Mutant filamentous fungi and methods for producing proteins using them

Abstract

To provide a filamentous fungus mutant with improved protein productivity, and to provide a method for producing a protein using the filamentous fungus.SOLUTION: There is provided a method for producing a mutant filamentous fungus. The method includes modifying XYR1 and ACE3 expression in a parent filamentous fungus. The modification of XYR1 is substitution, deletion, insertion, or addition of at least one amino acid residue in a region corresponding to positions 810 to 833 of SEQ ID NO: 1 to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical thereto, and functioning as a transcriptional activator of cellulase and hemicellulase, and the modification of ACE3 expression is expression enhancement of the partial polypeptide of the ACE3.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 Art

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

[0003] For industrial microbial culture, it is desirable that the carbon source be inexpensive and soluble. Conventionally, glucose has been widely used as the carbon source in microbial culture. However, in the presence of glucose, a control mechanism called catabolite repression causes a decrease or saturation in the productivity of substances by microorganisms. It has been reported that a wide-domain regulatory transcription factor CreA and CreB, CreC, CreD, etc. are involved in the catabolite repression of filamentous fungi such as the genus Aspergillus (Patent Document 1). Although it may be possible to regulate the catabolite repression of Aspergillus by controlling these transcription factors, sufficient results have not yet been obtained. Regarding Trichoderma, analysis of the mechanism of catabolite repression has been underway (Patent Document 2, Non-Patent Document 1). However, there are still many unclear points in the mechanism of catabolite repression of Trichoderma, and avoidance of repression has not been achieved.

[0004] Microorganisms sometimes require inducers for the production of enzymes and other proteins. For example, in Trichoderma, the expression of major cellulase genes cbh1, cbh2, egl1, and egl2 is induced by cellulose, cellobiose, etc., and inducers are essential for cellulase production (Non-Patent Literature 2). Generally, microcrystalline cellulose such as Avicel is used as an inducer for cellulase production. However, cellulose substrates are expensive and mostly insoluble, which puts a burden on industrial processes, making their use in industrial applications difficult in terms of cost and equipment.

[0005] Methods for producing cellulase using soluble lactose instead of cellulose as an inducer (Patent Document 3) and methods for synthesizing inducible sugars such as sophorose and gentiobiose from glucose by reacting Trichoderma-derived cellulase (including β-glucosidase, endoglucanase, and cellobiohydrolase) with glucose at high temperatures (Patent Document 4) have been disclosed. However, cellulase production using inducible sugars still suffers from disadvantages in terms of cost and process load.

[0006] To create microorganisms capable of expressing cellulase and xylanase without the use of inducers by modifying transcription factors, research is underway to analyze the cellulase expression mechanism. XYR1, ACE2, ACE3, and HAP2 / 3 / 5 have been reported as positive transcription factors involved in cellulase induction in Trichoderma (Non-Patent Literature 3). It has been reported that shortening the C-terminal 140 amino acids of XYR1 results in loss of cellulase productivity in Trichoderma, and that Trichoderma with the A824V mutation in XYR1 leads to deregulation of xylanase and increased cellulase production (Non-Patent Literature 3, 4). Non-Patent Literature 5 reports that combining the V821F mutation in XYR1 with enhanced expression of ACE2 in Trichoderma strains improved protein productivity in media using glucose and sucrose as carbon sources. Previously, it was believed that the XYR1 gene sequence contained a 20-amino acid intron upstream of the coding region of the fungal-specific transcription factor domain (Non-Patent Literature 6). Therefore, A824 and V821 of XYR1 disclosed in the above-mentioned document were initially corrected to A804 and V801, respectively. However, there have also been reports that this 20-amino acid region is translated into amino acids rather than being an intron. Recently, the latter finding is considered correct (for example, Non-Patent Literature 5 and 7). In this case, A824 and V821 disclosed in the above-mentioned document are considered to represent the correct amino acid numbers on the XYR1 sequence.

[0007] Non-patent document 8 suggests that ACE3 and XYR1 interact to regulate cellulase gene expression in Trichoderma lisee. Patent document 5 discloses a method to increase and decrease the productivity of cellulase and other enzymes in Trichoderma lisee by increasing and decreasing the expression of the tre77513(ACE3) gene. Patent documents 6 and non-patent documents 9 report that a filamentous fungus that upexpresses a modified ACE3 that retains all six cysteine ​​molecules in the N-side Zn(II)2Cys6 type DNA-binding domain and lacks the C-terminal amino acid improved cellulase expression even in the absence of an inducer. Non-patent document 9 also describes a filamentous fungus that upexpresses the C-terminally deficient ACE3 and co-expresses wild-type or A824V mutant XYR1; however, the effect of co-expression of XYR1 on cellulase expression in this filamentous fungus was only slightly observed in the presence of an inducer and not in the absence of an inducer. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2015-39349 [Patent Document 2] Special Publication No. 11-512930 [Patent Document 3] Patent No. 6169077 [Patent Document 4] Patent No. 5366286 [Patent Document 5] U.S. Patent No. 9512415 [Patent Document 6] International Public Gazette No. 2018 / 067599 [Non-patent literature]

[0009] [Non-Patent Document 1] Appl Environ Microbiol, 1997, 63:1298-1306 [Non-Patent Document 2] Curr Genomics, 2013, 14:230-249 [Non-Patent Document 3] BMC Genomics, 2015, 16:326 [Non-Patent Document 4] Biotech Biofuels, 2013, 6:62 [Non-Patent Document 5] Biotech Biofuels, 2017, 10:30 [Non-Patent Document 6] NCBI Reference Sequence: XP_006966092.1 [www.ncbi.nlm.nih.gov / protein / XP_006966092.1] [Non-Patent Document 7] Biotech Biofuels, 2020, 13:93 [Non-Patent Document 8] J Biol Chem, 2019, doi:10.1074 / jbc.RA119.008497 [Non-Patent Document 9] Biotech Biofuels, 2020, 13:137 [Overview of the project]

[0010] The present invention is a method for producing mutant filamentous fungi, This includes modifying the expression of XYR1 and ACE3 in parent filamentous fungi, The modification of XYR1 involves the substitution, deletion, insertion, or addition of at least one amino acid residue in the region corresponding to positions 810-833 of SEQ ID NO: 1 to a polypeptide consisting of an amino acid sequence identical to or at least 90% of the sequence of SEQ ID NO: 1, and functioning as a transcriptional activator of cellulase and hemicellulase. The modification of ACE3 expression is an enhancement of the expression of a polypeptide consisting of the amino acid sequence from positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto. To provide a method. [Brief explanation of the drawing]

[0011] [Figure 1] Relative protein productivity of mutant filamentous strains. [Figure 2] Protein composition analysis of cultures of mutant filamentous strains: Gel electrophoresis image. [Figure 3] Relative productivity of various proteins in mutant filamentous strains. [Figure 4] Relative productivity of various proteins in mutant filamentous strains. Detailed Description of the Invention

[0012] All patent documents, non-patent documents, and other publications cited in this specification are hereby incorporated by reference in their entirety.

[0013] In this specification, the identity of amino acid sequences and nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with Unit size to compare (ktup) set to 2.

[0014] In this specification, "at least 90% identity" with respect to amino acid sequences and nucleotide sequences means an identity of 90% or more, preferably 92% or more, more preferably 94% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 98% or more, and most preferably 99% or more.

[0015] In this specification, unless otherwise defined, “one or several” as used with respect to deletions, substitutions, additions or insertions of amino acids and nucleotides in amino acid sequences and nucleotide sequences may mean, for example, 1 to 20, preferably 1 to 16, more preferably 1 to 12, even more preferably 1 to 8, and even more preferably 1 to 4. In this specification, “addition” of an amino acid or nucleotide includes the addition of one or several amino acids or nucleotides to one end and both ends of a sequence. In this specification, “insertion” of an amino acid or nucleotide includes the insertion of an amino acid or nucleotide to the 5' or 3' end of a given position.

[0016] In this specification, the "corresponding position" or "corresponding region" on an amino acid sequence or nucleotide sequence can be determined by aligning the target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to give the greatest possible homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. Det. al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Clustal W can be used, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]), operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the alignment described above is considered a "corresponding position" to that arbitrary position. Furthermore, the region enclosed by the corresponding positions, or the region consisting of the corresponding motifs, is considered a corresponding region.

