACE3 that retains all 6 cysteines in the Zn (II) 2Cys6-type DNA binding region on the N side and lacks the amino acids at the C-terminus also increases the expression of cellulase under the condition of the absence of inducing substances. In non-patent document 9, a filamentous fungus that enhances the expression of the wild type or A824V mutant of the C-terminal deletion ACE3 and co-expresses XYR1 is also recorded, but the effect of the co-expression of the XYR1 of the filamentous fungus on the expression of cellulase can be slightly observed in the presence of inducing substances, but not in the absence of inducing substances.

[0008] (Patent Document 1) Japanese Patent Application Publication No. 2015-39349

[0009] (Patent Document 2) Japanese Patent Publication No. 11-512930

[0010] (Patent Document 3) Japanese Patent No. 6169077

[0011] (Patent Document 4) Japanese Patent No. 5366286

[0012] (Patent Document 5) U.S. Patent No. 9512415

[0013] (Patent Document 6) International Publication No. 2018 / 067599

[0014] (Non-patent document 1) Appl Environ Microbiol, 1997, 63: 1298-1306

[0015] (Non-patent document 2) Curr Genomics, 2013, 14: 230-249

[0016] (Non-patent document 3) BMC Genomics, 2015, 16: 326

[0017] (Non-patent document 4) Biotech Biofuels, 2013, 6:62

[0018] (Non-patent document 5) Biotech Biofuels, 2017, 10:30

[0019] (Non-patent document 6) NCBI Reference Sequence: XP_006966092.1 [www.ncbi.nlm.nih.gov / protein / XP_006966092.1]

[0020] (Non-patent document 7) Biotech Biofuels, 2020, 13:93

[0021] (Non-patent document 8) J Biol Chem, 2019, doi:10.1074 / jbc.RA119.008497

[0022] (Non-patent document 9) Biotech Biofuels, 2020, 13: 137 Summary of the invention

[0023] The present invention provides a method for producing a mutant filamentous fungus, comprising the steps of changing the expression of XYR1 and ACE3 of a parent filamentous fungus,

[0024] The alteration of XYR1 is a substitution, deletion, insertion or addition of at least one amino acid residue in the region corresponding to positions 810 to 833 of SEQ ID NO: 1, of a polypeptide consisting of an amino acid sequence at least 90% identical to SEQ ID NO: 1 and functioning as a transcriptional activator of cellulases and hemicellulases,

[0025] The change in ACE3 expression is the enhancement of the expression of a polypeptide consisting of the amino acid sequence at positions 107 to 734 of sequence number 3 or an amino acid sequence that is at least 90% identical to the amino acid sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the relative protein productivity of the mutant filamentous strain.

[0027] Figure 2 Analysis of protein composition of cultures of mutant filamentous strains: gel electrophoresis images.

[0028] Figure 3 are the relative productivity of various proteins of the mutant filamentous strains.

[0029] Figure 4 are the relative productivity of various proteins of the mutant filamentous strains. DETAILED DESCRIPTION

[0030] All patent documents, non-patent documents and other publications cited in this specification are incorporated herein by reference in their entirety.

[0031] In the present specification, the identity of amino acid sequences and nucleotide sequences can be calculated using the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it can be calculated by using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) and setting the Unit size to compare (ktup) to 2.

[0032] In the present specification, "at least 90% sequence identity" ("at least 90% identity") with respect to amino acid sequences and nucleotide sequences means an identity of 90% or more, preferably 92% or more, more preferably 94% or more, further preferably 95% or more, further preferably 96% or more, further preferably 98% or more, further preferably 99% or more.

[0033] In this specification, unless otherwise defined, the "one or more" used in the deletion, substitution, addition or insertion of amino acids and nucleotides in amino acid sequences and nucleotide sequences may refer to, for example, 1 to 20, preferably 1 to 16, more preferably 1 to 12, further preferably 1 to 8, further preferably 1 to 4. In this specification, "addition" of amino acids or nucleotides includes adding one or more amino acids or nucleotides to one end and both ends of the sequence. In addition, in this specification, "insertion" of amino acids or nucleotides includes inserting amino acids or nucleotides to the 5' side or 3' side of a specified position.

[0034] In the present specification, the "equivalent position" or "equivalent region" on an amino acid sequence or a nucleotide sequence can be determined by matching (aligning) the target sequence and the reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) in a manner that gives the greatest identity. The alignment of amino acid sequences or nucleotide sequences can be implemented using a known algorithm, and the method is well known to those skilled in the art. For example, the alignment can be performed by using the Clustal W multiple sequence alignment program (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. For example, Clustal W can be used on the website of the European Bioinformatics Institute (European Bioinformatics Institute: EBI [www.ebi.ac.uk / index.html]) and the DNA Database of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics. Through the above alignment, the position of the target sequence that matches an arbitrary position of the reference sequence is regarded as a "equivalent position" to the arbitrary position. In addition, a region sandwiched between corresponding positions or a region consisting of corresponding motifs is regarded as a corresponding region.

[0035] Those skilled in the art can make minor adjustments to the comparison of the amino acid sequences obtained above to optimize it. Such an optimal comparison is preferably determined by considering the similarity of the amino acid sequences and the frequency of the gaps inserted. Among them, the similarity of the amino acid sequence refers to the ratio (%) of the number of positions where the same or similar amino acid residues exist in the two sequences relative to the number of amino acid residues in the full length when two amino acid sequences are compared. Similar amino acid residues refer to amino acid residues that have similar properties to each other in terms of polarity or charge and undergo so-called conservative substitutions among the 20 amino acids that constitute proteins. Groups containing such similar amino acid residues are well known to those skilled in the art, and for example, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; leucine and isoleucine, etc. can be listed, but are not limited to these.

[0036] In the present specification, "amino acid residue" refers to the 20 amino acid residues constituting proteins, alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).

[0037] In this specification, "operably linked" between a regulatory region such as a promoter and a gene means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. The method of "operably linking" a gene and a regulatory region is well known to those skilled in the art.

[0038] In this specification, "upstream" and "downstream" of a gene refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene arranged downstream of a promoter" means that the gene exists on the 3' side of the promoter in the sense strand of DNA, and the upstream of a gene refers to the region on the 5' side of the gene in the sense strand of DNA.

[0039] In this specification, the term "endogenous" used for the function, property, or trait of a cell is to indicate that the function, property, or trait originally exists in the cell. In contrast, the term "exogenous" is used to indicate that the function, property, or trait does not originally exist in the cell but is introduced from the outside. For example, an "exogenous" gene or polynucleotide is a gene or polynucleotide introduced into a cell from the outside. An exogenous gene or polynucleotide may come from an organism of the same species as the cell into which it is introduced, or from an organism of a different species (i.e., a heterologous gene or polynucleotide).

[0040] The present invention relates to providing a mutant filamentous fungus and a method for producing the same, as well as a method for producing a protein using the mutant filamentous fungus. The present invention relates to improving the protein productivity of filamentous fungi. Currently, when filamentous fungi are cultured in the presence of glucose, protein production is sometimes inhibited due to catabolite repression. In particular, the expression of cellulase-based biomass decomposing enzymes such as cellulase and hemicellulase of filamentous fungi needs to be induced by cellulase-inducing substances such as cellulose, sophorose, and cellooligosaccharides (cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, etc.), and on the other hand, expression induction is inhibited in the presence of glucose.

[0041] The inventors of the present invention have found that the mutant filamentous fungi after mutating the expression combination of XYR1 and ACE3 do not use the currently required inducing substances in the production of cellulase and hemicellulase, and produce a large amount of proteins such as cellulase and hemicellulase. In the present invention, for filamentous fungi, by applying prescribed mutations to XYR1 and ACE3, which are transcriptional activators of cellulase and hemicellulase, respectively, the filamentous fungi can be given the ability to express cellulase and hemicellulase without cellulase inducing substances such as cellulose, and the filamentous fungi can highly express proteins such as cellulase and hemicellulase in the presence of non-inducing carbon sources for cellulase such as glucose. By mutating the expression combination of XYR1 and ACE3, the expression ability of cellulase and hemicellulase is significantly improved compared with the case where only either party is mutated. It can be considered that the mutations expressed by the XYR1 and ACE3 help to alleviate the catabolite repression or promote the transcription of cellulase and hemicellulase.

[0042] The filamentous fungi provided by the present invention can also efficiently produce proteins in an environment where a non-inducing carbon source for cellulase such as glucose is used as the main carbon source. In addition, even without using expensive cellulase inducing substances, the filamentous fungi can also efficiently produce proteins such as cellulase and hemicellulase. Utilizing the present invention, it is possible to achieve efficiency and cost reduction in protein production using filamentous fungi.

[0043] Therefore, in one embodiment, the present invention provides a mutant filamentous fungus and a method for producing the same. Basically, the method for producing a mutant filamentous fungus of the present invention comprises the step of changing the expression of XYR1 and ACE3 of the parent filamentous fungus. In this method, as long as the respective mutations can be achieved, the order of the expression of the mutant XYR1 and ACE3 is not limited.

[0044] As examples of parent filamentous fungi used in the present invention, there is no limitation, and filamentous fungi belonging to Eumycota and Oomycota can be listed. As more detailed examples, filamentous fungi such as Trichoderma, Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, Hypocrea, Acremonium, Chrysosporium, Myceliophthora, Piromyces, Talaromyces, Thermoascus, Thielavia can be listed. Wherein. Filamentous fungi of the genus Trichoderma are preferred.

[0045] Examples of the filamentous fungi of the genus Trichoderma include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride, etc., and preferably Trichoderma reesei and its mutants. For example, Trichoderma reesei QM9414 strain and its mutants, preferably Trichoderma reesei PC-3-7 strain (ATCC66589), Trichoderma reesei PCD-10 strain (FERM P-8172), Trichoderma reesei JN13 strain or their mutants, etc. are preferably used as parent filamentous fungi.

[0046] XYR1 (Xylanase regulator 1) is a transcriptional activator of cellulases and hemicellulases in filamentous fungi. XYR1 is a major factor controlling the expression of xylanase genes with a Zn(II)2Cs6 binuclear cluster domain, and is widely preserved in ascophytes excluding yeast, such as Trichoderma (XYR1), Fusarium (XYR1), Neurospora (XYR1), and Aspergillus (XLNR). XYR1 of Trichoderma reesei controls all xylanase xylose metabolism genes and cellulase genes. XYR1 of Trichoderma reesei is registered in the NCBI database (www.ncbi.nlm.nih.gov / ]) as NCBI Reference Sequence: XP_006966092.1.

[0047] It is currently believed that there is an intron of 20 amino acids in the upstream of the coding region of the fungal-specific transcription factor domain in the gene sequence of XYR1 (nucleotide region of 1024 to 1083 of sequence number 2), and the full length of XYR1 is believed to be 920 amino acids. In the above-mentioned NCBI database (non-patent document 6), XYR1 of XP_006966092.1 is also composed of an amino acid sequence of 920 amino acids (sequence number 51) and is defined as a polypeptide encoded by the nucleotide sequence of sequence number 2. On the other hand, there is currently a report that the nucleotide region of 1024 to 1083 of sequence number 2 is not an intron but is translated into amino acids. Recently, the latter is considered to be the correct structure of XYR1 (for example, non-patent documents 5 and 7). In this case, XYR1 has a 320 to 339 amino acid region encoded by the nucleotide region of 1024 to 1083 of sequence number 2, and a total length of 940 amino acids.

[0048] In consideration of the above matters, unless otherwise specified, the amino acid sequence of XYR1 disclosed in the present specification is represented by SEQ ID NO. 1 having a length of 940 amino acids, and the numbering of the amino acid residues of XYR1 (positions on the amino acid sequence) is represented by the numbering (positions) of the residues in the sequence of SEQ ID NO. 1. In addition, in consideration of the above matters, in the present specification, the amino acid residues after position 340 of SEQ ID NO. 1 can be interpreted as the amino acid residues at position [position 20 of SEQ ID NO. 1] of SEQ ID NO. 51. For example, positions 810 to 833 of SEQ ID NO. 1 are positions 790 to 813 of SEQ ID NO. 51, that is, position 810 of SEQ ID NO. 1 is position 790 of SEQ ID NO. 51, position 833 of SEQ ID NO. 1 is position 813 of SEQ ID NO. 51, and positions 821 and 824 of SEQ ID NO. 1 are positions 801 and 804 of SEQ ID NO. 51, respectively, and the same applies to other positions.

[0049] Therefore, as an example of the modified XYR1 (parent XYR1) in the present invention, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 can be cited. As another example of the parent XYR1, a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and functions as a transcriptional activator of cellulase and hemicellulase can be cited. As an example of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted into the amino acid sequence of SEQ ID NO: 1 can be cited.

