Mutated filamentous fungus, and method for producing protein using the same

Abstract

To control protein productivity in filamentous fungi.SOLUTION: A mutated filamentous fungus has enhanced or inhibited expression of a gene consisting of a nucleotide sequence of SEQ ID NO: 1 or a gene corresponding to the same.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Technologies have been developed to decompose cellulosic biomass to produce sugar, and then to produce useful resources such as biofuels from the sugar. Cellulosic biomass is composed mainly of cellulose, hemicellulose, and lignin, and saccharification enzymes capable of decomposing cellulose and hemicellulose, such as cellulase and xylanase, are used to decompose it.

[0003] As a producer of saccharifying enzymes, filamentous fungi such as Trichoderma have attracted attention. In particular, Trichoderma is useful as a producer of enzymes for biomass decomposition because it can simultaneously produce cellulase and xylanase. Patent Document 1 discloses that a culture obtained by culturing a filamentous fungal mutant in which tubulin function has been reduced or lost in the presence of a cellulase inducer is used as a biomass saccharifying agent. Patent Document 2 discloses a method for producing cellulase and / or xylanase using a filamentous fungal mutant in which Sre1 expression has been reduced or lost compared to the parent strain. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2018 / 025929 [Patent Document 2] International Publication No. 2017 / 018471 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention relates to the regulation of protein productivity in filamentous fungi. The present invention also relates to a mutant filamentous fungus in which protein productivity is controlled, and a method for producing a protein using the mutant filamentous fungus. [Means for solving the problem]

[0006] The present inventors have discovered a new gene involved in the control of protein productivity in filamentous fungi. By regulating the expression of this gene, it is possible to control the protein productivity of filamentous fungi.

[0007] Thus, the present invention provides a mutant filamentous fungus in which expression of a gene consisting of the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 98% identity thereto is enhanced or suppressed. The present invention also provides a method for producing a protein, which comprises culturing the mutant filamentous fungus. The present invention further provides a method for producing a mutant filamentous fungus, which comprises enhancing or suppressing expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 98% identity thereto, in a host filamentous fungus. The present invention further provides a method for reducing or improving protein productivity in a filamentous fungus, comprising enhancing or suppressing expression of a gene consisting of the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 98% identity thereto in a host filamentous fungus. Effect of the Invention

[0008] The present invention provides a newly discovered gene involved in the control of protein productivity in filamentous fungi, and a new technology for controlling protein productivity in filamentous fungi by regulating the expression of the gene. Based on the technology, the present invention provides mutant filamentous fungi with improved or reduced protein productivity. The mutant filamentous fungi with improved protein productivity are useful for protein production. [Brief description of the drawings]

[0009] [Figure 1]Improved protein production in mutant filamentous fungi. The dashed line with black circles indicates PCΔ32755, and the solid line with black circles indicates its parent strain, PC-3-7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In the present specification, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing an analysis using a homology analysis program of the genetic information processing software GENETY Ver. 12 with a unit size to compare (ktup) of 2.

[0011] As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 98.5% or more, even more preferably 99% or more, and even more preferably 99.5% or more.

[0012] As used herein, "an amino acid sequence in which one or several amino acids have been deleted, substituted, added, or inserted" refers to an amino acid sequence in which preferably one to ten, more preferably one to five, and even more preferably one to three amino acids have been deleted, substituted, added, or inserted. Furthermore, as used herein, "a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted" refers to a nucleotide sequence in which preferably one to thirty, more preferably one to fifteen, and even more preferably one to nine nucleotides have been deleted, substituted, added, or inserted. As used herein, "addition" of an amino acid or nucleotide includes addition of an amino acid or nucleotide to one or both ends of a sequence.

[0013] As used herein, "upstream" and "downstream" in relation to a gene refer to the upstream and downstream in the transcription direction of the gene. For example, the "upstream sequence" and "downstream sequence" of a gene refer to the sequences located on the 3' and 5' sides of the gene on the DNA sense strand, respectively.

