Modified filamentous fungus and method for producing protein using the same

Abstract

To provide modified filamentous fungi having improved protein productivity, and a method for producing protein using the modified filamentous fungi.SOLUTION: Modified filamentous fungi express modified ACE3. The modified ACE3 is a modified one deficient in substantially whole of Zn(II)2Cys6 DNA-binding domain of ACE3. There is also provided a method for producing protein that includes culturing the modified filamentous fungi.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to modified filamentous fungi and methods for producing proteins using the same. [Background technology]

[0002] Filamentous fungi are plant polysaccharide decomposing bacteria that produce a variety of cellulases and hemicellulases. Among them, Trichoderma is attracting attention as a microorganism for producing cellulase-based biomass decomposing enzymes because it is capable of simultaneously producing cellulases and hemicellulases in large quantities.

[0003] Carbon sources for industrial microbial cultivation are desirably inexpensive and soluble. Traditionally, glucose has been widely used as a carbon source in microbial cultivation. However, inducers may be required for the production of proteins such as enzymes by microorganisms. For example, inducers are generally essential for the production of cellulase by microorganisms. In Trichoderma, expression of major cellulase genes such as cbh1, cbh2, egl1, and egl2 is induced by inducers such as cellulose and cellobiose (Non-Patent Document 1). Without the use of inducers, for example, when glucose alone is used as the sole carbon source, saccharifying enzymes are generally hardly produced in Trichoderma.

[0004] Known microbial protein production methods using inducers include a cellulase production method using Avicel, a microcrystalline cellulose. Other methods include a cellulase production method using soluble lactose without using cellulose (Patent Document 1), and a method of inducing cellulase production by reacting Trichoderma-derived cellulases (including β-glucosidase, endoglucanase, and cellobiohydrolase) with glucose at high temperatures to synthesize inducible sugars such as sophorose and gentiobiose from glucose (Patent Document 2). However, cellulose substrates are expensive, and many are insoluble, placing a strain on industrial processes, making their use in industrial applications difficult in terms of cost and equipment. Furthermore, cellulase production using other inducible sugars still has disadvantages in terms of cost and process load.

[0005] Therefore, analysis of the cellulase expression mechanism is being conducted with the aim of creating microorganisms capable of expressing cellulases and xylanases without the use of inducers by modifying transcriptional regulatory factors. XYR1, ACE2, ACE3, HAP2 / 3 / 5, and other positive transcription factors involved in the induction and expression of cellulases in Trichoderma have been reported (Non-Patent Document 2). ACE3 is a transcription factor that controls promoters such as the major cellulase cbh1. ACE3 binds to promoters via a Zn(II)2Cys6-type DNA-binding domain (DBD) located at amino acids 120 to 160 on the N-terminus (Non-Patent Document 3). Furthermore, Non-Patent Document 3 suggests that ACE3 and XYR1 interact to regulate cellulase gene expression in Trichoderma reesei.

[0006] Patent Document 3 and Non-Patent Document 4 disclose methods for increasing or decreasing the productivity of cellulase and other substances in Trichoderma reesei by increasing or decreasing the expression of the tre77513 (ACE3) gene. Patent Document 4 and Non-Patent Document 5 report that a filamentous fungus that overexpresses a modified ACE3 that retains all six cysteines in the N-terminal Zn(II)2Cys6-type DNA-binding domain and has 7 to 17 amino acids deleted at the C-terminus exhibits improved cellulase expression even in the absence of an inducer. Non-Patent Document 5 also describes a filamentous fungus that overexpresses the C-terminally deleted ACE3 and coexpresses the wild-type or A824V mutant of XYR1. However, the effect of coexpression of XYR1 on cellulase expression in this filamentous fungus was observed slightly in the presence of an inducer, but not in the absence of an inducer.

[0007] Non-Patent Document 6 reports that xylanase deregulation occurs in Trichoderma harboring the A824V mutation in XYR1, resulting in increased cellulase production. Non-Patent Document 7 reports that combining the V821F mutation in XYR1 with enhanced expression of ACE2 in Trichoderma strains improves protein productivity in media containing glucose or sucrose as a carbon source. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6169077 [Patent Document 2] Patent No. 5366286 [Patent Document 3] U.S. Patent No. 9,512,415 [Patent Document 4] International Publication No. 2018 / 067599 [Non-patent literature]

[0009] [Non-Patent Document 1] Curr Genomics, 2013, 14:230-249 [Non-patent document 2] BMC Genomics, 2015, 16:326 [Non-patent document 3] J Biol Chem, 2019, doi:10.1074 / jbc.RA119.008497 [Non-patent document 4] Biotech Biofuels, 2014, 7:14 [Non-Patent Document 5] Biotech Biofuels, 2020, 13:137 [Non-patent document 6] Biotech Biofuels, 2013, 6:62 [Non-Patent Document 7] Biotech Biofuels, 2017, 10:30 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention relates to providing modified filamentous fungi and methods for producing the same, as well as methods for producing proteins using the modified filamentous fungi.

[0011] The inventors have discovered that modified filamentous fungi expressing an ACE3 variant lacking the Zn(II)2Cys6-type DNA-binding domain (hereinafter referred to simply as "DBD" in this specification) produce high levels of proteins such as cellulase and hemicellulase without the use of inducers that have traditionally been essential for the production of cellulase and hemicellulase.

[0012] Accordingly, the present invention provides a modified filamentous fungus comprising: expressing a modified ACE3, The ACE3 variant is a variant in which substantially all of the Zn(II)2Cys6-type DNA-binding domain of ACE3 has been deleted. Modified filamentous fungi are provided. The present invention also provides a method for producing a protein, which comprises culturing the modified filamentous fungus. The present invention also provides a method for producing a modified filamentous fungus, comprising the steps of: modifying a parent filamentous fungus to express an ACE3 variant; The ACE3 variant is a variant in which substantially all of the Zn(II)2Cys6-type DNA-binding domain of ACE3 has been deleted. A method is provided. [Effects of the Invention]

[0013] The modified filamentous fungus provided by the present invention can efficiently produce proteins even in an environment where a cellulase-non-inducing carbon source such as glucose is used as the main carbon source. Furthermore, the filamentous fungus can efficiently produce proteins such as cellulase and hemicellulase without using expensive cellulase-inducing substances. The present invention makes it possible to improve the efficiency and reduce costs of protein production using filamentous fungi. [Brief explanation of the drawings]

[0014] [Figure 1] Structure of ACE3 variants expressed by engineered filamentous fungi. [Figure 2] Structure of ACE3 variants expressed by engineered filamentous fungi. [Figure 3] Effect of ACE3 DBD deletion on protein productivity: (A) The values ​​on the vertical axis represent the relative protein productivity of modified filamentous fungi expressing ACE3 variants, and 1, 2, and 3 on the horizontal axis represent three modified filamentous fungal strains expressing the same ACE3 variant. (B) Gel electrophoresis image of modified filamentous fungal cultures. [Figure 4] Alignment of amino acid sequences of ACE3 variants derived from various Trichoderma species. [Figure 5] Protein production in engineered fungi expressing ACE3 variants from various Trichoderma species: (A) Relative protein productivity, (B) Gel electrophoresis of cultures of engineered fungi. [Figure 6]Effect of C-terminal deletion of ACE3 on protein productivity: (A) Relative protein productivity of engineered filamentous fungi expressing ACE3 variants, (B) Gel electrophoresis image of cultures of engineered filamentous fungi. [Figure 7] Effect of co-expression of mutant XYR1 and modified ACE3 on protein productivity: (A) Gel electrophoresis image of culture of the modified filamentous fungus, (B) Protein composition ratio of culture of the modified filamentous fungus. [Figure 8] Effect of mutant XYR1 expression in combination with DBD deletion and C-terminal deletion of ACE3 on protein productivity: (A) Gel electrophoresis image of cultures of the modified filamentous fungus, (B) Protein composition ratio of cultures of the modified filamentous fungus. DETAILED DESCRIPTION OF THE INVENTION

[0015] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0016] Herein, the identity of amino acid sequences and nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, the identity is calculated by performing analysis using the search homology program in the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with a unit size to compare (ktup) of 2.