[0050] The modification of the XYR1 of the present invention is to apply a mutation of at least one amino acid residue to the region presumed to be an α-helix in the acidic activation domain of the parent XYR1. In more detail, the modification of the XYR1 is performed by mutation (i.e., substitution, deletion, insertion or addition) of at least one amino acid residue in the region corresponding to positions 771 to 865 of sequence number 1 of a polypeptide consisting of the amino acid sequence of sequence number 1 as the parent XYR1 or an amino acid sequence at least 90% identical to the sequence thereof and functioning as a transcriptional activator of cellulases and hemicellulases. In a preferred embodiment, at least one amino acid residue to which the mutation is applied is located in the region corresponding to positions 810 to 833 of sequence number 1.

[0051] In a preferred embodiment, at least one of the above-mentioned amino acid residues is substituted. The substituted amino acid residue is at least one selected from Val, Ile, Leu, Ala, Gly, Thr and Glu, more preferably at least one selected from the following (1) and (2): (1) Val, Ile or Leu, (2) Ala or Gly, or at least one selected from Val, Ala, Thr and Glu, further preferably at least one selected from Val and Ala. Preferably, these amino acid residues are substituted by Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr, respectively.

[0052] Preferred examples of substitution of Val, Ile, Leu, Ala, Gly, Thr and Glu include the following:

[0053] Substitution of Val to Lys, Phe, Trp or Tyr;

[0054] Substitution of Ile to Phe, Trp or Tyr;

[0055] Substitution of Leu to Phe, Trp or Tyr;

[0056] Substitution of Ala to Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr;

[0057] Substitution of Gly to Val, Ile, Leu, Phe, Trp or Tyr;

[0058] Thr to Tyr substitution; and

[0059] Substitution of Glu to Tyr.

[0060] In a more preferred embodiment, the amino acid residue is substituted by at least one selected from the following substitutions:

[0061] Substitution of Gly at the position corresponding to position 812 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0062] Substitution of Val at the position corresponding to position 814 of SEQ ID NO: 1 with Phe, Trp or Tyr;

[0063] Substitution of Ala at the position corresponding to position 816 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0064] Substitution of Thr to Tyr at the position corresponding to position 817 of SEQ ID NO: 1;

[0065] Substitution of Ala at the position corresponding to position 820 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0066] Substitution of Val at the position corresponding to position 821 of SEQ ID NO: 1 with Lys, Phe, Trp or Tyr;

[0067] Substitution of Ala at the position corresponding to position 823 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0068] Substitution of Ala at the position corresponding to position 824 of SEQ ID NO: 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr;

[0069] Substitution of Glu at the position corresponding to position 825 of SEQ ID NO: 1 with Tyr;

[0070] Substitution of Ala at the position corresponding to position 826 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0071] Substitution of Ile at the position corresponding to position 827 of SEQ ID NO: 1 with Phe, Trp or Tyr;

[0072] Substitution of Ile at the position corresponding to position 830 of SEQ ID NO: 1 with Phe, Trp or Tyr; and

[0073] Substitution of Leu at the position corresponding to position 831 of SEQ ID NO: 1 with Phe, Trp or Tyr.

[0074] In a further preferred embodiment, the modification of XYR1 of the present invention is performed by substituting an amino acid residue at at least one position selected from the positions corresponding to positions 817, 821, 824, 825 and 826 of SEQ ID NO: 1 in the parent XYR1. The amino acid residues substituted at the positions corresponding to positions 817, 821, 824, 825 and 826 are as described above, respectively.

[0075] In another further preferred embodiment, the modification of XYR1 of the present invention is performed by substituting an amino acid residue at at least one position selected from the positions corresponding to positions 821 and 824 of SEQ ID NO: 1 in the parent XYR1. The amino acid residue substituted at the position corresponding to position 821 is preferably Lys, Phe, Trp or Tyr, more preferably Phe, and further preferably Lys or Tyr. The amino acid residue substituted at the position corresponding to position 824 is preferably Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr, more preferably Val, and further preferably Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr.

[0076] In another further preferred embodiment, the alteration of XYR1 of the present invention is performed by substituting an amino acid residue at at least one position selected from the positions corresponding to positions 817, 825 and 826 of SEQ ID NO: 1 of the parent XYR1. The amino acid residue substituted at the position corresponding to position 817 is preferably Tyr. The amino acid residue substituted at the position corresponding to position 825 is preferably Tyr. The amino acid residue substituted at the position corresponding to position 826 is preferably Val, Ile, Leu, Phe, Trp or Tyr, more preferably Val or Trp.

[0077] In one example, the amino acid residues at positions corresponding to positions 817, 821, 824, 825, and 826 of SEQ ID NO: 1 of the parent XYR1 are not the above-mentioned substituted amino acid residues. Preferably, the amino acid residues at positions corresponding to positions 817, 821, 824, 825, and 826 of SEQ ID NO: 1 of the parent XYR1 are the same as the amino acid residues at positions 817, 821, 824, 825, and 826 of SEQ ID NO: 1, respectively.

[0078] In another example, the amino acid residues at positions 812, 814, 816, 817, 820, 821, 823, 824, 825, 826, 827, 830 and 831 of parent XYR1 corresponding to positions 812, 814, 816, 817, 820, 821, 823, 824, 825, 826, 827, 830 and 831 of SEQ ID NO: 1 are not the aforementioned substituted amino acid residues, respectively. Preferably, the amino acid residues at positions corresponding to positions 812, 814, 816, 817, 820, 821, 823, 824, 825, 826, 827, 830 and 831 of SEQ ID NO: 1 are the same as the amino acid residues at positions 812, 814, 816, 817, 820, 821, 823, 824, 825, 826, 827, 830 and 831 of SEQ ID NO: 1, respectively.

[0079] In another example, the amino acid sequence of the region corresponding to positions 810 to 833 of SEQ ID NO: 1 of the parent XYR1 is identical to positions 810 to 833 of SEQ ID NO: 1.

[0080] As a method for mutating (substituting, deleting, inserting or adding) the amino acid residues of the polypeptide of the parent filamentous fungus, various mutation introduction techniques known in the art can be used. For example, in the genome of the parent filamentous fungus, a polynucleotide encoding the amino acid sequence to be mutated (parent XYR1) (hereinafter also referred to as the parent gene) can be changed to a polynucleotide encoding the mutated amino acid sequence (hereinafter also referred to as the mutant gene), and a polypeptide having the target mutation (mutated XYR1) can be expressed from the mutant gene.

[0081] As the method for importing the target mutation to the parental gene, for example, the method utilizing homologous recombination can be cited.For example, by homologous recombination, the parental gene in the genome of the parental filamentous fungus can be replaced with a mutant gene.In the example of the specific method of homologous recombination, first, a homologous recombination DNA construct comprising a mutant gene and a drug tolerance gene or a nutrient requirement gene as required is constructed, and is imported into the parental filamentous fungus by a common method.Then, using drug tolerance or nutrient requirement etc. as an index, the transformant having been imported into the homologous recombination construct on the genome is selected.As required, by genome analysis or enzyme activity analysis, it can also be confirmed that the resulting transformant has the target mutation.

[0082] The DNA construct for homologous recombination can be constructed by introducing site-directed mutations into the isolated parental gene. The introduction of site-directed mutations can be carried out by, for example, inverse PCR, annealing method, SOE (splicing by overlap extension)-PCR (Gene, 1989, 77 (1): p61-68) and other common methods in the field. Commercially available site-directed mutagenesis kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit, QuickChange Multi Site-Directed Mutagenesis Kit, etc.) can also be used.

[0083] The DNA construct can be introduced into the parent filamentous fungus using a vector commonly used for transformation of a plasmid, etc. The DNA construct or vector can be introduced into cells using common methods such as the protoplast method, the protoplast PEG method, and the competent cell method.

[0084] The parent gene can be separated from a filamentous bacterial strain of the same species as the parent filamentous fungus using a common method. Alternatively, chemical synthesis can also be performed based on the nucleotide sequence of the parent gene. As required, the parent gene can also be matched with the host (parent filamentous fungus) to be introduced and codon optimized. The codon information used by various organisms can be obtained from Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0085] The parent gene may be a polynucleotide encoding the parent XYR1 described above, i.e., a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical thereto and functioning as a transcriptional activator of cellulases and hemicellulases. Examples of such a polynucleotide include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence at least 90% identical thereto.

[0086] Site-directed mutagenesis of a parent gene can most commonly be performed using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer can be designed so as to anneal to a region of the parent gene containing a sequence encoding the amino acid residue to be mutated and to replace the sequence (codon) encoding the amino acid residue to be mutated with a sequence (codon) encoding the amino acid residue after the mutation. A person skilled in the art can appropriately identify and select the sequence (codon) encoding the amino acid residue before and after the mutation based on common textbooks, etc. Alternatively, site-directed mutagenesis can be performed by connecting the upstream and downstream DNA fragments of the target region containing the nucleotide mutation to be introduced obtained by amplification using two sets of primers respectively into one using SOE-PCR. The mutagenesis primer can be prepared using a known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids Research, 1989, 17: 7059-7071).

[0087] Alternatively, the parent filamentous fungus can be subjected to mutation treatment, and then a strain having the target mutation can be selected by genome analysis or enzyme activity analysis. Specific examples of mutation treatment include N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethylnitrosourea, ultraviolet light or radiation irradiation, etc. In addition, various alkylating agents and carcinogens can also be used as mutagens.

[0088] The change in ACE3 expression of the present invention enhances the expression of ACE3 or a portion of its polypeptide. ACE3 is a transcriptional activator of cellulases and hemicellulases of filamentous fungi. ACE3 is essential for the transcription of cellulase genes and a portion of xylanase genes during lactose induction. In addition, ACE3 is also partially involved in the transcriptional control of xyr1. ACE3 of Trichoderma reesei is registered as NCBI Reference Sequence: QEM24913.1 in the ncbi database (www.ncbi.nlm.nih.gov / ]), wherein ACE3 is composed of the amino acid sequence of sequence number 3 and is defined as a polypeptide encoded by the nucleotide sequence of sequence number 4. According to non-patent document 6, in the amino acid sequence of sequence number 3, positions 523 to 734 are regions presumed to interact with XYR1, positions 391 to 522 are regions presumed to be filamentous fungi-specific transcription factor regions, and positions 120 to 160 are regions presumed to be Zn(II)2Cys6-type DNA binding regions.

[0089] Therefore, as an example of the ACE3 or partial polypeptides expressed and enhanced by the present invention, a polypeptide or partial polypeptides thereof consisting of the amino acid sequence of sequence number 3 can be cited. As other examples of ACE3 or partial polypeptides thereof, a polypeptide or partial polypeptides thereof consisting of an amino acid sequence at least 90% identical to the amino acid sequence of sequence number 3 and functioning as a transcriptional activator of cellulase and hemicellulase can be cited. As an example of an amino acid sequence at least 90% identical to the sequence of sequence number 3, an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence of sequence number 3 can be cited.

[0090] In the present invention, the expression of the full-length polypeptide of ACE3 can be enhanced, but as long as the expression of at least a partial polypeptide thereof, preferably a polypeptide corresponding to the region of positions 107 to 734 of sequence number 3, can be enhanced. Therefore, as an example of the ACE3 whose expression is enhanced in the present invention, a polypeptide consisting of the amino acid sequence of positions 1 to 734 of sequence number 3 or an amino acid sequence that is at least 90% identical to the sequence thereof can be cited; as an example of a partial polypeptide of ACE3, a polypeptide consisting of the amino acid sequence of positions 107 to 734 of sequence number 3 or an amino acid sequence that is at least 90% identical to the sequence thereof can be cited.

[0091] It is preferred that the enhanced ACE3 or a partial polypeptide thereof expressed by the present invention retains all the 7th to 10th amino acid residues from the C-terminus of the wild-type ACE3 (amino acid residues in the region corresponding to positions 725 to 728 of sequence number 3). It is more preferred that the enhanced ACE3 or a partial polypeptide thereof expressed by the present invention retains all the 7th to 17th amino acid residues from the C-terminus of the wild-type ACE3 (amino acid residues in the region corresponding to positions 718 to 728 of sequence number 3). Alternatively, it is preferred that the enhanced ACE3 or a partial polypeptide thereof expressed by the present invention retains a region corresponding to 11 amino acids (positions 724 to 734 of sequence number 3) at the C-terminus of the wild-type ACE3. Among them, as long as the function as a transcriptional activator of cellulase and hemicellulase can be maintained, mutations of a portion of the amino acid residues in the region corresponding to positions 724 to 734 of sequence number 3 of the ACE3 or a partial polypeptide thereof are allowed (e.g., substitution, deletion, insertion or addition of one or more residues at positions 729 to 734 of sequence number 3).

