Modified filamentous fungi and methods for producing proteins using them

Abstract

Modified filamentous fungi with improved protein productivity, and proteins using the modified filamentous fungi. Providing a manufacturing method. [Solution] An ACE3 variant is expressed, and the ACE3 variant is Zn(II)2C of ACE3. A modified filamentous fungus in which substantially all of the ys6 type DNA binding domain is missing. A method for producing proteins, including culturing filamentous fungi.

Description

Technical Field

[0001] The present invention relates to a modified filamentous fungus and a method for producing a protein using the same.

Background Art

[0002] Filamentous fungi are decomposers of plant polysaccharides that produce various cellulases and hemicellulases. Among them, Trichoderma can produce cellulase and hemicellulase simultaneously and in large quantities, and thus has attracted attention as a microorganism for producing cellulase-based biomass degrading enzymes.

[0003] For industrial microbial culture, it is desirable that the carbon source is inexpensive and soluble. Conventionally, glucose has been widely used as a carbon source in microbial culture. On the other hand, an inducing substance may be required for the production of proteins such as enzymes by microorganisms. For example, an inducing substance is generally essential for the production of cellulase by microorganisms. In Trichoderma, the expression of major cellulase genes such as cbh1, cbh2, egl1, and egl2 is induced by inducing substances such as cellulose [[ID=2,7]] and cellobiose (Non-Patent Document 1). When no inducing substance is used, for example, when only glucose is used as a single carbon source, generally Trichoderma hardly produces glucoamylase. As a method for producing a microbiological protein using an inducing substance, a cellulase production method using avicel, which is microcrystalline cellulose, is known. Also, a cellulase production method using soluble lactose without using cellulose (Patent Document 1), a cellulase derived from Trichoderma

[0004] (Including β-glucosidase, endoglucanase, and cellobiohydrolase) and glucose By reacting glucose at high temperatures, it is possible to induce the formation of sophorose, gentiobiose, and other substances from glucose. A method for synthesizing a sugar and inducing cellulase production (Patent Document 2) has been disclosed. However, cellulose substrates are expensive and mostly insoluble, making them impractical for industrial processes. Because it involves lifting loads, its use in industrial applications is difficult in terms of cost and equipment requirements. Cellulase production using other inducible sugars still has disadvantages in terms of cost and process load. To carry.

[0005] Therefore, by modifying transcription factors, cellulase and xylamine can be produced without using inducible substances. To create microorganisms capable of expressing cellulase, research is underway to analyze the cellulase expression mechanism. XYR1 and ACE2 are positive transcription factors involved in cellulase-induced expression of Trichoderma. ACE3, HAP2 / 3 / 5, etc. have been reported (Non-Patent Document 2). ACE3 is the main ACE3 is a transcription factor that regulates promoters such as cbh1, which is a cellulase that requires cellulase. This is the Zn(II)2Cys6 type DNA binding domain located at amino acids 120-160 on the N-terminal side. It binds to the promoter via (DBD) (Non-Patent Document 3). Also, Non-Patent Document 3 states This is due to the interaction between ACE3 and XYR1 in relation to the cellulase gene expression of Trichoderma leese. It has been suggested that this should be regulated.

[0006] Patent Document 3 and Non-Patent Document 4 describe tre7751 in Trichoderma risei. By increasing and decreasing the expression of the ACE3 gene, the productivity of its cellulase and other enzymes can be controlled. Methods for increasing and decreasing are disclosed. Patent Document 4 and Non-Patent Document 5 describe the Z on the N side. n(II)2Cys6 type DNA-binding domain retains all six cysteine ​​molecules and has 7 at the C-terminus A filamentous fungus that enhances the expression of a modified ACE3 with a deficiency of 17 amino acids is an inducible substance. It has been reported that cellulase expression was improved even in the absence of the substance. Non-patent document 5 also states that Furthermore, the C-terminally deficient ACE3 is expressed to enhance its expression, and the wild-type or A824V mutant of XYR1 is also expressed. Co-expressing filamentous fungi are described. However, the co-expression of XYR1 in this filamentous fungus is... The effect on cellulase expression was observed only slightly in the presence of the inducible substance, It has not been observed in the absence of conductive materials.

[0007] Non-patent document 6 describes a Trichoderma with the XYR1 A824V mutation and xylanase It has been reported that deregulation occurs and cellulase production increases. Non-patent document 7 states: Combining the V821F mutation in XYR1 with enhanced ACE2 expression in Trichoderma strains This resulted in improved protein productivity in culture media using glucose and sucrose as carbon sources. This has been reported. [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. 9512415 [Patent Document 4] International Public Gazette No. 2018 / 067599 [Non-patent literature]

[0009] [Non-Patent Document 1] Curr Genomics, 2013, 14:230-249

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

【Non-Patent Document​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​It is a modified form with all parts missing. Provides modified filamentous fungi. Furthermore, the present invention provides a method for producing protein, which includes culturing the modified filamentous fungus. ru. Furthermore, the present invention is a method for producing modified filamentous fungi, This includes modifying the parent filamentous fungus to express an ACE3 variant, The ACE3 variant substantially modifies the Zn(II)2Cys6 type DNA binding domain of ACE3. It is a modified form with all parts missing. Provide a method. [Effects of the Invention]

[0013] The modified filamentous fungi provided by the present invention primarily utilize cellulase-non-inducible carbon sources such as glucose. It can efficiently produce proteins even in environments where carbon sources are essential. The bacteria produce cellulase, hemicellulase, etc., without the need for expensive cellulase-inducing substances. Proteins can be produced efficiently. According to the present invention, proteins using filamentous fungi This allows for increased efficiency and cost reduction in quality production. [Brief explanation of the drawing]

[0014] [Figure 1] Structure of the ACE3 variant expressed by the modified filamentous fungus. [Figure 2] Structure of the ACE3 variant expressed by the modified filamentous fungus. [Figure 3] Effect of ACE3 DBD deficiency on protein productivity: (A) The values ​​on the vertical axis represent the relative protein productivity of modified filamentous fungi expressing the ACE3 variant, and the horizontal axis (1, 2, 3) represents three modified filamentous fungal strains expressing the same ACE3 variant. (B) Gel electrophoresis image of cultures of modified filamentous fungi. [Figure 4] Amino acid sequence alignment of ACE3 variants derived from various Trichoderma species. [Figure 5]Protein production in modified filamentous fungi expressing ACE3 variants derived from various Trichoderma species: (A) Relative protein productivity, (B) Gel electrophoresis image of cultures of modified filamentous fungi. [Figure 6] Effect of ACE3 C-terminal deficiency on protein productivity: (A) Relative protein productivity of modified filamentous fungi expressing ACE3 variants, (B) Gel electrophoresis image of cultures of modified filamentous fungi. [Figure 7] Effect of co-expression of mutant XYR1 and ACE3 variant on protein productivity: (A) Gel electrophoresis image of modified filamentous fungal culture, (B) Protein composition ratio of modified filamentous fungal culture. [Figure 8] Effects of combined expression of mutant XYR1 on protein productivity and ACE3 DBD deficiency and C-terminal deficiency: (A) Gel electrophoresis image of modified filamentous fungal cultures, (B) Protein composition ratio of modified filamentous fungal cultures. [Modes for carrying out the invention]

[0015] All patent, non-patent, and other publications cited herein are entirely subject to the terms of their respective owners. The Body is incorporated herein by reference.

[0016] In this specification, the identity of amino acid sequences and nucleotide sequences is defined as Lipman-Pe Calculated using Arson's method (Science, 1985, 227:1435-1441) Specifically, the genetic information processing software Genetyx-Win (Ver. 5. 1.1; Homology analysis (search homology) in software development Using grams, we solve by setting Unit size to compare (ktup) to 2. It is calculated by performing an analysis.

[0017] In this specification, "at least 90% of the amino acid sequence and nucleotide sequence" "Identity" means 90% or more, preferably 92% or more, more preferably 94% or more, and further Preferably 95% or more, more preferably 96% or more, even more preferably 98% or more. Preferably, this refers to a level of identity of 99% or higher.

[0018] In this specification, unless otherwise defined, amino acid sequences and nucleotide sequences The term "1 or number" is used in relation to the deletion, substitution, addition, or insertion of amino acids and nucleotides. "pieces" means, for example, 1 to 20 pieces, preferably 1 to 16 pieces, more preferably 1 to 12 pieces, and further This may preferably mean 1 to 8, more preferably 1 to 4. In this specification, A The "addition" of a nucleotide or amino acid involves adding one or more amino acids to one end and both ends of the sequence. This includes the addition of an amino acid or nucleotide. Also, in this specification, an amino acid or nucleo "Insertion" of a nucleotide involves inserting an amino acid or nucleotide into the 5' or 3' position of a given location. It includes "entry".

[0019] In this specification, "corresponding position" or "phase" on an amino acid sequence or nucleotide sequence. The "corresponding region" is the maximum region between the target sequence and the reference sequence (for example, the amino acid sequence of sequence number 1). This can be determined by aligning them in a way that gives them homology. The alignment of amino acid sequences or nucleotide sequences is performed using a known algorithm. It can be performed by C Lustal W Multiple Alignment Program (Thompson, JDe t al,1994,Nucleic Acids Res.22:4673-4680 This can be done by using the default settings. Clustal W is an example. For example, the European Bioinformatics Institute tics Institute:EBI[www.ebi.ac.uk / index.h [tml]) and the DNA Data Bank of Japan (DDBJ) operated by the National Institute of Genetics [www [ddbj.nig.ac.jp / searches-j.html]) website It can be used above. Alignment as described above to any position in the reference array The position of the target array is considered to be the "corresponding position" to the arbitrary position. The area enclosed by the corresponding position, or the area consisting of the corresponding motif, is the corresponding area It is considered to be.

