WO2020075787A1

WO2020075787A1 - Mutant strain of trichoderma reesei, and protein manufacturing method

Info

Publication number: WO2020075787A1
Application number: PCT/JP2019/039933
Authority: WO
Inventors: 雄介加川; 紳吾平松; 山田　勝成
Original assignee: 東レ株式会社
Priority date: 2018-10-11
Filing date: 2019-10-10
Publication date: 2020-04-16
Also published as: JPWO2020075787A1

Abstract

The present invention is a mutant strain of Trichoderma reesei that has a mutation and in which a function of a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2 is eliminated or reduced, as well as a method to mass produce a protein by cultivating said mutant strain.

Description

Trichoderma reesei mutant and protein production method

The present invention relates to a mutant strain of Trichoderma reesei that improves protein production ability and a method for producing a protein using the mutant strain.

It is known that Trichoderma reesei has a high protein-producing ability, and studies on protein production using the filamentous fungus have been conducted so far. Trichoderma reesei is excellent in the ability to produce cellulase classified as a saccharifying enzyme among proteins, and for example, in order to further improve the production amount of cellulase, overexpression or deletion of a factor controlling cellulase production is performed. There is.

In Non-Patent Document 1, among factors controlling the production of cellulase of Trichoderma reesei, a mutant strain of Trichoderma reesei having a high cellulase producing ability by reducing the function of Cre1, which is a transcription factor that suppresses the production of cellulase. Has been acquired.

As mentioned above, the transcription factor, which is one of the factors controlling the protein production of Trichoderma reesei, has been elucidated, but I think that this is only a part of the control mechanism. Therefore, in the present invention, a novel factor that controls the protein production of Trichoderma reesei is searched, and a mutant of Trichoderma reesei that has enhanced protein production ability is obtained and a method for producing a protein using the mutant of Trichoderma reesei. The challenge is to provide.

The present inventor believes that the production amount of Trichoderma reesei protein can be further improved if a novel regulatory factor that improves protein production, which has not been known so far, can be elucidated. The present inventors have found that protein productivity can be improved by culturing a mutant strain of Trichoderma reesei having a mutation in the polypeptide having the amino acid sequence represented by the following, and have completed the present invention.

That is, the present invention includes the following (1) to (6).
(1) A mutant strain of Trichoderma reesei, which has a mutation in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is deleted or reduced.
(2) The mutation is a deletion, substitution, or addition mutation of an amino acid residue in the fungal transcription factor regulatory regulatory region domain of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, (1) The mutant strain described in.
(3) The mutation according to (1) or (2), wherein the mutation is a mutation at the 677th serine residue from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 to an amino acid residue other than serine. Mutant strain.
(4) A method for producing a protein, which comprises a step of culturing the mutant strain according to any one of (1) to (3).
(5) A method for producing cellulase, which comprises a step of culturing the mutant strain according to any one of (1) to (3).
(6) A method for producing sugar, which comprises the step of producing cellulase by the method for producing cellulase according to (5) and the step of saccharifying a cellulose-containing biomass using the cellulase obtained in the above step.

The mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is deficient or diminished, has an improved protein production ability and a higher protein content than the parent strain before the introduction of the mutation. It becomes possible to produce. Furthermore, when the protein to be produced is cellulase, an unexpected effect of improving various specific activities of cellulase is also obtained.

The present invention is characterized by further enhancing protein production ability by introducing a mutation into a parent strain of Trichoderma reesei, which is a microorganism originally excellent in protein production ability. Specifically, the present invention relates to a mutant strain of Trichoderma reesei, which is characterized in that the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is deleted or reduced.

The parent strain of Trichoderma reesei used in the present invention is not limited to a wild strain, and a mutant strain improved so as to enhance protein production ability can also be preferably used as the parent strain, for example, a mutation treatment with a mutagen or ultraviolet irradiation. The mutant strain having improved protein productivity can be used as the parent strain. Specific examples of the above-mentioned parent strains include Trichoderma reesei QM6a strain (NBRC31326), QM9414 strain (NBRC31329), PC-3-7 strain (ATCC66589), QM9123 strain (NBRC31327), RutC-30 strain (ATCC56765), CL-847. Strain (Enzyme. Microbiol. Technol. 10, 341-346 (1988)), MCG77 strain (Biotechnol. Bioeng. Symp. 8, 89 (1978)), MCG80 strain (Biotechnol. Bioeng. 12, 451-459 (1982)). ) And derivatives thereof. The QM6a strain, the QM9414 strain, and the QM9123 strain can be obtained from NBRC (NITE Biological Resource Center), and the PC-3-7 strain and the RutC-30 strain can be obtained from ATCC (American Type Culture Collection).

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a polypeptide having a total length of 774 amino acids possessed by Trichoderma reesei, and in the National Center for Biotechnology Information, it is also referred to as a predicted protein (EGR50030) possessed by the Trichoderma reesei QM6a strain. It is registered. The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a polypeptide whose function is unknown, but according to the Conserved Domain Architectural Retrieval Tool of National Center for Biotechnology Information, the 280th to 710th amino acids from the N-terminal side remain. The group is disclosed as having a fungal transcription factor regularity middle homology region domain. From this description, it is presumed that the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is at least involved in the transcriptional regulation of filamentous fungi. A specific example of the gene encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is the base sequence represented by SEQ ID NO: 1.