[0014] As used herein, the term "operably linked" between a regulatory region and a gene refers to a state in which the gene and the regulatory region are linked such that the gene can be expressed under the control of the regulatory region. Procedures for "operably linked" between a gene and a regulatory region are well known to those skilled in the art.

[0015] The present invention relates to a gene that has been newly found to be involved in the control of protein productivity in filamentous fungi, and to the control of protein productivity in filamentous fungi using said gene.

[0016] The gene used in the present invention for regulating protein productivity of a filamentous fungus (hereinafter also referred to as "the gene of the present invention") is a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a gene equivalent thereto. The gene consisting of the nucleotide sequence of SEQ ID NO: 1 is a gene encoding an amino acid sequence (SEQ ID NO: 2) identified as Protein iD: 32755 in the database JGI Genome Portal Trichoderma reesei v2.0 ([mycocosm.jgi.doe.gov / Trire2 / Trire2.home.html]) and as uncharacterized protein TRIREDRAFT_32755 in the NCBI Protein database ([www.ncbi.nlm.nih.gov / protein]). The amino acid sequence of SEQ ID NO: 2 is predicted to have the function of a transcriptional regulator.

[0017] An example of a "gene corresponding to" the gene consisting of the nucleotide sequence of SEQ ID NO: 1 used in the present invention is a gene consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 1 and functioning as a transcriptional regulator. Alternatively, a gene having a very high sequence identity with the gene consisting of the nucleotide sequence of SEQ ID NO: 1, for example, a gene consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added or inserted with respect to the nucleotide sequence of SEQ ID NO: 1; and a gene having a sequence identity of 98% or more, preferably 98.5% or more, more preferably 99% or more, and even more preferably 99.5% or more with the nucleotide sequence of SEQ ID NO: 1 are also examples of the "corresponding gene".

[0018] In one aspect, the present invention provides a mutant filamentous fungus in which the expression of the gene of the present invention described above has been modified. More specifically, the mutant filamentous fungus of the present invention has enhanced or suppressed expression of the gene of the present invention. By modifying the expression of the gene of the present invention, the mutant filamentous fungus of the present invention has modified protein productivity. In one embodiment, the mutant filamentous fungus of the present invention has suppressed expression of the gene of the present invention and has improved protein productivity. In another embodiment, the mutant filamentous fungus of the present invention has enhanced expression of the gene of the present invention and has reduced protein productivity.

[0019] As used herein, "enhanced expression" and "suppressed expression" of the gene of the present invention in a mutant filamentous fungus refer to enhanced and suppressed expression of the gene of the present invention in the mutant filamentous fungus, respectively, compared to the parent filamentous fungus of the mutant filamentous fungus (i.e., a host filamentous fungus in which the gene of the present invention is not modified). In a mutant filamentous fungus in which expression of the gene of the present invention is enhanced, the expression of the gene of the present invention is improved compared to the parent filamentous fungus, preferably 110% or more, more preferably 120% or more, and even more preferably 150% or more of the expression level of the parent filamentous fungus. On the other hand, in a mutant filamentous fungus in which expression of the gene of the present invention is suppressed, the expression of the gene of the present invention is reduced compared to the parent filamentous fungus, preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less of the expression level of the parent filamentous fungus. Even more preferably, in a mutant filamentous fungus in which expression of the gene of the present invention is suppressed, expression of the gene of the present invention is lost to an undetectable level (for example, below the expression level of a negative control or background in a gene expression analysis technique described below).

[0020] As used herein, "improved protein productivity" and "reduced protein productivity" in a mutant filamentous fungus refer to an improved and reduced protein production in the mutant filamentous fungus, respectively, compared to its parent filamentous fungus. The protein production by the mutant filamentous fungus of the present invention with improved protein productivity is improved to preferably 103% or more, more preferably 105% or more, and even more preferably 108% or more, compared to the protein production by its parent filamentous fungus. On the other hand, the protein production by the mutant filamentous fungus of the present invention with reduced protein productivity is reduced to preferably 95% or less, more preferably 90% or less, and even more preferably 80% or less, compared to the protein production by its parent filamentous fungus. Preferably, the protein productivity in the present specification is based on the protein production by the filamentous fungus after 2 to 7 days, and more preferably 72 to 120 hours, of culture.