[0020] Those skilled in the art will optimize the alignment of the amino acid sequence obtained above. Further fine-tuning is possible. Such optimal alignment is based on the similarity of amino acid sequences. It is preferable to determine this by considering factors such as sex and the frequency of inserted gaps. Here is the amino acid sequence Similarity means that when two amino acid sequences are aligned, both sequences are identical or This refers to the ratio (%) of the number of positions where similar amino acids exist relative to the total number of amino acids. The amino acids in question are among the 20 amino acids that make up proteins, and they differ in terms of polarity and charge from one another. This refers to amino acids that have similar properties and undergo so-called conservative substitutions. Such groups of similar amino acids are well known to those skilled in the art, for example, A Ruginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and Asparagine; leucine and isoleucine are examples, but the list is not limited to these. stomach.

[0021] In this specification, "amino acids" refers to the 20 amino acids that make up proteins, and ara Nin (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 (Hi) (s or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Ly (s or K), methionine (Met or M), phenylalanine (Phe or F), proli n (Pro or P), serine (Ser or S), threonine (Thr or T), trypt This refers to phan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). do.

[0022] In this specification, "operable linkage" between a regulatory region such as a promoter and a gene means The gene and the regulatory region are linked such that the gene can be expressed under the control of the regulatory region. This refers to the process of "operable linking" between a gene and a regulatory region. The procedure for this is well known to those skilled in the art.

[0023] In this specification, "upstream" and "downstream" with respect to a gene refer to the transcription direction of the gene. This refers to upstream and downstream. For example, "genes located downstream of a promoter" refers to DNA. This means that the gene is located on the 3' side of the promoter in the sense strand, and above the gene "Flow" refers to the 5' region of the gene in question within the DNA sense strand.

[0024] In this specification, the terms "inherent" and "nay" are used in reference to the function, properties, and characteristics of cells. "Native" is used to indicate that the function, properties, or traits in question are inherently present in the cell. It is used. In contrast, the term "external" refers to something that is not originally present in the cell in question, but is external. It is used to describe the function, characteristics, or traits introduced from another source. For example, "foreign" genes or A polynucleotide is a gene or polynucleotide introduced into a cell from an external source. Even if a foreign gene or polynucleotide originates from the same species of organism as the cell into which it is introduced, They may be derived from different organisms (i.e., different genes or polynucleotides).

[0025] This invention relates to improving the protein productivity of filamentous fungi. Conventionally, filamentous fungi are grown using a glycerinary stimuli. When cultured in the presence of coke, protein production is suppressed by catabolite inhibition. Yes, in particular, cellulase-based biomass such as cellulase and hemicellulase in filamentous fungi. The expression of digestive enzymes is typically associated with cellulose, sophorose, and cellooligosaccharides (cellobiose, cellooligosaccharides). Cellular ions such as triose, serotetraose, seropentaose, serohexaose, etc. It needs to be induced by an enzyme-inducing substance, while its expression induction is suppressed in the presence of glucose. They are controlled.

[0026] Inducible expression of cellulase-based biomass-degrading enzymes by the inducible substance in filamentous fungi is X Many transcriptions such as YR1, Cre1, ACE1, ACE2, ACE3, HAP2 / 3 / 5, etc. It is controlled by factors. ACE3 controls cellulase and hemicellulase in filamentous fungi. It is a transcription activator of the cellulase gene and part of the lactose-induced cellulase gene. ACE3 is a transcription activator of the cellulase gene and part of the cellulase gene. It is essential for the transcription of the xylanase gene. Furthermore, ACE3 regulates the transcription of the xyr1 gene. It is also partially involved in this. Trichoderma lisey's ACE3 is in the ncbi database ( [www.ncbi.nlm.nih.gov / ]) NCBI Referen It is registered as ce Sequence:QEM24913.1, and here ACE3 It consists of the amino acid sequence of SEQ ID NO: 1 and is encoded by the nucleotide sequence of SEQ ID NO: 6. It is defined as a polypeptide. Also, NCBI Reference Seque nce: XP_006966092.1 [www.ncbi.nlm.nih.go According to [v / protein / XP_006966092.1], the amino acids in sequence number 1 Of the sequence, positions 523-734 are the region that is presumed to interact with XYR1. The region from 391 to 522 is presumed to be a domain of filamentous fungi-specific transcription factors, and 120 The region up to position 160 is presumed to be the Zn(II)2Cys6 type DNA-binding domain (DBD). The gene encoding DBD is divided into two regions by an intron. On the other hand (Exon2) codes the region (C2) containing the two Cys on the N side of the DBD. And the other one (Exon3) codes the region (C4) that contains the remaining four Cys. Trichoderma species also possess ACE3 with a similar structure.

[0027] Deficiency of the C-terminal amino acid of ACE3 leads to the absence of inducible substances in filamentous fungi. It has been reported that production is possible (Non-Patent Document 5). However, Non-Patent Document 5 states that It has been disclosed that the effect of C-terminal deletion in DBD was abolished by C2 deletion in DBD. .

[0028] In response to this, the inventors have found that an ACE3 variant in which not only C2 but also C4 of DBD is missing, To give filamentous fungi the ability to express cellulase and hemicellulase without inducible substances, The filamentous fungus produces cellulase and hemicells in the presence of a non-cellulase-inducing carbon source such as glucose. We discovered that this AC makes it possible to highly express proteins such as lases. Furthermore, this AC The deletion of C2 and C4 in E3, when combined with C-terminal deletion, affects protein productivity. To improve synergistically.

[0029] Therefore, in one aspect, the present invention is an ACE3 modification that substantially lacks all of DBD. The present invention provides a modified filamentous fungus that expresses a body, and a method for producing the modified filamentous fungus. This involves causing the parent filamentous fungus to express an ACE3 variant that substantially lacks the DBD. It can be manufactured by modification. The modified filamentous fungus of the present invention manufactured is in the DB It manifests an ACE3 variant that is essentially devoid of all of D.

[0030] The DBD of ACE3 is the N-terminal side of the amino acid sequence of ACE3, specifically the first amino acid of sequence number 1. It is located in the region corresponding to amino acids 20 to 160, of which positions 120 to 131 are C2. And positions 132-160 are C4. In this specification, "substantially all missing data in DBD" "This means that the DBD has partial or complete absence of C2 and partial or complete absence of C4. This refers to a situation where DBD is missing all of C2 and part or all of C4. Here, “part of C4” preferably includes at least two Cys in C4. This refers to a region, more preferably a region containing at least three Cys in C4, and further Preferably, a region containing four Cys in C4, for example, positions 132-151 of SEQ ID NO: 1. This refers to the region corresponding to an amino acid. An example of "substantially complete deletion of DBD" in this specification. For example, 80% or more of the amino acid sequence of DBD, preferably 90% or more, more preferably One example is a state where over 95% is missing, and six Cys are missing during DBD. ru.

[0031] The parent filamentous fungus of the modified filamentous fungus of the present invention is ACE3 modified, which substantially lacks all of the above DBD. This filamentous fungus is prepared as the modified filamentous fungus of the present invention by undergoing modification to express a variant. It is preferable that the filamentous fungus inherently expresses ACE3. For example, the parent filamentous fungus Preferably, it expresses a native ACE3 or a variant thereof that has all of the DBD. More preferably, the parent filamentous fungus has all of DBD, native ACE3 or This is a bacterium that expresses the modified strain and also possesses cellulase activity.

[0032] Examples of filamentous fungi used in this invention include, but are not limited to, fungi (Eumycot). a) Examples include filamentous fungi belonging to the phylum Oomycota. More detailed examples include This includes the genus Trichoderma, and Aspergillus. *Penicillium* genus, *Neurospora* genus pora genus, Fusarium genus, Chrysosporium genus porium), Humicola genus, Emericel genus *L) * Hypocrea * Acremonium Myc) genus, Chrysosporium genus, Myc eliophthora, Piromyces, Talaromyces (Talaromyces), Thermoascus (Thermoascus), Thiera Examples include filamentous fungi of the genus Thielavia. Among these, the filaments of the genus Trichoderma... A fungus is preferred.

[0033] Examples of filamentous fungi of the genus Trichoderma (hereinafter also referred to as Trichoderma fungi) include Trichoderma Trichoderma reesei, Trichoderma longive Rachiatum (Trichoderma longibrachiatum), Trichoderma Trichoderma harzianum Ningii (Trichoderma koningii), Trichoderma viride (Tr Trichoderma viride, Trichoderma atroviride Examples include Trichoderma atroviride, and preferably Trichoderma risseyi. And its mutant strains are examples. For example, Trichoderma leesey QM9414 strain and its A mutant strain, preferably Trichoderma risey PC-3-7 strain (ATCC66589), Trichoderma lisey PCD-10 strain (FERM P-8172), Trichoderma li - Sei E1AB1 strain (hereinafter sometimes referred to as JN13 strain) or its variants, It can be preferably used as a parent filamentous fungus. Strain E1AB1 is Trichoderma risei For the PC-3-7 strain, the egl1 promoter was used to test Aspergillus acuritus (As This strain expresses β-glucosidase (BGL) derived from *Pergillus aculeatus* (Enz). See yme and Microbial Technology (2016) 82:89-95 and WO2013 / 115305. (See Examples 1-3).

[0034] The ACE3 variant expressed by the modified filamentous fungus of the present invention substantially eliminates the parent ACE3 of DBD. This can be obtained by modifying it so that a part is missing. The parent ACE3 is part of the DBD or This may be a native ACE3 or a variant thereof that has all of the above. Preferably, Parent ACE3 is a native ACE3 or a variant thereof that has at least DBD C4 These may include, for example. Preferably, the parent ACE3 is ACE3 derived from Trichoderma or the like. It is a modified form of the above. Examples of Trichoderma species include those mentioned above. However, preferably Trichoderma risei, Trichoderma halizium, and Trichoderma Luma Atroviride is one example.