In the present invention, a method for deleting or reducing the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 includes total deletion of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, fungal transcription factor regulatory middle There is a method of introducing a mutation that causes a partial deletion of the homology region domain and a partial deletion of the fungal transcription factor regulatory middle region region, specifically, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. Introduce frameshift mutations and stop codon mutations in the coding gene sequence by deleting, inserting, or replacing bases. How to, and the like.

A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a fungal transcription factor regulatory middle region domain is a deletion of the polypeptide or domain, a part of the polypeptide or the domain disappears, a part of the amino acid changes to a different amino acid Refers to a different amino acid, or a combination thereof. More specifically, it means that the sequence identity with the amino acid sequence represented by SEQ ID NO: 2 is 80% or less, preferably 50% or less, more preferably 20% or less, still more preferably 10% or less. % Or less, more preferably 5% or less, further preferably 3% or less, further preferably 1% or less, and most preferably 0%.

The function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is deficient or diminished by mutation such as deletion, substitution, or addition in the amino acid sequence located in the fungal transcription factor regulatory regrowth homology region domain. As a specific example, the 677th serine residue from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is preferably mutated to an amino acid residue other than serine, and particularly preferably mutated to phenylalanine. Is preferred.

A specific example of the nucleotide sequence encoding an amino acid sequence in which the 677th serine residue from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is mutated to an amino acid residue other than serine is represented by SEQ ID NO: 1. In the base sequence, a sequence in which cytosine, which is the 2588th base, is mutated to thymine, can be mentioned. Due to the mutation, the amino acid residue at the 677th position from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is mutated from serine to phenylalanine.

In addition, the function of the polypeptide may be reduced by introducing a mutation that reduces the expression of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or eliminates the expression. Alternatively, it may be due to a decrease or disappearance of the expression level of the polypeptide due to a mutation in the promoter or terminator region of the gene encoding the amino acid sequence represented by SEQ ID NO: 2. In general, a promoter and terminator region correspond to both sides of several hundred bases before and after a gene involved in transcription and include a promoter and terminator involved in transcription of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. Specific examples of the sequence include the base sequence represented by SEQ ID NO: 1.

For the introduction of mutations into the above-mentioned genes, existing gene mutation methods such as mutation treatment known to those skilled in the art or mutation treatment with ultraviolet irradiation, gene recombination such as homologous recombination using a selection marker, or mutation with a transposon are used. be able to.

The mutant strain of Trichoderma reesei lacking the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 can be obtained by the following method.

The mutant strain in which all of the functions of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 are deleted or reduced, spores of Trichoderma reesei, which is a parent strain, are nitrosoguanidine (NTG), ethylmethanesulfonic acid (EMS). ), A gene mutation treatment is performed using ultraviolet rays, etc., the gene of the obtained mutant strain is analyzed, and a mutant strain having the above mutation is screened to obtain the mutant strain.

Since the mutant strain of the present invention has improved protein production ability as compared with the parent strain before the mutation introduction, the culture solution of the mutant strain of the present invention is obtained before the mutation introduction under the same culture conditions. The protein concentration is increased as compared to the culture medium of the parent strain. When the protein is an enzyme, the specific activity of the enzyme increases. Here, the increase rate of the protein concentration and the increase rate of the specific activity of the enzyme are not particularly limited as long as they are increased, but are preferably 20% or more.

The mutant strain of the present invention is not only a mutant in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is deficient or diminished, but also the protein production amount is improved and / or the viscosity of the culture solution is decreased, so that May have a mutation that suppresses the decrease in the dissolved oxygen saturation level. Specific examples include mutations in the polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15.

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is a polypeptide possessed by Trichoderma reesei, and is registered in National Center for Biotechnology Information as EGR50654 of predicated protein possessed by the Trichoderma reesei QM6a strain. The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is a polypeptide whose function is unknown, but according to the Censored Domain Retrieval Tool of the National Center for Biotechnology Information, the remaining 95th to 277th amino acids from the N-terminal side. It is disclosed that the group has a Middle domain of eukaryotic initiation factor 4G domain (hereinafter referred to as MIF4G domain), and the 380th to 485th amino acid residues from the N-terminal side have a MA-3 domain. Both MIF4G and MA-3 domains are known to have a function of binding to DNA or RNA (Biochem. 44, 12265-12272 (2005), Mol. Cell. Biol. 1, 147-156 (2007). )). From these descriptions, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is presumed to have at least the function of binding to DNA and / or RNA.

A specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is the nucleotide sequence represented by SEQ ID NO: 4. A gene mutation in which the function of EGR50654 is reduced or deleted includes all deletions of MIF4G domain and / or MA-3 domain possessed by EGR50654, partial deletion of MIF4G domain and / or MA-3 domain, MIF4G domain and MA-3 domain. Gene mutations that change the configurational relationship with Furthermore, the function of the polypeptide can be reduced or deleted by introducing a mutation that reduces or eliminates the expression level of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3. As a specific example in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is lacking, a mutation in the base sequence represented by SEQ ID NO: 4 in which any of the 1039th to 1044th bases is deleted is Can be mentioned.

Compared with Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is not decreased or deleted due to the decrease or loss of the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3, Protein productivity is improved.