[0017] As used herein, "at least 90% identity" with respect to amino acid sequences and nucleotide sequences means identity of 90% or more, preferably 92% or more, more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0018] Unless otherwise defined herein, the term "one or several" used in reference to deletion, substitution, addition, or insertion of amino acids and nucleotides in an amino acid sequence or a nucleotide sequence can mean, for example, 1 to 20, preferably 1 to 16, more preferably 1 to 12, even more preferably 1 to 8, and even more preferably 1 to 4. As used herein, "addition" of an amino acid or nucleotide includes addition of one or several amino acids or nucleotides to one or both ends of a sequence. Furthermore, as used herein, "insertion" of an amino acid or nucleotide includes insertion of an amino acid or nucleotide to the 5' or 3' side of a given position.

[0019] As used herein, a "corresponding position" or "corresponding region" in an amino acid sequence or nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to maximize homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and procedures for doing so are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Clustal W is available, for example, on the websites of the European Bioinformatics Institute (EBI) [www.ebi.ac.uk / index.html] and the DNA Data Bank of Japan (DDBJ) [www.ddbj.nig.ac.jp / searches-j.html], operated by the National Institute of Genetics. A position of a target sequence that is aligned to any position of a reference sequence by the above-described alignment is considered to be a "position corresponding to" that position. In addition, a region between corresponding positions or a region consisting of a corresponding motif is also considered to be a corresponding region.

[0020] Those skilled in the art can further fine-tune the alignment of amino acid sequences obtained above to optimize it. Such optimal alignment is preferably determined taking into account the similarity of the amino acid sequences, the frequency of inserted gaps, and the like. Here, the similarity of amino acid sequences refers to the percentage (%) of the number of positions where identical or similar amino acids exist in both sequences when two amino acid sequences are aligned, relative to the total number of amino acids in the two sequences. Similar amino acids refer to amino acids among the 20 amino acids that constitute proteins that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Such groups of similar amino acids are well known to those skilled in the art, and examples include, but are not limited to, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; leucine and isoleucine.

[0021] As used herein, the term "amino acid" refers to the 20 amino acids that make up 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).

[0022] As used herein, the term "operably linked" between a gene and a regulatory region such as a promoter means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene to a regulatory region are well known to those skilled in the art.

[0023] As used herein, "upstream" and "downstream" in relation to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the 5' region of the gene on the DNA sense strand.

[0024] As used herein, the terms "original" and "native" when used with respect to a cellular function, property, or trait are used to indicate that the function, property, or trait is originally present in the cell. In contrast, the term "exogenous" is used to indicate that the function, property, or trait is not originally present in the cell but is introduced from outside. For example, a "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from outside. An exogenous gene or polynucleotide may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).

[0025] The present invention relates to improving protein productivity in filamentous fungi. Conventionally, when filamentous fungi are cultured in the presence of glucose, protein production is sometimes suppressed due to catabolite repression. In particular, the expression of cellulase-based biomass-degrading enzymes such as cellulases and hemicellulases in filamentous fungi usually needs to be induced by cellulase-inducing substances such as cellulose, sophorose, and cellooligosaccharides (cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, etc.), whereas induction of expression is suppressed in the presence of glucose.

[0026] Induction of cellulase-based biomass-degrading enzymes by inducers in filamentous fungi is regulated by numerous transcription factors, including XYR1, Cre1, ACE1, ACE2, ACE3, and HAP2 / 3 / 5. ACE3 is a transcriptional activator of cellulases and hemicellulases in filamentous fungi. ACE3 is essential for the transcription of cellulase genes and some xylanase genes during lactose induction. ACE3 is also partially involved in the transcriptional regulation of the xyr1 gene. Trichoderma reesei ACE3 is registered in the NCBI database (www.ncbi.nlm.nih.gov / ) as NCBI Reference Sequence: QEM24913.1, where ACE3 is defined as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and encoded by the nucleotide sequence of SEQ ID NO: 6. According to the NCBI Reference Sequence: XP_006966092.1 [www.ncbi.nlm.nih.gov / protein / XP_006966092.1], the amino acid sequence of SEQ ID NO: 1, positions 523-734, is predicted to interact with XYR1; positions 391-522 are predicted to be a fungus-specific transcription factor domain; and positions 120-160 are predicted to be a Zn(II)2Cys6-type DNA-binding domain (DBD). The gene encoding the DBD is divided into two regions by an intron. One (Exon 2) encodes the region containing the two N-terminal Cys residues of the DBD (C2), and the other (Exon 3) encodes the region containing the remaining four Cys residues (C4). ACE3s with similar structures also exist in other Trichoderma species.

[0027] It has been reported that deletion of the C-terminal amino acid of ACE3 enables protein production in filamentous fungi without inducers (Non-Patent Document 5). However, Non-Patent Document 5 also discloses that the effect of this C-terminal deletion was abolished by deletion of C2 in the DBD.

[0028] In contrast, the present inventors have found that an ACE3 variant lacking not only DBD C2 but also DBD C4 confers the ability to express cellulases and hemicellulases in filamentous fungi without inducers, and enables the filamentous fungi to highly express proteins such as cellulases and hemicellulases in the presence of a non-cellulase-inducing carbon source such as glucose. Furthermore, the deletion of C2 and C4 in ACE3 synergistically improves protein productivity when combined with a C-terminal deletion.

[0029] Therefore, in one aspect, the present invention provides modified filamentous fungi that express an ACE3 variant lacking substantially all of the DBD, and a method for producing the modified filamentous fungi. The modified filamentous fungi of the present invention can be produced by modifying a parent filamentous fungus so that it expresses an ACE3 variant lacking substantially all of the DBD. The produced modified filamentous fungi of the present invention express the ACE3 variant lacking substantially all of the DBD.

[0030] The DBD of ACE3 is located at the N-terminus of the amino acid sequence of ACE3, specifically in the region corresponding to amino acids 120 to 160 of SEQ ID NO: 1, with C2 at positions 120 to 131 and C4 at positions 132 to 160. As used herein, "substantially all of the DBD is deleted" refers to a DBD that is deleted in part or in whole of C2 and in part or in whole of C4, preferably a DBD that is deleted in whole of C2 and in part or in whole of C4. Here, "a part of C4" preferably refers to a region containing at least two Cys residues in C4, more preferably a region containing at least three Cys residues in C4, and even more preferably a region containing four Cys residues in C4, for example, a region corresponding to amino acids 132 to 151 of SEQ ID NO: 1. As used herein, an example of "substantially the entire DBD deleted" is a state in which 80% or more, preferably 90% or more, and more preferably 95% or more of the amino acid sequence of the DBD is deleted, and six Cys residues in the DBD are deleted.

[0031] The parent filamentous fungus of the modified filamentous fungus of the present invention is a filamentous fungus that is prepared by being modified to express an ACE3 variant lacking substantially all of the DBD. Preferably, the parent filamentous fungus inherently expresses ACE3. For example, the parent filamentous fungus preferably expresses native ACE3 or a variant thereof that has the entire DBD. More preferably, the parent filamentous fungus expresses native ACE3 or a variant thereof that has the entire DBD and has cellulase activity.

[0032] Examples of parent filamentous fungi that can be used in the present invention include, but are not limited to, filamentous fungi belonging to the phylum Eumycota and Oomycota. More specific examples include filamentous fungi of the genus Trichoderma, Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, Hypocrea, Acremonium, Chrysosporium, Myceliophthora, Piromyces, Talaromyces, Thermoascus, Thielavia, etc. Of these, filamentous fungi of the genus Trichoderma are preferred.

[0033] Examples of filamentous fungi of the genus Trichoderma (hereinafter also referred to as Trichoderma fungi) include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride, Trichoderma atroviride, etc., and preferably Trichoderma reesei and mutant strains thereof. For example, Trichoderma reesei strain QM9414 and its mutants, preferably Trichoderma reesei strain PC-3-7 (ATCC 66589), Trichoderma reesei strain PCD-10 (FERM P-8172), Trichoderma reesei strain E1AB1 (hereinafter sometimes referred to as JN13), or their mutants, can be used as parent filamentous fungi. Strain E1AB1 is a strain of Trichoderma reesei PC-3-7 that has been modified to express β-glucosidase (BGL) derived from Aspergillus aculeatus using the egl1 promoter (see Enzyme and Microbial Technology (2016) 82:89-95 and Examples 1 to 3 of WO2013 / 115305).