[0035] A preferred example of the parent ACE3 is the ACE3 consisting of the amino acid sequences of SEQ ID NOs: 1 to 4. or its variants are examples. Sequence ID 1 is the full-length ACE3 of Trichoderma lissey. This represents the amino acid sequence. Sequence ID 2 is a deletion of the DBD portion (C2) of Trichoderma risei. This represents the amino acid sequence of the ACE3 variant. Sequence ID 3 is from Trichoderma atroviride. This represents the amino acid sequence of a DBD (C2) deficient ACE3 variant. Sequence ID 4 is a tricode. This represents the amino acid sequence of the DBD (C2) deficient ACE3 variant of Rumah halidium.

[0036] Another preferred example of the parent ACE3 is the amino acid sequence of any of SEQ ID NOs: 1-4 Examples include polypeptides consisting of amino acid sequences that are at least 90% identical in sequence. Another preferred example of E3 is one amino acid sequence for any of the sequence numbers 1-4. A polypeptide is a polypeptide consisting of an amino acid sequence in which several amino acids are deleted, substituted, added, or inserted. These include the polypeptides of the parent ACE3, which are native ACE3s (e.g., sequences). It has a sequence that corresponds to part or all of DBD of number 1), preferably native A Like CE3, it can function as a transcriptional activator of cellulase and hemicellulase.

[0037] The ACE3 variant expressed by the modified filamentous fungus of the present invention is substantially deficient in DBD. Preferably, the ACE3 variant contains at least the amino acids at positions 120-160 of SEQ ID NO: 1. The region corresponding to acid is missing. Furthermore, the ACE3 variant is N-terminal compared to DBD. The region or the region closer to the C-terminus than the DBD may be missing. In one embodiment, The ACE3 variant lacks the region corresponding to amino acids 120-151 of SEQ ID NO: 1. In one embodiment, the ACE3 variant has amino acids at positions 1 to 151 of SEQ ID NO: 1. The corresponding region is missing. In one embodiment, the ACE3 variant is 1 of Sequence ID No. 1 The region corresponding to the 160th amino acid is missing. In one embodiment, the ACE3 modified The variant lacks the region corresponding to amino acids 1-200 of SEQ ID NO: 1. One embodiment In this case, the ACE3 variant lacks the region corresponding to amino acids 1-240 of SEQ ID NO: 1. I'm losing money.

[0038] In addition to the DBD defect described above, the ACE3 variant may also have a C-terminal region defect. An example of a C-terminal region deletion is the one disclosed in Non-Patent Document 5, which results in improved protein productivity. One example of a deficiency is the deletion of 7 to 17 amino acids at the C-terminus. Furthermore, the ACE3 variant has at least 7 amino acids at the C-terminus in the amino acid sequence of SEQ ID NO: 1. The region corresponding to amino acids (position 728 to C-terminus) up to 17 amino acids (position 718 to C-terminus) It may be missing. In one embodiment, the ACE3 variant is -7~- of Sequence ID No. 1. One of the amino acids selected from the group corresponding to the 17th ranked (718th-728th) amino acid. The above amino acids may be missing. In one embodiment, the ACE3 variant is sequence The region corresponding to the 11 amino acids at the C-terminus (positions 724 to C-terminus) in amino acid sequence number 1. The region may be missing. In one embodiment, the ACE3 variant is compared to the parent ACE3. It does not have a C-terminal deletion.

[0039] Preferably, the ACE3 variant includes the region containing the DBD described above, and the C-terminal region. Other amino acids are maintained. In a preferred example, the ACE3 variant is at least at position 280-701 of Sequence ID No. 1. It has a region corresponding to an amino acid. More preferably, the ACE3 variant is the same as SEQ ID NO: 1 It has a region corresponding to amino acids 280 to 717. More preferably, the ACE3 variant This has a region corresponding to amino acids 280-723 of SEQ ID NO: 1. It may have a region corresponding to amino acids 280-727 of SEQ ID NO: 1. In a more preferred example, the ACE3 variant is the amino acid at positions 260-701 of SEQ ID NO: 1. It has a region corresponding to acid. More preferably, the ACE3 variant is 26 of SEQ ID NO: 1. It has a region corresponding to amino acids 0 to 717. More preferably, the ACE3 variant is The ACE3 variant has a region corresponding to amino acids 260-723 of SEQ ID NO: 1. It may also have a region corresponding to amino acids 260-727 of SEQ ID NO: 1. In a more preferred example, the ACE3 variant is an amine at positions 250-701 of SEQ ID NO: 1. It has a region corresponding to an acid. More preferably, the ACE3 variant is 2 of SEQ ID NO. 1. It has a region corresponding to amino acids 50 to 717. More preferably, the ACE3 variant This has a region corresponding to amino acids 250-723 of SEQ ID NO: 1. It may have a region corresponding to amino acids 250-727 of SEQ ID NO: 1. In a more preferred example, the ACE3 variant is the A241-701 position of Sequence ID No. 1. It has a region corresponding to a amino acid. More preferably, the ACE3 variant is sequence number It has a region corresponding to amino acids 241 to 717 of ACE3. Preferably, the ACE3 modified The variant has a region corresponding to amino acids 241-723 of SEQ ID NO: 1. The variant may have a region corresponding to amino acids 241-727 of SEQ ID NO: 1. Preferably, the regions corresponding to each amino acid region of Sequence ID No. 1 listed above are, Each amino acid region of SEQ ID NO: 1 is sequence-identical by at least 90% or 100%. The % sequences are identical.

[0040] Alternatively, if there is no C-terminal region defect, the ACE3 variant is preferably sequence number 1. It has a region corresponding to amino acids 280-734 of SEQ ID NO: 1, and more preferably 260 It has a region corresponding to amino acid position ~734, and more preferably 250~73 of SEQ ID NO: 1 It has a region corresponding to the 4th amino acid, and more preferably the region from 241 to 734 of SEQ ID NO: 1. It has a region corresponding to an amino acid. Preferably, each amino acid region of SEQ ID NO: 1 listed above. Each of the regions corresponding to SEQ ID NO: 1 has at least 90 amino acid regions. The sequences are either % identical or 100% identical.

[0041] The aforementioned DBD is essentially the entirety of ACE3 modified, with the C-terminal region omitted where necessary. A method of modifying the parent filamentous fungus to express a variant (hereinafter also referred to as the target ACE3 variant). For example, introducing an exotic gene encoding the target ACE3 variant into the parent filamentous fungus. A method of introducing and expressing the gene that the parent filamentous fungus naturally possesses, which encodes ACE3, is used for the target A One method involves mutating the gene to encode the CE3 variant.

[0042] The gene encoding the target ACE3 variant (target gene) is genetically engineered or chemically modified. It can be synthesized scientifically. For example, the genomic DNA of filamentous fungi such as Trichoderma. Then, the DNA of the gene encoding parental ACE3 (parental gene) was isolated, and then the DNA encoding DBD was extracted. Delete part or all of the region, and if necessary, the region encoding the C-terminal region. This allows for the preparation of the target gene. An example of a parent gene is sequence numbers 6-9. Polynucleotides consisting of a nucleotide sequence or a sequence that is at least 90% identical thereto. This could be the case. Alternatively, based on known sequence information, a portion of the region that encodes the DBD or This involves chemically synthesizing a target gene that is completely or even more devoid of the region encoding the C-terminal region. This is possible. ACE3 sequence information is available in the ncbi database (www.ncbi.nlm. It is available from sources such as nih.gov / . If necessary, the target gene can guide it. The codon may be optimized to suit the host organism (parent filamentous fungus) it is introduced into. The information is from the Codon Usage Database ([www.kazusa.o It is available from [r.jp / codon / ]).

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

[0044] One method for mutating the gene encoding ACE3 in parent filamentous fungi is, for example, recombination. The methods used can be listed. For example, parental genes in the genome of parent filamentous fungi are homologous. By substitution or non-homologous recombination, the gene encoding the target ACE3 variant (target gene) is modified. ) can be replaced with. Alternatively, the DBD coding region of the parent gene in the genome can be homologous. By replacing the DBD deletion fragment with a modified or non-homologous recombination, the parental gene can be modified to target the desired gene. It may be mutated into a genetic variant.

[0045] An example of a specific recombination method is to first obtain the target gene or DBD deletion fragment, and, if necessary, Then, a recombinant DNA construct containing a drug resistance gene or a nutrient requirement gene is constructed. This is introduced into the parent filamentous fungus using a conventional method. Then, drug resistance or nutritional requirements are used as indicators. Then, select a transformed strain in which the recombinant construct has been incorporated into the genome. Furthermore, genome analysis and enzyme activity analysis confirm that the resulting transformed strain possesses the desired mutation. You may want to check this.

[0046] The introduction of DNA constructs into parent filamentous fungi is commonly performed using plasmids and other transformation methods. The vectors used can be used. DNA constructs and vectors can be introduced into cells. For example, methods such as protoplast method, protoplast PEG method, and competent cell method are available. Conventional methods can be used. As a vector for introducing the DNA construct, Any substance that can be stably maintained and proliferated within the principal cell is not particularly limited; for example, plus Commonly used vectors include mids, cosmids, phages, viruses, YACs, and BACs. These include plasmid vectors. Of these, plasmid vectors are preferred. Examples of introduction vectors include: Examples include pUC118.

