Filamentous fungal mutant and use thereof

Abstract

To provide a filamentous fungal mutant with reduced inhibition of enzyme production due to glucose, and to provide a method for producing polysaccharide-degrading enzymes, a method for producing sugars from biomass, and a method for saccharification of biomass, employing the filamentous fungus.SOLUTION: A filamentous fungal mutant strain exhibits decreased or lost expression of proteins selected from (a)-(c), relative to the original parent strain: (a) a protein composed of an amino acid sequence represented by SEQ ID NO: 2, (b) a protein composed of an amino acid sequence at least 80% identical to an amino acid sequence represented by SEQ ID NO: 2 and having β-glucosidase activity, and (c) a protein composed of an amino acid sequence, wherein one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 2, and having β-glucosidase activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a filamentous fungus mutant and to the production of a polysaccharide-degrading enzyme using said filamentous fungus. [Background technology]

[0002] Biomass is a renewable organic resource derived from living organisms, excluding fossil fuels. Among them, cellulosic biomass is attracting attention. Technologies are being developed around the world to produce sugar by decomposing cellulose, and then use the sugar to produce useful resources such as substitutes for petroleum resources and biofuels through chemical conversion or fermentation using microorganisms.

[0003] Cellulosic biomass is composed mainly of cellulose, hemicellulose, and lignin. It is known that such biomass is decomposed in a complex manner by the synergistic action of cellulase, which breaks down cellulose, hemicellulase, which breaks down hemicellulose, and xylanase. To effectively utilize cellulosic biomass, it is necessary to develop saccharification enzymes that can decompose cellulose and hemicellulose with high efficiency.

[0004] In order to efficiently break down cellulose into glucose, it is necessary for the various cellulases mentioned above to function in a comprehensive manner. Furthermore, since xylan is the second most abundant polysaccharide in plants after cellulose, filamentous fungi such as Trichoderma, which produce a variety of cellulases and xylanases, have attracted attention as plant polysaccharide decomposers (Non-Patent Document 1).

[0005] In particular, Trichoderma is capable of simultaneously producing cellulase and xylanase, and moreover produces these complex enzymes in large quantities, and therefore has been investigated as a host for cellulase production (Non-Patent Document 2). However, in order to industrially produce cellulase and xylanase using these filamentous fungi, it is necessary to develop techniques for inexpensive mass production and to create even more highly productive strains.

[0006] Generally, microcrystalline cellulose such as Avicel is used for cellulase production, but it is expensive and difficult to use for industrial purposes in terms of cost. In addition, many cellulose substrates are insoluble, and it is desirable to use glucose, which is an inexpensive and soluble carbon source, in terms of the burden on industrial processes. However, it is known that in the culture of filamentous fungi using glucose, productivity decreases or becomes saturated due to a control mechanism called catabolite repression. It is known that globally controlled transcription factors CreA, CreB, CreC, CreD, etc. are involved in catabolite repression in filamentous fungi of the genus Aspergillus (Patent Documents 1 and 2), and it is thought that catabolite repression can be regulated by controlling these factors, but it is thought that glucose inhibition is still insufficient to avoid. In filamentous fungi of the genus Trichoderma, mechanism analysis of catabolite repression is also being carried out (Patent Document 3, Non-Patent Document 3). Patent Document 4 discloses a method for producing cellulase and / or xylanase in which inhibition of enzyme production caused by glucose is suppressed, using a filamentous fungal mutant in which expression of the transcription gene Sre1 of cholesterol synthase group genes is reduced or lost compared to the parent strain. Patent Document 5 and Non-Patent Document 4 disclose methods for producing cellulase and / or xylanase in which inhibition of enzyme production caused by glucose is suppressed, using a filamentous fungal mutant in which tubulin function is reduced or lost.

[0007] On the other hand, Trichoderma reesei has Cel1b, which is a β-glucosidase and belongs to GH (glycoside hydrolase family) 1 (Non-Patent Document 5). Cel1b, together with Cel1a, which also belongs to GH1, functions as an enzyme necessary for cellulase induction in cells. Although the induction of cellulase is delayed when either Cel1a or Cel1b is deficient, the delay is particularly observed when Cel1a is deficient, and it has been reported that the simultaneous deficiency of Cel1a and Cel1b significantly delays cellulase induction or does not induce it at all (Non-Patent Documents 6, 7). However, the details of the relationship between Cel1b and cellulase production in the presence of high glucose concentrations are not clear. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2014-168424 A [Patent Document 2] JP 2015-39349 A [Patent Document 3] Special Publication No. 11-512930 [Patent Document 4] International Publication No. 2017 / 018471 [Patent Document 5] International Publication No. 2018 / 025929 [Non-patent literature]