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is a polypeptide possessed by Trichoderma reesei, and is registered as EGR44419 of predicated protein possessed by the strain of Trichoderma reesei QM6a in the National Center for Biotechnology Information. The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is a polypeptide whose function is unknown, but according to the Censored Domain Retrieval Tool of National Center for Biotechnology Information, amino acid residues 26 to 499 from the N-terminal side. Is disclosed to have a Sugar (and other) Transporter domain. According to this description, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is involved in sugar transport at least between the inside and outside of the bacterial cell. It is estimated that

A specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is the base sequence represented by SEQ ID NO: 6. A gene mutation in which the function of EGR44419 is reduced or deleted refers to a total deletion of the Sugar (and other) Transporter domain, a partial deletion of the Sugar (and other) Transporter domain, and a configurational relationship of the Sugar (and other) Transporter domain. The gene mutation which changes is mentioned. Furthermore, the function of the polypeptide can be reduced or deleted by introducing a mutation that reduces or eliminates the expression level of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5. A specific example in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is lacking is a mutation in which 11 bases are inserted at the 1415th position in the base sequence represented by SEQ ID NO: 6.

Compared with Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is not decreased or deleted due to the decrease or loss of the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5, The productivity of the protein and the specific activity of β-glucosidase are improved.

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7 is a polypeptide possessed by Trichoderma reesei, and in National Center for Biotechnology Information, the EGR registered as beta-adaptin large sub10 unit of Trichoderma reesei QM6a strain is registered. There is. The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7 constitutes an adapter protein that constitutes vesicles involved in intracellular and extracellular transport that bind to clathrin, which is widely conserved in eukaryotes. It is one of the proteins (Proc. Nati. Acad. Sci. USA. 101, 14108-14113 (2004)).

A specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7 is the nucleotide sequence represented by SEQ ID NO: 8. Examples of the EGR48910 gene mutation include mutation of cytosine, which is the 1080th base in the base sequence represented by SEQ ID NO: 8, to adenine.

Compared with Trichoderma reesei, which has no mutation in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7 by having a mutation in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7, liquid culture At that time, the viscosity of the culture solution decreases.

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 is a polypeptide possessed by Trichoderma reesei, and is registered in National Center for Biotechnology Information as EGR45828 of predicated protein possessed by the Trichoderma reesei QM6a strain. The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 is a polypeptide whose function is unknown, but according to the Censored Domain Retrieval Tool of National Center for Biotechnology Information, the 86th to 186th amino acid residues are located from the N-terminal side. Is disclosed as a heat shock factor (HSF) -type DNA-binding domain. The HSF-type DNA-binding domain is known to have the function of binding to the upstream region of the gene encoding HSF, which is a transcription factor that regulates the expression of heat shock proteins (Cell, 65 (3), 363). -366 (1991)).

A specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 is the base sequence represented by SEQ ID NO: 10. A gene mutation in which the function of EGR45828 is reduced or deleted refers to a total deletion of the HSF-type DNA-binding domain possessed by EGR45828, a partial deletion of the HSF-type DNA-binding domain, and a configurational relationship of the HSF-type DNA-binding domain. The gene mutation which changes is mentioned. Furthermore, the function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 9 can be reduced or deleted by introducing a mutation that reduces or eliminates the expression level of the polypeptide. A specific example in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 is deficient is a mutation causing a frame shift in which one guanine nucleotide is inserted at the 85th position in the nucleotide sequence represented by SEQ ID NO: 10. To be

Compared with Trichoderma reesei, in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 is reduced or deleted due to the decrease or loss of the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9, Protein productivity is improved.

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is a polypeptide possessed by Trichoderma reesei, and is also registered as a predicated protein (EGR47155) possessed by the Trichoderma reesei QM6a strain in the National Center for Biotechnology Information. . The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is a polypeptide of unknown function, but according to the Censored Domain Retrieval Tool of National Center for Biotechnology Information, the 362nd to 553rd amino acids from the N-terminal side are left. The group is disclosed as the TLD domain. The TLD domain has an unknown function. A specific example of the gene encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is the base sequence represented by SEQ ID NO: 12. Examples of the gene mutation in which the function of EGR47155 is reduced or deleted include a total deletion of the TLD domain possessed by EGR47155, a partial deletion of the TLD domain, and a gene mutation in which the configurational relationship of the TLD domain is changed. Furthermore, the function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11 can be reduced or deleted by introducing a mutation that reduces or eliminates the expression level of the polypeptide. A specific example in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is lacking is a frame in which 46 bases represented by SEQ ID NO: 13 are inserted at the 6th position in the base sequence represented by SEQ ID NO: 12. Examples include shift mutations.

Compared to Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is not reduced or deleted due to the decrease or loss of the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11, Protein productivity is improved.

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is a polypeptide possessed by Trichoderma reesei, and is also registered as a predicated protein (EGR48056) possessed by the Trichoderma reesei QM6a strain in National Center for Biotechnology Information. . The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is a polypeptide whose function is unknown, but according to the Censored Domain Retrieval Tool of National Center for Biotechnology Information, the remaining 130 to 172 amino acids from the N-terminal side. The group is disclosed as the F-box domain. The F-box domain is known to be a domain found in proteins that control the cell cycle (Proc. Natl. Acad. Sci., 95, 2417-2422 (1998)). A specific example of the gene encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is the base sequence represented by SEQ ID NO: 15. Examples of the gene mutation in which the function of EGR48056 is reduced or deleted include a total deletion of the F-box domain possessed by EGR48056, a partial deletion of the F-box domain, and a gene mutation in which the configurational relationship of the F-box domain is changed. Furthermore, the function of the polypeptide can be reduced or deleted by introducing a mutation that reduces or eliminates the expression level of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14. A specific example in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is lacking is a frameshift mutation in which one base of cytosine at the 499th position in the nucleotide sequence represented by SEQ ID NO: 15 is deleted. Compared with Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is not reduced or deleted due to the decrease or deletion of the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14, Protein productivity is improved.