[0034] The ACE3 variant expressed by the modified filamentous fungus of the present invention can be obtained by modifying a parent ACE3 so that substantially all of the DBD is deleted. The parent ACE3 can be a native ACE3 or a variant thereof, which has part or all of the DBD. Preferably, the parent ACE3 can be a native ACE3 or a variant thereof, which has at least C4 of the DBD. Preferably, the parent ACE3 is an ACE3 derived from a fungus of the genus Trichoderma or a variant thereof. Examples of Trichoderma include those described above, but preferably include Trichoderma reesei, Trichoderma haridium, and Trichoderma atroviride.

[0035] Preferred examples of the parent ACE3 include ACE3 consisting of the amino acid sequences of SEQ ID NOs: 1 to 4, or variants thereof. SEQ ID NO: 1 represents the amino acid sequence of full-length ACE3 from Trichoderma reesei. SEQ ID NO: 2 represents the amino acid sequence of a DBD portion (C2)-deleted ACE3 variant from Trichoderma reesei. SEQ ID NO: 3 represents the amino acid sequence of a DBD portion (C2)-deleted ACE3 variant from Trichoderma atroviride. SEQ ID NO: 4 represents the amino acid sequence of a DBD portion (C2)-deleted ACE3 variant from Trichoderma haridium.

[0036] Another preferred example of the parent ACE3 is a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 4. Another preferred example of the parent ACE3 is a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence of any one of SEQ ID NOs: 1 to 4. These parent ACE3 polypeptides have a sequence that corresponds to part or all of the DBD of native ACE3 (e.g., SEQ ID NO: 1), and preferably, like native ACE3, can function as a transcriptional activator of cellulase and hemicellulase.

[0037] The ACE3 mutant expressed by the modified filamentous fungus of the present invention lacks substantially the entire DBD. Preferably, the ACE3 mutant lacks at least a region corresponding to amino acids 120 to 160 of SEQ ID NO: 1. Furthermore, the ACE3 mutant may lack a region N-terminal to the DBD or a region C-terminal to the DBD. In one embodiment, the ACE3 mutant lacks a region corresponding to amino acids 120 to 151 of SEQ ID NO: 1. In one embodiment, the ACE3 mutant lacks a region corresponding to amino acids 1 to 151 of SEQ ID NO: 1. In one embodiment, the ACE3 mutant lacks a region corresponding to amino acids 1 to 160 of SEQ ID NO: 1. In one embodiment, the ACE3 mutant lacks a region corresponding to amino acids 1 to 200 of SEQ ID NO: 1. In one embodiment, the ACE3 mutant lacks a region corresponding to amino acids 1 to 240 of SEQ ID NO: 1.

[0038] In addition to the DBD deletion, the ACE3 variant may also have a deletion in the C-terminal region. Examples of deletions in the C-terminal region include deletions of 7 to 17 amino acids at the C-terminus, which are deletions that result in improved protein productivity as disclosed in Non-Patent Document 5. In one embodiment, the ACE3 variant may have a deletion in a region corresponding to at least 7 amino acids (from position 728 to the C-terminus) and up to 17 amino acids (from position 718 to the C-terminus) at the C-terminus of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the ACE3 variant may have a deletion of one or more amino acids selected from the group consisting of amino acids corresponding to positions -7 to -17 (718 to 728) of SEQ ID NO: 1. In one embodiment, the ACE3 variant may have a deletion in a region corresponding to 11 amino acids at the C-terminus (from position 724 to the C-terminus) of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the ACE3 variant does not have a deletion in the C-terminus compared to the parent ACE3.

[0039] Preferably, in the ACE3 variant, amino acids other than those in the region containing the DBD and the C-terminal region are maintained. In a preferred example, the ACE3 variant has at least a region corresponding to amino acids 280 to 701 of SEQ ID NO: 1. More preferably, the ACE3 variant has a region corresponding to amino acids 280 to 717 of SEQ ID NO: 1. More preferably, the ACE3 variant has a region corresponding to amino acids 280 to 723 of SEQ ID NO: 1. The ACE3 variant may have a region corresponding to amino acids 280 to 727 of SEQ ID NO: 1. In a more preferred example, the ACE3 variant has a region corresponding to amino acids 260 to 701 of SEQ ID NO: 1. Even more preferably, the ACE3 variant has a region corresponding to amino acids 260 to 717 of SEQ ID NO: 1. Even more preferably, the ACE3 variant has a region corresponding to amino acids 260 to 723 of SEQ ID NO: 1. The ACE3 variant may have a region corresponding to amino acids 260 to 727 of SEQ ID NO: 1. In a more preferred example, the ACE3 variant has a region corresponding to amino acids 250 to 701 of SEQ ID NO: 1. Even more preferably, the ACE3 variant has a region corresponding to amino acids 250 to 717 of SEQ ID NO: 1. Even more preferably, the ACE3 variant has a region corresponding to amino acids 250 to 723 of SEQ ID NO: 1. The ACE3 variant may have a region corresponding to amino acids 250 to 727 of SEQ ID NO: 1. In a more preferred example, the ACE3 variant has a region corresponding to amino acids 241 to 701 of SEQ ID NO: 1. Even more preferably, the ACE3 variant has a region corresponding to amino acids 241 to 717 of SEQ ID NO: 1. Even more preferably, the ACE3 variant has a region corresponding to amino acids 241 to 723 of SEQ ID NO: 1. The ACE3 variant may have a region corresponding to amino acids 241 to 727 of SEQ ID NO: 1. Preferably, the regions corresponding to each amino acid region of SEQ ID NO:1 listed above are at least 90% identical or 100% identical to the respective amino acid region of SEQ ID NO:1.

[0040] Alternatively, when there is no deletion of the C-terminal region, the ACE3 variant preferably has a region corresponding to amino acids 280 to 734 of SEQ ID NO: 1, more preferably has a region corresponding to amino acids 260 to 734 of SEQ ID NO: 1, even more preferably has a region corresponding to amino acids 250 to 734 of SEQ ID NO: 1, and even more preferably has a region corresponding to amino acids 241 to 734 of SEQ ID NO: 1. Preferably, the regions corresponding to each of the amino acid regions of SEQ ID NO: 1 listed above are at least 90% identical or 100% identical in sequence to the respective amino acid regions of SEQ ID NO: 1.

[0041] Methods for modifying a parent filamentous fungus to express an ACE3 variant (hereinafter also referred to as the desired ACE3 variant) lacking substantially all of the aforementioned DBD and, if necessary, the C-terminal region, include, for example, a method of introducing an exogenous gene encoding the desired ACE3 variant into the parent filamentous fungus and expressing it, and a method of mutating the gene encoding ACE3 that the parent filamentous fungus originally possesses into a gene encoding the desired ACE3 variant.

[0042] A gene encoding a desired ACE3 variant (target gene) can be synthesized by genetic engineering or chemical engineering. For example, a target gene can be prepared by isolating the DNA of a parent ACE3 gene (parent gene) from the genomic DNA of a filamentous fungus such as Trichoderma, followed by deleting part or all of the region encoding the DBD and, if necessary, the region encoding the C-terminal region. Examples of parent genes include polynucleotides consisting of the nucleotide sequences of SEQ ID NOS: 6 to 9 or sequences at least 90% identical thereto. Alternatively, a target gene can be chemically synthesized based on publicly known sequence information, from which part or all of the region encoding the DBD and the region encoding the C-terminal region have been deleted. ACE3 sequence information is available from the NCBI database (www.ncbi.nlm.nih.gov / ). If necessary, the target gene may be codon-optimized to match the host (parent filamentous fungus) into which it will be introduced. Information on codons used by various organisms is available from the Codon Usage Database (www.kazusa.or.jp / codon / ).

[0043] Methods for introducing a foreign gene encoding a desired ACE3 variant into a parent filamentous fungus include, for example, a method using recombination and a method using an expression vector. For example, a given region of the genome of the parent filamentous fungus can be replaced with the target gene by homologous or non-homologous recombination. Examples of expression vectors for filamentous fungi include the yeast expression vectors pNAN8142 (Biosci Biotechnol Biochem, 1996, 60:383-389) and pMA91 (Biosci Biotechnol Biochem, 1998, 62:1615-1618).

[0044] Methods for mutating the gene encoding ACE3 of a parent filamentous fungus include, for example, methods utilizing recombination. For example, the parent gene in the genome of the parent filamentous fungus can be replaced with a gene encoding the desired ACE3 variant (target gene) by homologous or non-homologous recombination. Alternatively, the DBD coding region of the parent gene in the genome can be replaced with a DBD deletion fragment by homologous or non-homologous recombination, thereby mutating the parent gene into the target gene.