[0021] The type of protein whose productivity is improved or decreased in the mutant filamentous fungus of the present invention is not particularly limited, but is preferably a secretory protein (preferably an enzyme) released outside the cells of the filamentous fungus, more preferably a saccharification enzyme involved in the decomposition of cellulosic biomass, and even more preferably at least one selected from the group consisting of cellulase and xylanase. In this specification, "cellulase" is a general term for enzymes that decompose cellulose, and includes endoglucanase (EC 3.2.1.4) that cleaves cellulose from the inside of the molecule; exoglucanase (cellobiohydrolase, EC 3.2.1.91) that decomposes cellulose from the reducing end or non-reducing end and releases cellobiose; and β-glucosidase (EC 3.2.1.21). In this specification, "xylanase" refers to an enzyme (EC 3.2.1.8) that hydrolyzes the β1-4 bond of xylan to produce xylose.

[0022] The expression level of a gene can be measured by a conventional method such as Northern blotting, quantitative PCR, microarray, etc. The expression level of a protein can be measured by a conventional method such as Western blotting, colorimetry, chromatography, etc.

[0023] The mutant filamentous fungus of the present invention can be produced by enhancing or suppressing the expression of the above-mentioned gene of the present invention in a host filamentous fungus (parent). In one embodiment, the mutant filamentous fungus of the present invention is a mutant filamentous fungus with reduced protein productivity produced by enhancing the expression of the gene of the present invention. In a preferred embodiment, the mutant filamentous fungus of the present invention is a mutant filamentous fungus with improved protein productivity produced by suppressing the expression of the gene of the present invention.

[0024] Means for suppressing gene expression in a host filamentous fungus include deleting or inactivating the gene, including deleting a part or the whole of the sequence of the gene, deleting the promoter region of the gene or inactivating the promoter by introducing a mutation, and the like.

[0025] Another method for suppressing gene expression is to reduce or eliminate the function of the expression product of the gene. Examples of the method for reducing the expression product include translation inhibition of the transcript of the gene by antisense oligonucleotides, siRNA, etc. Examples of the method for eliminating the function of the expression product include inactivation of the expressed peptide (e.g., inhibition of transcription factor function) by substitution or insertion of another sequence or introduction of mutation into the sequence of the gene, or by an inhibitor such as an aptamer or antibody.

[0026] Specific techniques for deleting, substituting, inserting or mutating the above gene sequences include, for example, mutagenesis using chemical mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, nitrous acid, etc., or physical mutagens such as ultraviolet light, X-rays, gamma rays, ion beams, etc., site-directed mutagenesis, and the method described by Dieffenbach et al. (Cold Spring Harbar Laboratory Press, New York, pp. 581-621, 1995). Site-specific mutagenesis techniques include homologous recombination, splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), ODA (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), Kunkel (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488), and genome editing using artificial DNA cleavage enzymes (artificial DNA nucleases or programmable nuclease). Alternatively, the Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio Inc.), Transformer TM Commercially available site-directed mutagenesis kits such as Site-Directed Mutagenesis Kit (Clonetech) and KOD-Plus-Mutagenesis Kit (Toyobo) can also be used.