[0009] [Non-Patent Document 1] Akihiko Kondo, Yoshihiko Amano, Hiroshi Tamaru, "The cutting edge of biomass decomposition enzyme research - with a focus on cellulase and hemicellulase", CMC Publishing, pp. 10-19 [Non-Patent Document 2] Wataru Ogasawara, Yosuke Shida, "Chemistry and Biology" vol.50, The Agricultural Chemical Society of Japan, Vol.50, No.8, pp.592-599, August 2012 [Non-Patent Document 3] Amore A1, Giacobbe S, Faraco V. Curr Genomics. 2013 Jun;14(4):230-49 [Non-Patent Document 4] Shibata N, Kakeshita H, Igarashi K, Takimura Y, Shida Y, Ogasawara W, Koda T, Hasunuma T, Kondo A. Biotechnol Biofuels. 2021 Feb;14(1):39 [Non-Patent Document 5] Guo B, Sato N, Biely P, Amano Y, Nozaki K. Appl Microbiol Biotechnol. 2016 Jun;100(11):4959-68 [Non-Patent Document 6] Zhou Q, Xu J, Kou Y, Lv X, Zhang X, Zhao G, Zhang W, Chen G, Liu W. Eukaryot Cell. 2012 Nov;11(11):1371-81 [Non-Patent Document 7] Xu J, Zhao G, Kou Y, Zhang W, Zhou Q, Chen G, Liu W. Eukaryot Cell. 2014 Aug;13(8):1001-13 Summary of the Invention [Problem to be solved by the invention]

[0010] An objective of the present invention is to construct a filamentous fungal mutant strain in which inhibition of enzyme production caused by glucose is suppressed, and to provide a method for producing a polysaccharide-degrading enzyme, a method for producing sugar from biomass, and a method for saccharification of biomass, using the filamentous fungus. [Means for solving the problem]

[0011] As a result of intensive research aimed at solving the above-mentioned problems, the inventors unexpectedly discovered that in a filamentous fungal mutant strain that has lost the expression of Cel1b, glucose inhibition in cellulase or xylanase production is dramatically suppressed, and that this strain is useful as a filamentous fungus for producing the enzymes, thereby completing the present invention.

[0012] That is, the present invention relates to the following. [1] A filamentous fungal mutant strain in which expression of a protein selected from the following (a) to (c) is reduced or lost compared to that of a parent strain: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:2; (b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity; (c) A protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity. [2] A method for producing cellulase and / or xylanase, comprising the steps of culturing the filamentous fungal mutant strain of [1] above in the presence of a cellulase inducer, producing and accumulating cellulase and / or xylanase in the culture, and recovering the cellulase and / or xylanase from the culture. [3] A method for producing cellulase and / or xylanase, comprising the steps of culturing the filamentous fungal mutant strain described in [1] above in the presence of a cellulase inducer and glucose to produce and accumulate cellulase and / or xylanase in the culture, and recovering the cellulase and / or xylanase from the culture. [4] A method for producing sugar from biomass, comprising culturing the filamentous fungal mutant strain described in [1] above in the presence of a cellulase inducer and using a culture obtained by this culture as a biomass saccharification agent. [5] A method for saccharifying biomass, comprising culturing the filamentous fungal mutant strain described in [1] above in the presence of a cellulase inducer and using a culture obtained as a biomass saccharification agent. Effect of the Invention

[0013] According to the present invention, a filamentous fungus is provided in which inhibition of enzyme production by glucose in the production of cellulase or xylanase is suppressed, and the use of the filamentous fungus makes it possible to produce cellulase and / or xylanase even under culture conditions in which high concentrations of glucose are present. Furthermore, the use of the filamentous fungus makes it possible to saccharify biomass to produce sugar. [Brief description of the drawings]

[0014] [Figure 1] A diagram showing protein productivity when cellulase expression in Trichoderma reesei was induced with a cellulase inducer. The dashed line with black circles indicates the PC-3-7 strain, and the solid line with black circles indicates the PCΔCel1b strain. [Diagram 2] 1 shows the protein productivity of strain PC-3-7 when cultured with cellulase inducer and 10% glucose added, with the dashed line and solid line showing the case with no glucose added. [Diagram 3] A graph showing protein productivity of the PCΔCel1b strain when cultured with cellulase inducer and 10% glucose added. The dashed line with black circles indicates the case without glucose addition, and the solid line with black circles indicates the case with glucose addition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In the present specification, the identity of amino acid sequences and nucleotide sequences is calculated by the Lipman-Pearson method (Lipman, DJ., Pearson. WR.: Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing analysis using the search homology program of genetic information processing software Genetyx-Win (Software Development) with Unit size to compare (ktup) set to 2.