[0092] Preferably, the expression enhancement of ACE3 or its partial polypeptide of the present invention refers to that the expression amount of the ACE3 or its partial polypeptide is improved. As a method for increasing the expression amount of the target polypeptide, a method for increasing the transcription amount of the gene encoding the polypeptide (hereinafter also referred to as the target gene) can be cited. As a method for increasing the transcription amount of the target gene, for example, it can be cited: after the regulatory region of the target gene on the genome of the parent filamentous fungus replaces or inserts a regulatory region (stressed regulatory region) that strongly promotes the transcription of the gene, the stressed regulatory region is operably connected to the target gene. Alternatively, the target gene fragment operably connected to the regulatory region (preferably the stressed regulatory region) as needed is introduced into the genome or plasmid of the parent filamentous fungus, and then the number of target genes that can be expressed is increased in the cell, so that the transcription amount of the target gene can be increased.

[0093] Examples of regulatory regions that can be used to increase the transcription level include genes whose transcription level does not decrease even under high glucose conditions, such as Trichoderma filamentous fungi, such as glyceraldehyde-3-phosphate dehydrogenase (gpd), pyruvate decarboxylase (pdc), enolase (eno), alcohol dehydrogenase (adh), triose phosphate isomerase (tpi), aldolase (fba), pyruvate kinase (pyk), citrate synthase (cit), α-ketoglutarate dehydrogenase (kdh), aldehyde dehydrogenase I (ald1), aldehyde dehydrogenase II (ald2), and aldehyde dehydrogenase II (ald3). II) (ald2), pyruvate dehydrogenase (pda), glucokinase (glk), actin (act1), translation elongation factor 1α (tef1) and other genes. Among them, as examples of preferred highly regulated regions, regulatory regions such as pdc (TRIREDRAFT_121534) and act1 (TRIREDRAFT_44504) can be cited.

[0094] The target gene can be isolated from a filamentous strain of the same species as the parent filamentous fungus by conventional methods. Alternatively, it can be chemically synthesized based on the nucleotide sequence of the gene of the parent filamentous fungus. As preferred examples of the target gene, there can be listed polynucleotides consisting of the nucleotide sequence of sequence number 4, polynucleotides consisting of a nucleotide sequence at least 90% identical to sequence number 4, and encoding a polypeptide that functions as a transcriptional activator of cellulase and hemicellulase, or partial polynucleotides thereof. As an example of a nucleotide sequence that is at least 90% identical to sequence number 4, a nucleotide sequence in which one or more nucleotides are deleted, substituted, added or inserted into the sequence of sequence number 4 can be used. As preferred examples of the partial polynucleotide, the above-mentioned polynucleotide encoding the partial polypeptide of ACE3 can be listed.

[0095] Compared with the parent filamentous fungus, the mutant strain of the present invention has an increased expression of ACE3 or a partial polypeptide thereof. Alternatively, compared with the parent filamentous fungus, the mutant strain of the present invention has an increased transcription amount of a gene encoding ACE3 or a partial polypeptide thereof. The expression amount of a gene or polypeptide can be quantified using known methods such as quantitative PCR, microarray, Western blotting, ELISA, HPLC, etc.

[0096] The mutant filamentous fungus of the present invention produced according to the above method comprises the mutant XYR1 obtained by the above-mentioned alteration of XYR1, and as described above, the expression of ACE3 or a partial polypeptide thereof is enhanced compared to the parent filamentous fungus.

[0097] Examples of the ACE3 or partial polypeptide thereof whose expression is enhanced in the mutant filamentous fungi of the present invention are as described above. Preferably, the mutant filamentous fungi of the present invention are introduced with a gene encoding a partial polypeptide of ACE3 (preferably a polypeptide consisting of the amino acid sequence at positions 107 to 734 of SEQ ID NO. 3 or an amino acid sequence at least 90% identical thereto) operably linked to a regulatory region (preferably a strong regulatory region), thereby enhancing the expression of the partial polypeptide of ACE3.

[0098] Preferably, the mutant XYR1 contained in the mutant filamentous fungus of the present invention is a polypeptide composed of an amino acid sequence in which at least one amino acid residue is substituted, deleted, inserted or added in the region corresponding to positions 771 to 865 of sequence number 1, preferably in the region corresponding to positions 810 to 833, with respect to the amino acid sequence of sequence number 1 or at least 90% of its sequence, and functions as a transcription activator of cellulase and hemicellulase.

[0099] More preferably, the mutant XYR1 contained in the mutant filamentous fungus of the present invention is a polypeptide consisting of an amino acid sequence in which the region corresponding to positions 771 to 865, preferably the region corresponding to positions 810 to 833 in SEQ ID NO: 1 or an amino acid sequence at least 90% identical thereto is substituted with at least one amino acid residue selected from the following:

[0100] Substitution of Val to Lys, Phe, Trp or Tyr;

[0101] Substitution of Ile to Phe, Trp or Tyr;

[0102] Substitution of Leu to Phe, Trp or Tyr;

[0103] Substitution of Ala to Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr;

[0104] Substitution of Gly to Val, Ile, Leu, Phe, Trp or Tyr;

[0105] Thr to Tyr substitution; and

[0106] Substitution of Glu to Tyr.

[0107] It is further preferred that the mutant XYR1 is a polypeptide consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and has at least one amino acid residue selected from the following (a) to (m), and functions as a transcriptional activator of cellulase and hemicellulase:

[0108] (a) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 812 of SEQ ID NO: 1;

[0109] (b) Phe, Trp or Tyr at the position corresponding to position 814 of SEQ ID NO: 1;

[0110] (c) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 816 of SEQ ID NO: 1;

[0111] (d) Tyr at the position corresponding to position 817 of sequence number 1;

[0112] (e) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 820 of SEQ ID NO: 1;

[0113] (f) Lys, Phe, Trp or Tyr at the position corresponding to position 821 of SEQ ID NO: 1;

[0114] (g) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 823 of SEQ ID NO: 1;

[0115] (h) Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr at the position corresponding to position 824 of SEQ ID NO: 1;

[0116] (i) Substitution of Glu at the position corresponding to position 825 of SEQ ID NO: 1 with Tyr;

[0117] (j) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 826 of SEQ ID NO: 1;

[0118] (k) Phe, Trp or Tyr at the position corresponding to position 827 of SEQ ID NO: 1;

[0119] (l) Phe, Trp or Tyr at the position corresponding to position 830 of SEQ ID NO: 1; and

[0120] (m) Phe, Trp or Tyr at the position corresponding to position 831 of SEQ ID NO: 1.

[0121] It is further preferred that the mutation XYR1 has at least one of (a), (c), (e), (g), (h), and (j) and / or has at least one of (b), (f), (k), (l), and (m). It is further preferred that the mutation XYR1 has at least one of (c), (e), (g), (h), and (j) and / or has at least one of (b) and (f).

[0122] In a further preferred embodiment, the mutant XYR1 is a polypeptide that is at least 90% identical to sequence number 1 and has an amino acid sequence of any one or more of (d), (f), (h), (i) and (j), and functions as a transcription activator of cellulase and hemicellulase.

[0123] In other further preferred embodiments, the mutant XYR1 is a polypeptide consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and has at least one amino acid residue selected from the following, and functions as a transcriptional activator of cellulases and hemicellulases:

[0124] Lys, Phe, Trp or Tyr, preferably Phe, more preferably Lys or Tyr, at the position corresponding to position 821 of SEQ ID NO: 1; and

[0125] Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr at the position corresponding to position 824 of SEQ ID NO: 1, preferably Val, more preferably Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr.

[0126] In other further preferred embodiments, the mutant XYR1 is a polypeptide consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and has at least one amino acid residue selected from the following, and functions as a transcriptional activator of cellulases and hemicellulases:

[0127] Tyr at the position corresponding to position 817 of sequence number 1;

[0128] Tyr at the position corresponding to position 825 of sequence number 1; and

[0129] Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 826 of SEQ ID NO: 1, preferably Val or Trp.

[0130] Even in the absence of cellulase-inducing substances such as cellulose, sophorose and cellooligosaccharides, the mutant filamentous fungi of the present invention can express cellulase-based biomass decomposing enzymes such as cellulase and hemicellulase. In addition, even in an environment where a cellulase-non-inducing carbon source such as glucose is used as the main carbon source, even in the absence of, for example, cellulase-inducing substances, the mutant filamentous fungi can efficiently produce proteins.

[0131] Therefore, in another embodiment, the present invention provides a method for producing a protein using the mutant filamentous fungus of the present invention. In the method for producing a protein of the present invention, the mutant filamentous fungus of the present invention is cultured. By culturing, the target protein is generated and accumulated in the culture. By isolating the target protein from the culture, the target protein can be produced.

[0132] As examples of the target protein produced, cellulase-based biomass decomposing enzymes such as cellulase and hemicellulase, exoglucanase, endoglucanase, β-glucosidase, protease, lipase, mannanase, arabinase, galactosidase, amylase, etc. can be listed, but are not limited to these. The target protein can be one protein or a mixture of multiple proteins. Preferably, the target protein is a cellulase-based biomass decomposing enzyme, more preferably a cellulase and / or a hemicellulase, and further preferably a cellulase and a hemicellulase. As examples of the hemicellulase, xylanase, β-xylosidase, α-arabinofuranosidase, etc. can be listed, among which xylanase is preferred.

[0133] Alternatively, the target protein may be a foreign protein that is not originally produced by the filamentous fungus. In this case, for the mutant filamentous fungus of the present invention, a gene encoding a foreign protein is inserted to produce a recombinant filamentous fungus, and the recombinant filamentous fungus is cultured to obtain a protein containing the foreign protein. In addition, by operably linking the gene encoding the foreign protein to a secretion signal peptide that functions in the filamentous fungus, the foreign protein can be secreted and produced in the culture.

[0134] The culture medium used for the protein production may be any of a synthetic medium and a natural medium as long as it contains components necessary for the growth of filamentous fungi and protein production, such as a carbon source, a nitrogen source, inorganic salts, and vitamins.

[0135] As a carbon source, any carbon source can be used as long as the mutant filamentous fungus can use it, for example, sugars such as glucose and fructose, sugar alcohols such as sorbitol, alcohols such as ethanol and glycerol, organic acids such as acetic acid, etc. These can be used alone or in combination.

[0136] Preferably, in the method for producing the protein of the present invention, the mutant filamentous fungus is cultured in an environment where a non-inductive carbon source for cellulase is the main carbon source. As non-inductive carbon sources for cellulase, glucose, fructose, sucrose, maltose, glycerol, etc. can be cited. Among them, glucose is preferred from the perspective of cost. In the case where the target protein to be produced is a cellulase-based biomass decomposing enzyme, the culture in this method can be carried out in the presence of cellulase-inducing substances such as cellulose, sophorose, and cellooligosaccharides, but even in the absence of the inducing substance, the target protein can be produced at a high level, and is not limited to the use or non-use of the inducing substance. In addition, in the present invention, in order to further reduce catabolite repression and efficiently produce proteins such as cellulase-based biomass decomposing enzymes, non-inductive carbon sources such as glucose can be added while the mutant filamentous fungus is cultured. At this time, from the perspective of culture efficiency and the ability to suppress foaming during culture, it is preferred to dissolve the non-inductive carbon source for cellulase, such as glucose, in ammonia water or an aqueous solution containing ammonium salts as a nitrogen source, and then add the dissolved solution for culture.

[0137] Examples of the nitrogen source include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolyzate.

[0138] Examples of the inorganic salt include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, potassium carbonate, and the like.

[0139] Examples of vitamins include biotin, thiamine, etc. If necessary, substances required for the growth of the filamentous fungi of the present invention may be added.

[0140] Cultivation is preferably carried out under aerobic conditions such as shaking culture and ventilation stirring culture. The culture temperature is preferably 10°C or more, more preferably 20°C or more, more preferably 25°C or more, and preferably 50°C or less, more preferably 42°C or less, more preferably 35°C or less. In addition, it is preferably 10 to 50°C, more preferably 20 to 42°C, more preferably 25 to 35°C. The pH during cultivation is 3 to 9, preferably 4 to 5. The culture time is 10 hours to 10 days, preferably 2 to 7 days.

[0141] After the culture, the target protein is separated from the culture by a common method. For example, the culture is recovered, and the bacterial cells are disrupted by ultrasound, pressure, etc. as needed, and filtration, centrifugation, ultrafiltration, salting out, dialysis, chromatography, etc. are appropriately combined to separate the target protein from the culture. The degree of separation of the target protein is not particularly limited. For example, the culture supernatant or its crude separation and purification can be obtained as a composition containing the target protein.