[0017] Those skilled in the art can further fine-tune the amino acid sequence alignment obtained above to optimize it. Such an optimal alignment is preferably determined by considering the similarity of the amino acid sequences and the frequency of inserted gaps. Here, similarity of amino acid sequences refers to the ratio (%) of the number of positions where identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned, relative to the total number of amino acid residues. Similar amino acid residues refer to amino acid residues among the 20 amino acid residues that make up a protein that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Groups consisting of such similar amino acid residues are well known to those skilled in the art, and examples include, but are not limited to, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; leucine and isoleucine, respectively.

[0018] In this specification, "amino acid residue" means the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0019] In this specification, "operable linkage" between a regulatory region such as a promoter and a gene means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operable linkage" between a gene and a regulatory region are well known to those skilled in the art.

[0020] In this specification, "upstream" and "downstream" with respect to a gene refer to the upstream and downstream regions in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter in the DNA sense strand, and "upstream of a gene" means the 5' region of the gene in the DNA sense strand.

[0021] In this specification, the term "inherent" used with respect to the function, properties, or traits of a cell is used to indicate that the function, property, or trait is inherently present in the cell. In contrast, the term "external" is used to indicate a function, property, or trait that is not inherently present in the cell but has been introduced from outside. For example, an "external" gene or polynucleotide is a gene or polynucleotide that has been introduced into a cell from outside. An external gene or polynucleotide may originate from the same species of organism as the cell into which it was introduced, or from a different species of organism (i.e., a different gene or polynucleotide).

[0022] This invention relates to providing mutant filamentous fungi, methods for producing the same, and methods for producing proteins using the mutant filamentous fungi. This invention relates to improving the protein productivity of filamentous fungi. Conventionally, when filamentous fungi are cultured in the presence of glucose, protein production is sometimes suppressed due to catabolite inhibition. In particular, the expression of cellulase-based biomass-degrading enzymes such as cellulase and hemicellulase in filamentous fungi needs to be induced by cellulase-inducible substances such as cellulose, sophorose, and cellooligosaccharides (cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, etc.), while expression induction is suppressed in the presence of glucose.

[0023] The inventors have discovered that mutant filamentous fungi modified by combining XYR1 and ACE3 expression produce high levels of cellulase, hemicellulase, and other proteins without using inducers that were conventionally essential for the production of cellulase and hemicellulase. In this invention, by making predetermined modifications to XYR1 and ACE3, which are transcription activators of cellulase and hemicellulase, respectively, the filamentous fungi are given the ability to express cellulase and hemicellulase without cellulase-inducing substances such as cellulose, and the filamentous fungi are enabled to highly express proteins such as cellulase and hemicellulase in the presence of a non-cellulase-inducing carbon source such as glucose. By modifying XYR1 and ACE3 expression in combination, the expression ability of cellulase and hemicellulase is significantly improved compared to when only one of them is modified. It is thought that the modification of XYR1 and ACE3 expression contributes to the mitigation of catabolite suppression or to the promotion of cellulase and hemicellulase transcription.

[0024] The filamentous fungi provided by the present invention can efficiently produce proteins even in environments where non-cellulase-inducing carbon sources such as glucose are the main carbon source. Furthermore, these filamentous fungi can efficiently produce proteins such as cellulase and hemicellulase without using expensive cellulase-inducing substances. According to the present invention, it is possible to improve the efficiency and reduce the cost of protein production using filamentous fungi.

[0025] Accordingly, in one embodiment, the present invention provides a mutant filamentous fungus and a method for producing the same. Basically, the method for producing the mutant filamentous fungus of the present invention includes modifying the expression of XYR1 and ACE3 in the parent filamentous fungus. In this method, the order in which the expression of XYR1 and ACE3 is modified is not limited, as long as each modification is achieved.

[0026] Examples of parent filamentous fungi used in the present invention include, but are not limited to, filamentous fungi belonging to the phyla Eumycota and Oomycota. More specific examples include filamentous fungi of the genera Trichoderma, Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, Hypocrea, Acremonium, Chrysosporium, Myceliophthora, Piromyces, Talaromyces, Thermoascus, and Thielavia. Of these, filamentous fungi of the genus Trichoderma are preferred.

[0027] Examples of filamentous fungi of the genus Trichoderma include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, and Trichoderma viride, with Trichoderma reesei and its mutants being preferred. For example, strain QM9414 of Trichoderma reesei and its mutants, preferably strain PC-3-7 (ATCC66589), strain PCD-10 (FERM P-8172), strain JN13 of Trichoderma reesei, or their mutants, can be preferably used as parent filamentous fungi.

[0028] XYR1 (Xylanase regulator 1) is a transcriptional activator of cellulase and hemicellulase in filamentous fungi. XYR1 is a major regulator of xylanase gene expression, possessing a Zn(II)2Cys6 binuclear cluster domain, and is widely conserved in ascomycetes excluding yeast, such as Trichoderma (XYR1), Fusarium (XYR1), Neurospora (XYR1), and Alpergillus (XLNR). XYR1 in Trichoderma lisey controls all xylanase / xylose metabolism genes and cellulase genes. XYR1 in Trichoderma lisey is registered in the NCBI database (www.ncbi.nlm.nih.gov / ) as NCBI Reference Sequence: XP_006966092.1.

[0029] Traditionally, the XYR1 gene sequence was thought to contain a 20-amino acid intron upstream of the coding region of the fungal-specific transcription factor domain (the nucleotide region 1024-1083 of SEQ ID NO: 2), and the total length of XYR1 was thought to be 920 amino acids. The ncbi database (Non-Patent Literature 6) also defines XYR1 XP_006966092.1 as a polypeptide consisting of a 920-amino acid sequence (SEQ ID NO: 51) encoded by the nucleotide sequence of SEQ ID NO: 2. On the other hand, it has been reported that the nucleotide region 1024-1083 of SEQ ID NO: 2 is translated into amino acids, not an intron. Recently, the latter is considered to be the correct structure of XYR1 (e.g., Non-Patent Literature 5 and 7). In that case, XYR1 has a 320-339 amino acid region encoded by the nucleotide region 1024-1083 of SEQ ID NO: 2, and its total length is 940 amino acids. In consideration of the above circumstances, unless otherwise specified, the amino acid sequence of XYR1 disclosed herein is represented by Sequence ID No. 1, which is 940 amino acids long, and the amino acid residue numbers (positions in the amino acid sequence) of XYR1 are represented by the residue numbers (positions) in the sequence of Sequence ID No. 1. Also in consideration of the above circumstances, in this specification, amino acid residues from position 340 onwards in Sequence ID No. 1 should be interpreted as the amino acid residue at position [position in Sequence ID No. 1 - 20] in Sequence ID No. 51. For example, positions 810-833 in Sequence ID No. 1 are positions 790-813 in Sequence ID No. 51, that is, position 810 in Sequence ID No. 1 is position 790 in Sequence ID No. 51, position 833 in Sequence ID No. 1 is position 813 in Sequence ID No. 51, positions 821 and 824 in Sequence ID No. 1 are positions 801 and 804 in Sequence ID No. 51, respectively, and the same applies to other positions.

[0030] Therefore, an example of the XYR1 (parent XYR1) to be modified in the present invention is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. Another example of the parent XYR1 is a polypeptide consisting of an amino acid sequence that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 1, and that functions as a transcriptional activator of cellulase and hemicellulase. An example of an amino acid sequence that is at least 90% identical in sequence to SEQ ID NO: 1 is an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 1.

[0031] The modification of XYR1 according to the present invention involves adding a mutation of at least one amino acid residue to a region presumed to be an α-helix in the acidic activation domain of the parent XYR1. More specifically, the modification of XYR1 is carried out by a mutation (i.e., substitution, deletion, insertion, or addition) of at least one amino acid residue in the region corresponding to positions 771-865 of SEQ ID NO: 1 of the parent XYR1 polypeptide, which consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical thereto, and functions as a transcriptional activator of cellulase and hemicellulase. In a preferred embodiment, the at least one amino acid residue to be mutated is located in the region corresponding to positions 810-833 of SEQ ID NO: 1.