[0045] In a specific example of a recombination method, a recombinant DNA construct containing a gene of interest or a DBD deletion fragment, and optionally a drug resistance gene or an auxotrophic gene, is first constructed and introduced into a parent filamentous fungus by standard methods. Next, transformants in which the recombinant construct has been integrated into the genome are selected using indicators such as drug resistance or auxotrophy. If necessary, the presence of the desired mutation in the resulting transformant may be confirmed by genome analysis or enzyme activity analysis.

[0046] A vector commonly used for transformation, such as a plasmid, can be used to introduce a DNA construct into a parent filamentous fungus. A DNA construct or vector can be introduced into cells using conventional methods, such as the protoplast method, the protoplast PEG method, or the competent cell method. The vector used to introduce the DNA construct is not particularly limited, as long as it can be stably maintained and propagated in host cells, and examples include commonly used vectors such as plasmids, cosmids, phages, viruses, YACs, and BACs. Among these, plasmid vectors are preferred. An example of a vector for introduction is pUC118.

[0047] In a preferred embodiment, the modified filamentous fungus of the present invention highly expresses a target ACE3 variant. A modified filamentous fungus that highly expresses a target ACE3 variant can be obtained by further modifying a parent filamentous fungus to enhance expression of the target ACE3 variant. An example of a method for improving the expression of the ACE3 variant is to increase the transcription level of a gene (target gene) encoding the target ACE3 variant. For example, a method for increasing the transcription level of a target gene can be achieved by substituting or inserting a regulatory region (strong regulatory region) that strongly promotes transcription of the target gene into the regulatory region of the target gene in the genome of the parent filamentous fungus, thereby operably linking the strong regulatory region to the target gene. Alternatively, if necessary, a target gene fragment operably linked to a regulatory region (preferably a strong regulatory region) can be introduced into the genome or plasmid of the parent filamentous fungus to increase the number of target genes that can be expressed in the cell, thereby increasing the transcription level of the target gene.

[0048] Examples of regulatory regions that can be used to increase the transcription level include those of genes whose transcription levels do not decrease even under high glucose conditions, such as, in the case of Trichoderma, the regulatory regions of genes 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), pyruvate dehydrogenase (PDA), glucokinase (GLK), actin (ACT1), and translation elongation factor 1α (TEF1). Among these, preferred examples of strong regulatory regions include the promoter of the pdc gene (TRIREDRAFT_121534) and the promoter of the act1 gene (TRIREDRAFT_44504).

[0049] Preferably, the expression level of the ACE3 variant of interest in the modified filamentous fungus of the present invention is improved compared to the expression level of ACE3 in the parent filamentous fungus. The expression level of the ACE3 variant of interest can be quantified as the amount of protein or the transcription level of the gene encoding it by known means such as quantitative PCR, microarray, Western blotting, ELISA, or HPLC.

[0050] Preferably, the modified filamentous fungus of the present invention further expresses XYR1 (Xylanase regulator 1). More preferably, the modified filamentous fungus of the present invention has been modified to highly express XYR1. XYR1 is a transcriptional activator of cellulase and hemicellulase in filamentous fungi. XYR1 is a key factor controlling xylanase gene expression, containing a Zn(II)2Cys6 binuclear cluster domain, and is widely conserved in ascomycetes excluding yeast, such as Trichoderma (XYR1), Fusarium (XYR1), Neurospora (XYR1), and Aspergillus (XLNR). XYR1 from 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, and is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5, encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10.

[0051] An example of XYR1 that is highly expressed by the modified filamentous fungus of the present invention is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5. Other examples of XYR1 include polypeptides consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5, and polypeptides consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 5. These polypeptides can function as transcriptional activators of cellulases and hemicellulases.

[0052] The XYR1 highly expressed by the modified filamentous fungus of the present invention may be a mutant XYR1. A preferred example of the mutant XYR1 is a mutant XYR1 in which at least one amino acid in the region corresponding to positions 810 to 833 of SEQ ID NO: 5 has been mutated (i.e., substituted, deleted, inserted, or added) in an XYR1 polypeptide (parent XYR1) consisting of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto. Examples of such a mutant XYR1 include the mutant XYR1 disclosed in PCT / JP2020 / 042489.

[0053] Preferably, the mutant XYR1 has at least one amino acid substitution selected from the group consisting of amino acids at positions corresponding to positions 817, 821, 824, 825 and 826 of SEQ ID NO: 5 relative to the parent XYR1.

[0054] More preferably, the mutant XYR1 consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5 and has any one or more amino acids selected from the group consisting of: Tyr at a position corresponding to position 817 of SEQ ID NO:5; Lys, Phe, Trp, or Tyr, preferably Phe, at a position corresponding to position 821 of SEQ ID NO: 5; Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, preferably Val, at a position corresponding to position 824 of SEQ ID NO: 5; Tyr at a position corresponding to position 825 of SEQ ID NO:5; Val, Ile, Leu, Phe, Trp, or Tyr at a position corresponding to position 826 of SEQ ID NO: 5;

[0055] More preferably, the mutant XYR1 is a mutant (V821F) in which Val at the position corresponding to position 821 of SEQ ID NO: 5 is substituted with Phe, or a mutant (A824V) in which Ala at the position corresponding to position 824 of SEQ ID NO: 5 is substituted with Val, relative to the parent XYR1.

[0056] The means for overexpressing XYR1 in the modified filamentous fungus can be the same as those for overexpressing the ACE3 variants described above. For example, a foreign gene encoding XYR1 operably linked to a strong regulatory region can be introduced and expressed in the modified filamentous fungus, or the XYR1 gene on the genome of the parent filamentous fungus can be operably linked to a strong regulatory region, or the number of XYR1 genes that can be expressed by the modified filamentous fungus can be increased.

[0057] The modified filamentous fungus of the present invention produced by the above procedures expresses the aforementioned ACE3 variant lacking substantially all of the DBD. Such modified filamentous fungus of the present invention has improved protein productivity in the absence of a cellulase inducer. The modified filamentous fungus can efficiently produce proteins even in an environment where a non-cellulase-inducing carbon source such as glucose is the primary carbon source, even in the absence of a cellulase inducer. Furthermore, the modified filamentous fungus can express cellulase-based biomass degrading enzymes such as cellulase and hemicellulase, even in the absence of cellulase inducers such as cellulose, sophorose, and cellooligosaccharides.

[0058] In a preferred embodiment, the modified filamentous fungus of the present invention expresses an ACE3 variant having a deletion in the C-terminal region as described above in addition to a deletion in substantially all of the DBD. The combination of the deletion in the DBD and the deletion in the C-terminal region synergistically improves protein productivity of the modified filamentous fungus.

[0059] In another preferred embodiment, the modified filamentous fungus of the present invention not only expresses an ACE3 variant lacking substantially all of the DBD, but also highly expresses XYR1 as described above. The combined expression of the ACE3 variant and XYR1 increases the cellulase content in the protein produced by the modified filamentous fungus, enabling efficient cellulase production.

[0060] In a more preferred embodiment, the modified filamentous fungus of the present invention expresses an ACE3 variant having substantially all of the DBD deleted and the C-terminal region deleted, and further highly expresses XYR1, thereby achieving both improved protein productivity and an increased cellulase content in the produced protein.

[0061] Therefore, in a further aspect, the present invention provides a method for producing a protein using the modified filamentous fungus of the present invention. In the protein production method according to the present invention, the modified filamentous fungus of the present invention is cultured. The culture results in the production and accumulation of a target protein in the culture. The target protein can be produced by isolating it from the culture.

[0062] Examples of target proteins to be produced include, but are not limited to, cellulase-based biomass-degrading enzymes such as cellulase and hemicellulase, exoglucanase, endoglucanase, β-glucosidase, protease, lipase, mannase, arabinase, galactase, and amylase. The target protein may be a single protein or a mixture of multiple proteins. Preferably, the target protein is a cellulase-based biomass-degrading enzyme, more preferably cellulase and / or hemicellulase, and even more preferably cellulase and hemicellulase. Examples of hemicellulases include xylanase, β-xylosidase, and α-arabinofuranosidase, with xylanase being preferred.

[0063] Alternatively, the target protein may be a heterologous protein that is not naturally produced by filamentous fungi. In this case, a gene encoding the heterologous protein is inserted into the modified filamentous fungus of the present invention to produce a recombinant filamentous fungus, and this recombinant filamentous fungus can be cultured to obtain a protein containing the heterologous protein. Furthermore, by operably linking the gene encoding the heterologous protein to a secretory signal peptide that functions in the filamentous fungus, the heterologous protein can be secreted and produced in the culture.