[0142] In addition, the present invention includes the following substances, production methods, uses, methods, etc. as illustrative embodiments. However, the present invention is not limited to these embodiments.

[0143] [1] A method for producing a mutant filamentous fungus, comprising the step of changing the expression of XYR1 and ACE3 of a parent filamentous fungus,

[0144] The alteration of XYR1 is a substitution, deletion, insertion or addition of at least one amino acid residue in the region corresponding to positions 810 to 833 of SEQ ID NO: 1, of a polypeptide consisting of an amino acid sequence at least 90% identical to SEQ ID NO: 1 and functioning as a transcriptional activator of cellulases and hemicellulases,

[0145] The change in ACE3 expression is the enhanced expression of a polypeptide consisting of the amino acid sequence at positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the amino acid sequence and functions as a transcriptional activator of cellulases and hemicellulases.

[0146] [2] The method as described in [1], wherein the at least one amino acid residue is preferably at least one selected from Val, Ile, Leu, Ala, Gly, Thr and Glu, more preferably at least one selected from the following (1) and (2): (1) Val, Ile or Leu, (2) Ala or Gly, or at least one selected from Val, Ala, Thr and Glu, further preferably at least one selected from Val and Ala, and the at least one amino acid residue is preferably substituted.

[0147] [3] The method according to [2], wherein the at least one amino acid residue is preferably at least one selected from the group consisting of amino acid residues at positions corresponding to positions 817, 821, 824, 825 and 826 of SEQ ID NO: 1.

[0148] [4] The method according to [2] or [3], wherein the at least one amino acid residue is preferably substituted by Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr.

[0149] [5] The method according to any one of [2] to [4], wherein the substitution of the at least one amino acid residue is preferably at least one selected from the following substitutions:

[0150] Substitution of Val to Lys, Phe, Trp or Tyr;

[0151] Substitution of Ile to Phe, Trp or Tyr;

[0152] Substitution of Leu to Phe, Trp or Tyr;

[0153] Substitution of Ala to Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr;

[0154] Substitution of Gly to Val, Ile, Leu, Phe, Trp or Tyr;

[0155] Thr to Tyr substitution; and

[0156] Substitution of Glu to Tyr,

[0157] More preferably, it is at least one selected from the following substitutions:

[0158] Substitution of Gly at the position corresponding to position 812 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0159] Substitution of Val at the position corresponding to position 814 of SEQ ID NO: 1 with Phe, Trp or Tyr;

[0160] Substitution of Ala at the position corresponding to position 816 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0161] Substitution of Thr to Tyr at the position corresponding to position 817 of SEQ ID NO: 1;

[0162] Substitution of Ala at the position corresponding to position 820 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0163] Substitution of Val at the position corresponding to position 821 of SEQ ID NO: 1 with Lys, Phe, Trp or Tyr;

[0164] Substitution of Ala at the position corresponding to position 823 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0165] Substitution of Ala at the position corresponding to position 824 of SEQ ID NO: 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr;

[0166] Substitution of Glu at the position corresponding to position 825 of SEQ ID NO: 1 with Tyr;

[0167] Substitution of Ala at the position corresponding to position 826 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr;

[0168] Substitution of Ile at the position corresponding to position 827 of SEQ ID NO: 1 with Phe, Trp or Tyr;

[0169] Substitution of Ile at the position corresponding to position 830 of SEQ ID NO: 1 with Phe, Trp or Tyr; and

[0170] Substitution of Leu at the position corresponding to position 831 of SEQ ID NO: 1 with Phe, Trp or Tyr.

[0171] [6] The method according to [5], wherein the substitution of the at least one amino acid residue is preferably at least one selected from the following substitutions:

[0172] Substitution of Thr to Tyr at the position corresponding to position 817 of SEQ ID NO: 1;

[0173] Substitution of Val at the position corresponding to position 821 of SEQ ID NO: 1 with Lys, Phe, Trp or Tyr;

[0174] Substitution of Ala at the position corresponding to position 824 of SEQ ID NO: 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr;

[0175] Substitution of Glu at the position corresponding to position 825 of SEQ ID NO: 1 with Tyr; and

[0176] Substitution of Ala at the position corresponding to position 826 of SEQ ID NO: 1 with Val, Ile, Leu, Phe, Trp or Tyr.

[0177] [7] The method according to any one of [1] to [6], wherein the amino acid sequence at positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence at least 90% identical thereto preferably retains all of the amino acid residues in the region corresponding to positions 725 to 728 of SEQ ID NO: 3, more preferably retains all of the amino acid residues in the region corresponding to positions 718 to 728 of SEQ ID NO: 3, or

[0178] It is preferred that the region corresponding to positions 724 to 734 of SEQ ID NO: 3 be retained; however, a part of the amino acid residues in this region may be mutated.

[0179] [8] The method according to any one of [1] to [7], wherein the expression of the polypeptide is preferably enhanced by increasing the transcription level of a gene encoding the polypeptide.

[0180] [9] The method as described in [8], wherein the increase in the transcription level of the gene encoding the above-mentioned polypeptide is preferably carried out by introducing the gene encoding the polypeptide operably linked to the regulatory region into the parent filamentous fungus.

[0181]

[10] The method according to any one of [1] to [9], wherein the XYR1 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 51 registered as XP_006966092.1, or a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51 and functions as a transcriptional activator of cellulase and hemicellulase,

[0182] The substitution of at least one amino acid residue is preferably at least one selected from the following substitutions:

[0183] Substitution of Thr to Tyr at the position corresponding to position 797 of SEQ ID NO: 51;

[0184] Substitution of Val at the position corresponding to position 801 of SEQ ID NO: 51 with Lys, Phe, Trp or Tyr;

[0185] Substitution of Ala at the position corresponding to position 804 of SEQ ID NO: 51 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr;

[0186] Substitution of Glu at the position corresponding to position 805 of SEQ ID NO: 51 with Tyr; and

[0187] Substitution of Ala at the position corresponding to position 806 of SEQ ID NO: 51 with Val, Ile, Leu, Phe, Trp or Tyr.

[0188]

[11] The method according to any one of [1] to

[10] , wherein the mutant filamentous fungus preferably has a lessened catabolite repression than the parent filamentous fungus, and more preferably expresses cellulase in the absence of a cellulase-inducing substance.

[0189]

[12] The method according to any one of [1] to

[11] , wherein the filamentous fungus is preferably a Trichoderma fungus.

[0190]

[13] The method as described in

[12] , wherein the Trichoderma fungus is preferably Trichoderma reesei or a mutant thereof.

[0191]

[14] A method for producing a protein, comprising the step of culturing a filamentous fungus obtained by the method described in any one of [1] to

[13] above.

[0192]

[15] The method as described in

[14] , wherein the protein is preferably cellulase and / or hemicellulase.

[0193]

[16] The method as described in

[14] or

[15] , wherein the above-mentioned culture is preferably carried out in the presence of glucose.

[0194]

[17] A mutant filamentous fungus comprising a mutant XYR1, and the expression of a partial polypeptide of ACE3 is enhanced compared to a parent filamentous fungus,

[0195] The mutant XYR1 is a polypeptide that is composed of an amino acid sequence in which at least one amino acid residue is substituted, deleted, inserted or added in the region corresponding to positions 810 to 833 of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical thereto, and functions as a transcriptional activator for cellulase and hemicellulase,

[0196] The partial polypeptide of ACE3 is a polypeptide consisting of the amino acid sequence at positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the amino acid sequence and functions as a transcriptional activator of cellulase and hemicellulase.

[0197]

[18] A mutant filamentous fungus as described in

[17] , wherein the amino acid sequence of positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto preferably retains all of the amino acid residues in the region corresponding to positions 725 to 728 of SEQ ID NO: 3, and more preferably retains all of the amino acid residues in the region corresponding to positions 718 to 728 of SEQ ID NO: 3, or

[0198] It is preferred that the region corresponding to positions 724 to 734 of SEQ ID NO: 3 be retained; however, a part of the amino acid residues in this region may be mutated.

[0199]

[19] The mutant filamentous fungus according to

[17] or

[18] , wherein the mutant XYR1 is preferably a polypeptide consisting of an amino acid sequence in which the region corresponding to positions 810 to 833 of SEQ ID NO: 1 or an amino acid sequence at least 90% identical thereto is substituted with at least one amino acid residue selected from the following:

[0200] Substitution of Val to Lys, Phe, Trp or Tyr;

[0201] Substitution of Ile to Phe, Trp or Tyr;

[0202] Substitution of Leu to Phe, Trp or Tyr;

[0203] Substitution of Ala to Val, Glu, He, Leu, Lys, Phe, Thr, Trp or Tyr; and

[0204] Substitution of Gly to Val, Ile, Leu, Phe, Trp or Tyr;

[0205] Thr to Tyr substitution; and

[0206] Substitution of Glu to Tyr.

[0207]

[20] The mutant filamentous fungus according to

[19] , wherein the mutant XYR1 is preferably a polypeptide consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and has at least one amino acid residue selected from the following (a) to (m), and functions as a transcriptional activator of cellulase and hemicellulase:

[0208] (a) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 812 of SEQ ID NO: 1;

[0209] (b) Phe, Trp or Tyr at the position corresponding to position 814 of SEQ ID NO: 1;

[0210] (c) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 816 of SEQ ID NO: 1;

[0211] (d) Tyr at the position corresponding to position 817 of sequence number 1;

[0212] (e) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 820 of SEQ ID NO: 1;

[0213] (f) Lys, Phe, Trp or Tyr at the position corresponding to position 821 of SEQ ID NO: 1;

[0214] (g) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 823 of SEQ ID NO: 1;

[0215] (h) Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr at the position corresponding to position 824 of SEQ ID NO: 1;

[0216] (i) Substitution of Glu at the position corresponding to position 825 of SEQ ID NO: 1 with Tyr;

[0217] (j) Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 826 of SEQ ID NO: 1;

[0218] (k) Phe, Trp or Tyr at the position corresponding to position 827 of SEQ ID NO: 1;

[0219] (l) Phe, Trp or Tyr at the position corresponding to position 830 of SEQ ID NO: 1; and

[0220] (m) Phe, Trp or Tyr at the position corresponding to position 831 of SEQ ID NO: 1.

[0221]

[21] The mutant filamentous fungus according to

[20] , wherein the mutant XYR1 is preferably a polypeptide consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and has at least one amino acid residue selected from the group consisting of:

[0222] Lys, Phe, Trp or Tyr, preferably Phe, more preferably Lys or Tyr, at the position corresponding to position 821 of SEQ ID NO: 1; and

[0223] Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr at the position corresponding to position 824 of SEQ ID NO: 1, preferably Val, more preferably Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr.

[0224]

[22] The mutant filamentous fungus according to

[20] or

[21] , wherein the mutant XYR1 is preferably a polypeptide consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and has at least one amino acid residue selected from the group consisting of:

[0225] Tyr at the position corresponding to position 817 of sequence number 1;

[0226] Tyr at the position corresponding to position 825 of sequence number 1; and

[0227] Val, Ile, Leu, Phe, Trp or Tyr at the position corresponding to position 826 of SEQ ID NO: 1, preferably Val or Trp.

[0228] Example

[0229] Hereinafter, the present invention will be described in more detail using examples.

[0230] Example 1 Construction of plasmid DNA for gene introduction

[0231] The following DNA fragments 1 to 5 were prepared by PCR using the genomic DNA of Trichoderma reesei as a template. Fragment 1: a promoter region of about 1.5 kbp upstream of the act1 gene (TRIREDRAFT_44504); Fragment 2: the xyr1 gene (SEQ ID NO. 2, about 3.0 kbp); Fragment 3: a polynucleotide encoding a partial polypeptide of ACE3 (nucleotides 107 to 734 of SEQ ID NO. 3) (nucleotide region 881 to 2918 of SEQ ID NO. 4, about 2.0 kbp); Fragment 4: a terminator region of about 0.6 kbp downstream of the cbh1 gene (TRIREDRAFT_44504); Fragment 5: a region of about 2.7 kbp of the pyr4 gene (TRIREDRAFT_74020).

[0232] Fragments 1 and 2 were combined to construct cassette 1: Pact1-XYR1. Fragments 1 and 3 were combined to construct cassette 2: Pact1-ACE3. Fragment 6 of about 0.5 kbp and fragment 7 of about 1.0 kbp were respectively configured upstream and downstream of fragment 5 as the same sequence for pop-out to prepare a transformation marker fragment. Fragment 4 and the transformation marker fragment were combined to construct cassette 3: Tcbh1-pyr4. The combination of DNA fragments was carried out according to the operating specifications of In-Fusion HD Cloning Kit (Takara Bio). The constructed cassette, the DNA fragments contained therein, and the primers used for the construction of the cassette are shown in Table 1.