[0016] Unless otherwise defined herein, the term "one or several" used in relation to the deletion, substitution, addition, or insertion of an amino acid or base in an amino acid sequence or base sequence can mean, for example, 1 to 12, preferably 1 to 8, and more preferably 1 to 4. Furthermore, in this specification, "addition" of an amino acid or base includes addition of one or several amino acids or bases to one or both ends of a sequence.

[0017] In this specification, unless otherwise defined, "stringent conditions" for hybridization are conditions that enable confirmation of genes having a nucleotide sequence with a sequence identity of about 80% or more or about 90% or more. Examples of "stringent conditions" include those described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001). Those skilled in the art can appropriately create stringent conditions by adjusting the salt concentration, temperature, etc. of the hybridization solution depending on the nucleotide sequence, concentration, length, etc. of the probe. As an example, the above-mentioned "stringent conditions" are preferably 5×SSC at 70° C. or more, more preferably 5×SSC at 85° C. or more, as hybridization conditions, and preferably 1×SSC at 60° C. or more, more preferably 1×SSC at 73° C. or more, as washing conditions. The above combinations of SSC and temperature conditions are merely examples, and a person skilled in the art would be able to achieve appropriate stringency by appropriately combining the above or other factors that determine hybridization stringency.

[0018] As used herein, the upstream and downstream of a gene refer to the regions following the 5' and 3' sides of the gene or region being considered as a target, respectively. Unless otherwise defined, the upstream and downstream of a gene are not limited to the upstream and downstream regions from the translation start point of the gene.

[0019] <Construction of mutant fungal strains> The filamentous fungal mutant strain of the present invention is a filamentous fungal strain in which expression of a protein selected from the following (a) to (c) (hereinafter also referred to as the protein of the present invention) is reduced or lost as compared to the parent strain: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:2; (b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity; (c) A protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity. The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is a β-glucosidase (EC 3.2.1.21) and is an enzyme belonging to GH (Glycoside hydrolase family) 1. The protein corresponds to Cel1b registered as Entry: Q7Z9M2 in the UniProt database. The identity of the amino acid sequence of the protein with other β-glucosidases is 75% with the β-glucosidase derived from Metarhizium robertsii ARSEF 23 and 74% with the β-glucosidase derived from Aspergillus fischeri NRRL 181.

[0020] In Trichoderma reesei, there are 10 kinds of β-glucosidases, of which two belong to GH1 (Cel1a and Cel1b) and eight belong to GH3. The amino acid sequence identity of Cel1a and Cel1b is 53%. Cel1a and Cel1b function as enzymes necessary for cellulase induction in cells, and the deficiency of either one of them delays the induction of cellulase. However, the deficiency of Cel1a in particular delays the induction of cellulase, and it has been reported that the simultaneous deficiency of Cel1a and Cel1b significantly delays the induction of cellulase or does not induce it at all (Non-Patent Documents 5 and 6). Thus, it was thought that the deficiency of Cel1b would reduce the protein productivity compared to the parent strain, but it was completely unexpected that in a filamentous fungal mutant strain that lost the expression of Cel1b, the protein productivity was not suppressed (Figure 1), and glucose inhibition in cellulase or xylanase production was dramatically suppressed (Figure 3).

[0021] Examples of amino acid sequences having 80% or more identity to the amino acid sequence shown in SEQ ID NO:2 include amino acid sequences having preferably 90% or more identity, more preferably 95% or more identity, more preferably 97%, more preferably 98% or more identity, or more preferably 99% or more identity.

[0022] "Expression" of a protein of the present invention means that a translation product (i.e., protein) is produced from a gene encoding the protein and is localized in a functional state at its site of action. Reduced expression of a protein of the present invention means that, as a result, the amount of the protein present in the cell of a filamentous fungus mutant strain is significantly reduced compared to that in the parent strain. Thus, means for reducing or eliminating expression of a protein of the present invention in a filamentous fungus mutant strain of the present invention include modifications at the gene level, transcription level, post-transcriptional regulation level, translation level, and post-translational modification level.

[0023] "The expression of the protein of the present invention is reduced compared to the parent strain" means that the expression level of the protein in the filamentous fungus is reduced compared to the parent strain, more specifically, the expression level of the protein in the fungus is usually reduced to 50% or less, preferably 20% or less, more preferably 10% or less compared to the parent strain, and thus the activity of the protein is also reduced. Most preferably, the expression level of the protein of the present invention is 0%, i.e., the expression of the protein is lost. The comparison of the expression levels of the proteins of the present invention is carried out based on the expression levels of the proteins. The expression level of the protein of the present invention can be measured by well-known immunological techniques such as Western blotting and immunohistochemical staining.