[0064] The medium used for producing the protein may be either a synthetic medium or a natural medium, as long as it contains components necessary for the growth of normal filamentous fungi and protein production, such as a carbon source, a nitrogen source, inorganic salts, and vitamins.

[0065] The carbon source may be any carbon source that can be assimilated by the modified filamentous fungus, and examples thereof include carbohydrates 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.

[0066] In the protein production method according to the present invention, the modified filamentous fungus is preferably cultured in an environment where a cellulase-non-inducing carbon source is used as the main carbon source. Examples of cellulase-non-inducing carbon sources include glucose, fructose, sucrose, maltose, and glycerol. Among these, glucose is preferred from the viewpoint of cost. When the target protein to be produced is a cellulase-based biomass degrading enzyme, the culture in this method may be carried out in the presence of a cellulase-inducing substance such as cellulose, sophorose, or cellooligosaccharide. However, high production of the target protein is possible even in the absence of the inducer, and the culture is not limited to the use or non-use of the inducer. Furthermore, in the present invention, the modified filamentous fungus may be cultured while feeding a non-inducing carbon source such as glucose in order to efficiently produce proteins such as cellulase-based biomass degrading enzymes while further reducing catabolite repression. In this case, it is preferable to dissolve the cellulase-non-inducing carbon source, such as glucose, in a nitrogen source such as aqueous ammonia or an aqueous solution containing an ammonium salt, and then culture the resulting solution in a feed stream, in terms of culture efficiency and suppressing foaming during culture.

[0067] Examples of nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate.

[0068] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0069] Examples of vitamins include biotin, thiamine, etc. Furthermore, substances required for growth by the modified filamentous fungus of the present invention can be added as needed.

[0070] Cultivation is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is preferably 10°C or higher, more preferably 20°C or higher, more preferably 25°C or higher, and preferably 50°C or lower, more preferably 42°C or lower, more preferably 35°C or lower. The temperature is also 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 cultivation time is 10 hours to 10 days, preferably 2 to 7 days.

[0071] After culturing, the target protein is separated from the resulting culture by a conventional method. For example, the culture is collected, and if necessary, cells are disrupted by ultrasonication, pressure, or the like, followed by an appropriate combination of filtration, centrifugation, ultrafiltration, salting out, dialysis, chromatography, or the like, 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 a crudely separated and purified product thereof can be obtained as a composition containing the target protein.

[0072] The present invention also encompasses the following substances, manufacturing methods, uses, methods, etc. as exemplary embodiments, but the present invention is not limited to these embodiments.

[0073] [1] A modified filamentous fungus, expressing a modified ACE3, The ACE3 variant is a variant in which substantially all of the Zn(II)2Cys6-type DNA-binding domain (DBD) of ACE3 has been deleted. Modified filamentous fungi. [2] The modified filamentous fungus according to [1], wherein the ACE3 is preferably a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 4 or an amino acid sequence that is at least 90% identical thereto. [3] The modified filamentous fungus according to [2], wherein the Zn(II)2Cys6-type DNA-binding domain (DBD) is preferably a region corresponding to amino acids 120 to 160 of SEQ ID NO:1. [4] Preferably, the ACE3 variant is any one of the following (1) to (4): (1) The DBD has a deletion of part or all of C2 and a deletion of part or all of C4, wherein C2 is a region corresponding to positions 120 to 131 of SEQ ID NO: 1, and C4 is a region corresponding to positions 132 to 160 of SEQ ID NO: 1, and the part of C4 preferably refers to a region containing at least two Cys in C4, more preferably refers to a region containing at least three Cys in C4, even more preferably refers to a region containing four Cys in C4, and even more preferably refers to a region corresponding to amino acids 132 to 151 of SEQ ID NO: 1; (2) 80% or more, preferably 90% or more, more preferably 95% or more of the amino acid sequence in the region corresponding to amino acids 120 to 160 of SEQ ID NO: 1 is deleted, and six Cys residues in the region are deleted; (3) A region corresponding to amino acids 120 to 151 of SEQ ID NO: 1 is deleted; (4) The region corresponding to amino acids 1 to 151 of SEQ ID NO: 1 is deleted. or, More preferably, the ACE3 variant is any one of the following (5) to (7): (5) A region corresponding to amino acids 1 to 160 of SEQ ID NO: 1 is deleted; (6) A region corresponding to amino acids 1 to 200 of SEQ ID NO: 1 is deleted; (7) A region corresponding to amino acids 1 to 240 of SEQ ID NO: 1 is deleted. [2] The modified filamentous fungus described in [2]. [5] The ACE3 variant, Preferably, a region corresponding to at least 7 amino acids and at most 17 amino acids at the C-terminus of the amino acid sequence of SEQ ID NO: 1 is deleted. Preferably, one or more amino acids selected from the group consisting of amino acids corresponding to amino acids at positions -7 to -17 of SEQ ID NO: 1 are deleted, or More preferably, the region corresponding to the C-terminal 11 amino acids in the amino acid sequence of SEQ ID NO: 1 is deleted. The modified filamentous fungus according to any one of [2] to [4]. [6] The ACE3 variant is Preferably, it has a region corresponding to amino acids 280 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 280 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 280 to 723 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 280 to 727 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 723 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 727 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 723 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 727 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 241 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 241 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 241 to 723 of SEQ ID NO: 1, or Preferably, it has a region corresponding to amino acids 241 to 727 of SEQ ID NO: 1. The modified filamentous fungus according to any one of [2] to [5]. [7] The modified filamentous fungus according to any one of claims [1] to [6], preferably having a gene that expresses the ACE3 variant introduced therein. [8] Preferably, the gene expressing the ACE3 variant is operably linked to a regulatory region that promotes transcription of the gene; Preferably, the regulatory region is selected from the group consisting of the pdc promoter and the act1 promoter. [7] The modified filamentous fungus described in [7]. [9] The filamentous fungus is Preferably, it is of the genus Trichoderma, More preferably, it is Trichoderma reesei or a mutant strain thereof. The modified filamentous fungus according to any one of [1] to [8].

[10] The modified filamentous fungus preferably expresses XYR1 or a mutant XYR1, more preferably a mutant XYR1; Preferably, the XYR1 or mutant XYR1 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the mutant XYR1 has at least one amino acid mutation in a region corresponding to positions 810 to 833 of SEQ ID NO: 5, relative to the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto; More preferably, the mutant XYR1 has at least one amino acid substitution selected from the group consisting of amino acids at positions 817, 821, 824, 825, and 826 of SEQ ID NO: 5 relative to the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto; More preferably, the mutant XYR1 consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5 and has any one or more amino acids selected from the group consisting of: Tyr at a position corresponding to position 817 of SEQ ID NO:5; Lys, Phe, Trp, or Tyr, preferably Phe, at a position corresponding to position 821 of SEQ ID NO: 5; Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, preferably Val, at a position corresponding to position 824 of SEQ ID NO: 5; Tyr at a position corresponding to position 825 of SEQ ID NO:5; Val, Ile, Leu, Phe, Trp or Tyr at a position corresponding to position 826 of SEQ ID NO: 5; The modified filamentous fungus according to any one of [1] to [9].

[0074]

[11] A method for producing a protein, comprising culturing the modified filamentous fungus according to any one of [1] to

[10] above.

[12] The method described in

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

[13] The method according to

[11] or

[12] , wherein the culture is preferably carried out in the presence of glucose.

[14] The method described in

[13] , wherein the culture is preferably carried out in the absence of a cellulase-inducing substance.

[0075]

[15] A method for producing a modified filamentous fungus, comprising: modifying a parent filamentous fungus to express an ACE3 variant; The ACE3 variant is a variant in which substantially all of the Zn(II)2Cys6-type DNA-binding domain (DBD) of ACE3 has been deleted. method.

[16] The method described in

[15] , wherein the ACE3 is preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical thereto.

[17] The method according to

[16] , wherein the Zn(II)2Cys6-type DNA binding domain (DBD) is preferably a region corresponding to amino acids 120 to 160 of SEQ ID NO: 1.

