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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/34—Sugars
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/885—Trichoderma

Definitions

• the present invention relates to a Trichoderma reesei mutant strain having improved protein-producing ability and a method for producing a protein using the mutant strain.
• Trichoderma reesei is known to have a high protein-producing ability, and the production of proteins using Trichoderma reesei has been studied so far. Trichoderma reesei has excellent ability to produce cellulase, which is particularly classified as a saccharifying enzyme among proteins. For example, in order to further improve the amount of cellulase production, overexpression or deletion of a factor that controls cellulase production, or cellulase production The study of culture conditions has been carried out.
• Non-Patent Document 1 a mutant of Trichoderma reesei having a high cellulase-producing ability is obtained by reducing the function of Cre1, which is a transcription factor that suppresses the production of cellulase, among the factors that control the production of cellulase of Trichoderma reesei. Have been.
• Non-Patent Document 2 describes a method of culturing Trichoderma reesei in a medium to which glucose or lactose is added as a method for improving the production of Trichoderma reesei cellulase.
• the transcription factor that is one of the factors controlling protein production of Trichoderma reesei has been elucidated, but this is considered to be only a part of the control mechanism. Therefore, in the present invention, a novel mechanism for controlling the production of Trichoderma reesei protein is searched for, a mutant strain of Trichoderma reesei with further enhanced protein production ability is obtained, and a method for producing a protein using the mutant strain of Trichoderma reesei is provided. The task is to
• the present inventor thought that if a gene capable of increasing the production amount of a protein, which had not been known before, could be identified, the production amount of Trichoderma reesei protein could be further improved.
• culturing a mutant strain of Trichoderma reesei in which the function of one or more polypeptides selected from the polypeptides consisting of the amino acid sequences represented by any of Nos. have found that they can be improved, and have completed the present invention.
• the present invention includes the following (1) to (15).
• (1) A mutant strain of Trichoderma reesei having a mutation in which the function of a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 4 to 6 is deleted or reduced.
• (2) The mutant strain according to (1), wherein the mutation is a mutation in which the HSF-type DNA-binding domain of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is deleted.
• the mutant strain according to (2), wherein the mutation is a frameshift mutation accompanying a mutation in a region N-terminal to the HSF-type DNA-binding domain.
• the mutation according to (6), wherein the mutation is a frameshift mutation caused by mutating the third glutamine residue from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 5 to an amino acid residue other than glutamine. Mutants.
• a method for producing cellulase comprising a step of culturing the mutant strain according to any one of (1) to (10) in a medium containing at least lactose.
• a method for producing sugar comprising a step of producing cellulase by the method of producing cellulase according to (13) or (14) and a step of saccharifying cellulose-containing biomass using the cellulase obtained in the step.
• the mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 4 to 6 has been deleted or reduced has improved protein production ability as compared to the parent strain before the introduction of the mutation.
• 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 increasing the protein-producing ability by introducing a mutation into a parent strain of Trichoderma reesei, a microorganism originally excellent in protein-producing ability.
• the present invention relates to a mutant strain of Trichoderma reesei, which is characterized in that it has a mutation in which the function of a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 4 to 6 is deleted or reduced. I have.
• the parent strain of Trichoderma reesei used in the present invention is not limited to a wild-type strain, and a mutant strain that has been improved so as to enhance protein production ability can also be preferably used as a parent strain.
• mutation treatment with a mutagen or ultraviolet irradiation can be used.
• the mutant strain with improved protein productivity can be used as the parent strain.
• Specific examples of the mutant strain used as the parent strain include known mutant strains belonging to Trichoderma reesei, QM6a strain (NBRC31326), QM9123 strain (ATCC24449), QM9414 strain (NBRC31329), PC-3-7 strain (ATCC66589), and QM9123.
• NBRC31327 RutC-30 strain (ATCC56765), CL-847 strain (Enzyme.Microbiol.Technol. 10, 341-346 (1988)), MCG77 strain (Biotechnol. Bioeng. Symp. 8, 89 (1978)). And MCG80 strain (Biotechnol. Bioeng. 12, 451-459 (1982)).
• the QM6a strain, QM9414 strain and QM9123 strain can be obtained from NBRC (NITE Biological Resource Center), and PC-3-7 strain and 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: 4 is a polypeptide belonging to Trichoderma reesei, and in National Center Center for Biotechnology Information, is also registered as a predicated R45 protein in Trichoderma reesei strain QM6a (Reg. .