[0032] In a preferred embodiment, at least one of the aforementioned amino acid residues is substituted. The substituted amino acid residue is at least one selected from the group consisting of Val, Ile, Leu, Ala, Gly, Thr, and Glu, more preferably at least one selected from the group consisting of (1) Val, Ile, or Leu, (2) Ala or Gly, or at least one selected from the group consisting of Val, Ala, Thr, and Glu, and even more preferably at least one selected from the group consisting of Val and Ala. Preferably, these amino acid residues are substituted with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, respectively.

[0033] Preferred examples of substitutions for Val, Ile, Leu, Ala, Gly, Thr, and Glu include the following: Substitution of Val with Lys, Phe, Trp, or Tyr; Substitution of Ile with Phe, Trp, or Tyr; Substitution of Leu with Phe, Trp, or Tyr; Substitution of Ala with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Gly with Val, Ile, Leu, Phe, Trp, or Tyr; Substitution of Thr for Tyr; and Substitution of Glu with Tyr.

[0034] In a more preferred embodiment, the amino acid residue substitution is at least one selected from the group consisting of: Substitution of Gly with Val, Ile, Leu, Phe, Trp, or Tyr at position 812 of sequence number 1; Substitution of Val with Phe, Trp, or Tyr at position 814 of sequence number 1; Substitution of Ala with Val, Ile, Leu, Phe, Trp, or Tyr at position 816 of sequence number 1; Substitution of Thr for Tyr at position 817 of sequence number 1; Substitution of Ala with Val, Ile, Leu, Phe, Trp, or Tyr at position 820 of sequence number 1; Substitution of Val with Lys, Phe, Trp, or Tyr at position 821 of sequence number 1; Substitution of Ala with Val, Ile, Leu, Phe, Trp, or Tyr at position 823 of sequence number 1; Substitution of Ala at position 824 of sequence number 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Glu with Tyr at position 825 of sequence number 1; Substitution of Ala at position 826 of sequence number 1 with Val, Ile, Leu, Phe, Trp, or Tyr; Substitution of Ile with Phe, Trp, or Tyr at position 827 of sequence number 1; Substitution of Ile with Phe, Trp, or Tyr at position 830 of sequence number 1; and, Substitution of Leu with Phe, Trp, or Tyr at position 831 of sequence number 1.

[0035] In a more preferred embodiment, the modification of XYR1 according to the present invention is carried out by the substitution of an amino acid residue in the parent XYR1 at at least one position selected from the group consisting of positions 817, 821, 824, 825, and 826 of SEQ ID NO: 1. The amino acid residues to be substituted at positions 817, 821, 824, 825, and 826 are as described above.

[0036] In another, more preferred embodiment, the modification of XYR1 according to the present invention is carried out by the substitution of an amino acid residue in the parent XYR1 at at least one position selected from the group consisting of positions 821 and 824 of SEQ ID NO: 1. The amino acid residue substituted at position 821 is preferably Lys, Phe, Trp, or Tyr, more preferably Phe, and even more preferably Lys or Tyr. The amino acid residue substituted at position 824 is preferably Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, more preferably Val, and even more preferably Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr.

[0037] In another, more preferred embodiment, the modification of XYR1 according to the present invention is carried out by the substitution of an amino acid residue in the parent XYR1 at at least one position selected from the group consisting of positions 817, 825, and 826 of SEQ ID NO: 1. The amino acid residue substituted at position 817 is preferably Tyr. The amino acid residue substituted at position 825 is preferably Tyr. The amino acid residue substituted at position 826 is preferably Val, Ile, Leu, Phe, Trp, or Tyr, and more preferably Val or Trp.

[0038] In one example, the amino acid residues at positions 817, 821, 824, 825, and 826 of SEQ ID NO: 1 in parent XYR1 are not the substituted amino acid residues described above. Preferably, the amino acid residues at positions 817, 821, 824, 825, and 826 of SEQ ID NO: 1 in parent XYR1 are the same as the amino acid residues at positions 817, 821, 824, 825, and 826 of SEQ ID NO: 1, respectively.

[0039] In another example, the amino acid residues at positions 812, 814, 816, 817, 820, 821, 823, 824, 825, 826, 827, 830, and 831 of Sequence ID No. 1 in parent XYR1 are not the substituted amino acid residues mentioned above. Preferably, the amino acid residues at positions 812, 814, 816, 817, 820, 821, 823, 824, 825, 826, 827, 830, and 831 of SEQ ID NO: 1 in parent XYR1 are the same as the amino acid residues at positions 812, 814, 816, 817, 820, 821, 823, 824, 825, 826, 827, 830, and 831 of SEQ ID NO: 1, respectively. In another example, the amino acid sequence of the region corresponding to positions 810-833 of SEQ ID NO: 1 in the parent XYR1 is the same as that of positions 810-833 in SEQ ID NO: 1.

[0040] Various mutagenesis techniques known in the art can be used as means to mutate (substitute, delete, insert, or add) amino acid residues in the polypeptide of the parent filamentous fungus. For example, in the genome of the parent filamentous fungus, the polynucleotide encoding the amino acid sequence to be mutated (parent XYR1) (hereinafter also referred to as the parent gene) can be changed to a polynucleotide encoding the mutated amino acid sequence (hereinafter also referred to as the mutant gene), and a polypeptide having the desired mutation (modified XYR1) can be expressed from the mutant gene.

[0041] One method for introducing a desired mutation into a parent gene is to utilize homologous recombination. For example, the parent gene in the genome of a parent filamentous fungus can be replaced with a mutant gene by homologous recombination. In a specific example of homologous recombination, first, a homologous recombination DNA construct containing the mutant gene and, if necessary, a drug resistance gene or a nutrient requirement gene is constructed, and this is introduced into the parent filamentous fungus by a conventional method. Next, a transformed strain in which the homologous recombination construct has been incorporated into the genome is selected using indicators such as drug resistance or nutrient requirements. If necessary, the obtained transformed strain may be confirmed to have the desired mutation by genome analysis or enzyme activity analysis.

[0042] Homologous recombination DNA constructs can be constructed by introducing site-directed mutations (SMTs) into isolated parental genes. SMTs can be introduced using standard methods in the field, such as inverse PCR, annealing, or SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68). Commercially available SMT kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit) can also be used.

[0043] For introducing DNA constructs into parent filamentous fungi, vectors commonly used for transformation, such as plasmids, can be used. For introducing DNA constructs and vectors into cells, conventional methods such as the protoplast method, protoplast PEG method, and competent cell method can be used.

[0044] The parent gene can be isolated by conventional methods from a strain of the same species as the parent filamentous fungus. Alternatively, it may be chemically synthesized based on the nucleotide sequence of the parent gene. If necessary, the parent gene may be codon-optimized to suit the host (parent filamentous fungus) into which it will be introduced. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0045] The parent gene may be any polynucleotide that encodes a polypeptide consisting of the amino acid sequence of the aforementioned parent XYR1, i.e., SEQ ID NO: 1, or an amino acid sequence that is at least 90% identical thereto, and that functions as a transcriptional activator of cellulase and hemicellulase. An example of such a polynucleotide is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is at least 90% identical thereto.

[0046] Site-directed mutagenesis into a parent gene can most commonly be performed using a mutation primer containing the nucleotide mutation to be introduced. This mutation primer should be designed to anneal to a region in the parent gene containing the sequence encoding the amino acid residue to be mutated, and to include a codon encoding the mutated amino acid residue in place of the original codon. Those skilled in the art can appropriately identify and select the pre-mutation and post-mutation amino acid residues based on standard textbooks. Alternatively, site-directed mutagenesis can be performed by ligating upstream and downstream DNA fragments containing the nucleotide mutation to be introduced, amplified using two sets of primers, into a single DNA fragment by SOE-PCR. Mutation primers can be prepared using well-known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids R4esearch, 1989, 17:7059-7071).

[0047] Alternatively, the parent filamentous fungus can be subjected to a mutagenesis treatment, and then strains with the desired mutation can be selected through genome analysis or enzyme activity analysis. Specific mutagenesis treatments include N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethylnitrosourea, and irradiation with ultraviolet light or radiation. Various alkylating agents and carcinogens can also be used as mutagens.