[0024] A filamentous fungal mutant in which expression of the protein of the present invention is reduced or lost as compared to a parent strain can be obtained by deleting or inactivating a gene encoding the protein on the chromosomal DNA of the parent filamentous fungus (hereinafter also referred to as the gene of the present invention). Here, the gene of the present invention means a DNA consisting of a transcription region including an ORF and a transcriptional regulatory region such as a promoter of the gene. Specific examples of the genes of the present invention include the following: (d) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1; (e) a polynucleotide having a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more identity to the nucleotide sequence shown in SEQ ID NO: 1, and encoding a protein having β-glucosidase activity; (f) a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1 and encodes a protein having β-glucosidase activity; (g) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (h) a polynucleotide encoding a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity; (i) A polynucleotide consisting of an amino acid sequence having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more to the amino acid sequence shown in SEQ ID NO: 2, and encoding a protein having β-glucosidase activity. Here, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1 is the cel1b gene.

[0025] Deletion or inactivation of the gene of the present invention may involve the introduction of a mutation in one or more bases in the base sequence of the gene, i.e., deletion of part or all of the base sequence of the gene, or substitution or insertion of another base sequence for the base sequence (in this case, the amino acid sequence of the protein of the present invention may be the same as or different from that of the parent strain).

[0026] Examples of regions into which a base mutation is introduced include the transcription region of the gene of the present invention and transcription regulatory regions such as the promoter and enhancer (transcription activation region) of the gene, preferably the transcription region.

[0027] An example of the transcriptional regulatory region of the gene of the present invention is a region up to 30 bases upstream from the 5' end of the transcriptional region of the gene on the chromosomal DNA. An example of the transcriptional activation region of the gene of the present invention is a region corresponding to -500 bases to -1000 bases upstream.

[0028] The introduction of base mutations into the transcription region is not limited by the type and number of bases, as long as the mutation reduces or eliminates the expression of the protein of the present invention, but examples of base deletion include deletion of a part of the transcription region of preferably 10 bases or more, more preferably 20 bases or more, even more preferably 100 bases or more, and particularly preferably 200 bases or more, and most preferably the entire transcription region. Examples of base substitution include substitution to introduce a nonsense codon by substituting a base within 150 bases, preferably within 100 bases, more preferably within 50 bases, particularly preferably within 30 bases, and most preferably within 20 bases from the 5' end of the transcription region. Examples of base insertion include adding a DNA fragment of 50 bases or more, preferably 100 bases or more, more preferably 200 bases or more, even more preferably 500 bases or more, and particularly preferably 1 kb or more, immediately after a base within 150 bases, preferably within 100 bases, more preferably within 50 bases, particularly preferably within 30 bases, and most preferably within 20 bases from the 5' end of the transcription region. Preferred embodiments of the addition of bases include the introduction of a drug resistance gene such as a hygromycin resistance gene or an aureobasidin resistance gene, or an auxotrophic gene that the filamentous fungus does not have, such as an acetamidase gene.

[0029] Methods for introducing a base mutation into the gene of the present invention on the chromosomal DNA of a filamentous fungus include, for example, a method using homologous recombination. A typical method using homologous recombination includes inserting a mutant gene in which a base has been deleted, substituted or inserted between the upstream and downstream regions of the gene of the present invention to create a DNA fragment having a drug resistance gene or an auxotrophic gene, and using the DNA fragment to cause homologous recombination at the locus of the gene in a host cell in which a base deletion or the like is to be introduced.

[0030] A specific method using homologous recombination is as follows: i) the DNA fragment for homologous recombination is introduced into a parent strain of filamentous fungus by a conventional method, and then a transformed strain in which the DNA fragment for homologous recombination has been integrated into the chromosomal DNA by homologous recombination is selected using drug resistance or auxotrophy as an indicator; ii) PCR is performed using the chromosomal DNA of the obtained transformed strain as a template. The primers used in this procedure are designed to amplify the site of the deletion, substitution or insertion of bases in the gene. A strain in which the original length of the gene is not amplified but a length reflecting the deletion, substitution or insertion of bases is amplified is selected; and iii) finally, a strain in which the mutant gene has been introduced only at the locus of the chromosomal DNA and not elsewhere can be obtained by Southern analysis.

[0031] Other methods for introducing a base mutation into the gene of the present invention on the chromosomal DNA of a parent strain include methods using bacteriophage or conjugation.