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is a polypeptide of which function is unknown, but according to the National Center for Biotechnology Information, Censored Domain, Architecture, Retriever, Toolbox, the 86th to 186th amino acid residues from the N-terminal side.
• the group is disclosed as a heat ⁇ shock ⁇ factor (HSF) -type ⁇ DNA-binding domain.
• HSF heat ⁇ shock ⁇ factor
• HSF-type @ DNA-binding domain has a function of binding to the upstream region of a gene encoding HSF which is a transcription factor that controls the expression of heat shock protein (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: 4 includes the base sequence represented by SEQ ID NO: 1.
• Methods for deficient or reducing the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 include a total deletion of the HSF-type DNA-binding domain, a partial deletion of the HSF-type DNA-binding domain, And a method for introducing a mutation that causes a total deletion of the polypeptide consisting of the amino acid sequence represented by the following. Specifically, a gene sequence encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 And a method of introducing a frameshift mutation or a stop codon mutation by deletion, insertion, substitution or the like of a base.
• ⁇ HSF-type ⁇ Deletion of a DNA-binding domain refers to the loss of all domains, the loss of some domains, all changes to different amino acids, some changes to different amino acids, or a combination thereof. More specifically, it indicates that the amino acid sequence represented by SEQ ID NO: 4 has a sequence identity of 80% or less with the amino acid sequence of the HSF-type DNA-binding domain shown above, and preferably 50% or less. Yes, more preferably 20% or less, further preferably 10% or less, more preferably 5% or less, further preferably 3% or less, further preferably 1% or less, and most preferably 0%.
• Mutation such as deletion, substitution, or addition in the amino acid sequence located in the HSF-type @ DNA-binding domain results in loss or reduction of the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4.
• Examples include mutations in the amino acid sequence represented by SEQ ID NO: 4 in which a part or the entirety of the 86th to 186th region from the N-terminal side corresponding to the HSF-type DNA-binding domain is deleted. .
• a specific example of such a mutation includes a mutation that causes a frame shift in which one base of guanine is inserted at the 85th position in the base sequence represented by SEQ ID NO: 1, and the mutation is represented by SEQ ID NO: 4
• the 30th amino acid residue from the N-terminal side of the amino acid sequence changes from histidine to threonine, and as a result of the frame shift, translation ends at the 90th amino acid residue from the N-terminal side, and HSF-typeHDNA-binding.
• the amino acid sequence constituting the domain disappears.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is a polypeptide belonging to Trichoderma reesei, and in National Center for Biotechnology Information, is also registered as a predicated EG5 strain in Trichoderma reesei strain QM6a as a registered trademark of EG47 strain.
• 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 National Center for Biotechnology Information, Censured Domain, Architecture, Retriever, Tool, the 362nd to 553rd amino acids from the N-terminal to the 553rd residue from the N-terminal side.
• the group is disclosed as a TLD domain.
• the TLD domain is of unknown function.
• a specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 includes a base sequence represented by SEQ ID NO: 2.
• Methods for reducing the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 include a total deletion of the TLD domain, a partial deletion of the TLD domain, and a total deletion of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5.
• a method for introducing a mutation that causes deletion is mentioned. Specifically, a gene sequence encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is deleted, inserted, or substituted with a base. Examples include a method of introducing a frameshift mutation or a stop codon mutation.
• ⁇ ⁇ Deficiency in a TLD domain refers to the loss of all, part of the domain, all changes to different amino acids, some changes to different amino acids, or a combination thereof. More specifically, it indicates that the amino acid sequence represented by SEQ ID NO: 5 has a sequence identity of 80% or less with the amino acid sequence of the TLD domain shown above, preferably 50% or less, and more preferably 50% or less. It is 20% or less, more preferably 10% or less, more preferably 5% or less, further preferably 3% or less, further preferably 1% or less, and most preferably 0%.
• polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is lost by mutation, such as deletion, substitution, or addition, occurring in the amino acid sequence located in the TLD domain.
• mutation in which the region at positions 362 to 553 from the N-terminal side corresponding to the TLD domain is partially or entirely deleted.
• a specific example of such a mutation is a frameshift mutation in which 46 bases represented by SEQ ID NO: 27 are inserted into the base sequence represented by SEQ ID NO: 2 at the sixth position. Due to the mutation, the third glutamine residue from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 5 is changed to arginine, translation ends at that position, and the amino acid sequence constituting the TLD domain disappears.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is a polypeptide belonging to Trichoderma reesei, and is registered in National Center for Biotechnology Information as a predicated PG4 strain, which is also a registered strain of Trichoderma reesei QM6a as a registered strain of QM6a.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is a polypeptide of unknown function, but according to the National Center for Biotechnology Information, Censured Domain, Architecture, Retriever, Toolle, the 130th to 172nd amino acid residues from the N-terminal side. The group is disclosed as an 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 a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 includes a base sequence represented by SEQ ID NO: 3.