[0047] In a preferred embodiment, the modified filamentous fungus of the present invention highly expresses the target ACE3 variant. Further modifications are made to the parent filamentous fungus to improve the expression of the target ACE3 variant. This allows us to obtain a modified filamentous fungus that highly expresses the target ACE3 variant. 3. As a means of improving the expression of the modified gene, the gene encoding the ACE3 modified gene (objective) Methods for increasing the transcription level of a target gene are mentioned. For example, this involves intensifying the transcription of a target gene in the regulatory region of the parent filamentous fungus genome. A regulatory region that promotes the action (strong regulatory region) is replaced or inserted, and the strong regulatory region is used in conjunction with the target gene. One example is to connect them in a movable manner. Alternatively, a control area (preferably strong) may be used as needed. A target gene fragment, operably linked to a regulatory region, is attached to the genome of the parent filamentous fungus or plus By introducing it into the mido solution and increasing the number of target genes that can be expressed in cells, the target gene This can improve the amount of transcription.

[0048] Examples of control regions that can be used to improve the amount of transcription include high glucose conditions. For genes whose transcription levels do not decrease even under these conditions, such as Trichoderma species, glyc eraldehyde-3-phosphate dehydrogenase(gpd) , pyruvate decarboxylase(PDC), enolase(eno), a lcohol dehydrogenase(adh), triose phosphat e isomerase(tpi), aldolase(fba), pyruvate kin ase(pyk), citrate synthase(cit), α-ketoglutar ate dehydrogenase(kdh), aldehyde dehydrogenase nase I(ald1), aldehyde dehydrogenase II(ald2) , pyruvate dehydrogenase (pda), glucokinase (g lk), actin(act1), translation elongation fact Examples include regulatory regions of genes such as or 1α(tef1). Of these, preferred strong regulation An example of such a region is the promoter of the pdc gene (TRIREDRAFT_121534). Examples include the promoter of the act1 gene (TRIREDRAFT_44504).

[0049] Preferably, the expression level of the target ACE3 variant in the modified filamentous fungus of the present invention is equal to that of the parent filamentous fungus. The expression level of ACE3 is improved compared to the expression level in the target ACE3 variant. Publicly available quantitative PCR, microarrays, Western blotting, ELISA, HPLC, etc. By means of knowledge, the amount of protein or the transcription amount of the gene encoding it can be quantified. It is possible.

[0050] Preferably, the modified filamentous fungus of the present invention further contains XYR1(Xylanase regul It expresses ator 1). More preferably, the modified filamentous fungus of the present invention highly expresses XYR1. It has been modified to do so. XYR1 is cellulase and hemicellulase in filamentous fungi. It is a transcription activator of Zn(II)2Cys6binuclear cl A major regulator of xylanase gene expression, possessing a uster domain, and Trichodel Ma (XYR1), Fusarium (XYR1), Neurospora (XYR1), Alpe It is widely conserved in ascomycetes, excluding yeasts such as Lugillus (XLNR). Trichoderma... Lisey's XYR1 is the xylanase / xylose metabolism gene and all of the cellulase genes. It controls. XYR1 of Trichoderma lisey is found in the ncbi database (www.n [cbi.nlm.nih.gov / ]) NCBI Reference Se It is registered as quence:XP_006966092.1, which is the array index. The amino acid sequence number 5 is encoded by a polynucleotide consisting of a sequence of 10 nucleotides. It is a polypeptide consisting of an acid sequence.

[0051] An example of XYR1 that is highly expressed in the modified filamentous fungus of the present invention is the amino acid sequence of SEQ ID NO: 5 Examples of polypeptides include the amino acids in SEQ ID NO: 5. A polypeptide consisting of an amino acid sequence that is at least 90% identical to the sequence, and the sequence number 5 amino acid sequences in which one or more amino acids are deleted, substituted, added, or inserted. Examples include polypeptides consisting of no acid sequences. These polypeptides are cellulase and It can function as a transcriptional activator of hemicellulase.

[0052] The XYR1 highly expressed in the modified filamentous fungus of the present invention may be mutant XYR1. A preferred example of YR1 is the amino acid sequence of SEQ ID NO: 5 or a sequence containing at least 90% of it. For a polypeptide of XYR1 (parent XYR1) consisting of identical amino acid sequences, the sequence number A mutation in at least one amino acid in the region corresponding to positions 810-833 of number 5 (i.e., Examples include mutant XYR1 (substitution, deletion, insertion, or addition). One example is the XYR1 mutation disclosed in PCT / JP2020 / 042489. ru.

[0053] Preferably, the mutant XYR1 is at position 817 and 821 of sequence number 5 relative to the parent XYR1. Select from the group consisting of amino acids at positions corresponding to the 824th, 825th, and 826th positions. At least one of the selected amino acids is substituted.

[0054] More preferably, the mutant XYR1 is at least 90% different from the amino acid sequence of SEQ ID NO: 5. Consists of identical amino acid sequences in a column, and is selected from one or more of the following groups. Contains amino acids: Tyr at position 817 of sequence number 5; Lys, Phe, Trp, or Tyr at position 821 of sequence number 5. Mashiku is Phe; Val, Glu, Ile, Leu, Ly at position 824 of sequence number 5 s, Phe, Thr, Trp, or Tyr, preferably Val; Tyr at position 825 of sequence number 5; Val, Ile, Leu, Phe, Tr at position 826 of sequence number 5 p or Tyr;

[0055] More preferably, the mutant XYR1 is related to the parent XYR1 at position 821 of sequence number 5. Either the Val at the corresponding position is replaced with Phe (V821F), or the A mutant in which Ala is replaced with Val at position 824 of column number 5 (A82 It is 4V.

[0056] One way to highly express XYR1 in modified filamentous fungi is to use the aforementioned ACE3 variant. The same means as for high expression can be used. For example, the strong control region is operably linked. The foreign gene encoding XYR1 is introduced and expressed in the modified filamentous fungus, or the parent To enable the activation of the XYR1 gene and strongly regulatory region in the genome inherently present in filamentous fungi, Alternatively, the number of XYR1 genes that can be expressed by the modified filamentous fungus can be increased.

[0057] The modified filamentous fungi of the present invention produced by the above procedure are substantially deficient in the aforementioned DBD. The modified ACE3 variant expresses the modified filamentous fungus of the present invention. Protein productivity is improved in the absence of glucose, etc. The modified filamentous fungus has improved protein productivity in the absence of glucose, etc. Even in environments where a non-cellulase-inducible carbon source is the primary carbon source, for example, cellulase-inducible substances Furthermore, the modified filamentous fungus can efficiently produce proteins even in the absence of [the specified element]. This occurs in the absence of cellulase-inducing substances such as cellulose, sophorose, and cellooligosaccharides. Even if present, they express cellulase-based biomass-degrading enzymes such as cellulase and hemicellulase. It is possible.

[0058] In a preferred embodiment, the modified filamentous fungus of the present invention has substantially all of the DBD defects. In addition, it expresses an ACE3 variant having the aforementioned C-terminal region defect. By combining this with the deletion of the C-terminal region, the protein productivity of the modified filamentous fungus is synergistically increased. To improve.

[0059] In another preferred embodiment, the modified filamentous fungus of the present invention lacks substantially all of the DBD In addition to expressing the compromised ACE3 variant, as mentioned above, it also highly expresses XYR1. By combining the expression of the ACE3 variant with XYR1, the tan produced in the modified filamentous fungus is obtained. The cellulase content in the protein increases, enabling efficient cellulase production.

[0060] In a more preferred embodiment, the modified filamentous fungus of the present invention substantially all of the DBD described above. An ACE3 variant having a defect in the C-terminal region and a defect in the C-terminal region is expressed, and further XYR1 is expressed in high expression This demonstrates that such modified filamentous fungi have improved protein productivity and that the produced proteins... This will also increase the cellulase content.

[0061] Therefore, in a further embodiment, the present invention uses the modified filamentous fungus of the present invention described above. The present invention provides a method for producing proteins. The modified filamentous fungus of the invention is cultured. Through cultivation, the target protein is produced and stored in the culture. The culture is then deposited. The target protein is produced by separating it from the culture. It is possible.

[0062] Examples of target proteins produced include cellulases such as cellulase and hemicellulase. Biomass-degrading enzymes, exoglucanase, endoglucanase, β-glucosidase proteases, lipases, manases, arabinases, galactases, amylases, etc. These are some examples, but are not limited to them. The target protein is a single type of protein. However, it may also be a mixture of multiple types of proteins. Preferably, the target protein is , a cellulase-type biomass-degrading enzyme, more preferably cellulase and / or hemicerase The hemicellulase is a cellulase, and more preferably a cellulase and a hemicellulase. Examples of enzymes include xylanase, β-xylosidase, and α-arabinofuranosidase. These include, among which xylanase is preferred.

[0063] Alternatively, the target protein may be a heterologous protein that filamentous fungi do not normally produce. i. In this case, a gene encoding a heterologous protein is inserted into the modified filamentous fungus of the present invention. By introducing this, recombinant filamentous fungi are produced, and by culturing these recombinant filamentous fungi, heterologous proteins are produced. A protein containing the above can be obtained. Furthermore, the gene encoding the heterologous protein By operably linking secretory signal peptides that function in filamentous fungi, the heterologous Proteins can be secreted and produced within the culture medium.

[0064] The culture medium used for the production of this protein contains a carbon source, nitrogen source, inorganic salts, vitamins, etc. As long as it contains the components necessary for the growth and protein production of normal filamentous fungi, synthetic media and natural media are acceptable. A slight misalignment is acceptable.

[0065] Any carbon source that the modified filamentous fungus can utilize can be used as a carbon source, for example. Carbohydrates such as glucose and fructose, sugar alcohols such as sorbitol, ethanol, Examples include alcohols such as glycerol and organic acids such as acetic acid. These can be used individually or in combination.

