Protein production method

Abstract

Provided is a method for producing a protein. The method of the present invention comprises culturing Talaromyces cellulolyticus in a medium, said Talaromyces cellulolyticus being capable of producing a target protein and having been modified so as to reduce the activity of protein Pep4, to thereby produce the target protein.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a protein. [Background technology]

[0002] Methods for producing proteins using various microorganisms such as coryneform bacteria, Bacillus bacteria, yeasts, and filamentous fungi have been reported.

[0003] For example, Non-Patent Document 1 discloses the production of host-derived cellulase using the filamentous fungus Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). Patent Document 1 discloses the production of antibodies using filamentous fungi. Patent Document 2 discloses the production of a multimeric protein having a lumen using filamentous fungi such as Talaromyces cellulolyticus.

[0004] Furthermore, Patent Documents 3 and 4 disclose the production of heterologous proteins using filamentous fungi in which the activity of endogenous proteases is attenuated, and Patent Document 5 discloses the production of heterologous proteins using filamentous fungi in which the activity of endogenous alkaline proteases is attenuated.

[0005] Furthermore, Patent Document 6 discloses the production of heterologous proteins using yeast lacking the activity of carboxypeptidase yscα, and also states that the yeast may further lack the activity of a peptidase selected from yscA, yscB, yscY, and yscS.

[0006] Furthermore, Patent Document 7 discloses the production of proteins using Talaromyces cellulolyticus that has been modified so that the activity of the protease YscB is reduced.

[0007] However, the relationship between the Pep4 protein and protein production in Talaromyces cellulolyticus is unknown. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication 2006-512891 [Patent Document 2] Patent Publication No. 2016-158599 [Patent Document 3] Special Publication 2015-512611 [Patent Document 4] Special Publication 2016-523552 [Patent Document 5] Special Publication 2000-507106 [Patent Document 6] Patent Publication No. 1990-104279 [Patent Document 7] WO2019 / 073954 [Non-patent literature]

[0009] [Non-Patent Document 1] Inoue H, et al., Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus. J Ind Microbiol Biotechnol. 2013 Aug;40(8):823-30. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a method for producing a protein. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the present inventors discovered that the protein production ability of Talaromyces cellulolyticus can be improved by modifying Talaromyces cellulolyticus so that the activity of the Pep4 protein is reduced, thereby completing the present invention.

[0012] That is, the present invention can be exemplified as follows. [1] A method for producing a target protein, comprising: Cultivating Talaromyces cellulolyticus capable of producing the target protein in a medium; Including, The method, wherein the Talaromyces cellulolyticus has been modified so that the activity of Pep4 protein is reduced compared to an unmodified strain. [2] The method as described above, wherein the activity of the Pep4 protein is reduced by reducing the expression of the pep4 gene or by disrupting the pep4 gene. [3] The method as described above, wherein the activity of the Pep4 protein is reduced by deleting the pep4 gene. [4] The method, wherein the Pep4 protein is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 71; (b) a protein comprising an amino acid sequence set forth in SEQ ID NO: 71, but which contains a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and which has protease activity; (c) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 71 and having protease activity. [5] The method as described above, wherein the Talaromyces cellulolyticus has been further modified to reduce the activity of YscB protein and / or CreA protein compared to an unmodified strain. [6] The method as described above, wherein the activity of the YscB protein and / or the CreA protein is reduced by reducing the expression of the yscB gene and / or the creA gene, or by disrupting the yscB gene and / or the creA gene. [7] The method as described above, wherein the activity of the YscB protein and / or the CreA protein is reduced by deletion of the yscB gene and / or the creA gene. [8] The method as described above, wherein the Talaromyces cellulolyticus is a modified strain derived from the Talaromyces cellulolyticus S6-25 strain (NITE BP-01685). [9] The method further comprises recovering the target protein.

[10] The method, wherein the target protein accumulates in the medium by the culturing.

[11] The method, wherein the target protein is expressed as a fusion protein with a signal peptide that functions in Talaromyces cellulolyticus.

[12] The above method, wherein the target protein is a heterologous protein.

[13] The above method, wherein the target protein is a human-derived protein.