The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16 is a polypeptide possessed by Trichoderma reesei, and in the National Center for Biotechnology Information, the glycosyltransferase family4 (76), which is also registered as triglycerylferaseferase76, glycotransferase family of Trichoderma reesei QM6a strain is registered in National Center for Biotechnology Information. Has been done. Glycosyltransferase family 41 is a protein (The EMBO Journal, 27, 2080-2788 (2008)) that is composed of a dimeric complex, and N in the process of nascent protein immediately after translation passing through the Golgi complex. -It has a function of transferring acetylgalactosamine (GalNAc) to a serine or threonine residue which is an amino acid residue (Biochemistry, Fourth edition, 11,280-281 (1995)). A specific example of the gene encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16 is the nucleotide sequence represented by SEQ ID NO: 17.

A gene mutation in which the function of EGR46476 is reduced or deleted is a total deletion of glycosyltransferase family 41, partial which EGR46476 has, a partial deletion of glycosyltranferase family mutated 41, a partial deletion of glycosyltranferase family mutated, and a partial deletion of glyceryl ferrycosylate. Can be mentioned. Furthermore, the function of the polypeptide can be reduced or deleted by introducing a mutation that reduces or eliminates the expression level of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16. As a specific example in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16 is deficient, a stop codon is inserted by mutating cytosine at position 6261 to adenine in the nucleotide sequence represented by SEQ ID NO: 17. Mutations are included. Compared with Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16 is not reduced or deleted due to the decrease or loss of the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16, Protein productivity is improved.

The present invention also relates to a method for producing a protein, which comprises a step of culturing the mutant strain.

The culture method is not particularly limited, and for example, liquid culture using a centrifuge tube, flask, jar fermenter, tank or the like, or solid culture using a plate or the like can be used. Trichoderma reesei is preferably cultivated under aerobic conditions, and among these culturing methods, a jar fermenter and a submerged culturing in which a tank is aerated and agitated are particularly preferable. The ventilation amount is preferably about 0.1 to 2.0 vvm, more preferably 0.3 to 1.5 vvm, and particularly preferably 0.5 to 1.0 vvm. The culture temperature is preferably about 25 to 35 ° C, more preferably 25 to 31 ° C. The pH condition in the culture is preferably pH 3.0 to 7.0, more preferably pH 4.0 to 6.0. The culturing time is carried out under the condition that the protein is produced until a recoverable amount of the protein is accumulated. Usually, it is 24 to 288 hours, preferably 24 to 240 hours, more preferably 36 to 240 hours, and further preferably 36 to 192 hours.

The protein produced in the present invention is not particularly limited, but it is possible to efficiently produce a protein secreted outside the cell, and among them, preferably an enzyme, more preferably cellulase, amylase, invertase, chitinase, pectinase. And the like, and more preferably cellulase.

The cellulase produced in the present invention contains various hydrolases, including enzymes having a degrading activity for xylan, cellulose, and hemicellulose. Specific examples are cellobiose hydrolase (EC 3.2.1.91), which produces cellobiose by hydrolysis of cellulose chains, and endoglucanase (EC 3.2.1.4, which hydrolyzes from the central part of the cellulose chain). ), Cellooligosaccharides and β-glucosidases that hydrolyze cellobiose (EC 3.2.1.21), xylanases (EC 3.2.1.8) characterized by acting on hemicellulose and especially xylan, xylooligosaccharides Examples include β-xylosidase (EC 3.2.1.37) that hydrolyzes.

The fact that the Trichoderma reesei mutant strain of the present invention has improved protein production ability as compared with the parent strain and that the specific activity of cellulase is improved is obtained by culturing the mutant strain and the parent strain under the same conditions. The resulting culture solution is compared with the protein concentration, the specific activity of β-glucosidase, the specific activity of β-xylosidase, and the specific activity of cellobiohydrolase, which are measured by the following methods, by comparison with one or more of them. Confirm.

The protein concentration was determined by diluting the supernatant obtained by centrifuging the cultures of the mutant strain and the parent strain at 15,000 × g for 10 minutes, and diluting them to 250 μL of Quick Start Bradford protein assay (Bio-Rad). After adding 5 μL of the supernatant and allowing it to stand at room temperature for 15 minutes, the absorbance used at 595 nm is measured. Using the bovine serum albumin solution as a standard solution, the concentration of protein contained in the saccharifying enzyme solution is calculated based on the calibration curve.

The β-glucosidase specific activity was determined by adding 10 μL of the enzyme diluent to 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside (manufactured by Sigma-Aldrich Japan) at 30 React at 10 ° C for 10 minutes. Next, 10 μL of 2 M sodium carbonate is added and mixed well to stop the reaction, and the increase in absorbance at 405 nm is measured. Finally, the specific activity is calculated with the activity of liberating 1 μmol of p-nitrophenol per minute as 1 U.

The β-xylosidase specific activity was obtained by adding 10 μL of the enzyme diluent to 90 μL of a 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside (manufactured by Sigma-Aldrich Japan) at 30 ° C. Then, the reaction is stopped by adding 10 μL of 2 M sodium carbonate and mixing well, and the increase in absorbance at 405 nm is measured. Finally, the specific activity is calculated with the activity of liberating 1 μmol of p-nitrophenol per minute as 1 U.