[0032] The filamentous fungal mutant of the present invention can also be obtained by subjecting a parent filamentous fungus to a mutation treatment and then selecting a strain in which the expression of the protein of the present invention is reduced or lost compared to the parent strain. Specific examples of the mutation treatment include treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl nitrosourea, ultraviolet light (New Edition Microorganism Experimental Methods, 1999, pp. 126-134, Kodansha Scientific), and irradiation with radiation. Various alkylating agents and carcinogens can also be used as mutagens.

[0033] Alternatively, the filamentous fungal mutant strain of the present invention can be obtained by genome editing using artificial DNA cleavage enzymes (artificial DNA nucleases or programmable nucleases).

[0034] It is also possible to reduce the expression of the protein of the present invention without introducing a mutation into the gene of the present invention. Examples of such methods include the introduction of a nucleic acid having an activity of degrading the transcription product of the gene encoding the protein, or a nucleic acid that inhibits the translation of the transcription product into a protein. Examples of such nucleic acids include nucleic acids that contain a base sequence complementary or substantially complementary to the base sequence of the mRNA encoding the protein, or a part thereof.

[0035] A base sequence that is substantially complementary to the base sequence of an mRNA encoding the protein of the present invention means a base sequence that has a degree of complementarity that allows it to bind to a target sequence of the mRNA and inhibit its translation under physiological conditions in the target filamentous fungus; specifically, for example, a base sequence that has an identity of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and most preferably about 97% or more in the overlapping region with a base sequence that is completely complementary to the base sequence of the mRNA (i.e., the base sequence of the complementary strand of the mRNA).

[0036] More specifically, examples of the nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA encoding the protein of the present invention include the polynucleotides shown in (d) to (i) above.

[0037] A suitable example of an mRNA encoding the protein of the present invention is an mRNA encoding Cel1b of Trichoderma reesei, which comprises the nucleotide sequence shown in SEQ ID NO:1.

[0038] The term "a portion of a base sequence complementary or substantially complementary to the base sequence of an mRNA encoding the protein of the present invention" refers to any portion that can specifically bind to the mRNA encoding the protein and inhibit translation of the protein from the mRNA, and is not particularly limited in length or position, but from the standpoint of sequence specificity, the portion that is complementary or substantially complementary to the target sequence contains at least 10 bases, preferably about 15 bases or more, and more preferably about 20 bases or more.

[0039] Specifically, preferred examples of nucleic acids containing a base sequence complementary or substantially complementary to the base sequence of mRNA encoding the protein of the present invention or a part thereof include any of the following (j) to (l): (j) antisense RNA against the mRNA encoding the protein of the present invention (k) siRNA (small interfering RNA) against the mRNA encoding the protein of the present invention (l) Ribozyme for mRNA encoding the protein of the present invention

[0040] The parent strain in the present invention is not limited as long as it is a filamentous fungus that expresses the protein of the present invention and has cellulase activity and / or xylanase activity, and examples of the filamentous fungi include filamentous fungi belonging to the phylum Eumycota and Oomycota. Specifically, the filamentous fungi include filamentous fungi of the genera Trichoderma, Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, and Hypocrea, with the filamentous fungi of the genus Trichoderma being preferred.

[0041] Examples of the filamentous fungi of the genus Trichoderma include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, and Trichoderma viride, among which Trichoderma reesei is preferred, and Trichoderma reesei strain PC-3-7 (ATCC 66589) is more preferred.

[0042] The parent filamentous fungus may be a wild-type strain, a strain artificially bred from the wild-type strain, or a mutant strain (mutant) or variant in which the base sequence in the genome has been substituted, added, deleted or modified.

[0043] A suitable example of the filamentous fungal mutant strain of the present invention is a filamentous fungus obtained by deleting the cel1b gene of Trichoderma reesei strain PC-3-7 (ATCC 66589) by homologous recombination to abolish expression of Cel1b, and specifically, Trichoderma reesei PCΔCel1b disclosed in the Examples described below.

[0044] The filamentous fungal mutant strain of the present invention constructed in this manner exhibits reduced inhibition by glucose in the production of cellulase or xylanase compared to the parent strain, due to reduced or absent expression of the protein of the present invention in the fungal cells compared to the parent strain. Therefore, by using the filamentous fungal mutant strain of the present invention, a decrease in cellulase or xylanase productivity can be suppressed even when glucose is present at a high concentration in the medium.