[0048] The modification of ACE3 expression according to the present invention involves enhancing the expression of ACE3 or its partial polypeptide. ACE3 is a transcriptional activator of cellulase and hemicellulase in filamentous fungi. ACE3 is essential for the transcription of the cellulase gene and some xylanase genes during lactose induction. ACE3 is also partially involved in the transcriptional regulation of xyr1. The ACE3 of Trichoderma risey is registered in the NCBI database (www.ncbi.nlm.nih.gov / ) as NCBI Reference Sequence: QEM24913.1, where ACE3 is defined as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 and encoded by the nucleotide sequence of SEQ ID NO: 4. According to Non-Patent Literature 6, of the amino acid sequence of SEQ ID NO: 3, positions 523-734 are presumed to be a region that interacts with XYR1, positions 391-522 are presumed to be a filamentous fungus-specific transcription factor domain, and positions 120-160 are presumed to be a Zn(II)2Cys6 type DNA binding domain.

[0049] Therefore, examples of ACE3 or its partial polypeptides whose expression is enhanced in the present invention include polypeptides consisting of the amino acid sequence of SEQ ID NO: 3, or partial polypeptides thereof. Another example of ACE3 or its partial polypeptide is a polypeptide or partial polypeptide consisting of an amino acid sequence that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 3, and that functions as a transcriptional activator of cellulase and hemicellulase. An example of an amino acid sequence that is at least 90% identical in sequence to SEQ ID NO: 3 is an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 3.

[0050] In the present invention, the expression of the full-length polypeptide of ACE3 may be enhanced, but it is sufficient to enhance the expression of a partial polypeptide, preferably a polypeptide containing at least the region corresponding to positions 107 to 734 of SEQ ID NO: 3. Therefore, examples of ACE3 whose expression is enhanced in the present invention include polypeptides consisting of the amino acid sequence from positions 1 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto, and examples of partial polypeptides of ACE3 include polypeptides consisting of the amino acid sequence from positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto.

[0051] Preferably, the ACE3 or partial polypeptide whose expression is enhanced according to the present invention retains all of the amino acid residues from the 7th to 10th C-terminus of wild-type ACE3 (amino acid residues in the region corresponding to positions 725 to 728 of SEQ ID NO: 3). More preferably, the ACE3 or partial polypeptide whose expression is enhanced according to the present invention retains all of the amino acid residues from the 7th to 17th C-terminus of wild-type ACE3 (amino acid residues in the region corresponding to positions 718 to 728 of SEQ ID NO: 3). Alternatively, preferably, the ACE3 or partial polypeptide whose expression is enhanced according to the present invention retains the region corresponding to the 11 amino acids at the C-terminus of wild-type ACE3 (positions 724 to 734 of SEQ ID NO: 3). However, mutations in some amino acid residues in the region corresponding to positions 724 to 734 of SEQ ID NO: 3 in the ACE3 or partial polypeptide (e.g., substitution, deletion, insertion, or addition of one or more residues at positions 729 to 734 of SEQ ID NO: 3) are permissible as long as the function as a transcriptional activator of cellulase and hemicellulase is maintained.

[0052] Preferably, the enhancement of ACE3 or its partial polypeptide expression in the present invention means an improvement in the expression level of the ACE3 or its partial polypeptide. Means for increasing the expression level of the target polypeptide include means for improving the transcription level of the gene encoding the polypeptide (hereinafter also referred to as the target gene). Means for improving the transcription level of the target gene include, for example, substituting or inserting a regulatory region that strongly promotes the transcription of the target gene (strong regulatory region) into the regulatory region of the target gene on the genome of the parent filamentous fungus, thereby operably linking the strong regulatory region to the target gene. Alternatively, the transcription level of the target gene can be improved by introducing the target gene fragment, which is operably linked to a regulatory region (preferably a strong regulatory region) as needed, into the genome or plasmid of the parent filamentous fungus to increase the number of target genes that can be expressed in the cell.

[0053] Examples of regulatory regions that can be used to improve the transcription level include those of genes whose transcription levels do not decrease even under high glucose conditions, such as the regulatory regions of genes in the Trichoderma genus, including glyceraldehyde-3-phosphate dehydrogenase (GPD), pyruvate decarboxylase (PDC), enolase (ENO), alcohol dehydrogenase (ADH), triose phosphate isomerase (TPI), aldolase (FBA), pyruvate kinase (PYK), citrate synthase (CIT), α-ketoglutarate dehydrogenase (KDH), aldehyde dehydrogenase I (ALD1), aldehyde dehydrogenase II (ALD2), pyruvate dehydrogenase (PDA), glucosekinase (GLK), actin (ACT1), and translation elongation factor 1α (TEF1). Examples of preferred high-control regions include pdc(TRIREDRAFT_121534) and act1(TRIREDRAFT_44504).

[0054] The target gene can be isolated by conventional methods from a strain of filamentous fungus of the same species as the parent filamentous fungus. Alternatively, it may be chemically synthesized based on the nucleotide sequence of the parent filamentous fungus gene. Preferred examples of the target gene include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, a polynucleotide comprising a nucleotide sequence that is at least 90% identical in sequence to SEQ ID NO: 4 and encoding a polypeptide that functions as a transcriptional activator of cellulase and hemicellulase, or partial polynucleotides thereof. An example of a nucleotide sequence that is at least 90% identical in sequence to SEQ ID NO: 4 is a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted in the sequence of SEQ ID NO: 4. A preferred example of the partial polynucleotide is a polynucleotide encoding the ACE3 partial polypeptide described above.

[0055] The expression level of ACE3 or its partial polypeptide in the mutant strain of the present invention is improved compared to the parent filamentous fungus. Alternatively, the transcription level of the gene encoding ACE3 or its partial polypeptide in the mutant strain of the present invention is improved compared to the parent filamentous fungus. The expression level of the gene or polypeptide can be quantified by known means such as quantitative PCR, microarray, Western blotting, ELISA, and HPLC.

[0056] The mutant filamentous fungus of the present invention produced by the above procedure contains a modified XYR1 obtained by the modification of XYR1 described above, and, as described above, the expression of ACE3 or its partial polypeptide is enhanced compared to the parent filamentous fungus.

[0057] Examples of ACE3 or its partial polypeptides whose expression is enhanced in the mutant filamentous fungi of the present invention are as described above. Preferably, the mutant filamentous fungi of the present invention are introduced with a gene encoding a partial polypeptide of ACE3 (preferably consisting of the amino acid sequence from positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto) which is operably linked to a regulatory region (preferably a strongly regulatory region), thereby enhancing the expression of the partial polypeptide of ACE3.

[0058] Preferably, the modified XYR1 contained in the mutant filamentous fungus of the present invention is a polypeptide that consists of an amino acid sequence in which at least one amino acid residue in the region corresponding to positions 771-865 of SEQ ID NO: 1, preferably the region corresponding to positions 810-833, is substituted, deleted, inserted, or added to SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical thereto, and functions as a transcriptional activator of cellulase and hemicellulase.

[0059] More preferably, the modified XYR1 contained in the mutant filamentous fungus of the present invention is a substitution of at least one amino acid residue selected from the group consisting of the following, in the region corresponding to positions 771-865 of SEQ ID NO: 1 or an amino acid sequence identical thereto by at least 90%, preferably in the region corresponding to positions 810-833: Substitution of Val with Lys, Phe, Trp, or Tyr; Substitution of Ile with Phe, Trp, or Tyr; Substitution of Leu with Phe, Trp, or Tyr; Substitution of Ala with Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Gly with Val, Ile, Leu, Phe, Trp, or Tyr; Substitution of Thr for Tyr; and Substitution of Glu with Tyr, It is a polypeptide consisting of an amino acid sequence that has undergone the following process, and that functions as a transcriptional activator of cellulase and hemicellulase.