[0066] Preferably, in the protein production method according to the present invention, a cellulase-non-inducible carbon source is mainly The modified filamentous fungus is cultured in an environment that provides a suitable carbon source. As a cellulase-non-inducible carbon source: Examples include glucose, fructose, sucrose, maltose, and glycerol. Of these, glucose is preferred in terms of cost. The target protein to be produced is cellulase. If it is a biomass-degrading enzyme, the culture in this method involves cellulose, sophorose, and cello The procedure may be carried out in the presence of cellulase-inducing substances such as oligosaccharides, but not in the presence of such inducing substances. High production of the target protein is possible even under these conditions, with or without the use of the inducible substance. Not limited to this. Furthermore, in the present invention, while further reducing catabolite suppression, efficiently... To produce proteins such as lullase-type biomass-degrading enzymes, non-adductive substances such as glucose are used. The modified filamentous fungus may be cultured while a conductive carbon source is continuously added. In this case, the cellulase is not attracted. A conductive carbon source, such as glucose, is included, along with a nitrogen source such as ammonia water or ammonium salt. Dissolving it in an aqueous solution and then adding the solution during cultivation improves cultivation efficiency and reduces foaming during cultivation. It is preferable because it can suppress urges.

[0067] Nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, and nitrogenous amines. Examples include compounds, peptones, and natural nitrogen sources such as soy hydrolysates.

[0068] Inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, Potassium carbonate and other substances can be used as examples.

[0069] Examples of vitamins include biotin and thiamine. Furthermore, as needed This allows for the addition of substances required for the growth of the modified filamentous fungus of the present invention.

[0070] The culture is preferably carried out under aerobic conditions such as shaking culture or aerated stirring culture. The culture temperature is Preferably 10°C or higher, more preferably 20°C or higher, and more preferably 25°C or higher. Furthermore, preferably 50°C or lower, more preferably 42°C or lower, and more preferably 35°C or lower. Furthermore, preferably 10-50°C, more preferably 20-42°C, and more preferably 25°C. The temperature is ~35°C. The pH during incubation is 3~9, preferably 4~5. The incubation time is 10 hours. The interval is 10 days, preferably 2 to 7 days.

[0071] After culturing, the target protein is separated from the obtained culture by a conventional method. For example, the culture The cells are collected and, if necessary, subjected to cell disruption treatment using ultrasound or pressure, followed by filtration, centrifugation, and limiting. By appropriately combining methods such as external filtration, salting out, dialysis, and chromatography, the culture can be analyzed. The target protein can be isolated. The degree of isolation of the target protein is not particularly limited. No. For example, culture supernatant or its crudely separated and purified product can be obtained as a composition containing the target protein. It is possible.

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

[0073] [1] Modified filamentous fungi, The ACE3 variant manifested, The ACE3 variant has the Zn(II)2Cys6 type DNA-binding domain (DBD) of ACE3. It is a modified version that has essentially lost all of its components. Modified filamentous fungi. [2] Preferably, the ACE3 has the amino acid sequence of any of SEQ ID NOs: 1 to 4 or A polypeptide having an amino acid sequence that is at least 90% identical in sequence to the one described in [1]. Modified filamentous fungi. [3] Preferably, the Zn(II)2Cys6 type DNA binding domain (DBD) is sequence number A modified filamentous fungus described in [2], corresponding to the amino acid positions 120-160 of No. 1. [4] Preferably, the ACE3 variant is any of the following (1) to (4): (1) A partial or complete absence of C2 in the DBD and a partial or complete absence of C4 It has, where C2 is the region corresponding to positions 120-131 of sequence number 1, and C4 This corresponds to the region from position 132 to 160 in sequence number 1, and a portion of C4 is preferred. "or" refers to a region in C4 that contains at least two Cys, and more preferably, a region in C4 that contains This refers to a region containing at least three Cys, and more preferably, a region containing four Cys in C4. This refers to a region, and more preferably, the region corresponding to amino acids 132-151 of SEQ ID NO: 1. Refers to a region; (2) 80% or less of the amino acid sequence in the region corresponding to amino acids 120-160 of SEQ ID NO: 1 Preferably 90% or more, more preferably 95% or more, of the above region is missing, and Six Cys values ​​are missing; (3) The region corresponding to amino acids 120-151 of SEQ ID NO: 1 is missing; (4) The region corresponding to amino acids 1-151 of sequence number 1 is missing. or, More preferably, the ACE3 variant is any of the following (5) to (7): (5) The region corresponding to amino acids 1-160 of SEQ ID NO: 1 is missing; (6) The region corresponding to amino acids 1-200 of SEQ ID NO: 1 is missing; (7) The region corresponding to amino acids 1-240 of sequence number 1 is missing. [2] Modified filamentous fungi as described. [5] The ACE3 variant is Preferably, at least 7 amino acids at the C-terminus of the amino acid sequence of SEQ ID NO: 1 to the maximum A region corresponding to 17 amino acids is missing. Preferably, from the group consisting of amino acids corresponding to the amino acids at positions -7 to -17 of SEQ ID NO: 1 One or more of the selected amino acids are missing, or More preferably, the 11 amino acids at the C-terminus of the amino acid sequence of SEQ ID NO: 1 The region is missing. A modified filamentous fungus described in any one of items [2] to [4]. [6] The ACE3 modified body 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 the amino acids at positions 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. A modified filamentous fungus described in any one of items [2] to [5]. [7] Preferably, a gene expressing the ACE3 variant is introduced, claim [1 A modified filamentous fungus described in any one of the items from ] to [6]. [8] Preferably, the gene expressing the ACE3 variant promotes the transcription of the gene. It is operably connected to the control area, Preferably, the control region is selected from the group consisting of the pdc promoter and the act1 promoter. Selected, [7] Modified filamentous fungi as described. [9] The filamentous fungus, Preferably, it is of the genus Trichoderma, More preferably, Trichoderma risei or a mutant thereof A modified filamentous fungus described in any one of items [1] to [8].

[10] The modified filamentous fungus preferably expresses XYR1 or mutant XYR1, more preferably Some express mutant XYR1, Preferably, the XYR1 or mutant XYR1 has the amino acid sequence of SEQ ID NO: 5 or a small fraction thereof. It is a polypeptide consisting of amino acid sequences that are at least 90% identical in sequence. Preferably, the mutant XYR1 is the amino acid sequence of SEQ ID NO: 5 or at least 90 times that sequence. For amino acid sequences with identical sequences, the region corresponding to positions 810-833 of sequence number 5 At least one amino acid is mutated, More preferably, the mutant XYR1 is the amino acid sequence of SEQ ID NO: 5 or at least For amino acid sequences that are 90% identical, positions 817, 821, and 824 of sequence number 5, Selected from the group consisting of amino acids at positions corresponding to 825 and 826, with a minimum number of amino acids selected. In both cases, one amino acid is substituted. More preferably, the mutant XYR1 is at least 90% different from the amino acid sequence of SEQ ID NO: 5. Consists of identical amino acid sequences and includes one or more selected from the following group. It contains the following amino acids: Tyr at position 817 of sequence number 5; Lys, Phe, Trp, or Tyr at position 821 of sequence number 5. Mashiku is Phe; Val, Glu, Ile, Leu, Ly at position 824 of sequence number 5 s, Phe, Thr, Trp, or Tyr, preferably Val; Tyr at position 825 of sequence number 5; Val, Ile, Leu, Phe, Tr at position 826 of sequence number 5 p or Tyr, A modified filamentous fungus described in any one of items [1] to [9].

[0074]

[11] Including culturing the modified filamentous fungus described in any one of the above items [1] to

[10] , A method for producing proteins.

[12] Preferably, the protein is cellulase and / or hemicellulase, [ 11) The method described.

[13] Preferably, the culture is carried out in the presence of glucose, as described in

[11] or

[12] . Method of loading.

[14] Preferably, the culture is carried out in the absence of a cellulase-inducing substance.

[13] Method of description.

[0075]

[15] A method for producing modified filamentous fungi, This includes modifying the parent filamentous fungus to express an ACE3 variant, The ACE3 variant has the Zn(II)2Cys6 type DNA-binding domain (DBD) of ACE3. It is a modified version that has essentially lost all of its components. method.

[16] Preferably, the ACE3 is the amino acid sequence of SEQ ID NO: 1 or at least The method described in

[15] , wherein the polypeptide consists of amino acid sequences that are 90% identical in sequence.

[17] Preferably, the Zn(II)2Cys6 type DNA binding domain (DBD) is sequence The method described in

[16] , which corresponds to the region of amino acids 120-160 of number 1.

[18] Preferably, the ACE3 variant is any of the following (1) to (4): (1) A partial or complete absence of C2 in the DBD and a partial or complete absence of C4 It has, where C2 is the region corresponding to positions 120-131 of sequence number 1, and C4 This corresponds to the region from position 132 to 160 in sequence number 1, and a portion of C4 is preferred. "or" refers to a region in C4 that contains at least two Cys, and more preferably, a region in C4 that contains This refers to a region containing at least three Cys, and more preferably, a region containing four Cys in C4. This refers to a region, and more preferably, the region corresponding to amino acids 132-151 of SEQ ID NO: 1. Refers to a region; (2) 80% or less of the amino acid sequence in the region corresponding to amino acids 120-160 of SEQ ID NO: 1 Preferably 90% or more, more preferably 95% or more, of the above region is missing, and Six Cys values ​​are missing; (3) The region corresponding to amino acids 120-151 of SEQ ID NO: 1 is missing; (4) The region corresponding to amino acids 1-151 of sequence number 1 is missing. or, More preferably, the ACE3 variant is any of the following (5) to (7): (5) The region corresponding to amino acids 1-160 of SEQ ID NO: 1 is missing; (6) The region corresponding to amino acids 1-200 of SEQ ID NO: 1 is missing; (7) The region corresponding to amino acids 1-240 of sequence number 1 is missing. The method described in

[16] .