[18] Preferably, the ACE3 variant is any one of the following (1) to (4): (1) The DBD has a deletion of part or all of C2 and a deletion of part or all of C4, wherein C2 is a region corresponding to positions 120 to 131 of SEQ ID NO: 1, and C4 is a region corresponding to positions 132 to 160 of SEQ ID NO: 1, and the part of C4 preferably refers to a region containing at least two Cys in C4, more preferably refers to a region containing at least three Cys in C4, even more preferably refers to a region containing four Cys in C4, and even more preferably refers to a region corresponding to amino acids 132 to 151 of SEQ ID NO: 1; (2) 80% or more, preferably 90% or more, more preferably 95% or more of the amino acid sequence in the region corresponding to amino acids 120 to 160 of SEQ ID NO: 1 is deleted, and six Cys residues in the region are deleted; (3) A region corresponding to amino acids 120 to 151 of SEQ ID NO: 1 is deleted; (4) The region corresponding to amino acids 1 to 151 of SEQ ID NO: 1 is deleted. or, More preferably, the ACE3 variant is any one of the following (5) to (7): (5) A region corresponding to amino acids 1 to 160 of SEQ ID NO: 1 is deleted; (6) A region corresponding to amino acids 1 to 200 of SEQ ID NO: 1 is deleted; (7) A region corresponding to amino acids 1 to 240 of SEQ ID NO: 1 is deleted.

[16] The method described in.

[19] The ACE3 variant, Preferably, a region corresponding to at least 7 amino acids and at most 17 amino acids at the C-terminus of the amino acid sequence of SEQ ID NO: 1 is deleted. Preferably, one or more amino acids selected from the group consisting of amino acids corresponding to amino acids at positions -7 to -17 of SEQ ID NO: 1 are deleted, or More preferably, the region corresponding to the C-terminal 11 amino acids in the amino acid sequence of SEQ ID NO: 1 is deleted. The method according to any one of

[16] to

[18] .

[20] The ACE3 variant, Preferably, it has a region corresponding to amino acids 280 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 280 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 280 to 723 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 280 to 727 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 723 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 260 to 727 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 723 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 250 to 727 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 241 to 701 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 241 to 717 of SEQ ID NO: 1. Preferably, it has a region corresponding to amino acids 241 to 723 of SEQ ID NO: 1, or Preferably, it has a region corresponding to amino acids 241 to 727 of SEQ ID NO: 1. The method according to any one of

[16] to

[19] .

[21] Preferably, the method described in any one of

[15] to

[20] , wherein the modification of the parent filamentous fungus comprises introducing a gene that expresses the ACE3 variant into the parent filamentous fungus.

[22] Preferably, the gene expressing the ACE3 variant is operably linked to a regulatory region that promotes transcription of the gene; Preferably, the regulatory region is selected from the group consisting of the pdc promoter and the act1 promoter.

[21] The method described in.

[23] The filamentous fungus is Preferably, it is of the genus Trichoderma, More preferably, it is Trichoderma reesei or a mutant strain thereof. The method according to any one of

[15] to

[22] . [Example]

[0076] The present invention will be explained in more detail below using examples.

[0077] Example 1 Construction of plasmid DNA for gene transfer The following DNA fragments 1 to 5 were prepared by PCR using Trichoderma reesei genomic DNA as a template: Fragment 1: approximately 1.5 kbp of the promoter region upstream of the act1 gene (TRIREDRAFT_44504); Fragment 2: a polynucleotide (SEQ ID NO:6, approximately 2.9 kbp) encoding the full-length ACE3 polypeptide (SEQ ID NO:1); Fragment 3: a polynucleotide (SEQ ID NO:7, approximately 2.0 kbp) encoding the DBD-deleted ACE3 polypeptide (SEQ ID NO:2); Fragment 4: a polynucleotide (SEQ ID NO:10, approximately 3.0 kbp) encoding the full-length XYR1 polypeptide (SEQ ID NO:5); Fragment 5: a terminator region approximately 0.6 kbp downstream of the cbh1 gene (TRIREDRAFT_44504); and Fragment 6: an approximately 2.7 kbp region of the pyr4 gene (TRIREDRAFT_74020).

[0078] Fragments 1 and 2 were ligated to construct cassette 1: Pact1-TrACE3 (1-734). Fragments 1 and 3 were ligated to construct cassette 2: Pact1-TrACE3 (1-629). Fragments 1 and 4 were ligated to construct cassette 3: Pact1-XYR1. A transformation marker fragment was prepared by placing approximately 0.5 kbp fragment 7 and approximately 1.0 kbp fragment 8 upstream and downstream of fragment 6, respectively, as homologous sequences for pop-out. Fragment 5 and the transformation marker fragment were ligated to construct cassette 4: Tcbh1-pyr4.

[0079] Cassette 5:ΔRCE1 was constructed by inserting an approximately 1.5 kbp region upstream (fragment 9) and an approximately 1.4 kbp region downstream (fragment 10) of the rce1 gene (TRIREDRAFT_72611) into the HincII restriction site of pUC118 (Takara Bio) while adding a smiI restriction site. Cassette 6:ΔACE1 was constructed by inserting an approximately 1.6 kbp region upstream (fragment 11) and an approximately 1.2 kbp region downstream (fragment 12) of the ace1 gene (TRIREDRAFT_75418) into the HincII restriction site of pUC118 (Takara Bio) while adding a smiI restriction site.

[0080] The DNA fragments were ligated according to the protocol of the In-Fusion HD Cloning Kit (Takara Bio). The constructed cassettes, the DNA fragments contained therein, and the primers used to construct the cassettes are shown in Table 1.

[0081] [Table 1]

[0082] A constitutive full-length ace3 expression plasmid, pUC-Pact1-TrACE3(1-734), was constructed by inserting a combined cassette 1 and cassette 4 between the upstream and downstream regions of the rce1 gene in cassette 5 by PCR using the primers listed in Table 2 (Plasmid 1). A constitutive full-length ace3 expression plasmid, pUC-Pact1-TrACE3(1-629), was constructed by inserting a combined cassette 2 and cassette 4 between the upstream and downstream regions of the rce1 gene in cassette 5 by PCR using the primers listed in Table 2 (Plasmid 2). Plasmid 1 expresses the full-length T. reesei ACE3 consisting of SEQ ID NO: 1, while plasmid 2 expresses a variant ACE3 lacking a portion of the DBD (C2) compared to SEQ ID NO: 1 (Figure 1).

[0083] The xyr1 gene constitutive expression plasmid pUC-Pact1-XYR1 (plasmid 3) was constructed by inserting a combination of cassettes 3 and 4 between the upstream and downstream regions of the ace1 gene in cassette 6 using PCR with the primers listed in Table 2. Plasmid 3 expresses the xylanase XYR1.

[0084] Using PCR with the primers listed in Table 3 and Plasmid 1 as a template, pUC-Pact1-TrACE3(161-734) (Plasmid 4), pUC-Pact1-TrACE3(201-734) (Plasmid 5), pUC-Pact1-TrACE3(241-734) (Plasmid 6), pUC-Pact1-TrACE3(281-734) (Plasmid 7), and pUC-Pact1-TrACE3(300-734) (Plasmid 8) were constructed. Plasmids 4 to 8 express ACE3 variants lacking a specific region containing the N-terminal DBD of SEQ ID NO: 1 (Figure 1).

[0085] A polynucleotide (SEQ ID NO: 8, approximately 2.0 kbp) encoding a variant of the ACE3 polypeptide from Trichoderma atroviride (SEQ ID NO: 3) and a polynucleotide (SEQ ID NO: 9, approximately 2.0 kbp) encoding a variant of the ACE3 polypeptide from Trichoderma harzianum (SEQ ID NO: 4) were artificially synthesized. The resulting fragments were ligated to Plasmid 1 by PCR using the primers listed in Table 4 to construct pUC-Pact1-TaACE3(1-630) (Plasmid 9) and pUC-Pact1-ThACE3(1-630) (Plasmid 10), respectively. Plasmids 9 and 10 express variants of ACE3 lacking a portion of the DBD (C2).