• Methods for reducing the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 include the entire deletion of the F-box domain, the partial deletion of the F-box domain, and the amino acid sequence represented by SEQ ID NO: 6.
• a method for introducing a mutation that causes a total deletion of the polypeptide is mentioned. Specifically, a base deletion or insertion into a gene sequence encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 And a method of introducing a frameshift mutation or a stop codon mutation by substitution or the like.
• Deletion of the F-box domain refers to the loss of all, part of the domain, all changes to different amino acids, some changes to different amino acids, or a combination thereof. More specifically, it indicates that the amino acid sequence represented by SEQ ID NO: 6 has a sequence identity of 80% or less, and preferably 50% or less, with the amino acid sequence of the F-box domain shown above. It is preferably 20% or less, more preferably 10% or less, further preferably 5% or less, more preferably 3% or less, further preferably 1% or less, and most preferably 0% or less. is there.
• Specific examples in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is lost due to mutation, such as deletion, substitution, or addition, occurring in the amino acid sequence located in the F-box domain include: In the base sequence represented by No. 3, there is a frameshift mutation in which the 499th cytosine is deleted by one base. Due to the mutation, the 167th amino acid residue from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 6 is changed from alanine to arginine, and thereafter, as a result of the frame shift, translation ends at the 193rd position from the N-terminal side. Therefore, the amino acid sequence constituting the F-box domain disappears.
• the function of the polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 4 to 6 may be reduced by reducing the expression level of the polypeptide or introducing a mutation that eliminates the expression. Specifically, 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 any of SEQ ID NOs: 4 to 6.
• a promoter and a terminator region correspond to a region of several hundred bases before and after a gene involved in transcription.
• Mutation into the above-mentioned gene can be performed by using a known gene mutation method, such as mutation treatment using a mutagen or ultraviolet irradiation known to those skilled in the art, gene recombination such as homologous recombination using a selection marker, or mutation using a transposon. Can be used.
• the mutant strain of the present invention may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 to 6 as long as at least one or more of the polypeptides has a defective or reduced function. All functions may be missing or reduced.
• the combination of polypeptides whose function is deficient or reduced is not particularly limited.
• mutant strain of Trichoderma reesei whose function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5 is deficient or reduced
• SEQ ID NO: 4 or 6 A mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented is deficient or reduced, and a mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 or 6 is deficient or reduced. Both are included in the mutant strain of the present invention.
• the mutant strain of the present invention may be a mutant strain in which the functions of all three polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 4 to 6 are deleted or reduced. Mutants in which all functions of the polypeptide consisting of the amino acid sequences represented by SEQ ID NOs: 4 to 6 have been deleted or reduced are compared with the parent strain Trichoderma reesei spores by nitrosoguanidine (NTG) and ethyl methanesulfonic acid. (EMS), by performing a gene mutation treatment using ultraviolet light or the like, analyzing the gene of the obtained mutant, and screening for a mutant having the above mutation.
• NTG nitrosoguanidine
• EMS ethyl methanesulfonic acid.
• the mutant strain of the present invention Since the mutant strain of the present invention has improved protein-producing ability as compared to the parent strain before the introduction of the mutation, the culture solution of the mutant strain of the present invention is obtained under the same culture conditions before the introduction of the mutation.
• the protein concentration is increased as compared to the culture of the parent strain.
• the protein is an enzyme, the specific activity of the enzyme increases.
• the rate of increase in the protein concentration or the rate of increase in the specific activity of the enzyme is not particularly limited as long as it increases, but is preferably 20% or more.
• the mutant strain of the present invention may be a protein having the amino acid sequence of SEQ ID NOs: 4 to 6, in addition to a mutation in which the function is deficient or reduced, as well as an increase in protein production and / or a decrease in the viscosity of the culture solution. You may have the mutation which suppresses the fall of dissolved oxygen saturation. Specifically, a gene mutation that reduces the function of the polypeptide represented by any one of SEQ ID NOs: 7, 9, and 11 can be given.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7 is a polypeptide belonging to Trichoderma reesei, and in National Center for Biotechnology Information, is a registered PG50 strain of Trimoderma reesei strain QM6a, which is registered as a predicated R50 strain in the strain QM6a.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7 is a polypeptide of unknown function, but according to the National Center for Biotechnology Information, Censated Domain, Architecture, Retriever, Toolbox, the 95th to 277th amino acid residues from the N-terminal side.