[19] The aforementioned ACE3 variant, Preferably, at least 7 amino acids at the C-terminus of the amino acid sequence of SEQ ID NO: 1 to the maximum A region corresponding to 17 amino acids is missing. Preferably, from the group consisting of amino acids corresponding to the amino acids at positions -7 to -17 of SEQ ID NO: 1 One or more of the selected amino acids are missing, or More preferably, the 11 amino acids at the C-terminus of the amino acid sequence of SEQ ID NO: 1 The region is missing. The method described in any one of items

[16] to

[18] .

[20] The ACE3 modified body, 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 the amino acids at positions 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 described in any one of items

[16] to

[19] .

[21] Preferably, the modification of the parent filamentous fungus involves the gene that expresses the ACE3 variant. A method according to any one of the items

[15] to

[20] , including introduction into a parent filamentous fungus.

[22] Preferably, the gene expressing the ACE3 variant promotes the transcription of the gene. It is operably connected to the control area, Preferably, the control region is selected from the group consisting of the pdc promoter and the act1 promoter. Selected, The method described in

[21] .

[23] The filamentous fungus, 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] Hereinafter, the present invention will be described more specifically with reference to examples.

[0077] Example 1 Construction of Plasmid DNA for Gene Transfer By PCR using the genomic DNA of Trichoderma reesei (T. reesei) as a template, the following DNA fragments 1 to 5 were prepared. Fragment 1: The promoter region of about 1.5 kbp upstream of the act1 gene (TRIREDRAFT_4 4504), Fragment 2: The polynucleotide (SEQ ID NO: 6, about 2.9 kbp) encoding the full-length polypeptide of ACE3 (SEQ ID NO: 1), Fragment 3: The polynucleotide (SEQ ID NO: 7, about 2.0 kbp) encoding the DBD-partially deleted polypeptide of ACE3 (SEQ ID NO: 2), Fragment 4: The polynucleotide (SEQ ID NO: 10, about 3.0 kbp) encoding the full-length polypeptide of XYR1 (SEQ ID NO: 5), Fragment 5: The terminator region of about 0.6 kbp downstream of the cbh1 gene (TRIREDRAFT_44504) , Fragment 6: About 2.7 kbp region of the pyr4 gene (TRIREDRAFT_74020). Fragments 1 and 2 were ligated to construct cassette 1: Pact1-TrACE3(1-734).

[0078] Fragments (SEQ ID NO: 1), Fragment 3: The polynucleotide (SEQ ID NO: 7, about 2.0 kbp) encoding the DBD-partially deleted polypeptide of ACE3 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. Upstream and downstream of fragment 6, fragment 7 of about 0.5 kbp and fragment 8 of about 1.0 kbp were respectively added, ​A transformed marker fragment arranged as a homologous sequence for Uto was prepared. Fragment 5 and the transformed marker fragment were ligated to construct cassette 4: Tcbh1-pyr4.

[0079] At the HincII restriction enzyme cleavage site of pUC118 (Takara Bio), while adding a smiI restriction enzyme site, a fragment of approximately 1.5 kbp upstream (fragment 9) and a fragment of approximately 1.4 kbp downstream (fragment 10) of the rce1 gene (TRIREDRAFT_72611) were inserted to construct cassette 5: ΔRCE1. Also, at the HincII restriction enzyme cleavage site of pUC118 (Takara Bio), while adding a smiI restriction enzyme site, a fragment of approximately 1.6 kbp upstream (fragment 11) and a fragment of approximately 1.2 kbp downstream (fragment 12) of the ace1 gene (TRIREDRAFT_75418) were inserted to construct cassette 6: ΔACE1.

[0080] Ligation of DNA fragments was carried out according to the protocol of In-Fusion HD Cloning Kit (Takara Bio). The constructed cassettes, the DNA fragments contained therein, as well as the primers used for cassette construction are shown in Table 1.

[0081]

Table 1

[0082] Between the upstream and downstream regions of the rce1 gene of cassette 5, a product obtained by ligating cassettes 1 and 4 by PCR using the primers in Table 2 was inserted to construct the full-length ace3 constitutive expression plasmid pUC-Pact1-TrACE3(1-734) (Plasmid 1). Between the upstream and downstream regions of the rce1 gene of cassette 5, a product obtained by ligating Insert the ligated 4 into the DBD partially deleted ace3 constitutive expression plasmid pUC-Pact1 -TrACE3(1-629) was constructed (plasmid 2). Plasmid 1 is sequence number Plasmid 1 expresses the full-length ACE3 of T. reesei, and plasmid 2 is associated with sequence number 1. This results in the expression of an ACE3 variant lacking a portion of DBD (C2) (Figure 1).

[0083] PCR was performed between the upstream and downstream regions of the ace1 gene in cassette 6 using the primers shown in Table 2. By inserting a combined cassette of 3 and 4, the xyr1 gene constitutive expression plasmid pUC is created. -Pact1-XYR1 was constructed (plasmid 3). Plasmid 3 is xylanase X It expresses YR1.

[0084] PCR using the primers in Table 3 with plasmid 1 as a template revealed pUC-Pact 1-TrACE3(161-734)(plasmid 4), pUC-Pact1-TrAC E3(201-734)(plasmid 5), pUC-Pact1-TrACE3(241 -734) (plasmid 6), pUC-Pact1-TrACE3 (281-734) ( Plasmid 7), pUC-Pact1-TrACE3(300-734)(Plasmid 8) Plasmids 4-8 contain the N-terminal DBD relative to Sequence ID 1. This results in the expression of an ACE3 variant lacking a specific region (Figure 1).

[0085] ACE3 polypeptide of Trichoderma atroviride A polynucleotide (sequence number 8, approximately 2.0 kbp) encoding a variant (sequence number 3) of the above. Modified ACE3 polypeptide of Trichoderma harzianum A polynucleotide (SEQ ID NO: 9, approximately 2.0 kbp) encoding the variant (SEQ ID NO: 4) was artificially synthesized in humans. The obtained fragment was ligated to plasmid 1 by PCR using the primers in Table 4. This resulted in the construction of pUC-Pact1-TaACE3(1-630) (plasmid 9 ) and pUC-Pact1-ThACE3(1-630) (plasmid 10), respectively. Plasmids 9-10 express ACE3 variants lacking a part (C2) of the DBD. .

[0086] By PCR using the primers in Table 4 with plasmid 9 or plasmid 10 as a template, pUC-Pact1-TaACE3(137-630) (plasmid 11) and pU C-Pact1-ThACE3(137-630) (plasmid 12) were constructed respectively. Plasmids 11 and 12 express ACE3 variants with a complete DBD deletion, lacking an additional 136 amino acids on the N-terminal side, corresponding to the ACE3 variants of SEQ ID NO: 3 and SEQ ID NO: 4, respectively. .

[0087] By PCR using the primers in Table 5 with plasmid 1 as a template, a plasmid (plasmid 13) expressing an ACE3 variant with a deletion of a predetermined region on the C-terminal side with respect to ACE3 of T. reesei (SEQ ID NO: 1) was constructed. By PCR using the primers in Table 5 with plasmid 2 or 4 as a template, plasmids (plasmids 14-17) expressing ACE3 variants with deletions of predetermined regions on the N-terminal side and C-terminal side with respect to ACE3 of T. reesei (SEQ ID NO: 1) were constructed. Also, by two-step PCR using the primers in Table 5 with plasmid 1 as a template, for ACE3 of T. reesei (SEQ ID NO: 1), Plasmids expressing ACE3 variants in which specific regions at the terminal and C-terminal ends are deleted. We constructed plasmids 18-19). The composition of ACE3 variants contained in plasmids 13-18. This is shown in Figure 2.

[0088] PCR using the primers in Table 5 with plasmid 3 as a template revealed pUC-Pact 1-XYR1(V821F) was constructed (plasmid 20). This is the amicin of sequence number 5. The mutant XYR1(V821F) in which the amino acid substitution V821F is made to the no-acid sequence is It is a plasmid that performs the following action.

[0089] Plasmids 1-20 constructed in this example, and the fragments (cassettes, plasmids) contained therein (and other similar products) and the primers used in their construction, as well as the ACE3 expressed by them, are shown. This is shown in 2-5.

[0090] [Table 2]

[0091] [Table 3]

[0092] [Table 4]

[0093] [Table 5]

[0094] The constructed plasmid was replicated. Competent cells: Escherichia coli DH5α Competent Cells (Takara Bio) plus the above-prepared product Mido was introduced and cultured in ampicillin-supplemented LB medium (37°C, 1 day). The bacterial cells after culture were then shown. From NucleoSpin TM Using Plasmid (Machley-Nagel) The material was recovered and purified.

[0095] Example 2: Preparation of modified filamentous fungi Trichoderma lisey E1AB1 (JN13) pyr4 gene knockout strain (JN13Δ The pyr4 strain was transformed by introducing a DNA fragment derived from the plasmid constructed in Example 1. The plasmid constructed in Example 1 was linearized by cleaving it at the smiI restriction enzyme site. , Protoplast PEG method (Biotechnol Bioeng, 2012, 109( The parent strain was transformed by introducing the gene according to 1):92-99). The transformant had the pyr4 gene. Using this as a marker, select medium (2% glucose, 1.1M sorbitol, 2% agar, 0 .2% KH2PO4(pH5.5), 0.06% CaCl2·2H2O, 0.06% Cs Cl2, 0.06% MgSO4·7H2O, 0.5% (NH4)2SO4, 0.1% Tra ce element1;% were all selected using w / v%). Trace elem The composition of ent1 is as follows: 0.5g FeSO4·7H2O, 0.2g CoC 12, 0.16g MnSO4·H2O, 0.14g ZnSO4·7H2O, dissolved in distilled water for 10 minutes. Dilute to 0 mL. From the selected transformants, identify the target gene fragment in the rce1 locus or The insertion of the protein into the ace1 locus was confirmed by PCR, and the desired transformant was obtained.