[0086] pUC-Pact1-TaACE3(137-630) (plasmid 11) and pUC-Pact1-ThACE3(137-630) (plasmid 12) were constructed by PCR using the primers listed in Table 4 and plasmid 9 or 10 as a template. Plasmids 11 and 12 express ACE3 variants with a complete deletion of the DBD, which further delete 136 amino acids at the N-terminus compared to the ACE3 variants of SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0087] A plasmid (plasmid 13) expressing a modified ACE3 in which a specific region on the C-terminus of T. reesei ACE3 (SEQ ID NO: 1) was deleted was constructed by PCR using the primers in Table 5 with plasmid 1 as a template. Plasmids 14 to 17 expressing modified ACE3 in which a specific region on the N-terminus and C-terminus of T. reesei ACE3 (SEQ ID NO: 1) was deleted were constructed by PCR using the primers in Table 5 with plasmid 2 or 4 as a template. Furthermore, two-step PCR using the primers in Table 5 with plasmid 1 as a template constructed plasmids (plasmids 18 to 19) expressing modified ACE3 in which a specific region on the N-terminus and C-terminus of T. reesei ACE3 (SEQ ID NO: 1) was deleted. The structures of the modified ACE3 contained in plasmids 13 to 18 are shown in Figure 2.

[0088] pUC-Pact1-XYR1(V821F) was constructed by PCR using the primers in Table 5 and Plasmid 3 as a template (Plasmid 20). This is a plasmid encoding mutant XYR1(V821F) in which the amino acid sequence of SEQ ID NO:5 has been substituted with V821F.

[0089] Tables 2 to 5 show the plasmids 1 to 20 constructed in this example, the fragments (cassettes, plasmids, etc.) contained therein, the primers used for their construction, and the ACE3 expressed by them.

[0090] [Table 2]

[0091] [Table 3]

[0092] [Table 4]

[0093] [Table 5]

[0094] The constructed plasmid was replicated. The plasmid prepared above was introduced into competent Escherichia coli DH5α Competent Cells (Takara Bio) and cultured in LB medium supplemented with ampicillin (37°C, 1 day). After the culture, the cells were purified by NucleoSpin TM Plasmids were recovered and purified using Plasmid (Machley-Nagel).

[0095] Example 2: Construction of modified filamentous fungi A pyr4 gene-deficient strain of Trichoderma reesei E1AB1 (JN13) (JN13Δpyr4 strain) was transformed with the DNA fragment derived from the plasmid constructed in Example 1. The plasmid constructed in Example 1 was linearized by cleavage at the smiI restriction enzyme site and then transformed into the parent strain by the protoplast PEG method (Biotechnol Bioeng, 2012, 109(1):92-99). Transformants were selected using the pyr4 gene as a marker on selective medium (2% glucose, 1.1 M 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; all % are w / v%). The composition of Trace element 1 is as follows: 0.5g FeSO4·7H2O, 0.2g CoCl2, 0.16g MnSO4·H2O, 0.14g ZnSO4·7H2O, and distilled water to a final volume of 100mL. PCR was used to confirm that the target gene fragment had been inserted into the rce1 or ace1 locus, and the desired transformants were obtained.

[0096] Double transformants expressing mutant XYR1(V821F) and ACE3 or its modified forms were prepared. The XYR1(V821F)-expressing strain containing plasmid 20 was cultured in PDA medium containing 0.2% 5-fluoroorotic acid (5-FOA) monohydrate, and strains that acquired 5-FOA resistance and grew were again selected. The growing strain was identified as JN13_XYR1(V821F)Δpyr4 strain. The resulting strain was then transformed again with the ACE3 expression plasmids (plasmids 1-19). These strains express mutant XYR1(V821F) and ACE3 or its modified forms.

[0097] Example 3 Cultivation of modified filamentous fungi The filamentous fungal strain obtained in Example 2 was cultured to produce a protein. In the preculture, 50 mL of medium was placed in a 500 mL flask, and 1 × 10 spores of the strain prepared in Example 2 were added. 5The cells were inoculated at a concentration of 100 cells / mL and cultured at 28°C with shaking at 220 rpm (Pris PRXYg-98R). The preculture medium had the following composition: 1% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Hypolypeptone N, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element 2, and 50 mM tartaric acid buffer (pH 4.0) (all percentages are w / v%). The composition of Trace element 2 was as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24 ·4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2, make up to 100mL with distilled water.

[0098] After two days of preculture, the main culture was carried out. 50 mL of medium was placed in a 500 mL flask and inoculated with 1% (v / v) of the preculture solution. The culture was carried out at 28°C and 220 rpm for 4 days. The medium composition for the main culture was as follows: 3% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Hypolypeptone N, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element 2, 1.28% diammonium hydrogen citrate, and 50 mM tartaric acid buffer (pH 4.0) (all percentages are w / v%).

[0099] Example 4 Effect of constitutive expression of DBD-deficient ACE3 on protein production The transformants prepared in Example 2 that constitutively express the full-length ACE3 derived from T. reesei or the DBD-deleted ACE3 variant were evaluated for protein productivity and composition of the produced proteins.

[0100] 1) Evaluation of protein productivity The protein concentration of the culture in Example 3 was measured by the Bradford assay. The Bradford assay used a Quick Start Protein Assay (BioRad) and calculated the protein amount based on a calibration curve prepared using bovine gamma globulin as a standard protein. The relative protein productivity of each strain was calculated by setting the protein productivity of JN13 in culture using glucose alone as a carbon source (glucose culture) as 1.

[0101] Figure 3(A) shows the relative protein productivity of each transformant. As shown in Figure 3(A), compared with the strain (“1-734”) expressing full-length ACE3 (TrACE3(1-734); SEQ ID NO: 1) and the strain (“1-629”) expressing a variant ACE3 lacking a portion of the DBD (C2) (TrACE3(1-629); SEQ ID NO: 2), the strains (“161-734”, “201-734”, and “241-734”) expressing ACE3 variants lacking the complete DBD (TrACE3(161-734), TrACE3(201-734), and TrACE3(241-734)), in which 160 to 240 amino acids on the N-terminus of SEQ ID NO: 1 are deleted, showed improved protein productivity. In particular, when 240 amino acids on the N-terminal side of SEQ ID NO: 1 were deleted ("241-734"), protein productivity was significantly improved, showing approximately 1.6 times the productivity compared to the DBD partial deletion strain ("1-629"). On the other hand, the effect of improving protein productivity was lost in strains ("281-734" and "300-734") expressing ACE3 variants (TrACE3(281-734) and TrACE3(300-734)) in which 280 or more amino acids on the N-terminal side of SEQ ID NO: 1 were deleted.

[0102] 2) Protein composition analysis The protein composition of the culture from 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. Appropriately diluted culture from Example 3 was mixed with buffer and treated at 99°C for 5 minutes, then applied to the gel and electrophoresed at 200 V for 35 minutes. From the resulting image file, the band intensity ratio was calculated using analysis software (Image Lab), and the composition ratio of the saccharifying enzymes produced was calculated.

[0103] The results of SDS-PAGE are shown in Figure 3(B). In the strains expressing the DBD-deficient ACE3 variants with improved protein productivity shown in Figure 3(A) ("161-734," "201-734," and "241-734" in Figure 3(B)), increases in the major cellulases CBH1, CBH2, and EG1 were confirmed. This indicates that the improvement in protein productivity due to the DBD-deficient ACE3 variants is mainly due to an increase in cellulase components.

[0104] Example 5 Effect of DBD deletion in other Trichoderma-derived ACE3 The amino acid sequence alignment of the ACE3 variants derived from T. reesei, T. atroviride, and T. harzianum that were expressed by the transformants prepared in Example 2 and that lacked the partial DBD (TrACE3(1-629): SEQ ID NO: 2, TaACE3(1-630): SEQ ID NO: 3, and ThACE3(1-630): SEQ ID NO: 4) and the ACE3 variants that completely lacked the DBD (TrACE3(241-734), TaACE3(131-630), and ThACE3(131-630)) to the full-length ACE3 of T. reesei (TrACE3(1-734): SEQ ID NO: 1) is shown in Figure 4. From the sequence alignment, it was found that the T. atroviride The sequences of the DBD-deleted ACE3 variants derived from T. atroviride and T. harzianum (TaACE3(1-630) and ThACE3(1-630)) were shown to correspond to the sequence of the DBD-deleted ACE3 variant derived from T. reesei (TrACE3(1-629)). Similarly, the sequences of the DBD-completely deleted ACE3 variants derived from T. atroviride and T. harzianum (TaACE3(131-630) and ThACE3(131-630)) were shown to correspond to the sequence of the DBD-deleted ACE3 variant derived from T. reesei (TrACE3(241-734)), which lacks the N-terminal 240 amino acids of SEQ ID NO: 1.