• MIF4G domain ⁇ eukaryotic ⁇ initiation ⁇ factor ⁇ 4G domain
• MA-3 domain ⁇ eukaryotic ⁇ initiation ⁇ factor ⁇ 4G domain
• MIF4G and MA are disclosed.
• -3 are known to have a function of binding to DNA or RNA (Bi Chem .. 44, 12265-12272 (2005), Mol. Cell. Biol. 1, 147-156 (2007) .
• a specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7 includes the base sequence represented by SEQ ID NO: 8.
• the gene mutation that reduces the function of EGR50654 is defined as a total deletion of the MIF4G domain and / or the MA-3 domain of EGR50654, a partial deletion of the MIF4G domain and / or the MA-3 domain, and a difference between the MIF4G domain and the MA-3 domain.
• the function of the polypeptide can also be reduced 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: 7.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 is a polypeptide belonging to Trichoderma reesei, and in National Center Center for Biotechnology Information, is a registered EGRP4 strain which is a registered strain of Trimoderma reesei strain QM6a and which is registered as a pre-registered EG44 strain of EG44.
• 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 National Center for Biotechnology Information, Censured Domain, Architecture, Retriever, Toolbox, the 26-th to 499-th amino acids from the N-terminal residue.
• the group is disclosed as having a "Sugar (and other) @Transporter domain.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 is capable of transporting sugar at least between the inside and outside of the bacterial body. It is presumed to be involved.
• a specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 includes the base sequence represented by SEQ ID NO: 10.
• the gene mutation that reduces the function of EGR44419 is defined as a total deletion of the Sugar (and other) @Transporter domain, a partial deletion of the Sugar (and @ other) @Transporter domain, and a change in the configuration of the Sugar (and @ other) @Transporter domain of EGR44419.
• the function of the polypeptide can also be reduced 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: 9.
• 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 in which 11 bases are inserted at the 1415th position in the base sequence represented by SEQ ID NO: 10.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is a polypeptide belonging to Trichoderma reesei, and in National Center for Biotechnology Information, is a beta-adaptinRegger which is registered as a beta-adaptinRegulin in the strain of Trichoderma reesei QM6a as a member of the strain QM6a.
• the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 constitutes an adapter protein that forms a vesicle involved in intracellular and extracellular transport that binds 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 the gene encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 includes the base sequence represented by SEQ ID NO: 12.
• the EGR48910 gene mutation includes a mutation of cytosine, which is the 1080th base in the base sequence represented by SEQ ID NO: 12, to adenine.
• the present invention also relates to a method for producing a protein, comprising a step of culturing the mutant strain.
• the ability to produce a protein is further improved by culturing the mutant strain in a medium containing an inducer.
• the inducer that improves the productivity of cellulase include lactose, glucose, cellulose, cellobiose, and xylan. Of these, lactose and / or glucose are preferred, and lactose is particularly preferred.
• the inducer When the inducer is added to the medium, it may be added at any timing, such as at the start of the culture or during the culture, but especially when lactose or glucose is added as the inducer, the protein is produced by the addition during the culture. This is preferable because the effect of improving the ability can be continued during the culture.
• the amount of lactose added is preferably about 1 g to 50 g per liter of culture solution per day, more preferably about 3 to 25 g, and particularly preferably about 6 to 20 g.
• the amount of added glucose is preferably about 1 to 200 g, more preferably about 5 to 100 g, particularly preferably about 20 to 80 g per liter of the culture solution per day.
• biomass containing them may be added as an inducer.
• biomass include plants such as seed plants, fern plants, moss plants, algae, and aquatic plants, as well as waste building materials.
• Seed plants are classified into gymnosperms and angiosperms, both of which can be preferably used.
• Angiosperms are further classified into monocotyledons and dicotyledons, and specific examples of monocotyledons include bagasse, switchgrass, napier grass, Elianthus, corn stover, corn cob, rice straw, straw, and the like.
• dicotyledonous plants beet pulp, eucalyptus, oak, birch and the like are preferably used.
• a pretreated one may be used.