The cellobiohydrolase specific activity was calculated by adding 10 μL of the enzyme diluent to 90 μL of 50 mM acetate buffer containing 1 mM of p-nitrophenyl-β-lactopyranoside (manufactured by Sigma-Aldrich Japan) in the culture supernatant. The reaction is allowed to proceed at 60 ° C. for 60 minutes, then 10 μL of 2 M sodium carbonate is added and mixed well to stop the reaction, and an increase in absorbance at 405 nm is measured. Finally, the specific activity is calculated with the activity of liberating 1 μmol of p-nitrophenol per minute as 1 U. The composition of the medium used in the step of culturing the mutant strain of the present invention is not particularly limited as long as it has a medium composition that allows Trichoderma reesei to produce a protein, and a known medium composition of Trichoderma spp. it can. As the nitrogen source, for example, polypeptone, gravy, CSL, soybean meal and the like can be used. In addition, an inducer for producing a protein may be added to the medium.

When the cellulase is produced according to the present invention, it can be cultured in a medium containing at least one or two or more inducers selected from the group consisting of lactose, cellulose and xylan. Further, as the cellulose or xylan, biomass containing cellulose or xylan may be added as an inducer. Specific examples of the biomass containing cellulose or xylan include seed plants, ferns, moss plants, algae, plants such as aquatic plants, and waste building materials. Seed plants are classified into gymnosperms and angiosperms, and both can be preferably used. Angiosperms are further classified into monocotyledonous plants and dicotyledonous plants, and specific examples of monocotyledonous plants include bagasse, switchgrass, napiergrass, Elianthus, corn stover, corncob, rice straw, and straw. As specific examples of the dicotyledon, beet pulp, eucalyptus, oak, birch, etc. are preferably used.

Also, as the biomass containing cellulose or xylan, pretreated one may be used. The pretreatment method is not particularly limited, but known methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, fine pulverization treatment, and steam treatment can be used. Pulp may be used as the biomass containing cellulose or xylan that has been subjected to such pretreatment.

The method for recovering the protein contained in the culture solution in which the mutant strain is cultured is not particularly limited, but the protein can be recovered by removing the bacterial cells of the mutant strain from the culture solution. Examples of the method for removing the bacterial cells include a centrifugal separation method, a membrane separation method, a filter press method and the like.

Further, when the mutant is cultivated, the bacterial cells are not removed from the culture solution, and when it is used as a protein solution, it is preferable to treat the mutant strain of Trichoderma reesei so that it cannot grow. . Examples of the method for treating the cells so that they cannot grow include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.

When the protein is an enzyme, the culture solution from which the bacterial cells have been removed or treated so as not to grow as described above can be directly used as an enzyme solution.

If the protein to be produced is cellulase, the cellulase can be used to saccharify the cellulose-containing biomass to produce sugar. In addition, the cellulase obtained by culturing the mutant strain has a particularly high specific activity of β-glucosidase as compared with the cellulase obtained by culturing the parent strain before the introduction of the mutation, and thus efficiently decomposes the cellulose-containing biomass. Thus, a sugar solution having a high glucose concentration can be obtained, and more sugar can be obtained.

As the cellulose-containing biomass used in the present invention, the same biomass as the cellulose-containing biomass described as the above-mentioned inducer or a pretreated biomass can be used.

The conditions for the saccharification reaction are not particularly limited, but the temperature for the saccharification reaction is preferably in the range of 25 to 60 ° C, and more preferably in the range of 30 to 55 ° C. The saccharification reaction time is preferably in the range of 2 hours to 200 hours. The pH of the saccharification reaction is preferably in the range of pH 3.0 to 7.0, and more preferably in the range of pH 4.0 to 6.0. In the case of cellulase derived from Trichoderma, the optimum pH for the reaction is 5.0. Furthermore, since the pH changes during the hydrolysis process, it is preferable to add a buffer solution to the reaction solution or to carry out the reaction while maintaining a constant pH using an acid or an alkali.

When the enzyme is separated and collected from the saccharified solution, the saccharified solution can be filtered through an ultrafiltration membrane or the like and collected on the non-permeate side. If necessary, the solid content can be collected from the saccharified solution as a pre-filtration step. It may be removed. The recovered enzyme can be used again in the saccharification reaction.

The present invention will be specifically described below with reference to examples.

<Reference Example 1> Protein concentration measurement conditions Protein concentration measurement reagent used: Quick Start Bradford protein assay, manufactured by Bio-Rad Measurement conditions Measurement temperature: room temperature Protein concentration measurement reagent: 250 μL
Filamentous culture medium: 5 μL
Reaction time: 5 minutes Absorbance: 595 nm
Standard product: BSA.

<Reference Example 2> Cellulase specific activity measurement conditions (β-glucosidase specific activity measurement conditions)
Substrate: p-nitrophenyl-β-glucopyranoside (manufactured by Sigma-Aldrich Japan)
Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside
Enzyme diluent: 10 μL
Reaction temperature: 30 ° C
Reaction time: 10 minutes Reaction terminator: 2M sodium carbonate 10 μL
Absorbance: 405 nm.

(Conditions for measuring β-xylosidase specific activity)
Substrate: p-nitrophenyl-β-xylopyranoside (manufactured by Sigma-Aldrich Japan)
Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside
Enzyme diluent: 10 μL
Reaction temperature: 30 ° C
Reaction time: 10 minutes Reaction terminator: 2M sodium carbonate 10 μL
Absorbance: 405 nm.

(Conditions for measuring cellobiohydrolase specific activity)
Substrate: p-nitrophenyl-β-lactopyranoside (manufactured by Sigma-Aldrich Japan)
Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-lactopyranoside
Enzyme diluent: 10 μL
Reaction temperature: 30 ° C
Reaction time: 10 minutes Reaction terminator: 2M sodium carbonate 10 μL
Absorbance: 405 nm.