[0045] <Production of cellulase and / or xylanase> Cellulase and / or xylanase can be produced by culturing the filamentous fungal mutant strain of the present invention in the presence of a cellulase inducer, producing and accumulating cellulase and / or xylanase in the culture, and collecting the cellulase and / or xylanase from the culture.

[0046] Here, the "cellulase inducer" is not limited as long as it is a substance that induces cellulase production in cellulase-producing filamentous fungi, and examples thereof include compounds selected from cellulose; sophorose; and cellooligosaccharides such as cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose.

[0047] Here, 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 carboxymethylated, aldehyded, or esterified derivatives. Furthermore, cellulose may be a glycoside β-glucoside, a complex with lignin and / or hemicellulose, or a complex with pectin or the like. Cellulose may be crystalline cellulose or non-crystalline cellulose.

[0048] The cellulase inducer can be added by any method such as lump-sum addition (batch method), divided addition (fed-batch method), or continuous addition (feed method). The amount of cellulase inducer added to the medium may be any amount that can induce cellulase and / or xylanase production by the filamentous fungus of the present invention, and varies depending on the addition method. The total amount of the cellulase inducer is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 1% by mass or more, and is preferably 40% by mass or less, more preferably 35% by mass or less, and more preferably 30% by mass or less, based on the medium. The amount is preferably 0.1 to 40% by mass, more preferably 0.5 to 35% by mass, and more preferably 1 to 30% by mass. Among these, the amount added in the case of adding all at once is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 1% by mass or more, and is preferably 16% by mass or less, more preferably 14% by mass or less, more preferably 12% by mass or less. Also, it is preferably 0.1 to 16% by mass, more preferably 0.5 to 14% by mass, more preferably 1 to 12% by mass.

[0049] The medium used in the method of the present invention may be either a synthetic medium or a natural medium, so long as it contains nutrients necessary for the growth of the filamentous fungus of the present invention and the production of cellulase and / or xylanase, such as a carbon source, a nitrogen source, inorganic salts, and vitamins.

[0050] The carbon source may be any carbon source that can be assimilated by the filamentous fungal mutant of the present invention, and specific examples thereof include the above-mentioned cellulase inducers, as well as carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, organic acids such as acetic acid, etc. These may be used alone or in combination. In the filamentous fungal mutant of the present invention, even when glucose is present in the medium at the start of culture, the productivity of cellulase or xylanase is not suppressed. In this case, the amount of glucose added is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 2.5% by mass or more, and preferably 15% by mass or less, more preferably 12% by mass or less, more preferably 10% by mass or less, based on the medium. Also, it is preferably 0.1 to 15% by mass, more preferably 0.5 to 12% by mass, more preferably 2.5 to 10% by mass. Also, the amount of cellulase inducer and glucose in the medium is preferably 10:1 to 1:4 in mass ratio, more preferably 4:1 to 1:2.

[0051] Examples of the nitrogen source include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate.

[0052] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0053] 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.

[0054] The culture is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is preferably 10° C. or higher, more preferably 20° C. or higher, more preferably 25° C. or higher, and preferably 50° C. or lower, more preferably 42° C. or lower, more preferably 35° C. or lower. Also, the culture temperature is preferably 10 to 50° C., more preferably 20 to 42° C., more preferably 25 to 35° C. The pH during the culture is 3 to 9, preferably 4 to 5. The culture time is 10 hours to 10 days, preferably 2 to 7 days.

[0055] After the cultivation is completed, the culture is recovered, and if necessary, the cells are disrupted by ultrasonication, pressure, or the like, and solid-liquid separation is performed by filtration, centrifugation, or the like, after which cellulase and / or xylanase can be obtained by an appropriate combination of ultrafiltration, salting out, dialysis, chromatography, or the like. The degree of separation and purification is not particularly limited. The culture supernatant or its crudely separated and purified product itself can be used as cellulase and xylanase.

[0056] In the present invention, "cellulase" is a general term for enzymes that degrade cellulose, and includes endoglucanases (EC 3.2.1.4), which cleave cellulose from the inside of the molecule; exoglucanases (cellobiohydrolases, EC 3.2.1.91), which degrade cellulose from the reducing or non-reducing ends and release cellobiose, and β-glucosidases (EC 3.2.1.21). "Xylanase" is an enzyme (EC 3.2.1.8) that hydrolyzes the β1-4 bond of xylan to produce xylose. As used herein, "cellulase and / or xylanase" means at least one selected from the group consisting of cellulase and xylanase.