[0105] Transformants constitutively expressing the above-mentioned ACE3 variants lacking the partial or complete DBD were evaluated for protein productivity using the same method as in Example 4, 1). The results are shown in Figure 5(A). Similar to the T. reesei ACE3, the protein productivity of the ACE3 variants lacking the complete DBD was improved in the T. atroviride and T. harzianum ACE3 strains compared to the strains expressing the ACE3 variant lacking the partial DBD. Furthermore, SDS-PAGE was performed using the same method as in Example 4, 2). As shown in Figure 5(B), increases in the major cellulases CBH1, CBH2, and EG1 were confirmed, as in Figure 3(B). Therefore, it was demonstrated that the improved cellulase productivity of the DBD-deficient ACE3 variants occurs not only in T. reesei ACE3 but also in ACE3 from other Trichoderma species.

[0106] Example 6 Synergistic effect of DBD deletion and constitutive expression of C-terminal deleted ACE3 The effect of C-terminal deletion of ACE3 on improving cellulase productivity has been reported (Patent Document 4 and Non-Patent Document 5). In this example, transformants constitutively expressing the ACE3 variants (TrACE3(1-723)) lacking 11 C-terminal amino acids relative to SEQ ID NO: 1, the ACE3 variant with C-terminal deletion and partial DBD deletion (TrACE3(1-618)), and the ACE3 variant with C-terminal deletion and complete DBD deletion (TrACE3(161-723)) prepared in Example 2 were evaluated for protein productivity using the same method as in Example 4, 1). The results are shown in Figure 6(A). Even with equivalent C-terminal deletions, the strain expressing the ACE3 variant with complete DBD deletion significantly improved protein productivity compared to the strain expressing the ACE3 variant without N-terminal deletion or with partial DBD deletion. Furthermore, Figures 3(A) and 6(A) reveal that the combination of DBD deletion and C-terminal deletion resulted in an unexpectedly high synergistic effect on protein productivity. Furthermore, SDS-PAGE was performed using the same method as in Example 4, 2), and as a result, as shown in Figure 6(B), increases in the major cellulases CBH1, CBH2, and EG1 were confirmed, as in Figure 3(B). Therefore, it was revealed that complete deletion of the DBD in ACE3 exerts a greater effect on improving cellulase productivity than C-terminal deletion, and furthermore, that the combination of complete DBD deletion and C-terminal deletion synergistically increases the effect.

[0107] Example 7: Combination effect of DBD-deficient ACE3 and constitutive expression of mutant XYR1 The mutant XYR1 (V821F)-expressing strain prepared in Example 2 and the double transformant constitutively expressing mutant XYR1 (V821F) and ACE3 or its modified form were evaluated for protein productivity using the same methods as in Example 4, 1) and 2).

[0108] Compared to strain JN13, mutant XYR1 (V821F) expression improved protein productivity. Regarding double transformants, co-expression of full-length ACE3 (TrACE3(1-734)) showed only a slight increase in protein productivity compared to mutant XYR1 (V821F) alone, whereas co-expression of a DBD-deleted ACE3 variant (TrACE3(1-629)) showed a 1.5-fold increase in protein productivity. Co-expression of V821F with DBD-deleted ACE3 variants (TrACE3(161-734), TrACE3(201-734), or TrACE3(241-734)) showed protein productivity comparable to that of V821F with DBD-deleted ACE3 variants (TrACE3(1-629)), and no further increase in production was observed.

[0109] The results of SDS-PAGE are shown in Figure 7(A). The composition ratios of the saccharifying enzymes produced are shown in Figure 7(B). In strains constitutively expressing mutant XYR1 and a DBD-completely deleted ACE3 variant, not only was protein productivity improved, but the content ratios of the major cellulases CBH1, CBH2, and EG1 were also increased. On the other hand, similar to the results of Example 4 (Figure 3), the effects of improving protein productivity and cellulase content ratio were lost in strains expressing ACE3 variants lacking 280 or more amino acids at the N-terminus. Therefore, coexpression of mutant XYR1 with a specific DBD-completely deleted ACE3 variant can further improve protein productivity and the cellulase content in the produced protein compared to coexpression of existing mutant XYR1 with ACE3 or its variant, demonstrating the possibility of more efficient cellulase production.

[0110] Example 8: Combination effect of DBD-deleted and C-terminal-deleted ACE3 with constitutive expression of mutant XYR1 Using the same procedures as in Example 7, the protein productivity of the double transformants constitutively expressing the mutant XYR1 (V821F) and the DBD- and C-terminal-deleted ACE3 variants prepared in Example 2 was evaluated using the same methods as in Examples 4, 1) and 2).

[0111] The results of SDS-PAGE are shown in Figure 8(A). The composition ratios of the saccharifying enzymes produced are shown in Figure 8(B). The strain constitutively expressing the mutant XYR1 and the ACE3 variant with a complete DBD deletion and a C-terminal deletion (TrACE3(161-723)) exhibited high protein productivity and the highest cellulase content ratio. The combination of the mutant XYR1 and the C-terminal deletion also demonstrated that the complete deletion of the ACE3 DBD was effective in improving the cellulase content ratio. On the other hand, the other C-terminally deleted ACE3 variants (TrACE3(1-723), TrACE3(1-618), TrACE3(1-595), TrACE3(1-455), TrACE3(300-522), or TrACE3(300-560)) did not result in increased protein production compared to the ACE3 variant with a partial DBD deletion (TrACE3(1-629)) without a C-terminal deletion.

[0112] As described above, constitutive expression of a mutant ACE3 lacking the Zn(II)2Cys6 DNA-binding domain (DBD) significantly improved microbial protein productivity in the absence of inducers. This mutant ACE3 was effective not only in T. reesei but also in other Trichoderma species. It was also effective in combination with known effective mutations, such as C-terminal deletions and the XYR1 mutation (e.g., V821F). Therefore, constitutive expression of the DBD-deficient ACE3 mutant demonstrated high activation of the cellulase promoter in microorganisms, enabling highly efficient protein production when cultured on a non-cellulase-inducing carbon source, such as glucose.

Claims

1. 1. A modified filamentous fungus comprising: expressing an ACE3 variant, the ACE3 variant lacks a region corresponding to amino acids 1 to 151 of SEQ ID NO: 1 and has a region corresponding to amino acids 241 to 723 of SEQ ID NO: 1; The ACE3 is a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 4 or an amino acid sequence having at least 90% sequence identity thereto. Modified filamentous fungi.

2. The modified filamentous fungus according to claim 1, wherein the ACE3 mutant is deleted from the region corresponding to amino acids 1 to 160 of SEQ ID NO:

1.

3. The modified filamentous fungus according to claim 1 or 2, wherein the ACE3 variant lacks a region corresponding to the C-terminal 11 amino acids in the amino acid sequence of SEQ ID NO:

1.

4. The modified filamentous fungus according to any one of claims 1 to 3, wherein a gene expressing the modified ACE3 has been introduced.

5. The modified filamentous fungus of claim 4, wherein the gene expressing the ACE3 variant is operably linked to a regulatory region that promotes transcription of the gene.

6. The modified filamentous fungus of any one of claims 1 to 5, wherein the filamentous fungus is of the genus Trichoderma.

7. A method for producing a protein, comprising culturing the modified filamentous fungus according to any one of claims 1 to 6.

8. The method of claim 7, wherein the protein is a cellulase and / or a hemicellulase.

9. The method according to claim 7 or 8, wherein the culturing is carried out in the presence of glucose.

10. 1. A method for producing a modified filamentous fungus, comprising: modifying a parent filamentous fungus to express an ACE3 variant; the ACE3 variant lacks a region corresponding to amino acids 1 to 151 of SEQ ID NO: 1 and has a region corresponding to amino acids 241 to 723 of SEQ ID NO: 1; The ACE3 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto. method.

11. The method according to claim 10, wherein the ACE3 variant lacks a region corresponding to amino acids 1 to 160 of SEQ ID NO:

1.

12. The method according to claim 10 or 11, wherein the ACE3 variant lacks a region corresponding to the C-terminal 11 amino acids in the amino acid sequence of SEQ ID NO:

1.

13. The method according to any one of claims 10 to 12, wherein the modification of the parent filamentous fungus comprises introducing a gene that expresses the ACE3 variant into the parent filamentous fungus.

14. The method of claim 13, wherein the gene expressing the ACE3 variant is operably linked to a regulatory region that promotes transcription of the gene.

15. The method according to any one of claims 10 to 14, wherein the filamentous fungus is of the genus Trichoderma.

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