• the pretreatment method is not particularly limited, and for example, a known method such as an acid treatment, a sulfuric acid treatment, a dilute sulfuric acid treatment, an alkali treatment, a hydrothermal treatment, a subcritical treatment, a fine pulverizing treatment, and a steaming treatment can be used. Pulp may be used as biomass containing pretreated cellulose and xylan.
• the composition of the medium used in the step of culturing the mutant strain of Trichoderma reesei of the present invention is not particularly limited as long as the medium composition allows Trichoderma reesei to produce a protein, and a well-known medium composition of filamentous fungi of the genus Trichoderma is employed. can do.
• a well-known medium composition of filamentous fungi of the genus Trichoderma is employed. can do.
• the nitrogen source for example, polypeptone, broth, CSL, soybean meal and the like can be used.
• the above-mentioned inducer for producing a protein may be added to the medium.
• the culture method is not particularly limited, and for example, the culture can be performed by liquid culture using a centrifuge tube, flask, jar fermenter, tank, or the like, or solid culture using a plate or the like.
• Trichoderma reesei is preferably cultured under aerobic conditions, and among these culture methods, a jar fermenter or a deep submerged culture in which aeration and stirring are performed in a tank are particularly preferable.
• the ventilation rate 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 cultivation 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, and more preferably pH 4.0 to 6.0.
• the cultivation is performed under conditions that produce the protein 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, even more preferably 36 to 192 hours.
• the protein produced by the present invention is not particularly limited, but can efficiently produce a protein secreted outside the cells, and is preferably an enzyme, more preferably a cellulase, an amylase, an invertase, a chitinase, and a pectinase. And more preferably cellulase.
• the cellulase produced by the present invention contains various hydrolases, including enzymes having a decomposing activity on xylan, cellulose, and hemicellulose.
• hydrolases including enzymes having a decomposing activity on xylan, cellulose, and hemicellulose.
• cellobiohydrolase EC 3.2.1.91
• endoglucanase EC43.2.1.4
• ⁇ -glucosidase which hydrolyzes cellooligosaccharides and cellobiose
• xylanase characterized by acting on hemicellulose and particularly xylan
• xylooligosaccharide ⁇ -xylosidase which hydrolyzes
• Trichoderma reesei mutant of the present invention has improved protein-producing ability and improved cellulase specific activity compared to the parent strain can be obtained by culturing the mutant strain and the parent strain under the same conditions. The culture was confirmed by comparing the protein concentration measured by the following method and the specific activity of at least one selected from the group consisting of ⁇ -glucosidase specific activity, ⁇ -xylosidase specific activity and cellobiohydrolase specific activity. I do.
• the protein concentration was determined by centrifuging the cultures of the mutant strain and the parent strain at 15,000 ⁇ g for 10 minutes, and adding a cellulase solution diluted with 250 ⁇ L of Quick Start Protein Bradford Protein Assay (Bio-Rad) to the supernatant. Add 5 ⁇ L and measure the absorbance at 595 nm after standing at room temperature for 15 minutes. Using the bovine serum albumin solution as a standard solution, the concentration of the protein contained in the saccharifying enzyme solution is calculated based on the calibration curve.
• the ⁇ -glucosidase specific activity was determined by adding 10 ⁇ L of the enzyme diluent to 90 ⁇ L of 50 mM acetate buffer containing 1 mM p-nitrophenyl- ⁇ -glucopyranoside (Sigma-Aldrich Japan) to the culture supernatant. The reaction was allowed to proceed at 10 ° C. for 10 minutes, then 10 ⁇ L of 2M sodium carbonate was added and mixed well to stop the reaction, and the increase in absorbance at 405 nm was measured. Finally, the specific activity is calculated based on the activity of releasing 1 ⁇ mol of p-nitrophenol per minute as 1 U.
• ⁇ -xylosidase specific activity was determined by adding 10 ⁇ L of enzyme diluent to 90 ⁇ L of 50 mM acetate buffer containing 1 mM p-nitrophenyl- ⁇ -xylopyranoside (manufactured by Sigma-Aldrich Japan) to the culture supernatant. For 30 minutes, then add 10 ⁇ L of 2M sodium carbonate and mix well to stop the reaction, and measure the increase in absorbance at 405 nm. Finally, the specific activity is calculated based on the activity of releasing 1 ⁇ mol of p-nitrophenol per minute as 1 U.