[0027] On the other hand, means for enhancing the expression of a gene in a host filamentous fungus include a method of introducing a polynucleotide encoding the gene from the outside into the host so that the gene can be expressed; a method of improving the transcription amount of the gene by modifying the control region of the gene on the host genome, and the like. For example, a vector or DNA fragment containing a gene of interest may be introduced into the host filamentous fungus. If necessary, the gene of interest to be introduced may be operably linked to a control region. The control region linked to the gene of interest is a sequence for expressing the introduced gene in the host into which the vector or DNA fragment has been introduced, and examples of the control region include an expression control region such as a promoter or a terminator, and an origin of replication. The type of the control region can be appropriately selected depending on the type of host into which the vector or DNA fragment is introduced. The gene of interest and the control region contained in the vector or DNA fragment may be introduced into the nucleus of the host, or may be introduced into the host genome. Alternatively, the gene of interest contained in the vector or DNA fragment may be introduced into the host genome and operably linked to a high expression promoter on the genome. Alternatively, a vector or DNA fragment containing a high expression promoter may be introduced into a host, and the high expression promoter may be operably linked to a gene of interest on the host genome. Preferably, the vector is an expression vector.

[0028] To introduce a vector or a DNA fragment into a host filamentous fungus, a general transformation method such as electroporation, transformation, transfection, conjugation, protoplast-PEG, particle gun, Agrobacterium, etc. Means for introducing a vector or a DNA fragment into the genome of a host include homologous recombination and genome editing using an artificial DNA cleavage enzyme.

[0029] The host filamentous fungus (parent) of the mutant filamentous fungus of the present invention is not particularly limited as long as it is a filamentous fungus carrying the above-mentioned gene of the present invention. Examples of the host filamentous fungus include filamentous fungi of the genera Trichoderma, Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, and Hypocrea. Preferably, the host filamentous fungus is a filamentous fungus that expresses saccharification enzymes such as cellulase and xylanase, and more preferably, it is a fungus of the genus Trichoderma. Examples of the Trichoderma fungus include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, and Trichoderma viride, of which Trichoderma reesei is preferred, and Trichoderma reesei PC-3-7 strain or a mutant thereof is more preferred.

[0030] Thus, in a preferred embodiment, the mutant filamentous fungus of the present invention is a mutant Trichoderma fungus in which the expression of the above-mentioned gene of the present invention is enhanced or suppressed. In a more preferred embodiment, the mutant filamentous fungus of the present invention is a mutant strain of Trichoderma reesei, preferably Trichoderma reesei PC-3-7 strain, in which the expression of the above-mentioned gene of the present invention is enhanced or suppressed.

[0031] By regulating the expression of the gene of the present invention in a host filamentous fungus by the above-mentioned procedure, a mutant filamentous fungus with improved or reduced protein productivity can be produced. Accordingly, another aspect of the present invention is a method for controlling protein productivity in a filamentous fungus by regulating the expression of the gene of the present invention in a host filamentous fungus. One embodiment of the method is a method for improving protein productivity, which comprises suppressing the expression of the gene of the present invention in a host filamentous fungus. Another embodiment of the method is a method for reducing protein productivity, which comprises enhancing the expression of the gene of the present invention in a host filamentous fungus.

[0032] The mutant filamentous fungus having improved protein productivity according to the present invention is useful in microbiological protein production. Thus, the present invention also provides a method for producing a protein, comprising culturing the mutant filamentous fungus of the present invention having improved protein productivity. The type of target protein produced by this method is not particularly limited, but is preferably a secreted protein (preferably an enzyme), more preferably a saccharification enzyme involved in the decomposition of cellulosic biomass, and even more preferably at least one selected from the group consisting of cellulase and xylanase.

[0033] The medium and culture conditions for culturing the mutant filamentous fungus of the present invention can be appropriately selected depending on the type of host of the mutant filamentous fungus and the type of target protein. In general, the medium and culture conditions usually used for the host of the mutant filamentous fungus can be used.

[0034] The mutant filamentous fungus of the present invention is preferably cultured under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is preferably 10 to 50° C., more preferably 20 to 42° C., and even more preferably 25 to 35° C. The pH of the culture is preferably 3 to 9, and more preferably 4 to 5. The culture time is preferably 10 hours to 10 days, and more preferably 2 to 7 days.

[0035] The medium for the culture may be either a synthetic medium or a natural medium, so long as it contains substances necessary for the growth of the mutant filamentous fungus of the present invention, such as a carbon source, a nitrogen source, inorganic salts, and vitamins.