[0060] More preferably, the modified XYR1 is at least 90% identical in sequence to sequence number 1, and (a) to (m) below: (a) Val, Ile, Leu, Phe, Trp, or Tyr at position 812 of sequence number 1; (b) Phe, Trp, or Tyr at position 814 of Sequence ID No. 1; (c) Val, Ile, Leu, Phe, Trp, or Tyr at position 816 of Sequence ID No. 1; (d) Tyr at position 817 of sequence number 1; (e) Val, Ile, Leu, Phe, Trp, or Tyr at the position corresponding to position 820 of sequence number 1; (f) Lys, Phe, Trp, or Tyr at position 821 of sequence number 1; (g) Val, Ile, Leu, Phe, Trp, or Tyr at position 823 of Sequence ID No. 1; (h) Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr at position 824 of Sequence ID No. 1; (i) Substitution of Glu with Tyr at position 825 of Sequence ID No. 1; (j) Val, Ile, Leu, Phe, Trp, or Tyr at position 826 of sequence number 1; (k) Phe, Trp, or Tyr at position 827 of sequence number 1; (l) Phe, Trp, or Tyr at position 830 of sequence number 1; and, (m) Phe, Trp, or Tyr at position 831 of sequence number 1 It is a polypeptide consisting of an amino acid sequence having at least one amino acid residue selected from the group comprising the following, and which functions as a transcriptional activator of cellulase and hemicellulase. More preferably, the modified XYR1 has one or more of (a), (c), (e), (g), (h), (j) and / or one or more of (b), (f), (k), (l), (m). More preferably, the modified XYR1 has one or more of (c), (e), (g), (h), (j) and / or one or more of (b), (f).

[0061] In a more preferred embodiment, the modified XYR1 is a polypeptide that is at least 90% identical in sequence to SEQ ID NO: 1 and comprises an amino acid sequence having one or more of (d), (f), (h), (i), and (j), and functions as a transcriptional activator of cellulase and hemicellulase.

[0062] In another, more preferred embodiment, the modified XYR1 is at least 90% sequence-identical to sequence number 1, and also: Lys, Phe, Trp, or Tyr at the position corresponding to position 821 of sequence number 1, preferably Phe, more preferably Lys or Tyr; and, This polypeptide consists of an amino acid sequence having at least one amino acid residue selected from the group consisting of Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, preferably Val, and more preferably Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, at a position corresponding to position 824 of Sequence ID No. 1, and functions as a transcriptional activator of cellulase and hemicellulase.

[0063] In another, more preferred embodiment, the modified XYR1 is at least 90% sequence-identical to sequence number 1, and also: Tyr at position 817 of sequence number 1; Tyr at position 825 of sequence number 1, and Val, Ile, Leu, Phe, Trp, or Tyr at position 826 of Sequence ID No. 1, preferably Val or Trp, It is a polypeptide consisting of an amino acid sequence having at least one amino acid residue selected from the group comprising the following, and which functions as a transcriptional activator of cellulase and hemicellulase.

[0064] The mutant filamentous fungi of the present invention can express cellulase-based biomass-degrading enzymes such as cellulase and hemicellulase even in the absence of cellulase-inducing substances such as cellulose, sophorose, and cellooligosaccharides. Furthermore, these mutant filamentous fungi can efficiently produce proteins even in environments where non-cellulase-inducing carbon sources such as glucose are the main carbon source, for example, even in the absence of cellulase-inducing substances.

[0065] Therefore, in a further embodiment, the present invention provides a method for producing a protein using the mutant filamentous fungus of the present invention described above. In the method for producing a protein according to the present invention, the mutant filamentous fungus of the present invention is cultured. Through culture, the target protein is generated and accumulated in the culture. The target protein can be produced by separating the target protein from the culture.

[0066] Examples of target proteins to be produced include, but are not limited to, cellulase-based biomass-degrading enzymes such as cellulase and hemicellulase, exoglucanase, endoglucanase, β-glucosidase, protease, lipase, mannase, arabinase, galactase, and amylase. The target protein may be a single protein or a mixture of multiple proteins. Preferably, the target protein is a cellulase-based biomass-degrading enzyme, more preferably cellulase and / or hemicellulase, and even more preferably cellulase and hemicellulase. Examples of hemicellulases include xylanase, β-xylosidase, and α-arabinofuranosidase, of which xylanase is preferred.

[0067] Alternatively, the target protein may be a heterologous protein that the filamentous fungus does not normally produce. In this case, recombinant filamentous fungus can be created by inserting a gene encoding the heterologous protein into the mutant filamentous fungus of the present invention, and a protein containing the heterologous protein can be obtained by culturing this recombinant filamentous fungus. Furthermore, by operably linking the gene encoding the heterologous protein to a secretory signal peptide that functions in the filamentous fungus, the heterologous protein can be secreted and produced in the culture.

[0068] The culture medium used for the production of the protein may be either a synthetic or natural medium, as long as it contains the components necessary for the growth and protein production of typical filamentous fungi, such as a carbon source, nitrogen source, inorganic salts, and vitamins.

[0069] Any carbon source that the mutant filamentous fungus can utilize can be used as a carbon source, such as carbohydrates like glucose and fructose, sugar alcohols like sorbitol, alcohols like ethanol and glycerol, and organic acids like acetic acid. These can be used individually or in combination.

[0070] Preferably, in the protein production method according to the present invention, the mutant filamentous fungus is cultured in an environment where a cellulase-non-inducible carbon source is the main carbon source. Examples of cellulase-non-inducible carbon sources include glucose, fructose, sucrose, maltose, and glycerol. Of these, glucose is preferred in terms of cost. When the target protein to be produced is a cellulase-based biomass-degrading enzyme, the culture in this method may be carried out in the presence of cellulase-inducible substances such as cellulose, sophorose, and cello-oligosaccharides, but high production of the target protein is possible even in the absence of such inducible substances, and the method is not limited to the use or non-use of such inducible substances. Furthermore, in the present invention, in order to efficiently produce proteins such as cellulase-based biomass-degrading enzymes while further reducing catabolite inhibition, the mutant filamentous fungus may be cultured while continuously adding a non-inducible carbon source such as glucose. In this case, it is preferable to dissolve the cellulase-non-inducible carbon source, such as glucose, in an aqueous solution containing ammonia water or an ammonium salt, which is a nitrogen source, and to culture by continuously adding the solution, in order to improve culture efficiency and suppress foaming during culture.

[0071] Examples of nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, peptone, and natural nitrogen sources such as soybean hydrolysates.

[0072] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0073] Examples of vitamins include biotin and thiamine. Furthermore, substances required for the growth of the filamentous fungi of this invention can be added as needed.

[0074] Culturing is preferably carried out under aerobic conditions such as shaking culture or aeration stirring 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, and more preferably 35°C or lower. It is also preferably 10-50°C, more preferably 20-42°C, and more preferably 25-35°C. The pH during cultivation is 3-9, preferably 4-5. The cultivation time is 10 hours to 10 days, preferably 2-7 days.

[0075] After culturing, the target protein is separated from the obtained culture by conventional methods. For example, the culture can be collected, subjected to cell disruption treatment such as ultrasound or pressurization as needed, and the target protein can be separated from the culture by appropriately combining filtration, centrifugation, ultrafiltration, salting out, dialysis, chromatography, etc. The degree of separation of the target protein is not particularly limited. For example, the culture supernatant or its crude separated and purified product can be obtained as a composition containing the target protein.

[0076] The present invention also includes, as exemplary embodiments, the following substances, manufacturing methods, uses, methods, etc. However, the present invention is not limited to these embodiments.