• the specific activity of cellobiohydrolase was determined by adding 10 ⁇ L of enzyme diluent to 90 ⁇ L of 50 mM acetate buffer containing 1 mM p-nitrophenyl- ⁇ -lactopyranoside (Sigma-Aldrich Japan) to the culture supernatant. Reaction was carried out at 60 ° C. for 60 minutes, then 10 ⁇ L of 2M sodium carbonate was added and mixed well to stop the reaction, and the increase in absorbance at 405 nm was measured. Finally, the specific activity is calculated based on the activity of releasing 1 ⁇ mol of p-nitrophenol per minute as 1 U.
• the method of 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 cells of the mutant strain from the culture solution.
• Examples of the method for removing cells include a centrifugal separation method, a membrane separation method, a filter press method and the like.
• the mutant is used as a protein lysate without removing the cells from the culture solution in which the mutant has been cultured, it is preferable to treat the mutant so that the mutant cannot grow in the culture.
• the method for treating cells so that they cannot grow include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.
• the culture solution that has been treated so that the cells are not removed or grown as described above can be used as it is as the enzyme solution.
• a sugar can be produced by saccharifying cellulose-containing biomass using the cellulase.
• the cellulase obtained by culturing the mutant strain has a higher specific activity of ⁇ -glucosidase than the cellulase obtained by culturing the parent strain before the introduction of the mutation.
• a sugar solution having a high glucose concentration can be obtained, and more sugar can be obtained.
• the same biomass as the biomass containing cellulose described as the inducer or the pretreated biomass can be used.
• the conditions of the saccharification reaction are not particularly limited, but the temperature of the saccharification reaction is preferably in the range of 25 to 60 ° C, more preferably in the range of 30 to 55 ° C.
• the saccharification reaction time is preferably in the range of 2 to 200 hours.
• the pH of the saccharification reaction is preferably in the range of pH 3.0 to 7.0, more preferably in the range of pH 4.0 to 6.0. In the case of a cellulase derived from the genus Trichoderma, the optimum pH for the reaction is 5.0. is there. Furthermore, since the pH changes during the hydrolysis process, it is preferable to add a buffer to the reaction solution or to carry out the reaction while maintaining a constant pH using an acid or an alkali.
• the saccharified solution can be filtered through an ultrafiltration membrane or the like and collected on the non-permeate side. You may remove it.
• the recovered enzyme can be used again for the saccharification reaction.
• 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 ⁇ L Culture solution of filamentous fungi: 5 ⁇ L Reaction time: 5 minutes Absorbance: 595 nm Standard product: BSA.
• the enzyme solution was added so that the protein concentration was 0.8 mg, and the solution was made up with sterilized water so as to be 1 mL in total, and placed in a 2 mL tube.
• the saccharification reaction was performed at 50 ° C. for 24 hours, the supernatant obtained by centrifuging the saccharified product was collected as a saccharified solution, and 1/10 of a 1N NaOH solution of the collected saccharified solution was added to carry out the enzyme reaction. Stopped.
• the glucose concentration in the saccharified solution after the termination of the reaction was measured by UPLC shown below.
• Glucose was quantitatively analyzed using the ACQUITY UPLC system (Waters) under the following conditions. Quantitative analysis was performed based on a calibration curve prepared using a glucose standard.
• Example 1 Preparation of mutant strain of Trichoderma reesei defective in function of polypeptide consisting of amino acid sequence represented by SEQ ID NO: 4 (method of preparing mutant strain)
• a mutant of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is deficient, has a gene represented by SEQ ID NO: 1 encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 It is destroyed by replacing with acetamide as a selection marker and acetamidase (AmdS) gene (amdS) which can degrade acetamide as a selection marker gene.
• AmdS acetamidase
• a DNA fragment consisting of the gene sequence represented by SEQ ID NO: 13 was prepared, and the DNA fragment was transformed into Trichoderma reesei ATCC 66589 strain.
• a mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is deleted is prepared. According to this method, a mutant of Trichoderma reesei lacking the nucleotide sequence represented by SEQ ID NO: 1 is obtained.
• PCR was performed using genomic DNA extracted from Trichoderma rereei strain QM9414 according to a standard method and oligo DNAs represented by SEQ ID NOs: 14 and 15, and the resulting amplified fragment was treated with restriction enzymes AflII and NotI. The fragment is referred to as an upstream DNA fragment.
• PCR was performed using the oligo DNAs represented by SEQ ID NOs: 16 and 17, and the obtained amplified fragment was treated with restriction enzymes MluI and SpeI to obtain a downstream DNA fragment.
• the upstream and downstream DNA fragments are introduced into the plasmid into which amdS has been inserted using AflII and NotI and MluI and SpeI restriction enzymes, respectively, to construct a mutation-introducing plasmid.