[0036] The carbon source may be any carbon source that can be assimilated by the mutant filamentous fungus of the present invention, and examples thereof include carbohydrates such as glucose, fructose, and cellulase, alcohols such as ethanol and glycerol, and organic acids such as acetic acid. Examples of the nitrogen source include ammonium salts such as ammonia and ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate. These carbon and nitrogen sources can be used alone or in combination. These carbon and nitrogen sources can be added to the medium by any method, such as lump addition (batch method), divided addition (fed-batch method), or continuous addition (feed method).

[0037] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, potassium carbonate, etc. Examples of vitamins include biotin, thiamine, etc. Furthermore, substances required for the growth of the filamentous fungus of the present invention can be added as necessary.

[0038] When the target protein is a saccharifying enzyme involved in the decomposition of cellulosic biomass, such as cellulase or xylanase, it is preferable to culture the mutant filamentous fungus of the present invention in the presence of a cellulase inducer. The cellulase inducer is not limited as long as it induces cellulase production in the cellulase-producing filamentous fungus, and examples of the cellulase inducer include compounds selected from cellulose, sophorose, and cellooligosaccharides such as cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose.

[0039] The cellulose includes polymers in which glucose is polymerized through β-1,4-glucoside bonds and derivatives thereof. The degree of polymerization of glucose is not particularly limited. Examples of derivatives include derivatives obtained by carboxymethylation, aldehyde conversion, esterification, or the like. Furthermore, the cellulose may be a β-glucoside which is a glycoside, lignocellulose which is a complex with lignin and / or hemicellulose, or a complex with pectin or the like. The cellulose may be crystalline cellulose or non-crystalline cellulose.

[0040] The cellulase inducer can be added to the medium by any method, such as adding all at once (batch method), adding in portions (fed-batch method), or adding continuously (feed method). The amount of cellulase inducer added to the medium may be any amount that can induce the production of the target saccharifying enzyme by the mutant filamentous fungus of the present invention, and may vary depending on the method of addition, but may be preferably 0.1 to 40% by mass, more preferably 0.5 to 35% by mass, and even more preferably 1 to 30% by mass in total relative to the medium. For example, when added all at once, the amount added is preferably 0.1 to 16% by mass, more preferably 0.5 to 14% by mass, and even more preferably 1 to 12% by mass.

[0041] The mutant filamentous fungus of the present invention is cultured according to the above procedure to produce a target protein. After culturing, the target protein is recovered from the culture. If necessary, the recovered target protein may be further purified. The method for recovering or purifying the target protein from the culture is not particularly limited, and may be performed according to a known recovery or purification method. For example, the culture is recovered, and if necessary, a bacterial cell disruption treatment is performed using ultrasonic waves or pressure, and then cell components are removed by decantation, filtration, centrifugation, or the like, and the remaining fraction containing the target protein is recovered. If necessary, the recovered fraction may be concentrated and subjected to a method such as crystallization, ion exchange, or solvent extraction, or a combination thereof, to purify the target protein.

[0042] In the protein production method according to the present invention, the mutant filamentous fungus may be cultured and the target protein may be collected by any of a batch, semi-batch or continuous method. For example, when the target protein is a secretory protein, the mutant filamentous fungus after culture can be separated from the culture without disrupting the cells and reused for protein production.

[0043] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed in this specification, but the present invention is not limited to these embodiments.