[14] The above method, wherein the target protein is an antibody-related molecule. [Brief explanation of the drawings]

[0013] [Figure 1] A diagram (photograph) showing the results of Trastuzumab degradation by the culture supernatants of the T. cellulolyticus control strain and the Δpep4 strain. [Figure 2] FIG. 1 shows the results of Trastuzumab production by a Trastuzumab-expressing strain derived from the T. cellulolyticus F09 ΔyscB strain and its pep4 gene-disrupted strain (photograph). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in detail below.

[0015] The method of the present invention is a method for producing a target protein using Talaromyces cellulolyticus. The Talaromyces cellulolyticus used in this method is also referred to as the "microorganism of the present invention."

[0016] <1> The microorganism of the present invention The microorganism of the present invention is a Talaromyces cellulolyticus strain capable of producing a target protein that has been modified to reduce the activity of the Pep4 protein. In describing the microorganism of the present invention, the microorganism of the present invention or the Talaromyces cellulolyticus strain used to construct the microorganism may be referred to as a "host."

[0017] <1-1> Talaromyces cellulolyticus The microorganism of the present invention is Talaromyces cellulolyticus. The former name of Talaromyces cellulolyticus is Acremonium cellulolyticus. That is, Acremonium cellulolyticus was reclassified as Talaromyces cellulolyticus in a phylogenetic revision (FEMS Microbiol. Lett., 2014, 351:32-41). Specific examples of Talaromyces cellulolyticus include the C1 strain (JP 2003-135052), the CF-2612 strain (JP 2008-271927), the TN strain (FERM BP-685), the S6-25 strain (NITE BP-01685), the Y-94 strain (FERM BP-5826, CBS 136886), and their derivatives. "Talaromyces cellulolyticus" collectively refers to fungi classified as Talaromyces cellulolyticus at least at any point before, at the time of, or after the filing of this application. That is, for example, strains once classified as Talaromyces cellulolyticus, such as the strains exemplified above, will be treated as belonging to Talaromyces cellulolyticus even if the phylogenetic classification is changed in the future.

[0018] The S6-25 strain was originally deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, Japan, 292-0818) on August 8, 2013, and transferred to international deposit under the Budapest Treaty on November 15, 2013, and assigned accession number NITE BP-01685. The S6-25 strain was derived from the TN strain (FERM BP-685) and has high cellulase productivity. The TN strain was derived from the Y-94 strain (FERM BP-5826, CBS 136886) (JP 2011-193773).

[0019] These strains can be obtained, for example, from the depository institutions where they have been deposited. The Y-94 strain can be obtained, for example, from the CBS-KNAW Collections (Netherlands).

[0020] The microorganism of the present invention can be obtained by modifying Talaromyces cellulolyticus, such as the strains exemplified above. That is, the microorganism of the present invention may be, for example, a modified strain derived from the strain exemplified above. Specifically, the microorganism of the present invention may be, for example, a modified strain derived from the S6-25 strain or the Y-94 strain. More specifically, the microorganism of the present invention may be, for example, a modified strain derived from the S6-25 strain. The order of modifications to construct the microorganism of the present invention is not particularly limited.

[0021] <1-2> Target protein productivity The microorganism of the present invention has the ability to produce a target protein. "A microorganism capable of producing a target protein" refers to a microorganism capable of producing a target protein. Specifically, a "microorganism capable of producing a target protein" may be a microorganism capable of expressing a target protein when cultured in a medium and accumulating the target protein in the culture to an extent that the protein can be recovered. "Accumulation in the culture" may specifically refer to accumulation in the medium, on the cell surface, within the cell, or a combination thereof. Accumulation of a target protein outside the cell (e.g., in the medium or on the cell surface) is also referred to as "secretion" or "secretory production" of the target protein. In other words, the microorganism of the present invention may have the ability to secrete and produce the target protein (the ability to secrete and produce the target protein). The target protein may particularly accumulate in the medium. The amount of the target protein accumulated in the culture may be, for example, 10 μg / L or more, 1 mg / L or more, 100 mg / L or more, or 1 g / L or more. The microorganism of the present invention may have the ability to produce one type of target protein, or may have the ability to produce two or more types of target proteins.