[0077] [1] A method for producing mutant filamentous fungi, This includes modifying the expression of XYR1 and ACE3 in parent filamentous fungi, The modification of XYR1 involves the substitution, deletion, insertion, or addition of at least one amino acid residue in the region corresponding to positions 810-833 of SEQ ID NO: 1 to a polypeptide consisting of an amino acid sequence identical to or at least 90% of the sequence of SEQ ID NO: 1, and functioning as a transcriptional activator of cellulase and hemicellulase. The modification of ACE3 expression involves enhancing the expression of a polypeptide consisting of the amino acid sequence from positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto, and which functions as a transcriptional activator of cellulase and hemicellulase. method. [2] The at least one amino acid residue is Preferably, it is at least one selected from the group consisting of Val, Ile, Leu, Ala, Gly, Thr, and Glu. More preferably, (1) and (2): (1) at least one selected from the group consisting of Val, Ile, or Leu, (2) Ala or Gly, or at least one selected from the group consisting of Val, Ala, Thr, and Glu. More preferably, at least one selected from the group consisting of Val and Ala. Furthermore, preferably, at least one amino acid residue is substituted. [1] The method described. [3] Preferably, the method according to [2], wherein the at least one amino acid residue is selected from the group consisting of amino acid residues at positions corresponding to positions 817, 821, 824, 825 and 826 of Sequence ID No. 1. [4] Preferably, the method according to [2] or [3], wherein each of the at least one amino acid residues is substituted with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr. [5] The substitution of at least one amino acid residue is Preferably, the following: Substitution of Val with Lys, Phe, Trp, or Tyr; Substitution of Ile with Phe, Trp, or Tyr; Substitution of Leu with Phe, Trp, or Tyr; Substitution of Ala with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Gly with Val, Ile, Leu, Phe, Trp, or Tyr; Substitution of Thr for Tyr; and Substitution of Glu with Tyr, At least one selected from the group consisting of, More preferably, the following: Substitution of Gly with Val, Ile, Leu, Phe, Trp, or Tyr at position 812 of sequence number 1; Substitution of Val with Phe, Trp, or Tyr at position 814 of sequence number 1; Substitution of Ala with Val, Ile, Leu, Phe, Trp, or Tyr at position 816 of sequence number 1; Substitution of Thr for Tyr at position 817 of sequence number 1; Substitution of Ala with Val, Ile, Leu, Phe, Trp, or Tyr at position 820 of sequence number 1; Substitution of Val with Lys, Phe, Trp, or Tyr at position 821 of sequence number 1; Substitution of Ala with Val, Ile, Leu, Phe, Trp, or Tyr at position 823 of sequence number 1; Substitution of Ala at position 824 of sequence number 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Glu with Tyr at position 825 of sequence number 1; Substitution of Ala at position 826 of sequence number 1 with Val, Ile, Leu, Phe, Trp, or Tyr; Substitution of Ile with Phe, Trp, or Tyr at position 827 of sequence number 1; Substitution of Ile with Phe, Trp, or Tyr at position 830 of sequence number 1; and, Substitution of Leu with Phe, Trp, or Tyr at position 831 of sequence number 1, At least one selected from the group consisting of The method described in any one of items [2] to [4]. [6] The substitution of at least one amino acid residue is Preferably, the following: Substitution of Thr for Tyr at position 817 of sequence number 1; Substitution of Val with Lys, Phe, Trp, or Tyr at position 821 of sequence number 1; Substitution of Ala at position 824 of sequence number 1 with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Glu to Tyr at position 825 of sequence number 1; and, Substitution of Ala at position 826 of sequence number 1 with Val, Ile, Leu, Phe, Trp, or Tyr. At least one selected from the group consisting of The method described in [5]. [7] The amino acid sequence at positions 107-734 of sequence number 3, or an amino acid sequence that is at least 90% identical thereto, Preferably, all amino acid residues in the region corresponding to positions 725-728 of SEQ ID NO: 3 are retained, and more preferably, all amino acid residues in the region corresponding to positions 718-728 of SEQ ID NO: 3 are retained. or, Preferably, it retains the region corresponding to positions 724-734 of SEQ ID NO: 3, however, some amino acid residues in that region may be mutated. The method described in any one of items [1] to [6]. [8] Preferably, the method according to any one of [1] to [7], wherein the enhancement of polypeptide expression is carried out by increasing the transcription level of the gene encoding the polypeptide. [9] Preferably, the method of [8], wherein the increase in the transcription level of the polypeptide-encoding gene is achieved by introducing the polypeptide-encoding gene, which is operably linked to a regulatory region, into the parent filamentous fungus.

[10] If XYR1 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 51, which is registered as XP_006966092.1, or a polypeptide consisting of an amino acid sequence that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 51 and functions as a transcriptional activator of cellulase and hemicellulase, The substitution of at least one amino acid residue is preferably as follows: Substitution of Thr for Tyr at position 797 of sequence number 51; Substitution of Val with Lys, Phe, Trp, or Tyr at position 801 of sequence number 51; Substitution of Ala with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr at position 804 of sequence number 51; Substitution of Glu with Tyr at position 805 of sequence number 51; and, Substitution of Ala with Val, Ile, Leu, Phe, Trp, or Tyr at position 806 of sequence number 51. At least one selected from the group consisting of The method described in any one of items [1] to [9].

[11] The mutant filamentous fungus, Preferably, catabolite inhibition is milder compared to filamentous fungi. More preferably, cellulase is expressed in the absence of a cellulase-inducing substance. The method described in any one of items [1] to

[10] .

[12] Preferably, the method according to any one of [1] to

[11] , wherein the filamentous fungus is a Trichoderma.

[13] Preferably, the method according to

[12] , wherein the Trichoderma species is Trichoderma risei or a mutant thereof.

[0078]

[14] A method for producing protein, comprising culturing a filamentous fungus produced by any one of the methods described in [1] to

[13] above.

[15] Preferably, the method according to

[14] , wherein the protein is cellulase and / or hemicellulase.

[16] Preferably, the method according to

[14] or

[15] , wherein the culture is carried out in the presence of glucose.

[0079]

[17] A mutant filamentous fungus containing a modified XYR1, and having enhanced expression of the ACE3 partial polypeptide compared to the parent filamentous fungus, The modified XYR1 is a polypeptide that consists of an amino acid sequence in which at least one amino acid residue in the region corresponding to positions 810-833 of SEQ ID NO: 1 is substituted, deleted, inserted, or added to SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical thereto, and functions as a transcriptional activator of cellulase and hemicellulase. The ACE3 partial polypeptide consists of the amino acid sequence from positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto, and is a polypeptide that functions as a transcriptional activator of cellulase and hemicellulase. Mutant filamentous fungi.

[18] Preferably, the amino acid sequence at positions 107 to 734 of SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical thereto, Preferably, all amino acid residues in the region corresponding to positions 725-728 of SEQ ID NO: 3 are retained, and more preferably, all amino acid residues in the region corresponding to positions 718-728 of SEQ ID NO: 3 are retained. or, Preferably, the mutant filamentous fungus according to

[17] retains the region corresponding to positions 724-734 of Sequence ID No. 3, wherein some amino acid residues in the region may be mutated.

[19] Preferably, the modified XYR1 is a substitution of at least one amino acid residue selected from the group consisting of the following in the region corresponding to positions 810-833 of SEQ ID NO: 1 or an amino acid sequence identical thereto by at least 90%: Substitution of Val with Lys, Phe, Trp, or Tyr; Substitution of Ile with Phe, Trp, or Tyr; Substitution of Leu with Phe, Trp, or Tyr; Substitution of Ala with Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; and, Substitution of Gly with Val, Ile, Leu, Phe, Trp, or Tyr; Substitution of Thr for Tyr; and Substitution of Glu with Tyr, It is a polypeptide consisting of an amino acid sequence that has been modified and functions as a transcriptional activator of cellulase and hemicellulase. The mutant filamentous fungus described in

[17] or

[18] .

[20] Preferably, the modified XYR1 is at least 90% identical in sequence to sequence number 1, and the following (a) to (m): (a) Val, Ile, Leu, Phe, Trp, or Tyr at position 812 of sequence number 1; (b) Phe, Trp, or Tyr at position 814 of Sequence ID No. 1; (c) Val, Ile, Leu, Phe, Trp, or Tyr at position 816 of Sequence ID No. 1; (d) Tyr at position 817 of sequence number 1; (e) Val, Ile, Leu, Phe, Trp, or Tyr at the position corresponding to position 820 of sequence number 1; (f) Lys, Phe, Trp, or Tyr at position 821 of sequence number 1; (g) Val, Ile, Leu, Phe, Trp, or Tyr at position 823 of Sequence ID No. 1; (h) Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr at position 824 of Sequence ID No. 1; (i) Substitution of Glu with Tyr at position 825 of Sequence ID No. 1; (j) Val, Ile, Leu, Phe, Trp, or Tyr at position 826 of sequence number 1; (k) Phe, Trp, or Tyr at position 827 of sequence number 1; (l) Phe, Trp, or Tyr at position 830 of sequence number 1; and, (m) Phe, Trp, or Tyr at position 831 of sequence number 1 A polypeptide comprising an amino acid sequence having at least one amino acid residue selected from the group consisting of the following, and which functions as a transcriptional activator of cellulase and hemicellulase.

[19] The mutant filamentous fungus described.

[21] Preferably, the modified XYR1 is at least 90% identical in sequence to sequence number 1, and the following: Lys, Phe, Trp, or Tyr at the position corresponding to position 821 of sequence number 1, preferably Phe, more preferably Lys or Tyr; and, Val, Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr at the position corresponding to position 824 of Sequence ID No. 1, preferably Val, more preferably Glu, Ile, Leu, Lys, Phe, Thr, Trp or Tyr, A polypeptide comprising an amino acid sequence having at least one amino acid residue selected from the group consisting of the following, and which functions as a transcriptional activator of cellulase and hemicellulase.