[0044] [1] A mutant filamentous fungus in which expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a gene equivalent thereto is enhanced or suppressed. [2] Preferably, the gene corresponding to the gene consisting of the nucleotide sequence of SEQ ID NO: 1 is A gene that consists of a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 1 and functions as a transcriptional regulator; A gene having a sequence identity of 98% or more, more preferably 98.5% or more, even more preferably 99% or more, and even more preferably 99.5% or more to a gene consisting of the nucleotide sequence of SEQ ID NO: 1; or A gene consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added or inserted relative to the nucleotide sequence of SEQ ID NO: 1. The mutant filamentous fungus described in [1]. [3] The mutant filamentous fungus according to [1] or [2], preferably in which expression of the gene is suppressed and protein productivity is improved. [4] The mutant filamentous fungus according to [3], wherein the gene is preferably deleted or inactivated. [5] The mutant filamentous fungus according to [3], preferably, wherein the expression product of the gene is reduced or has no function. [6] The mutant filamentous fungus according to [1] or [2], preferably in which expression of the gene is enhanced and protein productivity is reduced. [7] The protein, Preferably it is a secreted protein, More preferably, it is a saccharification enzyme involved in the decomposition of cellulosic biomass, More preferably, it is at least one selected from the group consisting of cellulase and xylanase. The mutant filamentous fungus according to any one of [3] to [6]. [8] The filamentous fungus, Preferably, the fungus is of the genus Trichoderma. More preferably, it is Trichoderma reesei. The mutant filamentous fungus according to any one of [1] to [7].

[0045] [9] A method for producing a protein, comprising culturing the mutant filamentous fungus according to any one of [3] to [5] above.

[10] The protein, Preferably it is a secreted protein, More preferably, it is a saccharification enzyme involved in the decomposition of cellulosic biomass, More preferably, it is at least one selected from the group consisting of cellulase and xylanase. The method described in [9].

[11] The method described in [9] or

[10] , wherein the filamentous fungus is preferably cultured in the presence of a cellulase inducer.

[12] The filamentous fungus, Preferably, the fungus is of the genus Trichoderma. More preferably, it is Trichoderma reesei. The method according to any one of [9] to

[11] .

[13] The method according to any one of [9] to

[12] , further comprising recovering the protein from the culture.

[0046]

[14] A method for producing a mutant filamentous fungus, comprising enhancing or suppressing expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a gene equivalent thereto in a host filamentous fungus.

[15] Preferably, the gene corresponding to the gene consisting of the nucleotide sequence of SEQ ID NO: 1 is A gene that consists of a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 1 and functions as a transcriptional regulator; A gene having a sequence identity of 98% or more, more preferably 98.5% or more, even more preferably 99% or more, and even more preferably 99.5% or more to a gene consisting of the nucleotide sequence of SEQ ID NO: 1; or A gene consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added or inserted relative to the nucleotide sequence of SEQ ID NO: 1. The method described in

[14] .

[16] The method according to

[14] or

[15] , preferably comprising suppressing expression of the gene, and wherein the mutant filamentous fungus has improved protein productivity.

[17] The method according to

[16] , preferably comprising deleting or inactivating the gene.

[18] The method according to

[16] , preferably comprising reducing or eliminating the function of the expression product of the gene.

[19] The method according to

[14] or

[15] , preferably comprising enhancing expression of the gene, and wherein the mutant filamentous fungus has reduced protein productivity.

[20] The protein Preferably it is a secreted protein, More preferably, it is a saccharification enzyme involved in the decomposition of cellulosic biomass, More preferably, it is at least one selected from the group consisting of cellulase and xylanase. The method according to any one of

[16] to

[19] .

[21] The filamentous fungus, Preferably, the fungus is of the genus Trichoderma. More preferably, it is Trichoderma reesei. The method according to any one of

[14] to

[20] .

[0047]

[22] A method for improving protein productivity in a filamentous fungus, comprising suppressing expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a gene equivalent thereto in a host filamentous fungus.

[23] The method according to

[22] , preferably comprising deleting or inactivating the gene.

[24] The method according to

[22] , preferably comprising reducing or eliminating the function of the expression product of the gene.

[25] A method for reducing protein productivity in a filamentous fungus, comprising enhancing expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a gene equivalent thereto in a host filamentous fungus.