• the mutation-introducing plasmid is treated with restriction enzymes AflII and SpeI, and the obtained DNA fragment represented by SEQ ID NO: 13 is used to transform Trichoderma reesei ATCC 66589 strain.
• the molecular biological technique is performed as described in Molecular Cloning, Laboratory Manual, 1st, 2nd, 3rd (1989). Transformation is performed using the protoplast-PEG method, which is a standard technique, specifically as described in Gene, 61, 165-176 (1987).
• Trichoderma reesei mutant obtained according to the method described above was used as the Trichoderma reesei mutant I in the following protein production tests and experiments for measuring protein concentration and cellulase specific activity.
• Example 2 Preparation of mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is deficient (Method for preparing mutant strain)
• a mutant of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is deficient, has a gene represented by SEQ ID NO: 2 encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 It is destroyed by replacing with acetamide as a selection marker and acetamidase (AmdS) gene (amdS) which can degrade acetamide as a selection marker gene.
• AmdS acetamidase
• 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 ATCC 66589 strain.
• a mutant strain of Trichoderma reesei that lacks the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is prepared. According to this method, a mutant of Trichoderma reesei lacking the nucleotide sequence represented by SEQ ID NO: 2 is obtained.
• a DNA fragment obtained by treating the synthesized DNA fragment represented by SEQ ID NO: 19 with restriction enzymes AflII and NotI is defined as an upstream DNA fragment.
• PCR was performed using genomic DNA extracted from Trichoderma reesei QM9414 strain according to a conventional method and oligo DNAs represented by SEQ ID NOs: 20 and 21, and the resulting amplified fragment was subjected to downstream a DNA fragment treated with restriction enzymes MluI and SpeI. DNA fragments. Then, the upstream and downstream DNA fragments are introduced into the plasmid into which amdS has been inserted using AflII and NotI and MluI and SpeI restriction enzymes, respectively, to construct a mutation-introducing plasmid.
• the plasmid for mutation introduction is treated with restriction enzymes AflII and SpeI, and the resulting DNA fragment represented by SEQ ID NO: 18 is used to transform Trichoderma reesei ATCC 66589 strain.
• the molecular biological technique is performed as described in Molecular Cloning, Laboratory Manual, 1st, 2nd, 3rd (1989). Transformation is performed using the protoplast-PEG method, which is a standard technique, specifically as described in Gene, 61, 165-176 (1987).
• Trichoderma reesei mutant obtained by the above-described method was used as the Trichoderma reesei mutant II in the following protein production tests and experiments for measuring protein concentration and cellulase specific activity.
• Example 3 Preparation of Trichoderma reesei Mutant Deficient in Function of Polypeptide Consisting of Amino Acid Sequence Represented by SEQ ID NO: 6
• a mutant of Trichoderma reesei which lacks the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, has a gene represented by SEQ ID NO: 3 which encodes the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 It is destroyed by replacing with acetamide as a selection marker and acetamidase (AmdS) gene (amdS) which can degrade acetamide as a selection marker gene.
• AmdS acetamidase
• a DNA fragment consisting of the gene sequence represented by SEQ ID NO: 22 was prepared, and the DNA fragment was transformed into Trichoderma reesei ATCC 66589 strain.
• a mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is deficient, is prepared. According to this method, a mutant of Trichoderma reesei lacking the nucleotide sequence represented by SEQ ID NO: 3 is obtained.
• PCR was performed using genomic DNA extracted from Trichoderma rereei strain QM9414 according to a standard method and oligo DNAs represented by SEQ ID NOS: 23 and 24, and the resulting amplified fragment was treated with restriction enzymes AflII and NotI. The fragment is referred to as an upstream DNA fragment.
• PCR was performed using the oligo DNAs represented by SEQ ID NOS: 25 and 26, and the obtained amplified fragment was treated with restriction enzymes MluI and XhoI to obtain a downstream DNA fragment.
• the upstream and downstream DNA fragments are introduced into the plasmid into which amdS has been inserted using AflII and NotI, and MluI and XhoI restriction enzymes, respectively, to construct a mutation-introducing plasmid.
• the plasmid for mutation introduction is treated with restriction enzymes AflII and SpeI, and the obtained DNA fragment represented by SEQ ID NO: 22 is used to transform Trichoderma reesei ATCC 66589 strain.