<Reference Example 3> Saccharification test of cellulose-containing biomass As the cellulose-containing biomass, Arbocel (registered trademark) B800 (manufactured by Rettenmeyer) or bagasse powdered to an average particle size of 100 μm was used. As the enzyme solution, 1 mL of the culture solution of Trichoderma reesei or Trichoderma reesei mutant strain was collected, centrifuged, the supernatant from which the bacterial cells were removed was collected, and the filtrate filtered with a 0.22 μm filter was used. I was there.

(Saccharification reaction)
100 μL of 1 M sodium acetate buffer as a buffer for saccharification reaction, 2 μL of 50 g / L erythromycin solution as a preventive against the growth of various bacteria, Arbocel (registered trademark) B800 (manufactured by Rettenmeier Co., Ltd.) or powdered to an average particle size of 100 μm 0.1 g of each bagasse was used. Further, the enzyme solution obtained by the flask culture using Arbocel (registered trademark) B800 is 450 μL when Arbocel (registered trademark) B800 is the saccharification target, and the powdered bagasse is the saccharification target. Was used at 400 μL. The enzyme solution obtained by the flask culture using lactose was added in an amount of 350 μL when Arbocel (registered trademark) B800 was used as the saccharification target, and 400 μL when powdered bagasse was the saccharification target, so that the total amount was 1 mL. A 2 mL tube was filled with sterile water. The saccharification reaction is performed at a temperature of 50 ° C. for 24 hours, the supernatant obtained by centrifuging the saccharified product is recovered as a saccharification solution, and 1/10 amount of 1N NaOH solution of the recovered saccharification solution is added to conduct an enzymatic reaction Stopped. The glucose concentration in the saccharified solution after the reaction was stopped was measured by UPLC shown below.

(Measurement of glucose concentration)
Glucose was quantitatively analyzed using the ACQUITY UPLC system (Waters) under the following conditions. Quantitative analysis was performed based on a calibration curve prepared from a glucose standard.
Column: AQUITY UPLC BEH Amide 1.7 μm 2.1 × 100 mm Column
Separation method: HILIC
Mobile phase: Mobile phase A: 80% acetonitrile, 0.2% TEA aqueous solution, mobile phase B: 30% acetonitrile, 0.2% TEA aqueous solution, and the following gradient was followed. The gradient was a linear gradient that reached the mixing ratio corresponding to the time below.
Starting condition: (A99.90%, B0.10%), 2 minutes after the start: (A96.70%, B3.30%), 3.5 minutes after the start: (A95.00%, B5.00%) 3.55 minutes after the start: (A99.90%, B0.10%), and 6 minutes after the start: (A99.90%, B0.10%).
Detection method: ELSD (evaporative light scattering detector)
Flow rate: 0.3 mL / min
Temperature: 55 ° C.

<Example 1> Preparation of mutant strain of Trichoderma reesei deficient in polypeptide comprising amino acid sequence represented by SEQ ID NO: 2 Trichoderma deficient in function of polypeptide comprising amino acid sequence represented by SEQ ID NO: 2 The mutant strain of Resei is an acetamidase gene capable of degrading the gene represented by SEQ ID NO: 1 encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 as a selection marker and acetamide as a selection marker gene. It is destroyed by replacing it with (amdS). In order to delete the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a DNA fragment consisting of the gene sequence represented by SEQ ID NO: 18 was prepared, and the DNA fragment was transformed into Trichoderma reesei QM9414 strain. A mutant strain of Trichoderma reesei lacking the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is prepared. By this method, a mutant strain of Trichoderma reesei lacking the nucleotide sequence represented by SEQ ID NO: 1 can be obtained. In order to introduce a DNA fragment consisting of the nucleotide sequence represented by SEQ ID NO: 1 above and downstream of the amdS-containing DNA sequence, a portion homologous to the gene sequence of Trichoderma reesei QM9414 strain should be added. Create a mutation-introducing plasmid.

Specifically, PCR was performed using genomic DNA extracted from Trichoderma reesei QM9414 strain according to a standard method and oligo DNAs represented by SEQ ID NOs: 19 and 20, and the obtained amplified fragment was treated with restriction enzymes PacI and NotI. Let the DNA fragment be an upstream DNA fragment. PCR is carried out using the oligo DNAs represented by SEQ ID NOs: 21 and 22, and the obtained amplified fragment is treated with restriction enzymes MluI and SpeI to obtain a downstream DNA fragment. Then, the upstream and downstream DNA fragments are introduced into the plasmid into which amdS has been inserted by using the restriction enzymes PacI and NotI, and MluI and SpeI, respectively, to construct a mutation-introducing plasmid. Then, the mutation-introducing plasmid is treated with restriction enzymes PacI and SpeI, and Trichoderma reesei QM9414 strain is transformed with the obtained DNA fragment represented by SEQ ID NO: 18. The molecular biological method is performed as described in Molecular cloning, laboratory manual, 1st, 2nd, 3rd (1989). Further, the transformation is carried out by using the standard method, protoplast-PEG method, specifically, as described in Gene, 61, 165-176 (1987).

(Preparation and evaluation of mutants)
The mutant strain of Trichoderma reesei obtained according to the method described above was used as Trichoderma reesei mutant strain I in the following protein production test and experiments for protein concentration and cellulase specific activity measurement.