[26] Preferably, the gene corresponding to the gene consisting of the nucleotide sequence of SEQ ID NO: 1 is A gene that consists of a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 1 and functions as a transcriptional regulator; A gene having a sequence identity of 98% or more, more preferably 98.5% or more, even more preferably 99% or more, and even more preferably 99.5% or more to a gene consisting of the nucleotide sequence of SEQ ID NO: 1; or A gene consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added or inserted relative to the nucleotide sequence of SEQ ID NO: 1. The method according to any one of

[22] to

[25] .

[27] The protein Preferably it is a secreted protein, More preferably, it is a saccharification enzyme involved in the decomposition of cellulosic biomass, More preferably, it is at least one selected from the group consisting of cellulase and xylanase. The method according to any one of

[22] to

[26] .

[28] The filamentous fungus, Preferably, the fungus is of the genus Trichoderma. More preferably, it is Trichoderma reesei. The method according to any one of

[22] to

[27] . EXAMPLES

[0048] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0049] Example 1: Construction of mutant filamentous fungi (1) Construction of plasmid DNA for gene deletion A gene deletion plasmid was constructed to delete the gene encoding TRIREDRAFT_32755 (hereinafter referred to as the "32755 gene", SEQ ID NO: 1) on the genomic DNA of Trichoderma reesei strain PC-3-7 (ATCC 66589) by homologous recombination with a uridine synthase gene (pyr4) expression cassette.

[0050] Using the genomic DNA of the PC-3-7 strain as a template, the upstream and downstream sequences of the 32755 gene and the pyr4 expression cassette (a fragment containing the promoter, structural gene, and terminator of the pyr4 gene) were amplified by PCR using the primers shown in Table 1. The obtained upstream and downstream sequences and the pyr4 expression cassette were inserted into the HincII restriction enzyme cleavage fragment of pUC118 using Gibson Assembly Master Mix (New England Biolabs) to construct a plasmid for gene deletion.

[0051] The obtained plasmid was used to transform competent cells of E. coli DH5α (Takara Bio). From the ampicillin-resistant transformants obtained, strains carrying the target plasmid were selected based on colony PCR and the restriction enzyme cleavage pattern of the extracted plasmid. The selected transformants were cultured in LB medium supplemented with ampicillin (37°C, overnight). The plasmid was recovered from the obtained cells using a QIAGEN Plasmid Midi Kit (QIAGEN) and purified. The obtained gene-deficient plasmid was named pUCΔ32755-pyr4. pUCΔ32755-pyr4 contains the upstream and downstream sequences of the 32755 gene derived from the genomic DNA of Trichoderma reesei, and a pyr4 expression cassette placed between them.

[0052] [Table 1]

[0053] (2) Creation of gene-deficient mutant fungal strains A uridine-requiring strain of Trichoderma reesei PC-3-7 (ATCC 66589) was transformed with pUCΔ32755-pyr4 constructed in (1) above. The plasmid was introduced by the protoplast-PEG method. Transformants were selected for their ability to synthesize uridine in a synthetic medium that did not contain uridine. From the resulting transformants, candidates for the 32755 gene deletion strain in which pyr4 was inserted at the position of the 32755 gene were selected by colony PCR. From the resulting candidate strains, strains in which only one copy of the pyr4 cassette was introduced at the desired position by double crossover were further selected by Southern analysis. This recombinant strain with one copy of pyr4 was obtained as the 32755 gene deletion mutant filamentous fungal strain (PCΔ32755).