[20] The mutant filamentous fungus described.

[22] Preferably, the modified XYR1 is at least 90% identical in sequence to sequence number 1, and the following: Tyr at position 817 of sequence number 1; Tyr at position 825 of sequence number 1, and Val, Ile, Leu, Phe, Trp, or Tyr at position 826 of Sequence ID No. 1, preferably Val or Trp, A polypeptide comprising an amino acid sequence having at least one amino acid residue selected from the group consisting of the following, and which functions as a transcriptional activator of cellulase and hemicellulase. The mutant filamentous fungus described in

[20] or

[21] . [Examples]

[0080] The present invention will be described in more detail below using examples.

[0081] Example 1: Construction of plasmid DNA for gene transfer The following DNA fragments 1-5 were prepared by PCR using the genomic DNA of Trichoderma leese as a template: Fragment 1: approximately 1.5 kbp of the promoter region upstream of the act1 gene (TRIREDRAFT_44504), Fragment 2: approximately 3.0 kbp of the xyr1 gene (sequence number 2), Fragment 3: polynucleotide encoding a partial polypeptide of ACE3 (positions 107-734 of sequence number 3) (nucleotide region 881-2918 of sequence number 4, approximately 2.0 kbp), Fragment 4: approximately 0.6 kbp of the terminator region downstream of the cbh1 gene (TRIREDRAFT_44504), and Fragment 5: approximately 2.7 kbp of the pyr4 gene (TRIREDRAFT_74020). Cassette 1: Pact1-XYR1 was constructed by joining fragments 1 and 2. Cassette 2: Pact1-ACE3 was constructed by joining fragments 1 and 3. A transformation marker fragment was prepared by placing fragment 6 (approximately 0.5 kbp) and fragment 7 (approximately 1.0 kbp) as homologous sequences for pop-out upstream and downstream of fragment 5, respectively. Cassette 3: Tcbh1-pyr4 was constructed by joining fragment 4 to the transformation marker fragment. DNA fragment joining was performed according to the In-Fusion HD Cloning Kit (Takara Bio) protocol. Table 1 shows the constructed cassettes, the DNA fragments contained therein, and the primers used to construct the cassettes. Cassettes 1 and 3 were joined and inserted into the HincII restriction enzyme cleavage site of pUC118 (Takara Bio) to construct the xyr1 constitutive expression plasmid pUC-Pact1-XYR1. Similarly, cassettes 2 and 3 were joined and inserted into the HincII restriction enzyme cleavage site of pUC118 (Takara Bio) to construct the ace3 constitutive expression plasmid pUC-Pact1-ACE3.

[0082] [Table 1]

[0083] pUC-Pact1-XYR1(V821F) and pUC-Pact1-XYR1(A824V) were constructed by PCR using the primers in Table 2 with pUC-Pact1-XYR1 as a template. pUC-Pact1-XYR1(V821F) is a plasmid encoding mutant XYR1(V821F) in which the amino acid substitution of V821F is made to the amino acid sequence of SEQ ID NO: 2. pUC-Pact1-XYR1(A824V) is a plasmid encoding mutant XYR1(A824V) in which the amino acid substitution of A824V is made to the amino acid sequence of SEQ ID NO: 2.

[0084] [Table 2]

[0085] The constructed plasmids pUC-Pact1-ACE3, pUC-Pact1-XYR1(V821F), and pUC-Pact1-XYR1(A824V) were replicated. The plasmids were introduced into competent cells Escherichia coli DH5α Competent Cells (Takara Bio), cultured in ampicillin-supplemented LB medium (37°C, 1 day), and NucleoSpin was extracted from the cultured cells. TM Plasmid (Machley-Nagel) was used to recover and purify the plasmid.

[0086] Example 2: Preparation of filamentous fungal mutant strains The Trichoderma lisey JN13Δpyr4 strain was transformed by introducing the plasmid constructed in Example 1. Plasmid introduction was performed by protoplast PEG (Biotechnol Bioeng, 2012, 109(1):92-99). Transformants were selected using the pyr4 gene as a marker in selective medium (2% glucose, 1.1M sorbitol, 2% agar, 0.2% KH2PO4 (pH 5.5), 0.06% CaCl2·2H2O, 0.06% CsCl2, 0.06% MgSO4·7H2O, 0.5% (NH4)2SO4, 0.1% Trace element 1; % all are w / v). Trace element 1 was composed as follows: 0.5g FeSO4·7H2O, 0.2g CoCl2, 0.16g MnSO4·H2O, and 0.14g ZnSO4·7H2O were mixed to 100mL with distilled water. From the resulting transformants, the insertion of the target gene fragment was confirmed by PCR, and the JN13_ACE3 strain, into which pUC-Pact1-ACE3 was introduced, and the JN13_XYR1(V821F) strain, into which pUC-Pact1-XYR1(V821F) was introduced, were obtained. The JN13_ACE3 strain highly expresses ACE3 via the ace3 constitutive expression plasmid pUC-Pact1-ACE3. The JN13_XYR1(V821F) strain expresses mutant XYR1(V821F).

[0087] To re-transform the transformed strains, strains that re-acquired 5-FOA resistance and grew using PDA medium containing 0.2% 5-fluoroorotic acid (5-FOA) monohydrate were selected. Specifically, spores of the JN13_ACE3 strain were inoculated onto 5-FOA-containing medium, and the resulting strain was obtained as the JN13_ACE3Δpyr4 strain. The obtained strains were transformed with the above-mentioned pUC-Pact1-XYR1(V821F) or pUC-Pact1-XYR1(A824V) to obtain the JN13_XYR1(V821F)+ACE3 strain and the JN13_XYR1(A824V)+ACE3 strain. These strains highly express ACE3 and express mutant XYR1(V821F or A824V).

[0088] Example 3: Culture of filamentous fungal mutant strains The filamentous fungal strain obtained in Example 2 was cultured to produce protein. In the pre-culture, 50 mL of culture medium was placed in a 500 mL flask, and spores of the strain prepared in Example 2 were added in a 1 × 10⁶ solution. 5 The cells were inoculated to a concentration of 1 / mL and cultured with shaking at 28°C and 220 rpm (PRXYg-98R, manufactured by PRIS). The culture medium composition was as follows: 1% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% high polypeptone N, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element 2, 50 mM tartrate buffer (pH 4.0) (all percentages are w / v%). The composition of Trace element 2 was as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24 Mix 4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2 with distilled water to make up 100mL.

[0089] After a two-day pre-culture, the main culture was performed. 50 mL of culture medium was placed in a 500 mL flask, and 1% (v / v%) of the pre-culture solution was inoculated. The culture was incubated at 28°C and 220 rpm for four days. The culture medium composition was as follows: 3% cellulose or 3% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% high polypeptone N, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element 2, 1.28% diammonium hydrogen citrate, 50 mM tartaric acid buffer (pH 4.0) (all percentages are w / v%).

[0090] Example 4: Protein concentration measurement The protein concentration of the cultures in Example 3 was measured using the Bradford method. In the Bradford method, the Quick Start Protein Assay (BioRad) was used, and the protein amount was calculated based on a calibration curve created using bovine γ-globulin as the standard protein. Figure 1 shows the relative protein productivity of each strain, with the protein productivity of the JN13 strain (parent strain) in culture using glucose alone as the carbon source (glucose culture) set to 1. Compared to culture in the presence of cellulose, the protein productivity of the parent strain JN13 in glucose culture was significantly lower. This is presumed to be because when glucose alone is used as the carbon source, there are no inducers, and the expression of cellulases and xylanases is not induced by transcriptional activation. In the XYR1(V821F)+ACE3 and XYR1(A824V)+ACE3 strains, which were modified in both XYR1 and ACE3, protein productivity in glucose culture was significantly improved compared to the parent strain, and even compared to the XYR1(V821F) and ACE3 strains in which only one of XYR1 or ACE3 was modified.