<Example 2> Culture test of Trichoderma reesei mutant strain (pre-culture)
The spores of the Trichoderma reesei mutant strain prepared in Example 1 were diluted with physiological saline to a concentration of 1.0 × 10 ⁷ / mL, and 2.5 mL of the diluted spore solution was attached to a 1 L baffle shown in Table 1. 250 mL of the preculture medium placed in the flask is inoculated and cultured for 72 hours at 28 ° C. and 120 rpm in a shaking culture machine. As a control, Trichoderma reesei QM9414 strain is used, and the same experiment operation is performed below.

(Main culture)
Arbocel B800 (Rettenmeyer) is added to the main culture medium shown in Table 2 and a 5 L jar fermenter (manufactured by Biot) is used for deep culture examination.

Inoculate 2.5 mL of the main culture medium supplemented with Arbocel B800 with 200 mL of the preculture liquid of the Trichoderma reesei QM9414 strain and the mutant strain of Trichoderma reesei prepared in Example 1.

Regarding the culture conditions, after inoculating the main culture medium with the pre-culture medium, deep culture is performed under the culture conditions of 28 ° C., 700 rpm, and aeration rate of 100 mL / min while controlling the pH to 5.0.

(Collection of culture solution)
After 120 hours from the start of culturing, 20 mL of each culture solution is collected. A part of the collected culture solution is centrifuged for 10 minutes at 15,000 × g and 4 ° C. to obtain a supernatant. The supernatant is filtered through a 0.22 μm filter, and the filtrate is used as a cellulase solution in the following experiment.

(Measurement of protein concentration)
Under the conditions of Reference Example 1, the protein concentration in the culture solution collected 120 hours after the start of culture is measured. As a result, the protein concentration in the culture broth of the mutant strain of Trichoderma reesei lacking the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was compared with that in the culture broth of the Trichoderma reesei QM9414 strain. Get higher

(Measurement of enzyme activity)
Under the conditions of Reference Example 2, the specific activities of β-glucosidase, β-xylosidase, and cellobiohydrolase are measured as the specific activities of cellulase in the culture solution collected 120 hours after the start of the culture. The specific activity is calculated by measuring the increase in absorbance at 405 nm and assuming 1 U as the activity of releasing 1 μmol of substrate per minute. As a result, the three types of specific activities in the culture solution of the mutant strain of Trichoderma reesei lacking the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 were found to be the specific activities in the culture solution of Trichoderma reesei QM9414 strain. Will be higher than

(Flask culture)
The spores of Trichoderma reesei mutant strain I prepared in Example 1 were diluted with physiological saline to a concentration of 1.0 × 10 ⁷ / mL, and 0.1 mL of the diluted spore solution was added to Arbocel (Table 3). (Registered trademark) B800 (Rettenmeyer) or lactose was inoculated into a 10 mL flask medium placed in a 50 mL baffled flask and cultured for 120 hours at 28 ° C. and 120 rpm in a shaking culture machine.

Also, the parent strain of the mutant strain I, the parent strain of Trichoderma reesei QM9414, was cultured for 120 hours according to the above method as a comparison target.

(Collection of culture solution)
120 hours after the start of flask culture, 1 mL of culture solution was collected. The culture was centrifuged at 15,000 xg and 4 ° C for 10 minutes to obtain a supernatant. The supernatant was filtered with a 0.22 μm filter, and the filtrate was used in various experiments described below.

(Measurement of protein concentration)
In the culture using Arbocel (registered trademark) B800, when the protein concentration contained in the culture medium in which the Trichoderma reesei QM9414 strain was cultured was 1, the relative value of the protein concentration contained in the culture medium of Trichoderma reesei mutant I Was 1.2, and it was confirmed that the protein-producing ability of the mutant strain was higher than that of the parent strain.

Even when culturing with lactose, if the protein concentration contained in the culture solution of Trichoderma reesei QM9414 strain was set to 1, the relative value of the protein concentration contained in the culture solution of Trichoderma reesei mutant I was 1.2. Yes, it was confirmed that the protein-producing ability of the mutant strain was improved over that of the parent strain.

(Measurement of various specific activities of cellulase)
In the culture using Arbocel (registered trademark) B800, when various specific activities of cellulase in the culture solution in which the Trichoderma reesei QM9414 strain was cultivated were set to 1, various ratios of the cellulase in the culture solution in which the Trichoderma reesei mutant strain I was cultured were set. The relative activities were β-glucosidase specific activity of 1.4, β-xylosidase specific activity of 1.5, and cellobiohydrolase specific activity of 1.8, which is an unexpected improvement of various specific activities of cellulase. It was confirmed that the effect of was obtained.

Even when culturing with lactose, when the various specific activities of cellulase in the culture solution in which the Trichoderma reesei QM9414 strain was cultivated were set to 1, the relative values of the various specific activities of the cellulase in the culture solution in which the Trichoderma reesei mutant strain I was cultivated were , Β-glucosidase specific activity is 1.4, β-xylosidase specific activity is 2.0, cellobiohydrolase specific activity is 1.6, and unexpected effects that various specific activities of cellulase are improved can be obtained. It was confirmed.

(Saccharification reaction test)
According to the method described in Reference Example 3, the saccharification reaction test of the cellulose-containing biomass was performed using the culture solution of Trichoderma reesei mutant strain I 120 hours after the start of the flask culture as cellulase. Arbocel® B800 or powdered bagasse was used as the cellulose-containing biomass.