[0054] Example 2 Protein production using mutant filamentous fungi (1) Cultivation of mutant fungal strains The enzyme productivity of the transformants was evaluated by the following culture. For preculture, 50 mL of medium was added to a 500 mL flask, and the 5 Spores of Trichoderma reesei strain PC-3-7 (parent strain) and PCΔ32755 prepared in Example 1 were inoculated at a concentration of spores / mL and cultured at 28°C and 220 rpm with shaking (PRIS PRXYg-98R). The medium composition was as follows: 1% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% hypopeptone N, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% trace elements, 50 mM tartrate buffer (pH 4.0). The composition of the trace elements was as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24·4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2 are mixed with distilled water to make a final volume of 100mL. After pre-cultivation for 2 days, main culture was carried out using a jar fermenter (BMZ manufactured by Biot). The above pre-culture solution was inoculated at 5% (v / v%) and cultured for 5 days. 10% powdered cellulose (KC Flock (registered trademark) W-400G, Nippon Paper Industries Co., Ltd.) was used as a carbon source, and other medium components were as follows: 0.42% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Hypolypeptone N, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element, 0.2% Antifoam PE-L. The jar fermenter settings were as follows: temperature: 28°C, aeration: 0.5vvm, pH 4.5 (adjusted with 5% ammonia water), and agitation speed was constant at 700 rpm. The main culture was carried out for 5 days (120 hours).

[0055] (2) Evaluation of protein productivity The protein concentration in the culture supernatant obtained in (1) was examined. Protein concentration was measured by measuring the absorbance of the culture supernatant at 595 nm using a Bio-Rad Protein Assay (Bio-Rad), and the protein concentration (mg / mL) in the culture supernatant was calculated based on a calibration curve using bovine γ-globulin as the standard protein.

[0056] The results of measuring the protein concentration are shown in Figure 1. As shown in Figure 1, the mutant filamentous fungus PCΔ32755, in which the 32755 gene was deleted, had improved protein production compared to the parent strain (PC-3-7). The improvement in protein production in these mutant filamentous fungi compared to the parent strain was up to 8% in PCΔ32755 (cultured for 72 hours).

Claims

1. A mutant filamentous fungus in which the expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 98% identity thereto is enhanced or suppressed.

2. The mutant filamentous fungus according to claim 1, wherein expression of the gene is suppressed and protein productivity is improved.

3. The mutant filamentous fungus according to claim 2, wherein the gene is deleted or inactivated.

4. The mutant filamentous fungus of claim 1 , wherein expression of the gene is enhanced and protein productivity is reduced.

5. The mutant filamentous fungus according to claim 2, wherein the protein is at least one selected from the group consisting of cellulases and xylanases.

6. The mutant filamentous fungus described in claim 4, wherein the protein is at least one selected from the group consisting of cellulase and xylanase.

7. The mutant filamentous fungus according to any one of claims 1 to 6, wherein the filamentous fungus belongs to the genus Trichoderma.

8. A method for producing a protein, comprising culturing the mutant filamentous fungus according to claim 2.

9. The method according to claim 8, wherein the protein is at least one selected from the group consisting of cellulases and xylanases.

10. The method of claim 8, wherein the filamentous fungus is cultured in the presence of a cellulase inducer.

11. The method according to any one of claims 8 to 10, wherein the filamentous fungus is a fungus of the genus Trichoderma.

12. The method of any one of claims 8 to 10, further comprising recovering the protein from the culture.

13. The method of claim 11, further comprising recovering the protein from the culture.

14. A method for producing a mutant filamentous fungus, comprising enhancing or suppressing expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 98% identity thereto in a host filamentous fungus.

15. The method of claim 14, comprising suppressing expression of the gene, and wherein the mutant filamentous fungus has improved protein productivity.

16. 16. The method of claim 15, comprising deleting or inactivating the gene.

17. 15. The method of claim 14, comprising enhancing expression of the gene, and wherein the mutant filamentous fungus has reduced protein productivity.

18. A method for improving protein productivity in a filamentous fungus, comprising suppressing the expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 98% identity thereto in a host filamentous fungus.

19. 19. The method of claim 18, comprising deleting or inactivating the gene.

20. A method for reducing protein productivity in a filamentous fungus, comprising enhancing expression of a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 98% identity thereto in a host filamentous fungus.

21. The method according to any one of claims 15 to 20, wherein the protein is at least one selected from the group consisting of cellulase and xylanase.

22. The method according to any one of claims 14 to 20, wherein the filamentous fungus is a fungus of the genus Trichoderma.

23. The method described in claim 21, wherein the filamentous fungus is a Trichoderma fungus.

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