• the molecular biological technique is performed as described in Molecular Cloning, Laboratory Manual, 1st, 2nd, 3rd (1989). Transformation is performed using the protoplast-PEG method, which is a standard technique, specifically as described in Gene, 61, 165-176 (1987).
• Trichoderma reesei mutant obtained according to the method described above was used as the Trichoderma reesei mutant III in the following protein production tests and in experiments for measuring protein concentration and cellulase specific activity.
• Example 4 Protein production test using Trichoderma reesei mutant (preculture) Each of the spores of the mutant strain of Trichoderma reesei prepared in Examples 1 to 3 was diluted with physiological saline to a concentration of 1.0 ⁇ 10 7 / mL, and 2.5 mL of the diluted spore solution was added to 1 L shown in Table 1. The cells are inoculated into 250 mL of a preculture medium placed in a baffled flask, and cultured in a shaking incubator at 28 ° C. and 120 rpm for 72 hours. Using Trichoderma reesei ATCC 66589 strain as a control, the same experimental operation is performed.
• Arbocel (registered trademark) B800 is added to the main culture medium shown in Table 2, and the submerged culture is examined using a 5 L jar fermenter (Biot).
• the culture conditions after inoculating the preculture medium into the main culture medium, deep culture is performed while controlling the pH to 5.0 under the culture conditions of 28 ° C, 700 rpm, and aeration rate of 100 mL / min.
• Trichoderma reesei ATCC 66589 strain which is the parent strain of the mutant strains I to III before the introduction of the mutation, was cultured for 120 hours in accordance with the above-mentioned method as a comparison target.
• Mutant strain II 1.2
• mutant strain III 1.1
• ⁇ -xylosidase specific activity was 1.1 for both Trichoderma reesei mutants I, II, and III
• cellobiohydrolase specific activity was also: Mutants I, II, and III were 1.1, and it was confirmed that an unexpected effect of improving various specific activities of cellulase was obtained.
• saccharification reaction test According to the method described in Reference Example 3, a saccharification reaction test of cellulose-containing biomass was performed using a culture solution of Trichoderma reesei mutant I, mutant II, and mutant III 120 hours after the start of flask culture as cellulase. Arbocel® B800 or powdered bagasse was used as the cellulose-containing biomass.
• Example 5 Preparation of mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NOS: 4, 5, and 6 was deficient
• the strain QM9414-A which is a transgenic strain of Trichoderma reesei QM9414
• spores of the QM9414-A strain were inoculated so as to have a density of 1.0 ⁇ 10 5 spores per 1 mL of the preculture medium shown in Table 1, and 15 mL of the preculture medium was cultured for half a day, followed by centrifugation. Collected.
• the collected spores are suspended in a trismaleic acid buffer (pH 6.0) so as to form a 10 mL spore solution, and dissolved therein with a trismaleic acid buffer (pH 6.0) so as to be 1.0 g / L.
• a gene mutation treatment was performed at 28 ° C. for 100 minutes.
• the spores subjected to the gene mutation treatment were collected by centrifugation, washed three times with a tris-maleic acid buffer (pH 6.0), and finally suspended in 10 mL of a tris-maleic acid buffer (pH 6.0). The spores were treated with gene mutation.
• the spores treated with the gene mutation are added to an agar medium prepared by adding microcrystalline cellulose, and the halo size of the QM9414-B strain having a large halo is determined using the size of the halo, which is the region of microcrystalline cellulose degradation by cellulase generated around the colony as an index. Was selected.
• Example 6 Protein production test using Trichoderma reesei strain QM9414-B With respect to the mutant strain QM9414-B obtained in Example 5, (pre-culture), (main culture), ( Culture was carried out according to (addition of liquid sugar during main culture) and (collection of culture solution), and the protein concentration was measured under the conditions of Reference Example 1. As a control, the parent strain QM9414-A was used and cultured in the same manner as QM9414-B, and the protein concentration was measured under the conditions of Reference Example 1.
• the relative value of the QM9414-B strain was improved by 1.2 times compared to the QM9414-A strain.
• the protein concentration of the QM9414-A strain did not change compared to the 120 hours after the start of the culture, but the protein concentration of the QM9414-B strain was This was 1.3 times higher than the protein concentration at 120 hours.
• the relative value of the protein concentration of the QM9414-B strain was 1.8 when the protein concentration of the QM9414-A strain at 120 hours from the start of culture was 1, and the protein concentration of the QM9414-A strain at 185 hours after the start of culture was 1. Assuming that the concentration was 1, the relative value of the protein concentration of the QM9414-B strain was 2.7.

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