[0233] Cassettes 1 and 3 were combined and inserted into the HincII restriction site of pUC118 (Takara Bio) to construct the xyr1 constant expression plasmid pUC-Pact1-XYR1. In addition, cassettes 2 and 3 were combined and inserted into the HincII restriction site of pUC118 (Takara Bio) to construct the ace3 constant expression plasmid pUC-Pact1-ACE3.

[0234] [Table 1]

[0235]

[0236] pUC-Pact1-XYR1(V821F) and pUC-Pact1-XYR1(A824V) were constructed by PCR using the primers in Table 2 with pUC-Pact1-XYR1 as a template. pUC-Pact1-XYR1(V821F) is a plasmid encoding a mutant XYR1(V821F) in which the amino acid sequence of SEQ ID NO. 2 was substituted with the amino acid V821F. pUC-Pact1-XYR1(A824V) is a plasmid encoding a mutant XYR1(A824V) in which the amino acid sequence of SEQ ID NO. 2 was substituted with the amino acid A824V.

[0237] [Table 2]

[0238]

[0239] The constructed plasmids pUC-Pact1-ACE3, pUC-Pact1-XYR1(V821F) and pUC-Pact1-XYR1(A824V) were replicated. The plasmids were introduced into competent cells Escherichia coli DH5α Competent Cells (Takara Bio), cultured in LB medium supplemented with ampicillin (37°C, 1 day), and stained with NucleoSpin. TM Plasmid (MACHEREY NAGEL), the plasmid is recovered from the cultured bacterial cells and purified.

[0240] Example 2 Preparation of filamentous fungal mutants

[0241] The Trichoderma reesei JN13Δpyr4 strain was transformed by introducing the plasmid constructed in Example 1. Plasmid introduction was performed using the protoplast PEG method (Biotechnol Bioeng, 2012, 109(1): 92-99). Using the pyr4 gene as a marker, transformants were selected in a selection medium (2% glucose, 1.1M sorbitol, 2% agar, 0.2% KH2PO4 (pH 5.5), 0.06% CaCl2·2H2O, 0.06% CsCl2, 0.06% MgSO4·7H2O, 0.5% (NH4)2SO4, 0.1% trace element 1; % are all w / v%). The composition of trace element 1 is as follows: 0.5 g FeSO4·7H2O, 0.2 g CoCl2, 0.16 g MnSO4·H2O, and 0.14 g ZnSO4·7H2O were fixed to 100 mL with distilled water. From the obtained transformants, the insertion of the target gene fragment was confirmed by PCR, thereby obtaining the JN13_ACE3 strain into which pUC-Pact1-ACE3 was introduced and the JN13_XYR1(V821F) strain into which pUC-Pact1-XYR1(V821F) was introduced. The JN13_ACE3 strain highly expresses ACE3 using the ace3 constant expression plasmid pUC-Pact1-ACE3. The JN13_XYR1(V821F) strain expresses mutant XYR1(V821F).

[0242] In order to transform the transformed strain again, a PDA medium containing 0.2% 5-fluoroorotic acid (5-FOA) monohydrate was used to select the strain that gained 5-FOA tolerance and grew again. That is, the spores of the JN13_ACE3 strain were spread on a medium containing 5-FOA, and the growing strain was obtained as the JN13_ACE3Δpyr4 strain. For the obtained strain, the JN13_XYR1(V821F)+ACE3 strain and the JN13_XYR1(A824V)+ACE3 strain were obtained by transforming the above-mentioned pUC-Pact1-XYR1(V821F) or pUC-Pact1-XYR1(A824V). These strains highly expressed ACE3 and expressed mutant XYR1(V821F or A824V).

[0243] Example 3 Cultivation of filamentous fungal mutants

[0244] The filamentous strain obtained in Example 2 was cultured to produce protein. In the pre-culture, 50 mL of the culture medium was added to a 500 mL flask at a concentration of 1×10 5 Spores of the strain prepared in Example 2 were cultured at 28°C and 220 rpm with shaking (PRXYg-98R manufactured by PRECI). The composition of the culture medium is as follows. 1% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% polypeptone N, 0.05% Bacto yeast extract, 0.1% Tween 80, 0.1% trace element 2, 50 mM tartaric acid buffer (pH 4.0) (% are all w / v%). The composition of trace element 2 is as follows. 6 mg H3BO3, 26 mg (NH4)6Mo7O 24 ·4H2O, 100mg FeCl3·6H2O, 40mgCuSO4·5H2O, 8mg MnCl2·4H2O, 200mg ZnCl2, make up to 100mL.

[0245] After 2 days of pre-culture, the main culture was carried out. 50 mL of culture medium was added to a 500 mL flask, 1% (v / v%) of pre-culture solution was inoculated, and culture was carried out at 28°C and 220 rpm for 4 days. The composition of the culture medium is as follows. 3% cellulose or 3% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% polypeptone N, 0.05% Bacto yeast extract (Yeast extract), 0.1% Tween 80, 0.1% trace element 2 (Traceelement 2), 1.28% diammonium hydrogen citrate, 50 mM tartaric acid buffer (pH 4.0) (% are all w / v%).

[0246] Example 4 Protein concentration determination

[0247] The protein concentration of the culture of Example 3 was measured by the Bradford method. In the Bradford method, the Quick Start Protein Assay Kit (BioRad) was used. A calibration curve was prepared using bovine gamma globulin as a standard protein, and the protein amount was calculated based on the prepared calibration curve. The protein productivity of the JN13 strain (parent strain) in the culture using only glucose as the carbon source (glucose culture) was set to 1, and the relative protein production of each strain at this time was shown in Figure 1 . The protein production of the parent strain JN13 in glucose culture decreased significantly compared to culture in the presence of cellulose. This is presumed to be because, when only glucose is used as a carbon source, there is no inducing substance, and the expression of the cellulase group and the xylanase group is not induced by transcriptional activation. In the XYR1(V821F)+ACE3 strain and the XYR1(A824V)+ACE3 strain in which both XYR1 and ACE3 were changed, the protein productivity in glucose culture was significantly improved compared to the parent strain and compared to the XYR1(V821F) strain and the ACE3 strain in which only one of XYR1 and ACE3 was changed.

[0248] Example 5 Protein composition analysis

[0249] The protein composition of the culture of Example 3 was analyzed. Mini PROTEAN TGX Stain-Free Gels (Any KD, 15 well, BIORAD) were used for the analysis. Precision Plus Protein Unstained standards were used as standards. The culture of Example 3 after appropriate dilution was mixed with the buffer (Buffer), treated at 99°C for 5 minutes, and the treated sample was applied to the gel and electrophoresed at 200V for 35 minutes. According to the obtained image file ( Figure 2 ), using analysis software (Image Lab), the band intensity ratio was calculated, and the composition ratio of the produced proteins was calculated. The production amount of each protein was calculated based on the composition ratio and the protein concentration obtained in Example 4. Next, the protein productivity when only XYR1 was mutated (XYR1 (V821F) strain) was set to 1, and the relative productivity of each protein in each strain was calculated.

[0250] The composition ratio of the proteins produced by each strain is shown in Table 3, and the relative productivity is shown in Figure 3 . Under glucose culture, almost no cellulase and xylanase (BXL1, CBH1, CBH2+EG1, XYN1+XYN2) were produced in the JN13 strain (parent strain). In the XYR1 single mutant strain (XYR1(V821F)), mainly xylanases such as XYN1+XYN2 (XYN1+XYN2) were produced. Regarding the ACE3 high expression strain (ACE3), the ratio of cellulases such as CBH1, CBH2 and EG1 increased, but as Figure 1 As shown, the total production of protein is less than that of the XYR1 single mutant strain, and is at the same level as the parent strain. In the strains (XYR1 (V821F) + ACE3 and XYR1 (A824V) + ACE3) that combine XYR1 mutation and ACE3 high expression, cellulase and xylanase are produced even under glucose culture. In addition, in XYR1 (V821F) + ACE3 and XYR1 (A824V) + ACE3, compared with the XYR1 single mutant strain and the ACE3 high expression strain, the composition ratio of the product is closer to the parent strain in the presence of cellulose, and the productivity of CBH1, CBH2 and EG1, which are the main cellulases, is greatly improved.

[0251] [Table 3]

[0252]

[0253] Example 6 Preparation of filamentous fungal mutants

[0254] According to the same steps as in Example 1, a plasmid pUC-Pact1-XYR1 (T817Y) expressing a mutant XYR1 with an amino acid substitution of T817Y was constructed by PCR using primers of sequence numbers 23 and 24 shown in Table 4 with pUC-Pact1-XYR1 as a template. According to the same steps, plasmids expressing mutant XYR1 with amino acid substitutions of V821Y, V821K, A824I, A824L, A824F, A824W, A824Y, A824T, A824K, A824E, E825Y, A826V and A826W were constructed respectively using the primers shown in Table 4. According to the same method as in Example 2, the constructed plasmid was transformed into the JN13_ACE3Δpyr4 strain to obtain a filamentous fungus mutant strain into which ACE3 and the above-mentioned mutant XYR1 were introduced.

[0255] [Table 4]

[0256]

[0257] The mutant strains obtained above, as well as the JN13_XYR1(V821F)+ACE3 strain, JN13_XYR1(A824V)+ACE3 strain, XYR1 single mutant strain (JN13_XYR1(V821F)) and ACE3 high expression strain (JN13_ACE3) prepared in Example 2 were cultured with glucose according to the method described in Example 3, and then the protein concentration of the culture was measured and the protein composition was analyzed according to the methods described in Examples 4 and 5.

[0258] The composition ratio of the proteins produced by each strain is shown in Table 5, and the relative productivity is shown in Figure 4 .exist Figure 4In the above, BXL1 is β-xylosidase BXL1, CBH1 is cellulase CBH1, CBH2+EG1 is cellulase CBH2 and EG1, XYN1+XYN2 is xylanase XYN1 and XYN2, and other refers to other proteins. The filamentous fungus mutant strains that highly express ACE3 and express mutant XYR1 (T817Y, V821Y, V821K, A824I, A824L, A824F, A824W, A824Y, A824T, A824K, A824E, E825Y, A826V and A826W) prepared in this example all produce proteins with the same composition as the JN13_XYR1 (V821F) + ACE3 strain and the JN13_XYR1 (A824V) + ACE3 strain prepared in Example 2. In addition, compared with the XYR1 single mutant (JN13_XYR1(V821F)) and the ACE3 high-expression strain (JN13_ACE3), the productivity of CBH1, CBH2 and EG1, which are the main cellulases, was significantly improved in these filamentous fungal mutants under glucose culture.

[0259] [Table 5]

[0260]