[0057] The decomposition or saccharification of cellulose or xylan, and the production of monosaccharides using the filamentous fungal mutant of the present invention can be carried out by known methods. That is, the culture obtained by culturing the above-mentioned filamentous fungal mutant strain of the present invention in the presence of a cellulase inducer is used as a biomass saccharification agent, and this is coexisted with a cellulose- or xylan-containing substance (biomass) in an aqueous medium, and heated with stirring or shaking, whereby the biomass can be decomposed or saccharified to produce monosaccharides. As the cellulose or xylan-containing substance, those listed as the cellulase inducers contained in the medium can be used. In the decomposition or saccharification of biomass, the pH and temperature of the reaction solution may be within a range in which cellulase or xylanase is not inactivated. Generally, when the reaction is carried out at normal pressure, the temperature is in the range of 5 to 95°C and the pH is in the range of 1 to 11. The biomass decomposition or saccharification process may be carried out in a batch or continuous manner.

[0058] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A filamentous fungal mutant strain in which expression of a protein selected from the following (a) to (c) is reduced or lost as compared to a parent strain: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:2; (b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity; (c) A protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity. <2> the expression of said protein is lost; <1> A filamentous fungal mutant of. <3> the protein is Ce1b; <1> A filamentous fungal mutant of. <4> The gene encoding the protein has been deleted or inactivated <1> ~ <3> 2. The filamentous fungal mutant strain according to claim 1 . <5> The gene encoding the protein is selected from the group consisting of: <4> Mutant strains of filamentous fungi: (d) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1; (e) a polynucleotide having a base sequence having 80% or more identity to the base sequence shown in SEQ ID NO: 1 and encoding a protein having β-glucosidase activity; (f) a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO:1 and encodes a protein having β-glucosidase activity; (g) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (h) a polynucleotide encoding a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity; (i) A polynucleotide having an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having β-glucosidase activity. <6> The filamentous fungus belongs to the genus Trichoderma. <1> ~ <5> 2. The filamentous fungal mutant strain according to claim 1 . <7> The filamentous fungus is Trichoderma reesei, <1> ~ <5> 2. The filamentous fungal mutant strain according to claim 1 . <8> <1> ~ <7> A method for producing cellulase and / or xylanase, comprising the steps of culturing a filamentous fungal mutant strain described in any one of claims 1 to 5 in the presence of a cellulase inducer to produce and accumulate cellulase and / or xylanase in the culture, and recovering the cellulase and / or xylanase from the culture. <9> <1> ~ <7> A method for producing cellulase and / or xylanase, comprising the steps of culturing a filamentous fungal mutant strain described in any one of claims 1 to 5 in the presence of a cellulase inducer and glucose to produce and accumulate cellulase and / or xylanase in the culture, and recovering the cellulase and / or xylanase from the culture. <10> The medium contains a cellulase inducer in a total amount of 0.1 to 40% by mass, preferably 0.5 to 35% by mass, and more preferably 1 to 30% by mass, and contains glucose in a total amount of 0.1 to 15% by mass, preferably 0.5 to 12% by mass, and more preferably 2.5 to 10% by mass. <9> A method for producing cellulase and / or xylanase. <11> The mass ratio of the cellulase inducer to glucose is 10:1 to 1:4, preferably 4:1 to 1:2. <10> A method for producing cellulase and / or xylanase. <12> <1> ~ <7> 2. A method for producing sugar from biomass, comprising culturing the filamentous fungus mutant according to any one of claims 1 to 10 in the presence of a cellulase inducer and using a culture obtained by culturing the filamentous fungus mutant according to any one of claims 1 to 10 in the presence of a cellulase inducer as a biomass saccharification agent. <13> <1> ~ <7> 2. A method for saccharifying biomass, comprising culturing the filamentous fungal mutant according to claim 1 in the presence of a cellulase inducer and using a culture obtained by this culture as a biomass saccharification agent. EXAMPLES

[0059] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0060] 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 (hereinafter referred to as cel1b, SEQ ID NO: 1) encoding TRIREDRAFT_22197 (1,4-β-glucosidase) in the genomic DNA of Trichoderma reesei strain PC-3-7 (ATCC 66589) by homologous recombination with an expression cassette for the uridine synthase gene (pyr4).

[0061] Using the genomic DNA of the PC-3-7 strain as a template, the upstream and downstream sequences of the cel1b 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.

[0062] 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 pUCΔcel1b-pyr4. pUCΔcel1b-pyr4 contains the upstream and downstream sequences of the cel1b gene derived from the genomic DNA of Trichoderma reesei, and a pyr4 expression cassette placed between them.