[0096] Dual transformants expressing mutant XYR1 (V821F) and ACE3 or its variants. A PD containing 0.2% 5-fluoroorotic acid (5-FOA) monohydrate was prepared. The XYR1(V821F) expression strain, into which plasmid 20 was introduced using medium A, was cultured again. We selected strains that had acquired 5-FOA resistance and were able to grow. The resulting strain was named JN13_XYR1(V821 F) The strain was obtained as Δpyr4. The obtained strain was treated with the above ACE3 expression plasmid. (Plasmids 1-19) were re-transformed. These strains were mutant XYR1 (V821F ) and express ACE3 or a variant thereof.

[0097] Example 3: Culture of modified filamentous fungi The filamentous fungal strain obtained in Example 2 was cultured to produce protein. In the pre-culture, 500 mL Place 50 mL of culture medium in a flask and add 1 × 10 spores of the strain prepared in Example 2. 5 pieces / mL The cells were inoculated in this manner and cultured with shaking at 28°C and 220 rpm (PRXYg- from Pliss). 98R). The pre-culture medium composition is as follows: 1% glucose, 0.14% (NH4 )2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgS O4·7H2O, 0.1% high polypeptone N, 0.05% Bacto Yeast e xtract, 0.1% Tween 80, 0.1% Trace element2, 50 mM tartaric acid buffer (pH 4.0) (all percentages are w / v%). Trace el The composition of ement2 is as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7 O 24 ·4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8m Dissolve 200 mg of MnCl2·4H2O and 200 mg of ZnCl2 in distilled water to make up 100 mL.

[0098] After a two-day pre-culture, the main culture was performed. 50 mL of culture medium was placed in a 500 mL flask. The pre-culture solution was inoculated at 1% (v / v%) and cultured at 28°C and 220 rpm for 4 days. Main culture The culture medium composition is as follows: 3% glucose, 0.14% (NH4)2SO4, 0.2 % KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0 0.1% High Polypeptone N, 0.05% Bacto Yeast Extract, 0 0.1% Tween 80, 0.1% Trace element 2, 1.28% citric acid Diammonium oxyhydrogen, 50 mM tartaric acid buffer (pH 4.0) (all percentages are w / v) %).

[0099] Example 4: Effects of constitutive expression of DBD-deficient ACE3 on protein production The full-length ACE3 or DBD-deficient ACE3 modified from T. reesei, prepared in Example 2. Regarding transformants that constitutively express the body, protein productivity and the combination of proteins produced. I evaluated success.

[0100] 1) Evaluation of protein productivity The protein concentration of the culture from Example 3 was measured using the Bradford method. In the rd method, the Quick Start Protein Assay (BioRad) is used. The protein content was calculated based on a calibration curve created using cy-gamma globulin as the standard protein. Protein synthesis of JN13 in cultures using glucose alone as the carbon source (glucose culture) The relative protein productivity of each strain was calculated, with the total productivity set to 1.

[0101] Figure 3(A) shows the relative protein productivity of each transformant. As shown in Figure 3(A), the full length ACE3 (TrACE3(1-734); SEQ ID NO: 1) expressing strain ("1-734"), and A modified ACE3 with a missing part of DBD (C2) (TrACE3(1-629); sequence number) Compared to strain 2) ("1-629"), the N-terminal side of 160-24 A DBD-deficient ACE3 variant lacking 0 amino acids (TrACE3(161-734)) strains expressing TrACE3(201-734) and TrACE3(241-734) In "161-734", "201-734", and "241-734", the protein Productivity improved. In particular, the N-terminal 240 amino acids were missing from SEQ ID NO: 1. In cases where ("241-734") protein productivity is significantly improved, DBD partial knockout strain It showed approximately 1.6 times the productivity compared to (1-629). On the other hand, compared to Sequence ID No. 1 ACE3 variant with more than 280 amino acids missing from the N-terminus (TrACE3(281-734 Strains expressing ) and TraACE3(300-734) ("281-734" and "30 At 0-734"), the effect of improving protein productivity disappeared.

[0102] 2) Protein composition analysis The protein composition of the culture from Example 3 was analyzed. Mini PROTEAN was used for the analysis. TGX Stain-Free Gels(Any KD, 15well, BIORA D) was used as the standard. Precision Plus Protein Unstained standards were used. The culture from Example 3 was diluted as appropriate and Bu Apply the mixture of ffer, treated at 99°C for 5 minutes, to the gel, and then run at 200V for 35 minutes. Intercalated electrophoresis was performed. The resulting image files were analyzed using analysis software (ImageLab). The band intensity ratio was calculated, and the composition ratio of the produced saccharifying enzymes was determined.

[0103] The results of SDS-PAGE are shown in Figure 3(B). The protein productivity shown in Figure 3(A) Improved DBD-deficient ACE3 variant expression strains (in Figure 3(B), “161-734”, “201 In "-734" and "241-734"), the major cellulases are CBH1 and CB Increases in H2 and EG1 were observed. In other words, the protein produced by the DBD-deficient ACE3 variant... It was shown that the improvement in chlorine productivity was mainly due to an increase in cellulase components.

[0104] Example 5: DBD deficiency effect in ACE3 from other Trichoderma species The transformants prepared in Example 2 express T. reesei and T. atrovirid A DBD partially deficient ACE3 variant derived from e and T.harzianum (TrACE3(1 -629): Sequence ID 2, TaACE3(1-630): Sequence ID 3, and ThACE3 (1-630): Sequence ID 4), and DBD complete deficiency ACE3 variant (TrACE3(2 41-734, TaACE3 (131-630) and ThACE3 (131-630)) For T. reesei total length ACE3 (TrACE3(1-734): Sequence ID 1) The amino acid sequence alignment is shown in Figure 4. From the sequence alignment, T. atro A partially deficient DBD ACE3 variant (TaA) derived from viride and T.harzianum. The sequences of CE3(1-630) and ThACE3(1-630) are derived from T. reesei. This corresponds to the sequence of the next DBD partial-missing ACE3 variant (TrACE3(1-629)). This was shown. Similarly, DBs derived from T. atroviride and T. hazianum D Completely Deficient ACE3 Modified (TaACE3 (131-630) and ThACE3 (131 The sequence -630)) is a T. ree with the N-terminal 240 amino acids of sequence number 1 missing. The sequence of the sei-derived DBD partial deletion ACE3 variant (TrACE3(241-734)) It was shown to be equivalent.

[0105] The above are derived from T. reesei, T. atroviride, and T. hazianum. Regarding transformants that constitutively express the DBD portion or a completely deficient ACE3 variant, Example 4, Protein productivity was evaluated using the same method as in 1). The results are shown in Figure 5(A). T.atr In ACE3 of oviride and T.harzianum, T.reesei Similar to ACE3, compared to expression lines of a partially deficient DBD ACE3 variant, DBD complete deficient Protein productivity was improved in strains expressing the ACE3 variant. Also, similar to Examples 4 and 2). The results of performing SDS-PAGE using this method are shown in Figure 5(B), similar to Figure 3(B). Increases in the major cellulases CBH1, CBH2, and EG1 were observed. Therefore Therefore, the improvement in cellulase productivity by DBD-deficient ACE3 variants is due to T. reesei's AC It has been shown that this occurs not only in E3 but also in ACE3 of other Trichoderma species.

[0106] Example 6: Synergistic effect of DBD deficiency and constitutive expression of C-terminally deficient 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, the C-terminus 11 of the sequence number 1 prepared in Example 2 is used. ACE3 variant lacking amino acids (TrACE3(1-723)), C-terminal deletion and D ACE3 variant with BD portion missing (TrACE3(1-618)), C-terminus missing and D This organism constitutively expresses an ACE3 variant (TrACE3(161-723)) with a complete BD defect. Protein productivity was evaluated in the transformants using the same method as in Example 4, 1). Results This is shown in Figure 6(A). Even with equivalent C-terminal defects, there is no N-terminal defect or DBD portion. Compared to the expression strain of the partially deficient ACE3 variant, the expression strain of the completely deficient DBD ACE3 variant showed Protein productivity was significantly improved. Furthermore, Figures 3(A) and 6(A) show that DBD deficiency The combination of this and the C-terminal deletion yielded an unexpectedly high synergistic effect on protein productivity. It was found that this was done. Also, SDS-PAGE was performed using the same method as in Example 4, 2). As a result, as shown in Figure 6(B), the major cellulase, CBH1, was found to be similar to that in Figure 3(B). Increases in CBH2 and EG1 were observed. Therefore, complete DBD in ACE3 The deletion exhibits a greater effect in improving cellulase productivity than the C-terminal deletion, and furthermore, DB It has been revealed that the effect is synergistically increased by combining complete D deletion with C-terminal deletion. It happened.

[0107] Example 7: Combination effect of DBD-deficient ACE3 and constitutive expression of mutant XYR1 The mutant XYR1(V821F) expression strain prepared in Example 2, and the mutant XYR1(V821 Regarding a double transformant that constitutively expresses F) and ACE3 or a variant thereof, Example 4, Protein productivity was evaluated using the same methods as in 1) and 2).