[0261] As described above, by combining XYR1 mutation and ACE3 high expression, the productivity of not only xylanase but also CBH1, CBH2 and EG1, which are the main cellulases, is greatly improved compared to XYR1 mutation alone. The effective mutation of XYR1 is not limited to V821F and A824V. The mutant XYR1 showing a glucose blind phenotype (usually, the property of increasing protein productivity even with glucose that causes catabolite repression) obtained by applying at least one amino acid mutation to the region in the acidic activation region of XYR1 is effective in the production of cellulase and xylanase under conditions without inducing substances. Therefore, by using the combination of the above-mentioned XYR1 mutation and ACE3 high expression, even when using a non-inducing carbon source for cellulase such as glucose, it is shown that the promoter of the cellulase / xylanase gene is activated in the same way as when using a cellulase inducing substance such as cellulose, and protein production is efficiently induced. Sequence Listing <110> Kao Co., Ltd. <120> Mutant filamentous fungi and method for producing protein using the same <130> KS1701 <150> JP 2019-208236 <151> 2019-11-18 <160> 51 <170> PatentIn Version 3.5 <210> 1 <211> 940 <212> PRT <213> Trichoderma reesei <400> 1 Met Leu Ser Asn Pro Leu Arg Arg Tyr Ser Ala Tyr Pro Asp Ile Ser 1 5 10 15 Ser Ala Ser Phe Asp Pro Asn Tyr His Gly Ser Gln Ser His Leu His 20 25 30 Ser Ile Asn Val Asn Thr Phe Gly Asn Ser His Pro Tyr Pro Met Gln 35 40 45 His Leu Ala Gln His Ala Glu Leu Ser Ser Ser Arg Met Ile Arg Ala 50 55 60 Ser Pro Val Gln Pro Lys Gln Arg Gln Gly Ser Leu Ile Ala Ala Arg 65 70 75 80 Lys Asn Ser Thr Gly Thr Ala Gly Pro Ile Arg Arg Arg Ile Ser Arg 85 90 95 Ala Cys Asp Gln Cys Asn Gln Leu Arg Thr Lys Cys Asp Gly Leu His 100 105 110 Pro Cys Ala His Cys Ile Glu Phe Gly Leu Gly Cys Glu Tyr Val Arg 115 120 125 Glu Arg Lys Lys Arg Gly Lys Ala Ser Arg Lys Asp Ile Ala Ala Gln 130 135 140 Gln Ala Ala Ala Ala Ala Ala Gln His Ser Gly Gln Val Gln Asp Gly 145 150 155 160 Pro Glu Asp Gln His Arg Lys Leu Ser Arg Gln Gln Ser Glu Ser Ser 165 170 175 Arg Gly Ser Ala Glu Leu Ala Gln Pro Ala His Asp Pro Pro His Gly 180 185 190 His Ile Glu Gly Ser Val Ser Ser Phe Ser Asp Asn Gly Leu Ser Gln 195 200 205 His Ala Ala Met Gly Gly Met Asp Gly Leu Glu Asp His His Gly His 210 215 220 Val Gly Val Asp Pro Ala Leu Gly Arg Thr Gln Leu Glu Ala Ser Ser 225 230 235 240 Ala Met Gly Leu Gly Ala Tyr Gly Glu Val His Pro Gly Tyr Glu Ser 245 250 255 Pro Gly Met Asn Gly His Val Met Val Pro Pro Ser Tyr Gly Ala Gln 260 265 270 Thr Thr Met Ala Gly Tyr Ser Gly Ile Ser Tyr Ala Ala Gln Ala Pro 275 280 285 Ser Pro Ala Thr Tyr Ser Ser Asp Gly Asn Phe Arg Leu Thr Gly His 290 295 300 Ile His Asp Tyr Pro Leu Ala Asn Gly Ser Ser Pro Ser Trp Gly Val 305 310 315 320 Ser Leu Ala Ser Pro Ser Asn Gln Phe Gln Leu Gln Leu Ser Gln Pro 325 330 335 Ile Phe Lys Gln Ser Asp Leu Arg Tyr Pro Val Leu Glu Pro Leu Leu 340 345 350 Pro His Leu Gly Asn Ile Leu Pro Val Ser Leu Ala Cys Asp Leu Ile 355 360 365 Asp Leu Tyr Phe Ser Ser Ser Ser Ala Gln Met His Pro Met Ser 370 375 380 Pro Tyr Val Leu Gly Phe Val Phe Arg Lys Arg Ser Phe Leu His Pro 385 390 395 400 Thr Asn Pro Arg Arg Cys Gln Pro Ala Leu Leu Ala Ser Met Leu Trp 405 410 415 Val Ala Ala Gln Thr Ser Glu Ala Ser Phe Leu Thr Ser Leu Pro Ser 420 425 430 Ala Arg Ser Lys Val Cys Gln Lys Leu Leu Glu Leu Thr Val Gly Leu 435 440 445 Leu Gln Pro Leu Ile His Thr Gly Thr Asn Ser Pro Ser Pro Lys Thr 450 455 460 Ser Pro Val Val Gly Ala Ala Ala Leu Gly Val Leu Gly Val Ala Met 465 470 475 480 Pro Gly Ser Leu Asn Met Asp Ser Leu Ala Gly Glu Thr Gly Ala Phe 485 490 495 Gly Ala Ile Gly Ser Leu Asp Asp Val Ile Thr Tyr Val His Leu Ala 500 505 510 Thr Val Val Ser Ala Ser Glu Tyr Lys Gly Ala Ser Leu Arg Trp Trp 515 520 525 Gly Ala Ala Trp Ser Leu Ala Arg Glu Leu Lys Leu Gly Arg Glu Leu 530 535 540 Pro Pro Gly Asn Pro Pro Ala Asn Gln Glu Asp Gly Glu Gly Leu Ser 545 550 555 560 Glu Asp Val Asp Glu His Asp Leu Asn Arg Asn Asn Thr Arg Phe Val 565 570 575 Thr Glu Glu Glu Arg Glu Glu Arg Arg Arg Ala Trp Trp Leu Val Tyr 580 585 590 Ile Val Asp Arg His Leu Ala Leu Cys Tyr Asn Arg Pro Leu Phe Leu 595 600 605 Leu Asp Ser Glu Cys Ser Asp Leu Tyr His Pro Met Asp Asp Ile Lys 610 615 620 Trp Gln Ala Gly Lys Phe Arg Ser His Asp Ala Gly Asn Ser Ser Ile 625 630 635 640 Asn Ile Asp Ser Ser Met Thr Asp Glu Phe Gly Asp Ser Pro Arg Ala 645 650 655 Ala Arg Gly Ala His Tyr Glu Cys Arg Gly Arg Ser Ile Phe Gly Tyr 660 665 670 Phe Leu Ser Leu Met Thr Ile Leu Gly Glu Ile Val Asp Val His His 675 680 685 Ala Lys Ser His Pro Arg Phe Gly Val Gly Phe Arg Ser Ala Arg Asp 690 695 700 Trp Asp Glu Gln Val Ala Glu Ile Thr Arg His Leu Asp Met Tyr Glu 705 710 715 720 Glu Ser Leu Lys Arg Phe Val Ala Lys His Leu Pro Leu Ser Ser Lys 725 730 735 Asp Lys Glu Gln His Glu Met His Asp Ser Gly Ala Val Thr Asp Met 740 745 750 Gln Ser Pro Leu Ser Val Arg Thr Asn Ala Ser Ser Arg Met Thr Glu 755 760 765 Ser Glu Ile Gln Ala Ser Ile Val Val Ala Tyr Ser Thr His Val Met 770 775 780 His Val Leu His Ile Leu Leu Ala Asp Lys Trp Asp Pro Ile Asn Leu 785 790 795 800 Leu Asp Asp Asp Asp Leu Trp Ile Ser Ser Glu Gly Phe Val Thr Ala 805 810 815 Thr Ser His Ala Val Ser Ala Ala Glu Ala Ile Ser Gln Ile Leu Glu 820 825 830 Phe Asp Pro Gly Leu Glu Phe Met Pro Phe Phe Tyr Gly Val Tyr Leu 835 840 845 Leu Gln Gly Ser Phe Leu Leu Leu Leu Ile Ala Asp Lys Leu Gln Ala 850 855 860 Glu Ala Ser Pro Ser Val Ile Lys Ala Cys Glu Thr Ile Val Arg Ala 865 870 875 880 His Glu Ala Cys Val Val Thr Leu Ser Thr Glu Tyr Gln Arg Asn Phe 885 890 895 Ser Lys Val Met Arg Ser Ala Leu Ala Leu Ile Arg Gly Arg Val Pro 900 905 910 Glu Asp Leu Ala Glu Gln Gln Gln Arg Arg Arg Glu Leu Leu Ala Leu 915 920 925 Tyr Arg Trp Thr Gly Asn Gly Thr Gly Leu Ala Leu 930 935 940 <210> 2 <211> 2950 <212> DNA <213> Trichoderma reesei <400> 2 atgttgtcca atcctctccg tcgctattct gcctaccccg acatctcctc ggcgtcattt 60 gacccgaact accatggctc acagtcgcat ctccactcga tcaacgtcaa cacattcggc 120 aacagccacc cctatcccat gcagcacctc gcacagcatg cggagctttc gagttcacgc 180 atgataaggg ccagtccggt gcagccaaag cagcgccagg gctctcttat tgctgccagg 240 aagaattcaa cgggtactgc tgggcccatt cggcggagga tcagtcgcgc ttgtgaccag 300 tgcaaccagc ttcgtaccaa gtgcgatggc ttacacccat gtgcccattg tataggtatg 360 tcccttttcc tctacacagt gatgctgcgc tcaagcacat gtactgatcg atcttgttta 420 gaattcggcc ttggatgcga atatgtccga gagagaaaga agcgtggcaa agcttcgcgc 480 aaggatattg ctgcccagca agccgcggcg gctgcagcac aacactccgg ccaggtccag 540 gatggtccag aggatcaaca tcgcaaactc tcacgccagc aaagcgaatc ttcgcgtggc 600 agcgctgagc ttgcccagcc tgcccacgac ccgcctcatg gccacattga gggctctgtc 660 agctccttca gcgacaatgg cctttcccag catgctgcca tgggcggcat ggatggcctg 720 gaagatcacc atggccacgt cggagttgat cctgccctgg gccgaactca gctggaagcg 780 tcatcagcaa tgggcctggg cgcatacggt gaagtccacc ccggctatga gagccccggc 840 atgaatggcc atgtgatggt gcccccgtcg tatggcgcgc agaccaccat ggccgggtat 900 tccggtatct cgtatgctgc gcaagccccg agtccggcta cgtatagcag cgacggtaac 960 tttcgactca ccggtcacat ccatgattac ccgctggcaa atgggagctc gccctcatgg 1020 ggagtctcgc tggcctcgcc ttcgaaccag ttccagcttc agctctcgca gcccatcttc 1080 aagcaaagcg atttgcgata tcctgtgctt gagcctctgc tgcctcacct gggaaacatc 1140 ctccccgtgt ctttggcgtg cgatctgatt gacctgtact tctcctcgtc ttcatcagca 1200 cagatgcacc caatgtcccc atacgttctg ggcttcgtct tccggaagcg ctccttcttg 1260 caccccacga acccacgaag gtgccagccc gcgctgcttg cgagcatgct gtgggtggcg 1320 gcacagacta gcgaagcgtc cttcttgacg agcctgccgt cggcgaggag caaggtctgc 1380 cagaagctgc tcgagctgac cgttgggctt cttcagcccc tgatccacac cggcaccaac 1440 agcccgtctc ccaagactag ccccgtcgtc ggtgctgctg ccctgggagt tcttggggtg 1500 gccatgccgg gctcgctgaa catggattca ctggccggcg aaacgggtgc ttttggggcc 1560 atagggagcc ttgacgacgt catcacctat gtgcacctcg ccacggtcgt ctcggccagc 1620 gagtacaagg gcgccagcct gcggtggtgg ggtgcggcat ggtctctcgc cagagagctc 1680 aagcttggcc gtgagctgcc gcctggcaat ccacctgcca accaggagga cggcgagggc 1740 cttagcgaag acgtggatga gcacgacttg aacagaaaca acactcgctt cgtgacggaa 1800 gaggagcgcg aagagcgacg gcgagcatgg tggctcgttt acatcgtcga caggcacctg 1860 gcgctctgct acaaccgccc cttgtttctt ctggacagcg agtgcagcga cttgtaccac 1920 ccgatggacg acatcaagtg gcaggcaggc aaatttcgca gccacgatgc agggaactcc 1980 agcatcaaca tcgatagctc catgacggac gagtttggcg atagtccccg ggcggctcgc 2040 ggcgcacact acgagtgccg cggtcgtagc atttttggct acttcttgtc cttgatgaca 2100 atcctgggcg agattgtcga tgtccaccat gctaaaagcc acccccggtt cggcgttgga 2160 ttccgctccg cgcgggattg ggacgagcag gttgctgaaa tcacccgaca cctggacatg 2220 tatgaggaga gcctcaagag gttcgtggcc aagcatctgc cattgtcctc aaaggacaag 2280 gagcagcatg agatgcacga cagtggagcg gtaacagaca tgcaatctcc actctcggtg 2340 cggaccaacg cgtccagccg catgacggag agcgagatcc aggccagcat cgtggtggct 2400 tacagcaccc atgtgatgca tgtcctccac atcctccttg cggataagtg ggatcccatc 2460 aaccttctag acgacgacga cttgtggatc tcgtcggaag gattcgtgac ggcgacgagc 2520 cacgcggtat cggctgccga agctattagc cagattctcg agtttgaccc tggcctggag 2580 tttatgccat tcttctacgg cgtctatctc ctgcagggtt ccttcctcct cctgctcatc 2640 gccgacaagc tgcaggccga agcgtctcca agcgtcatca aggcttgcga gaccattgtt 2700 agggcacacg aagcttgcgt tgtgacgctg agcacagagt atcaggtaag ccctatcagt 2760 tcaaacgtct atcttgctgt gaatcaaaga ctgacttgga catcagcgca actttagcaa 2820 ggttatgcga agcgcgctgg ctctgattcg gggccgtgtg ccggaagatt tagctgagca 2880 gcagcagcga cgacgcgagc ttcttgcact ataccgatgg actggtaacg gaaccggtct 2940 ggccctctaa 2950 <210> 3 <211> 734 <212> PRT <213> Trichoderma reesei <400> 3 Met Ala Thr Ala Ala Ala Ala Ala Ala Gly Gly Ala Ala Val Ala Ala 1 5 10 15 Gly Ala Asp Thr Gly Ala Ala Gly Ser Ser Ser Thr Gly Pro Pro Gly 20 25 30 Leu Pro Gly Leu Pro Gly Thr Arg Thr Gly Ser Val Ala Met Gly Ser 35 40 45 Ala Ala Pro Ala Gln Gly Ser Val Ala Ala Ala Ala Gly Gly Pro Pro 50 55 60 Ala Ala Gly Ala Gly Ala Gly Ala Val His Ala Leu Thr Thr Ser Pro 65 70 75 80 Glu Ser Ala Ser Ala Ser Gln Pro Gly Ser Pro Thr Ala Ser Thr Thr 85 90 95 Pro Pro Gln Asn Ser Leu Val Ser Ala Ala Thr Ser Phe His His His 100 105 110 Pro Arg Gly Arg Leu Val Ser Arg Ala Cys Asp Arg Cys Arg Arg Arg 115 120 125 Lys Ala Lys Cys Glu Tyr Leu Ser Ala Val Asp Ser Cys Thr His Cys 130 135 140 Arg Asp Ala His Val Gln Cys Thr Phe Asp Leu Pro Leu Ala Arg Arg 145 150 155 160 Gly Pro Lys Ala Arg Lys Lys Ser Asp Gln Pro Gly Gln Pro Pro Pro 165 170 175 Asp Pro Ser Ser Leu Ser Thr Ala Ala Arg Pro Gly Gln Met Pro Pro 180 185 190 Pro Leu Thr Phe Ser Gly Pro Ala Val Ala Ala Leu Gln Pro Phe Ala 195 200 205 Ser Ser Ser Leu Ser Pro Asp Ala Ala Trp Glu Pro Val Glu Pro Leu 210 215 220 Ser Ile Asp Asn Gly Leu Pro Arg Gln Pro Leu Gly Asp Leu Pro Gly 225 230 235 240 Leu Ser Thr Ile Gln Asn Ile Ser Thr Arg Gln Arg Trp Ile His Leu 245 250 255 Ala Asn Ala Met Thr Leu Arg Asn Thr Thr Leu Glu Arg Val Ser Lys 260 265 270 Arg Cys Ile Asp Leu Phe Phe Asp Tyr Leu Tyr Pro Leu Thr Pro Leu 275 280 285 Val Tyr Glu Pro Ala Leu Arg Asp Val Leu Ala Tyr Ile Phe Ser Gln 290 295 300 Pro Leu Pro Gly Val Asn Gln Pro Ser Pro Leu Ser Gln Leu Thr Pro 305 310 315 320 Asp Pro Thr Thr Gly Thr Thr Pro Leu Asn Ala Ala Glu Ser Trp Ala 325 330 335 Gly Phe Gly Gln Pro Ser Gly Ser Arg Thr Val Gly Ser Arg Leu Ala 340 345 350 Pro Trp Ala Asp Ser Thr Phe Thr Leu Val Thr Ala Val Cys Ala Glu 355 360 365 Ala Ala Phe Met Leu Pro Lys Asp Ile Phe Pro Glu Gly Glu Ser Val 370 375 380 Ser Glu Ile Leu Leu Glu Ala Ser Arg Asp Cys Leu His Gln His Leu 385 390 395 400 Glu Ala Asp Leu Glu Asn Pro Thr Ala Asn Ser Ile Ala Ile Arg Tyr 405 410 415 Phe His Ser Asn Cys Leu His Ala Ala Gly Lys Pro Lys Tyr Ser Trp 420 425 430 His Ile Phe Gly Glu Ala Ile Arg Leu Ala Gln Val Met Gln Leu His 435 440 445 Glu Glu Ala Ala Leu Glu Gly Leu Val Pro Ile Glu Ala Glu Phe Arg 450 455 460 Arg Arg Cys Phe Trp Ile Leu Tyr Leu Gly Asp Lys Ser Ala Ala Ile 465 470 475 480 Leu Asn Asn Arg Pro Ile Thr Ile His Lys Tyr Cys Phe Asp Ala Gly 485 490 495 Ile Thr Thr Leu Tyr Pro Ser Gly Ile Glu Asp Glu Phe Leu Ser Thr 500 505 510 Ala Ser Glu Pro Pro Arg Lys Ser Phe Ile Ser Gly Phe Asn Ala Asn 515 520 525 Val Arg Leu Trp Gln Ser Ala Ala Asp Leu Leu Leu Glu Ile Arg Val 530 535 540 Leu Gln Asp Gln Met Met Gln His Phe Arg Gly Thr Met Pro Pro Asn 545 550 555 560 His Val Leu Pro Ser Ala Asp Arg Gln His Leu Asp Ser Leu Tyr Val 565 570 575 Arg Phe Ile Thr Cys Leu Asp Asp Leu Pro Pro Tyr Leu Gln Ser Cys 580 585 590 Thr Leu Ala Met Ala Ala Met Ala Glu Gly Asn Gly Ser Ala Glu Ser 595 600 605 Lys Gln Tyr Val Ile Gln Cys Ile Asn Leu Gln Val Thr Phe His Cys 610 615 620 Leu Arg Met Val Ile Thr Gln Lys Phe Glu Asp Leu Ser Tyr Phe Ala 625 630 635 640 Pro Gly Val Glu Gln Ala Asp Leu Arg Lys Ser Glu Ile Val Arg Asp 645 650 655 Met Leu Arg Val Met Asn Glu Ala Pro Phe Trp Gly Leu Gln Ala Asn 660 665 670 Gly Glu Pro Asn Val Glu Lys Ile Arg Leu Ile Gly Ala Ser Leu Leu 675 680 685 Ala Ile Ile His Arg Asn Gln Asp Ser Pro Leu Ala Thr Arg Ala Arg 690 695 700 