[0063] [Table 1]

[0064] (2) Creation of gene-deficient fungal mutants A uridine-requiring strain of Trichoderma reesei PC-3-7 (ATCC 66589) was transformed with pUCΔcel1b-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 cel1b gene-deficient strains in which pyr4 had been inserted at the position of the cel1b gene were selected by colony PCR. From the resulting candidate strains, strains in which only one copy of the pyr4 cassette had been introduced at the desired position by double crossover were selected by Southern analysis. This recombinant strain with one copy of pyr4 was obtained as a cel1b gene-deficient filamentous fungal mutant (PCΔcel1b).

[0065] Example 2 Protein production using filamentous fungal mutant strains (1) Cultivation of mutant fungi 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 PCΔcel1b prepared in Example 1 were inoculated at 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 100mg FeCl3·4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2 were diluted with distilled water to make 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)) 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).

[0066] (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. As a result, it was found that the PCΔcel1b strain had improved protein productivity compared to the PC-3-7 strain (parent strain) (Figure 1).

[0067] Example 3 Cultivation of filamentous fungal mutants in the presence of glucose (1) Cultivation of mutant fungi In the culture experiment in which glucose was added to the medium, 10% crystalline cellulose (FD101 (registered trademark), Asahi Kasei) or 10% FD101 (Asahi Kasei) + 10% glucose was used as the carbon source. Other medium components were the same as in Example 2 (1). The settings of the jar fermenter were as follows: temperature: 28°C, aeration: 1.0 vvm, pH 4.5 (adjusted with 5% ammonia water), and agitation speed was constant at 900 rpm. The main culture was carried out for 5 days (120 hours).

[0068] (2) Evaluation of protein productivity The protein concentration in the culture supernatant obtained in (1) was examined in the same manner as in Example 2(2). As a result, it was found that the PCΔcel1b strain produced protein (Figure 3) without a decrease in protein (cellulase) production (Figure 2), as did the PC-3-7 strain (parent strain), even when high concentrations of glucose were added during medium adjustment in batch culture.

Claims

1. A filamentous fungal mutant strain in which expression of a protein selected from the following (a) to (c) is reduced or eliminated compared to the parent strain: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having β-glucosidase activity; (c) A protein having the amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted, added or inserted, and having β-glucosidase activity.

2. A filamentous fungal mutant strain as described in claim 1, wherein the reduction or loss of expression of the protein is a reduction or loss of a translation product being produced from the gene encoding the protein and being localized in a functional state at its site of action.

3. The filamentous fungal mutant strain according to claim 1, in which expression of said protein is lost.

4. The filamentous fungal mutant strain of claim 1, wherein the protein is CeIb.

5. The filamentous fungal mutant according to claim 1, wherein the gene encoding said protein has been deleted or inactivated.

6. The filamentous fungal mutant according to claim 5, wherein the gene encoding the protein is represented by any one of the following (d) to (i): (d) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1; (e) a polynucleotide consisting of a base sequence having 80% or more identity to the base sequence shown in SEQ ID NO: 1 and encoding a protein having β-glucosidase activity; (f) a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 and encodes a protein having β-glucosidase activity; (g) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (h) a polynucleotide encoding a protein having β-glucosidase activity and consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 2; (i) A polynucleotide that consists of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and encodes a protein having β-glucosidase activity.

7. The mutant filamentous fungus according to claim 1, wherein the filamentous fungus belongs to the genus Trichoderma.

8. The mutant filamentous fungus according to claim 1, wherein the filamentous fungus is Trichoderma reesei.

9. A method for producing cellulase and / or xylanase, comprising the steps of culturing the filamentous fungal mutant strain according to any one of claims 1 to 8 in the presence of a cellulase inducer to produce and accumulate cellulase and / or xylanase in the culture, and recovering the cellulase and / or xylanase from the culture.

10. A method for producing cellulase and / or xylanase, comprising the steps of culturing the filamentous fungal mutant according to any one of claims 1 to 8 in the presence of a cellulase inducer and glucose to produce and accumulate cellulase and / or xylanase in the culture, and recovering the cellulase and / or xylanase from the culture.

11. The method for producing cellulase and / or xylanase according to claim 10, wherein the medium contains a total of 0.1 to 40% by mass of cellulase inducers and 0.5 to 15% by mass of glucose.

12. A method for producing sugar from biomass, comprising culturing the filamentous fungal mutant according to any one of claims 1 to 8 in the presence of a cellulase inducer and using a culture obtained by culturing the filamentous fungal mutant according to any one of claims 1 to 8 in the presence of a cellulase inducer as a biomass saccharifying agent.

13. A method for saccharifying biomass, comprising culturing the filamentous fungal mutant according to any one of claims 1 to 8 in the presence of a cellulase inducer and using a culture obtained thereby as a biomass saccharifying agent.

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