[0108] Compared to the JN13 strain, the mutant XYR1(V821F) expression strain showed improved protein productivity. Regarding the double transformants, compared to the mutant XYR1(V821F) monoexpression strain, the full-length The improvement in protein productivity in ACE3 (TrACE3(1-734)) co-expressing strains was slight. However, the protein of the DBD partial-deficient ACE3 variant (TrACE3(1-629)) co-expressing strain Quality productivity improved by approximately 1.5 times. V821F and DBD complete missing ACE3 modification (Tr ACE3(161-734), TraACE3(201-734), or TraACE3(24 The protein productivity of co-expression strains with 1-734)) is compared to V821F and DBD partial knockout (T It is equivalent to the co-expressing strain with rACE3(1-629), and further increases in production have been confirmed. I couldn't do it.

[0109] The results of SDS-PAGE are shown in Figure 7(A). The composition ratio of the produced saccharifying enzymes is also shown in Figure 7(A). This is shown in 7(B). In strains constitutively expressing mutant XYR1 and DBD completely deficient ACE3 variants... This not only improves protein productivity, but also the major cellulases CBH1 and CBH2. The EG1 content ratio was increasing. On the other hand, similar to the results of Example 4 (Figure 3), the N-terminal side In strains expressing ACE3 variants lacking 280 amino acids or more, protein productivity and se The effect of improving the lurase content ratio disappeared. Therefore, mutant XYR1 and specific DBD complete Co-expression with a completely deficient ACE3 variant shows that existing mutant XYR1 and ACE3 or its variants are being expressed together. Compared to co-expression with the body, protein productivity and cellulase content in the produced protein were improved. This allows for a greater increase in quantity, thus enabling more efficient cellulase production. It was shown.

[0110] Example 8: Combination effect of DBD-deficient and C-terminally deficient ACE3 with constitutive expression of mutant XYR1 Using the same procedure as in Example 7, the mutant XYR1 (V821F) prepared in Example 2 and DBD and Regarding the dual transformant constitutively expressing both a C-terminally deficient ACE3 variant and a C-terminally deficient ACE3 variant, see Examples 4, 1) Protein productivity was evaluated using the same method as in (2).

[0111] The results of SDS-PAGE are shown in Figure 8(A). The composition ratio of the produced saccharifying enzymes is also shown in Figure 8(A). This is shown in 8(B). Mutant XYR1 and DBD complete deletion and C-terminal deletion ACE3 variant (Tr Strains that constitutively express ACE3(161-723) have high protein productivity and are the most It also showed a high cellulase content ratio. It was found that complete deficiency of ACE3 DBD is effective in improving the cellulase content ratio. On the other hand, other C-terminally deficient ACE3 variants (TrACE3(1-723), TrACE3(1- 618), TrACE3(1-595), TrACE3(1-455), TrACE3( 300-522), or TraACE3 (300-560), DB without C-terminal defects. Compared to the D-partially deficient ACE3 variant (TrACE3(1-629)), protein production amount No increase was observed.

[0112] As described above, A is completely lacking in the Zn(II)2Cys6 type DNA-binding domain (DBD). Constitutive expression of the CE3 variant enhances microbial protein productivity in the absence of inducible substances. It has been significantly improved. Such ACE3 variants are not only found in T. reesei, but also in other species. It is also effective against Lycoderma species, and is effective against known effective mutations such as C-terminal deletion and mutation X. It was also effective in combination with YR1 (e.g., V821F). Therefore, the above DB Constitutive expression of a D-deficient ACE3 variant leads to the non-inducible carbon of cells such as glucose. Under culture conditions using the source, cellulase promoters in microorganisms are highly activated, resulting in high efficiency. It was demonstrated that protein production can be achieved.

Claims

1. A modified filamentous fungus, The ACE3 variant manifested, The ACE3 variant is Zn(II) of ACE3 2 Cys 6 This is a modified variant lacking the DNA-binding domain region. The ACE3 is a polypeptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 4 or an amino acid sequence that is at least 90% identical thereto. The Zn(II) 2 Cys 6 The DNA-binding domain region corresponds to the amino acid positions 120-151 of SEQ ID NO:

1. Modified filamentous fungi.

2. The modified filamentous fungus according to claim 1, wherein the ACE3 variant lacks the region corresponding to the amino acids at positions 120 to 160 of SEQ ID NO:

1.

3. The modified filamentous fungus according to claim 1, wherein the ACE3 variant lacks the region corresponding to amino acids 1 to 151 of SEQ ID NO:

1.

4. The modified filamentous fungus according to claim 2, wherein the ACE3 variant lacks the region corresponding to amino acids 1 to 160 of SEQ ID NO:

1.

5. The modified filamentous fungus according to any one of claims 1 to 4, wherein the ACE3 variant has a region corresponding to amino acids 260 to 701 of SEQ ID NO:

1.

6. The modified filamentous fungus according to claim 5, wherein the ACE3 variant has a region corresponding to amino acids 260 to 717 of SEQ ID NO:

1.

7. The modified filamentous fungus according to claim 6, wherein the ACE3 variant has a region corresponding to amino acids 260 to 723 of SEQ ID NO:

1.

8. The modified filamentous fungus according to claim 5, wherein the ACE3 variant has a region corresponding to the amino acids at positions 250 to 701 of SEQ ID NO:

1.

9. The modified filamentous fungus according to claim 8, wherein the ACE3 variant has a region corresponding to amino acids 250 to 717 of SEQ ID NO:

1.

10. The modified filamentous fungus according to claim 9, wherein the ACE3 variant has a region corresponding to amino acids 250 to 723 of SEQ ID NO:

1.

11. The modified filamentous fungus according to claim 5, wherein the ACE3 variant has a region corresponding to amino acids 241 to 701 of SEQ ID NO:

1.

12. The modified filamentous fungus according to claim 11, wherein the ACE3 variant has a region corresponding to amino acids 241 to 717 of SEQ ID NO:

1.

13. The modified filamentous fungus according to claim 1 or 2, wherein the ACE3 variant has a region corresponding to amino acids 241 to 723 of SEQ ID NO:

1.

14. The modified filamentous fungus according to any one of claims 1 to 13, wherein the ACE3 variant lacks a region corresponding to at least seven amino acids at the C-terminus of the amino acid sequence of Sequence ID No.

1.

15. The modified filamentous fungus according to claim 14, wherein the ACE3 variant lacks a region corresponding to the 11 amino acids at the C-terminus of the amino acid sequence of Sequence ID No.

1.

16. A modified filamentous fungus according to any one of claims 1 to 15, wherein a gene expressing the ACE3 variant has been introduced.

17. The modified filamentous fungus according to claim 16, wherein the gene expressing the ACE3 variant is operably linked to a regulatory region that promotes the transcription of the gene.

18. The modified filamentous fungus according to any one of claims 1 to 17, wherein the filamentous fungus is of the genus Trichoderma.

19. A method for producing a protein, comprising culturing a modified filamentous fungus according to any one of claims 1 to 18.

20. The method according to claim 19, wherein the protein is cellulase and / or hemicellulase.

21. The method according to claim 19 or 20, wherein the culture is carried out in the presence of glucose.

22. A method for producing modified filamentous fungi, This includes modifying the parent filamentous fungus to express an ACE3 variant, The ACE3 variant is Zn(II) of ACE3 2 Cys 6 This is a modified variant lacking the DNA-binding domain region. The ACE3 is 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. The Zn(II) 2 Cys 6 A method in which the DNA-binding domain region corresponds to the amino acid region 120-151 of SEQ ID NO:

1.

23. The method according to claim 22, wherein the ACE3 variant lacks the region corresponding to the amino acids at positions 120 to 160 of SEQ ID NO:

1.

24. The method according to claim 22, wherein the ACE3 variant lacks the region corresponding to amino acids 1 to 151 of SEQ ID NO:

1.

25. The method according to claim 23, wherein the ACE3 variant lacks the region corresponding to amino acids 1 to 160 of SEQ ID NO:

1.

26. The method according to any one of claims 22 to 25, wherein the ACE3 variant has a region corresponding to the amino acids at positions 260 to 701 of SEQ ID NO:

1.

27. The method according to claim 26, wherein the ACE3 variant has a region corresponding to amino acids 260 to 717 of SEQ ID NO:

1.

28. The method according to claim 27, wherein the ACE3 variant has a region corresponding to amino acids 260 to 723 of SEQ ID NO:

1.

29. The method according to claim 26, wherein the ACE3 variant has a region corresponding to the amino acids at positions 250 to 701 of SEQ ID NO:

1.

30. The method according to claim 29, wherein the ACE3 variant has a region corresponding to the amino acids at positions 250 to 717 of SEQ ID NO:

1.

31. The method according to claim 30, wherein the ACE3 variant has a region corresponding to the amino acids at positions 250 to 723 of SEQ ID NO:

1.

32. The method according to claim 26, wherein the ACE3 variant has a region corresponding to the amino acids at positions 241 to 701 of SEQ ID NO:

1.

33. The method according to claim 32, wherein the ACE3 variant has a region corresponding to amino acids 241 to 717 of SEQ ID NO:

1.

34. The method according to claim 22 or 23, wherein the ACE3 variant has a region corresponding to amino acids 241 to 723 of SEQ ID NO:

1.

35. The method according to any one of claims 22 to 34, wherein the ACE3 variant lacks a region corresponding to at least seven amino acids at the C-terminus of the amino acid sequence of Sequence ID No.

1.

36. The method according to claim 35, wherein the ACE3 variant lacks a region corresponding to the 11 amino acids at the C-terminus of the amino acid sequence of Sequence ID No.

1.

37. The method according to any one of claims 22 to 36, wherein the modification of the parent filamentous fungus includes introducing a gene that expresses the ACE3 variant into the parent filamentous fungus.

38. The method according to claim 37, wherein the gene expressing the ACE3 variant is operably linked to a regulatory region that promotes transcription of the gene.

39. The method according to any one of claims 22 to 38, wherein the filamentous fungus is of the genus Trichoderma.

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