Ser Asp Phe Ser Val Leu Leu Asp Ile Leu Thr Arg Leu Asp Ser Lys 705 710 715 720 Ala Ser Asp Gln Leu Arg Asn Thr Ser Thr Thr Val Val Gly 725 730 <210> 4 <211> 2918 <212> DNA <213> Trichoderma reesei <400> 4 atggccacag cggccgcggc agcagctggc ggcgcggcgg ttgctgcggg tgcagacaca 60 ggtgcgttga gtcccgtccc gtccgctcgc gttccctccc agctgccagc ccgcgtgggt 120 ggcactggaa cgcagtgcag cgcaatcagt gcagtgcggc ccccccaact aacgctgccc 180 cccgtggctc ctcggccaca caggcgctgc aggctccagc tctacaggcc ctccaggcct 240 tccagggctt ccaggcaccc ggacaggctc cgtggcgatg ggctcagcag ctccggccca 300 gggctctgta gctgcagctg caggcggccc tccagctgct ggcgctggcg ctggcgctgt 360 ccacgccctc accacctcgc ccgagtctgc ctcggcctcg cagcccggct cgccaaccgc 420 ctcaaccacg ccgccgcaga actcactcgt gtcggctgca acctcgttcc accaccatcc 480 cagaggccgt ctggtgagca gagcctgcga ccgctgccgc cggcgcaagg ccaaggtcag 540 tctagcccct ttgctgttgc ttgcatctct gttgtcattg ctcctcctc tgctgctgct 600 gatgctgctg ctcctcctc tcctcctcct cccgtctc tggtccctgg tccctgctct 660 tcatatgtcc ttactgcccg tgtctccctct cccgttccc gttccccctc ctcccgtcct 720 cttctcctgc gtgtctgtca tgcgtacaaa gcatacatac aatacatcag catacatggc 780 aagcgttgtg ttgtgttgag agttgtgtgt attgtattgc actgccttca caactcgttc 840 atactgctgc agcctcaccc caacaccgac ctcgctttc atgctgcgct actcccccgt 900 cttacacctg gatactctct ccttgccacc actgaccaat gctcttcccc gcccaaagtg 960 cgagtacctc agcgctgtcg atagctgcac gcactgccgc gatgcccacg tgcagtgcac 1020 ttcgacctg cccctggcgc gacgcggccc caaagcgagg aagaagagcg accagcccgg 1080 ccagccgcct cctgatccga gctcgctctc caccgcggct cgacccggcc agatgccgcc 1140 gccgctgacc ttctccggcc ccgcagtagc cgcgctgcag cccttcgcct cgtcgtcgct 1200 gtcgcccgac gcggcctggg agcccgtcga gccgctcagc attgacaacg gcctgccccg 1260 gcagccgctg ggcgacctgc ccggcctctc caccatccag aacatctcga cgcgccagcg atggatacac ctggccaacg ccatgacgct gcgcaacacg acgctagagc gcgtctcgaa gcgatgtatc gacctcttct tcgactacct ctaccccctc acccccctgg tgtacgagcc 1440 ggccctccgg gacgtgctcg catacatctt ctcccagccc ttgcctggcg tcaaccaacc atcgccgctg tcacagctca cgccagaccc gaccaccggc accacccccc tcaacgctgc 1560 cgagtcgtgg gccggctttg gccagcccag cggctcgcga accgtcggca gcaggctggc 1620 tccctgggcc gactcgacct tcaccctggt cacggccgtc tgcgcagagg cagcattcat 1680 gctacccaag gacattttcc ccgaaggaga atccgtctct gagatcttgc tcgaagcctc tcgggactgc ctgcaccagc acctcgaggc cgacctggag aatccgacgg ccaactcgat tgccattcgc tacttccact ccaactgcct ccacgctgcg gggaagccca agtactcgtg gcacatattt ggcgaggcca tccgcctggc gcaggtcatg cagctgcacg aggaggctgc 1920 cctcgagggg ctcgtcccca tcgaggcaga gttccgccgt cgctgctttt ggatcctgta 1980 cttgggcgac aagtcagccg ctatactcaa caatcggccc atcaccatcc acaagtactg 2040 cttcgacgcc ggcatcacca cgctataccc gtcgggtatc gaggacgagt tcctgagcac 2100 ggcgtccgag ccgccccgga agagcttcat atccggcttc aacgcaaatg tgcggctctg 2160 gcagtccgcg gctgatttgc tgctggaaat ccgcgtgctg caagatcaga tgatgcagca 2220 ctttcgaggg accatgcccc cgaaccatgt gctgccctcc gccgacaggc agcatctcga 2280 ttctctctat gtccgcttca tcacctgctt ggacgatctc ccgccgtacc tccagtcgtg 2340 cactctggcg atggcagcga tggcagaagg caacgggtct gccgagtcca agcagtacgt 2400 gatacagtgc atcaacctgc aggtgacgtt tcactgtctg cgcatggtaa ttacgcagaa 2460 attcgaagac ctctcttatt ttgctcctgg cgttgagcag gctgatctca gaaagtcgga 2520 gattgtgcga gacatgctga gggtgatgaa cgaggcgccc ttttggggcc tgcaggccaa 2580 tggcgagcca aacgtgagtc gtttccttgt ctcttctctt ttctgcacac ccttttcttc 2640 gacgaccccc cctctctctt tatatccctg cggatatgta tatcatcaag cctcggcact 2700 tgttgctaat ctgtcctgat tatgttgtct ggatgctgca ggttgaaaag attcgcctta 2760 tcggagctag tttgctggcc atcatccatc gcaaccagga ttcacccttg gctacgcgag 2820 ccaggagcga cttttccgtg cttttggata ttctcacgcg gctggactcg aaggcgtcgg 2880 accaactgag gaatacgtcc actaccgttg ttggctaa 2918 <210> 5 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 5 acggggtacg cgtgaaattg 20 <210> 6 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 6 tgtgactgat taatgtatga 20 <210> 7 <211> 35 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 7 cattaatcag tcacaatgtt gtccaatcct ctccg 35 <210> 8 <211> 35 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 8 tttcgccacg gagctttaga gggccagacc ggttc 35 <210> 9 <211> 35 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 9 cattaatcag tcacaatgct gcgctactcc cccgt 35 <210> 10 <211> 35 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 10 tttcgccacg gagctttagc caacaacggt agtgg 35 <210> 11 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 11 agctccgtgg cgaaagcctg 20 <210> 12 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 12 ctcggctacg ttgtcatcgt 20 <210> 13 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 13 caaaccagcc aaggtaggta 20 <210> 14 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 14 ttggttcttg gtttggaggg 20 <210> 15 <211> 35 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 15 gacaacgtag ccgagaagta ccgcgcgctt gacaa 35 <210> 16 <211> 35 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 16 accttggctg gtttgctgaa tgcccggtgg taagc 35 <210> 17 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 17 aagtaccgcg cgcttgacaa 20 <210> 18 <211> 20 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 18 ttggttcttg gtttggaggg 20 <210> 19 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 19 cacgcgttct cggctgccga agctatt 27 <210> 20 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 20 agccgagaac gcgtggctcg tcgccgt 27 <210> twenty one <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> twenty one tcggctgtcg aagctattagccagatt 27 <210> twenty two <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> twenty two agcttcgaca gccgataccg cgtggct 27 <210> twenty three <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> twenty three acggcgtaca gccacgcggt atcggct 27 <210> twenty four <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> twenty four gtggctgtac gccgtcacga atccttc 27 <210> 25 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 25 cacgcgtact cggctgccga agctatt 27 <210> 26 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 26 agccgagtac gcgtggctcg tcgccgt 27 <210> 27 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 27 cacgcgaagt cggctgccga agctatt 27 <210> 28 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 28 agccgacttc gcgtggctcg tcgccgt 27 <210> 29 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 29 tcggctatcg aagctattagccagatt 27 <210> 30 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 30 agcttcgata gccgataccg cgtggct 27 <210> 31 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 31 tcggctctcg aagctattagccagatt 27 <210> 32 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 32 agcttcgaga gccgataccg cgtggct 27 <210> 33 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 33 tcggctttcg aagctattagccagatt 27 <210> 34 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 34 agcttcgaaa gccgataccg cgtggct 27 <210> 35 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 35 tcggcttggg aagctattagccagatt 27 <210> 36 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 36 agcttcccaa gccgataccg cgtggct 27 <210> 37 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 37 tcggcttacg aagctattagccagatt 27 <210> 38 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 38 agcttcgtaa gccgataccg cgtggct 27 <210> 39 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 39 tcggctaccg aagctattagccagatt 27 <210> 40 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 40 agcttcggta gccgataccg cgtggct 27 <210> 41 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 41 tcggctaagg aagctattagccagatt 27 <210> 42 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 42 agcttcctta gccgataccg cgtggct 27 <210> 43 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 43 tcggctgagg aagctattagccagatt 27 <210> 44 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 44 agcttcctca gccgataccg cgtggct 27 <210> 45 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 45 gctgcctacg ctattagcca gattctc 27 <210> 46 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 46 aatagcgtag gcagccgata ccgcgtg 27 <210> 47 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 47 gccgaagtca ttagccagat tctcgag 27 <210> 48 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 48 gctaatgact tcggcagccg ataccgc 27 <210> 49 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 49 gccgaatgga ttagccagat tctcgag 27 <210> 50 <211> 27 <212> DNA <213> Synthesis <220> <223> Oligonucleotide primers <400> 50 gctaatccat tcggcagccg ataccgc 27 <210> 51 <211> 920 <212> PRT <213> Trichoderma reesei <400> 51 Met Leu Ser Asn Pro Leu Arg Arg Tyr Ser Ala Tyr Pro Asp Ile Ser 1 5 10 15 Ser Ala Ser Phe Asp Pro Asn Tyr His Gly Ser Gln Ser His Leu His 20 25 30 Ser Ile Asn Val Asn Thr Phe Gly Asn Ser His Pro Tyr Pro Met Gln 35 40 45 His Leu Ala Gln His Ala Glu Leu Ser Ser Ser Arg Met Ile Arg Ala 50 55 60 Ser Pro Val Gln Pro Lys Gln Arg Gln Gly Ser Leu Ile Ala Ala Arg 65 70 75 80 Lys Asn Ser Thr Gly Thr Ala Gly Pro Ile Arg Arg Arg Ile Ser Arg 85 90 95 Ala Cys Asp Gln Cys Asn Gln Leu Arg Thr Lys Cys Asp Gly Leu His 100 105 110 Pro Cys Ala His Cys Ile Glu Phe Gly Leu Gly Cys Glu Tyr Val Arg 115 120 125 Glu Arg Lys Lys Arg Gly Lys Ala Ser Arg Lys Asp Ile Ala Ala Gln 130 135 140 Gln Ala Ala Ala Ala Ala Ala Gln His Ser Gly Gln Val Gln Asp Gly 145 150 155 160 Pro Glu Asp Gln His Arg Lys Leu Ser Arg Gln Gln Ser Glu Ser Ser 165 170 175 Arg Gly Ser Ala Glu Leu Ala Gln Pro Ala His Asp Pro Pro His Gly 180 185 190 His Ile Glu Gly Ser Val Ser Ser Phe Ser Asp Asn Gly Leu Ser Gln 195 200 205 His Ala Ala Met Gly Gly Met Asp Gly Leu Glu Asp His His Gly His 210 215 220 Val Gly Val Asp Pro Ala Leu Gly Arg Thr Gln Leu Glu Ala Ser Ser 225 230 235 240 Ala Met Gly Leu Gly Ala Tyr Gly Glu Val His Pro Gly Tyr Glu Ser 245 250 255 Pro Gly Met Asn Gly His Val Met Val Pro Pro Ser Tyr Gly Ala Gln 260 265 270 Thr Thr Met Ala Gly Tyr Ser Gly Ile Ser Tyr Ala Ala Gln Ala Pro 275 280 285 Ser Pro Ala Thr Tyr Ser Ser Asp Gly Asn Phe Arg Leu Thr Gly His 290 295 300 Ile His Asp Tyr Pro Leu Ala Asn Gly Ser Ser Pro Ser Trp Gly Gln 305 310 315 320 Ser Asp Leu Arg Tyr Pro Val Leu Glu Pro Leu Leu Pro His Leu Gly 325 330 335 Asn Ile Leu Pro Val Ser Leu Ala Cys Asp Leu Ile Asp Leu Tyr Phe 340 345 350 Ser Ser Ser Ser Ser Ala Gln Met His Pro Met Ser Pro Tyr Val Leu 355 360 365 Gly Phe Val Phe Arg Lys Arg Ser Phe Leu His Pro Thr Asn Pro Arg 370 375 380 Arg Cys Gln Pro Ala Leu Leu Ala Ser Met Leu Trp Val Ala Ala Gln 385 390 395 400 Thr Ser Glu Ala Ser Phe Leu Thr Ser Leu Pro Ser Ala Arg Ser Lys 405 410 415 Val Cys Gln Lys Leu Leu Glu Leu Thr Val Gly Leu Leu Gln Pro Leu 420 425 430 Ile His Thr Gly Thr Asn Ser Pro Ser Pro Lys Thr Ser Pro Val Val 435 440 445 Gly Ala Ala Ala Leu Gly Val Leu Gly Val Ala Met Pro Gly Ser Leu 450 455 460 Asn Met Asp Ser Leu Ala Gly Glu Thr Gly Ala Phe Gly Ala Ile Gly 465 470 475 480 Ser Leu Asp Asp Val Ile Thr Tyr Val His Leu Ala Thr Val Val Ser 485 490 495 Ala Ser Glu Tyr Lys Gly Ala Ser Leu Arg Trp Trp Gly Ala Ala Trp 500 505 510 Ser Leu Ala Arg Glu Leu Lys Leu Gly Arg Glu Leu Pro Pro Gly Asn 515 520 525 Pro Pro Ala Asn Gln Glu Asp Gly Glu Gly Leu Ser Glu Asp Val Asp 530 535 540 Glu His Asp Leu Asn Arg Asn Asn Thr Arg Phe Val Thr Glu Glu Glu 545 550 555 560 Arg Glu Glu Arg Arg Arg Ala Trp Trp Leu Val Tyr Ile Val Asp Arg 565 570 575 His Leu Ala Leu Cys Tyr Asn Arg Pro Leu Phe Leu Leu Asp Ser Glu 580 585 590 Cys Ser Asp Leu Tyr His Pro Met Asp Asp Ile Lys Trp Gln Ala Gly 595 600 605 Lys Phe Arg Ser His Asp Ala Gly Asn Ser Ser Ile Asn Ile Asp Ser 610 615 620 Ser Met Thr Asp Glu Phe Gly Asp Ser Pro Arg Ala Ala Arg Gly Ala 625 630 635 640 His Tyr Glu Cys Arg Gly Arg Ser Ile Phe Gly Tyr Phe Leu Ser Leu 645 650 655 Met Thr Ile Leu Gly Glu Ile Val Asp Val His His Ala Lys Ser His 660 665 670 Pro Arg Phe Gly Val Gly Phe Arg Ser Ala Arg Asp Trp Asp Glu Gln 675 680 685 Val Ala Glu Ile Thr Arg His Leu Asp Met Tyr Glu Glu Ser Leu Lys 690 695 700 Arg Phe Val Ala Lys His Leu Pro Leu Ser Ser Lys Asp Lys Glu Gln 705 710 715 720 His Glu Met His Asp Ser Gly Ala Val Thr Asp Met Gln Ser Pro Leu 725 730 735 Ser Val Arg Thr Asn Ala Ser Ser Arg Met Thr Glu Ser Glu Ile Gln 740 745 750 Ala Ser Ile Val Val Ala Tyr Ser Thr His Val Met His Val Leu His 755 760 765 Ile Leu Leu Ala Asp Lys Trp Asp Pro Ile Asn Leu Leu Asp Asp Asp 770 775 780 Asp Leu Trp Ile Ser Ser Glu Gly Phe Val Thr Ala Thr Ser His Ala 785 790 795 800 Val Ser Ala Ala Glu Ala Ile Ser Gln Ile Leu Glu Phe Asp Pro Gly 805 810 815 Leu Glu Phe Met Pro Phe Phe Tyr Gly Val Tyr Leu Leu Gln Gly Ser 820 825 830 Phe Leu Leu Leu Leu Ile Ala Asp Lys Leu Gln Ala Glu Ala Ser Pro 835 840 845 Ser Val Ile Lys Ala Cys Glu Thr Ile Val Arg Ala His Glu Ala Cys 850 855 860 Val Val Thr Leu Ser Thr Glu Tyr Gln Arg Asn Phe Ser Lys Val Met 865 870 875 880 Arg Ser Ala Leu Ala Leu Ile Arg Gly Arg Val Pro Glu Asp Leu Ala 885 890 895 Glu Gln Gln Gln Arg Arg Arg Glu Leu Leu Ala Leu Tyr Arg Trp Thr 900 905 910 Gly Asn Gly Thr Gly Leu Ala Leu 915 920