[0091] Example 5: Protein composition analysis The protein composition of the culture from Example 3 was analyzed. Mini PROTEAN TGX Stain-Free Gels (Any KD, 15-well, BIORAD) were used for the analysis. Precision Plus Protein Unstained standards were used as the standard. The culture from Example 3, diluted as appropriate, was mixed with Buffer and treated at 99°C for 5 minutes. The mixture was applied to a gel and electrophoresis was performed at 200V for 35 minutes. From the obtained image file (Figure 2), the band intensity ratio was calculated using analysis software (Image Lab), and the composition ratio of the produced proteins was calculated. The production amount of each protein was determined from this composition ratio and the protein concentration obtained in Example 4. Next, the relative production of each protein in each strain was calculated, with the protein production of only the XYR1 mutation (XYR1(V821F) strain) set to 1.

[0092] Table 3 shows the composition ratio of proteins produced by each strain, and Figure 3 shows the relative productivity. Under glucose culture conditions, the JN13 strain (parent strain) produced almost no cellulases and xylanases (BXL1, CBH1, CBH2+EG1, XYN1+XYN2). The XYR1 single mutant (XYR1(V821F)) mainly produced xylanases such as XYN1+XYN2 (XYN1+XYN2). The ACE3 high-expression strain (ACE3) showed an increased ratio of cellulases such as CBH1, CBH2, and EG1, however, as shown in Figure 1, the total protein production was lower than that of the XYR1 single mutant and was about the same as that of the parent strain. Strains combining XYR1 mutation and ACE3 high expression (XYR1(V821F)+ACE3, and XYR1(A824V)+ACE3) produced cellulases and xylanases even under glucose culture conditions. Furthermore, in the XYR1(V821F)+ACE3 and XYR1(A824V)+ACE3 strains, the composition ratio of the products was closer to that of the parent strain in the presence of cellulose compared to the XYR1 mono-mutant and ACE3 high-expression strains, and the productivity of the major cellulases CBH1, CBH2, and EG1 was significantly improved.

[0093] [Table 3]

[0094] Example 6: Preparation of filamentous fungal mutant strains Plasmids expressing mutant XYR1 with the T817Y amino acid substitution were constructed by PCR using primers SEQ ID NOs. 23 and 24 shown in Table 4, with pUC-Pact1-XYR1 as the template, following the same procedure as in Example 1. Plasmids expressing mutant XYR1 with the V821Y, V821K, A824I, A824L, A824F, A824W, A824Y, A824T, A824K, A824E, E825Y, A826V, and A826W amino acid substitutions were constructed using the same procedure as in Example 2. The constructed plasmids were transformed into the JN13_ACE3Δpyr4 strain using the same method as in Example 2 to obtain filamentous fungal mutants into which ACE3 and the above mutant XYR1 were introduced.

[0095] [Table 4]

[0096] The mutant strains obtained above, as well as the JN13_XYR1(V821F)+ACE3 strain, JN13_XYR1(A824V)+ACE3 strain, XYR1 single mutant (JN13_XYR1(V821F)), and ACE3 high-expression strain (JN13_ACE3) prepared in Example 2, were cultured in glucose according to the method described in Example 3, and then the protein concentration of the cultures and the protein composition analysis were performed according to the methods described in Examples 4 and 5.

[0097] Table 5 shows the composition ratio of the proteins produced by each strain, and Figure 4 shows the relative productivity. In Figure 4, BXL1 is β-xylosidase BXL1, CBH1 is cellulase CBH1, CBH2+EG1 is cellulase CBH2 and EG1, XYN1+XYN2 is xylanase XYN1 and XYN2, and others are other proteins. The filamentous fungal mutant strains prepared in this example, which highly express ACE3 and express mutant XYR1 (T817Y, V821Y, V821K, A824I, A824L, A824F, A824W, A824Y, A824T, A824K, A824E, E825Y, A826V, and A826W), all produced proteins with the same composition as the JN13_XYR1(V821F)+ACE3 strain and JN13_XYR1(A824V)+ACE3 strain prepared in Example 2. Furthermore, all of these filamentous fungal mutant strains showed significantly improved productivity of the major cellases CBH1, CBH2, and EG1 under glucose culture conditions compared to the XYR1 single mutant (JN13_XYR1(V821F)) and the ACE3 high-expression strain (JN13_ACE3).

[0098] [Table 5]

[0099] As described above, combining XYR1 mutations with high ACE3 expression significantly improved the productivity of not only xylanase but also the major cellulases CBH1, CBH2, and EG1 compared to XYR1 mutations alone. Effective XYR1 mutations were not limited to V821F and A824V; mutant XYR1 exhibiting a glucose-blind phenotype (a property that improves protein productivity even when using glucose, which normally causes catabolite repression), with at least one amino acid mutation in the region presumed to be the α-helix in the acidic activation domain of XYR1, was effective in cellulase and xylanase production in the absence of inducers. Therefore, it was shown that the above combination of XYR1 mutations and high ACE3 expression can efficiently induce protein production by activating the promoters of cellulase / xylanase genes, even when using non-cellulase-inducing carbon sources such as glucose, similar to when using cellulase-inducing substances such as cellulose.

Claims

1. A method for producing mutant filamentous fungi, This includes modifying the expression of XYR1 and ACE3 in parent filamentous fungi, The following modifications to XYR1 affect polypeptides that consist of an amino acid sequence identical to or at least 90% of SEQ ID NO: 1, and that function as transcriptional activators of cellulase and hemicellulase: Substitution of Thr to Tyr at position 817 of sequence number 1; Substitution of Val at position 821 of sequence number 1 with Lys or Tyr; Substitution of Ala at position 824 of sequence number 1 with Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Glu with Tyr at position 825 of sequence number 1; and, Substitution of Ala with Val or Trp at position 826 of sequence number 1, The substitution is of at least one amino acid residue selected from the group consisting of the following: The modification of ACE3 expression is an enhancement of the expression of a polypeptide that functions as a transcriptional activator of cellulase and hemicellulase, comprising an amino acid sequence selected from the group consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of position 107 to 734 of SEQ ID NO: 3, an amino acid sequence of a polypeptide encoded in the nucleotide region of nucleotide NO: 4 from 881 to 2918, and an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of a polypeptide encoded in the nucleotide region of nucleotide NO: 4, The mutant filamentous fungus expresses cellulase and / or hemicellulase in the absence of cellulase-inducing substances. method.

2. The method according to claim 1, wherein the enhancement of polypeptide expression is performed by increasing the transcription level of the gene encoding the polypeptide.

3. The method according to claim 1 or 2, wherein the mutant filamentous fungus expresses cellulase in the absence of a cellulase-inducing substance.

4. The method according to any one of claims 1 to 3, wherein the filamentous fungus is a Trichoderma species.

5. The method according to claim 4, wherein the Trichoderma species is Trichoderma risei.

6. A method for producing a protein, comprising culturing a mutant filamentous fungus produced by the method described in any one of claims 1 to 5.

7. The method according to claim 6, wherein the protein is cellulase and / or hemicellulase.

8. The method according to claim 6 or 7, wherein the culture is carried out in the presence of glucose.

9. The method according to any one of claims 6 to 8, wherein the culture is carried out in the absence of a cellulase-inducing substance.

10. A mutant filamentous fungus containing a modified XYR1, and exhibiting enhanced expression of the ACE3 partial polypeptide compared to the parent filamentous fungus. The modified XYR1, with respect to SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical thereto, is as follows: Substitution of Thr to Tyr at position 817 of sequence number 1; Substitution of Val at position 821 of sequence number 1 with Lys or Tyr; Substitution of Ala at position 824 of sequence number 1 with Glu, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr; Substitution of Glu with Tyr at position 825 of sequence number 1; and, Substitution of Ala with Val or Trp at position 826 of sequence number 1, A polypeptide comprising an amino acid sequence in which at least one amino acid residue selected from the group consisting of the following is substituted, and which functions as a transcriptional activator of cellulase and hemicellulase, The ACE3 partial polypeptide is a polypeptide that consists of an amino acid sequence selected from the group consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of position 107 to 734 of SEQ ID NO: 3, an amino acid sequence of a polypeptide encoded in the nucleotide region of nucleotides 881 to 2918 of the nucleotide sequence of SEQ ID NO: 4, and an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of a polypeptide encoded in the nucleotide region of nucleotides 881 to 2918 of the nucleotide sequence of SEQ ID NO: 4, and functions as a transcriptional activator of cellulase and hemicellulase. The mutant filamentous fungus expresses cellulase and / or hemicellulase in the absence of cellulase-inducing substances. Mutant filamentous fungi.

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