As a result, in the saccharification reaction when Arbocel (registered trademark) B800 was used as the saccharification target, the cellulase of Trichoderma reesei QM9414 strain obtained by performing flask culture using Arbocel (registered trademark) B800 was used. When the glucose concentration contained in the saccharified solution is set to 1, the relative glucose concentration value of the saccharified solution when using the cellulase of Trichoderma reesei mutant I is 1.8, and it was obtained by flask culture using lactose. The relative value of the glucose concentration contained in the saccharified solution when using the cellulase was also 1.6.

In the saccharification reaction when powdered bagasse was used as the saccharification target, the glucose concentration contained in the saccharification solution when the cellulase of Trichoderma reesei QM9414 strain was used, which was obtained by performing flask culture using Arbocel (registered trademark) B800. When the cellulase of Trichoderma reesei mutant I is used, the relative value of the glucose concentration of the saccharified solution is 1.2, and the cellulase obtained by culturing the flask with lactose is used. The relative value of the glucose concentration contained in the saccharified solution was also 1.3.

From these results, it was confirmed that the cellulase produced by Trichoderma reesei mutant strain I has a higher enzymatic activity than the cellulase produced by the parent strain, and as a result, the ability to produce glucose from the cellulose-containing biomass is superior.

<Example 3> Preparation of mutant strain of Trichoderma reesei having a mutation in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2
The QM9414-E strain, which is a passage strain of the Trichoderma reesei QM9414 strain, was subjected to gene mutation treatment to obtain the mutant strain QM9141-F. For the gene mutation treatment, spores of the QM9414-E strain were inoculated so as to give 1.0 × 10 ⁵ spores per mL of the preculture medium shown in Table 1, 15 mL of the preculture medium was cultured for half a day, and then centrifuged to recover the spores. did. Then, the collected spores were suspended in Tris-maleic acid buffer (pH 6.0) to give a 10 mL spore solution, and dissolved therein with Tris-maleic acid buffer (pH 6.0) to 1.0 g / L. 0.5 mL of the prepared NTG solution was added, and gene mutation treatment was performed at 28 ° C. for 100 minutes. The spores subjected to gene mutation treatment were recovered by centrifugation, washed with Tris-maleic acid buffer (pH 6.0) three times, and finally suspended in 10 mL of Tris-maleic acid buffer (pH 6.0). The spores were treated with gene mutation.

The gene-mutated spores were added to an agar medium prepared by adding crystalline cellulose, and the size of halo, which is a region for degrading crystalline cellulose by cellulase generated around the colony, was used as an index, and the QM9414-F strain having a large halo was identified. Selected.

Genetic analysis of the QM9414-E and QM9414-F strains revealed that the QM9414-E strain retained the nucleotide sequence represented by SEQ ID NO: 1, but the QM9414-F strain represented by SEQ ID NO: 1. The 2588th cytosine in the nucleotide sequence was mutated to thymine. The mutation is a mutation for mutating the 677th serine residue of the amino acid sequence represented by SEQ ID NO: 2 encoded by the base sequence represented by SEQ ID NO: 1 into a phenylalanine residue.

Example 4 Protein production test using a mutant strain of Trichoderma reesei lacking the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (measurement of protein concentration and various specific activities of cellulase)
The QM9414-F strain obtained in Example 3 was cultured in the same manner as in Example 2, and under the conditions of Reference Example 1 and Reference Example 2, the protein concentration and cellulase in the culture medium 120 hours after the start of the culture. Was measured for specific activity. The QM9414-E strain was used as a control. The results are shown in Table 3.

As a result, the QM9414-F strain had a 2.6 times higher protein concentration in the culture solution than the QM9414-E strain. In addition, various specific activities of the QM9414-F strain were 3.1 times higher for β-glucosidase, 1.5 times higher for β-xylosidase, and 2.0 times higher for cellobiohydrolase than the QM9414-E strain. It was

(Saccharification reaction test)
According to the method described in Reference Example 3, the saccharification reaction test of the cellulose-containing biomass was performed using the culture solution 120 hours after the start of the culture of the Trichoderma reesei QM9414-F strain as cellulase. Arbocel® B800 or powdered bagasse was used as the cellulose-containing biomass.

As a result, when the glucose concentration contained in the saccharified solution using Trichoderma reesei QM9414-E cellulase was set to 1, the glucose concentration of the saccharified solution using Trichoderma reesei QM9414-F cellulase was determined. The relative value was 1.1 when Arbocel (registered trademark) B800 was the saccharification target, and 1.2 in the saccharification reaction when powdered bagasse was the saccharification target.

From these results, it was confirmed that the cellulase produced by the Trichoderma reesei QM9414-E strain has a higher enzymatic activity than the cellulase produced by the parent strain, and thus has an excellent ability to produce glucose from the cellulose-containing biomass.

Claims

A mutant strain of Trichoderma reesei, which has a mutation in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is deleted or reduced.
2. The mutation according to claim 1, wherein the mutation is a deletion, substitution or addition mutation of an amino acid residue in the fungal transcription factor regulatory regulatory middle region domain of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2. Mutant strain.
The mutant strain according to claim 1 or 2, wherein the mutation is a mutation at the 677th serine residue from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 to an amino acid residue other than serine.
A method for producing a protein, comprising a step of culturing the mutant strain according to any one of claims 1 to 3.
A method for producing cellulase, comprising the step of culturing the mutant strain according to any one of claims 1 to 3.
A method for producing sugar, which comprises the step of producing cellulase by the method for producing cellulase according to claim 5 and the step of saccharifying a cellulose-containing biomass using the cellulase obtained in the step.