Claims

1. A method for producing a mutant filamentous fungus, the method being characterized by: The method comprises the steps of changing the expression of XYR1 and ACE3 of the parent filamentous bacteria, The alteration of XYR1 is a substitution selected from the following for a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and functioning as a transcriptional activator of cellulases and hemicellulases: Substitution of Thr to Tyr at the position corresponding to position 817 of SEQ ID NO: 1; Substitution of Val at the position corresponding to position 821 of SEQ ID NO: 1 with Lys, Phe or Tyr; Substitution of Ala at the position corresponding to position 824 of SEQ ID NO: 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr; Substitution of Glu at the position corresponding to position 825 of SEQ ID NO: 1 with Tyr; and Ala at the position corresponding to position 826 of SEQ ID NO: 1 is substituted with Val or Trp, The change in ACE3 expression is the enhanced expression of a polypeptide consisting of the amino acid sequence at positions 107 to 734 of SEQ ID NO:

3.

2. The method according to claim 1, characterized in that: The expression of the polypeptide is enhanced by increasing the transcription amount of the gene encoding the polypeptide.

3. The method according to claim 1 or 2, characterized in that: The mutant filamentous fungus expresses cellulase in the absence of a cellulase-inducing substance.

4. The method according to claim 1 or 2, characterized in that: The filamentous fungus is a Trichoderma fungus.

5. The method according to claim 4, characterized in that: The Trichoderma fungus is Trichoderma reesei.

6. A method for producing a protein, characterized in that: The method comprises the step of culturing the mutant filamentous bacteria obtained by the method according to any one of claims 1 to 5.

7. The method according to claim 6, characterized in that: The protein is cellulase and / or hemicellulase.

8. The method according to claim 6 or 7, characterized in that: The cultivation is carried out in the presence of glucose.

9. A mutant filamentous fungus, characterized in that: The mutant filamentous fungus comprises a mutant XYR1, and compared with the parent filamentous fungus, the expression of a portion of the ACE3 polypeptide is enhanced. The mutant XYR1 is a polypeptide consisting of an amino acid sequence in which the amino acid sequence of SEQ ID NO: 1 is substituted, and functions as a transcriptional activator of cellulase and hemicellulase. The substitution is selected from: Substitution of Thr to Tyr at the position corresponding to position 817 of SEQ ID NO: 1; Substitution of Val at the position corresponding to position 821 of SEQ ID NO: 1 with Lys, Phe or Tyr; Substitution of Ala at the position corresponding to position 824 of SEQ ID NO: 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr; Substitution of Glu at the position corresponding to position 825 of SEQ ID NO: 1 with Tyr; and Substitution of Ala at the position corresponding to position 826 of SEQ ID NO: 1 with Val or Trp, The partial polypeptide of ACE3 is a polypeptide consisting of the amino acid sequence at positions 107 to 734 of sequence number 3.

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