[0022] The microorganism of the present invention may be one that inherently has the ability to produce a target protein, or may be one that has been modified to have the ability to produce a target protein. Typically, the microorganism of the present invention may inherently have the ability to produce cellulase (the ability to produce cellulase). Alternatively, the microorganism of the present invention may be one that has been modified so that its inherent ability to produce a target protein is enhanced. A microorganism capable of producing a target protein can be obtained, for example, by imparting the ability to produce a target protein to the above-mentioned Talaromyces cellulolyticus, or by enhancing the ability of the above-mentioned Talaromyces cellulolyticus to produce a target protein. The ability to produce a target protein can be imparted or enhanced, for example, by introducing a gene construct for expressing the target protein, introducing other modifications that improve the ability to produce a target protein, or a combination thereof.

[0023] The microorganism of the present invention has the ability to produce a target protein due to the presence of at least a gene construct for expressing the target protein. Specifically, the microorganism of the present invention may have the ability to produce a target protein by having a gene construct for expressing the target protein, or by having a gene construct for expressing the target protein in combination with other properties. That is, the microorganism of the present invention has a gene construct for expressing the target protein. The microorganism of the present invention may have one copy of the gene construct for expressing the target protein, or two or more copies of the gene construct for expressing the target protein. The microorganism of the present invention may have a gene construct for expressing one type of target protein, or two or more types of target proteins. The copy number and number of types of the gene construct for expressing the target protein may be interpreted as the copy number and number of types of the target protein gene, respectively.

[0024] In the microorganism of the present invention, the gene construct for expressing a target protein may be present on an extrachromosomally autonomously replicating vector such as a plasmid, or may be integrated into the chromosome. That is, the microorganism of the present invention may, for example, have a gene construct for expressing a target protein on a vector, in other words, a vector containing a gene construct for expressing a target protein. Furthermore, the microorganism of the present invention may, for example, have a gene construct for expressing a target protein on a chromosome. When the microorganism of the present invention has gene constructs for expressing two or more target proteins, these gene constructs may be retained in the microorganism of the present invention so as to be able to produce the target proteins. For example, these gene constructs may all be retained on a single expression vector, or all may be retained on a chromosome. Furthermore, these gene constructs may be retained separately on multiple expression vectors, or they may be retained separately on a single or multiple expression vectors and on the chromosome.

[0025] The microorganism of the present invention may inherently have a gene construct for expressing a target protein, or may be modified to have a gene construct for expressing a target protein. Typically, the microorganism of the present invention may inherently have a gene construct for expressing cellulase. Furthermore, the microorganism of the present invention may have a gene construct for expressing a target protein introduced into it, instead of or in addition to the gene construct for expressing a target protein that it inherently has. A microorganism having a gene construct for expressing a target protein can be obtained by introducing the gene construct for expressing a target protein into Talaromyces cellulolyticus as described above.

[0026] A "gene construct for expressing a target protein" refers to a gene expression system configured to express a target protein. A gene construct for expressing a target protein is also referred to as a "target protein expression system," "target protein expression unit," or "target protein expression cassette." A gene construct for expressing a target protein contains, from 5' to 3', a promoter sequence and a nucleotide sequence encoding the target protein. The promoter sequence is also simply referred to as a "promoter." A nucleotide sequence encoding an amino acid sequence is also referred to as a "gene." For example, a nucleotide sequence encoding a target protein is also referred to as a "gene encoding the target protein" or a "target protein gene." The target protein gene may be linked downstream of the promoter so that the target protein is expressed under the control of the promoter. Furthermore, a gene construct for expressing a target protein may have control sequences (such as operators and terminators) effective for expressing the target protein at appropriate positions so that they can function. In this specification, unless otherwise specified, the terms "expression of a target protein gene," "expression of a target protein," "production of a target protein," and "production of a target protein" are used interchangeably. A gene construct for expressing a target protein can be designed appropriately depending on various conditions such as the type of target protein.

[0027] The promoter is not particularly limited as long as it functions in Talaromyces cellulolyticus. A "promoter that functions in Talaromyces cellulolyticus" refers to a promoter that has promoter activity, i.e., gene transcription activity, in Talaromyces cellulolyticus.

[0028] The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be the native promoter of the target protein gene or a promoter of another gene. The promoter may be an inducible promoter or a constitutive promoter. Examples of promoters include promoters of microbial cellulase genes. Specific examples of promoters include promoters of cellulase genes from Talaromyces cellulolyticus. Cellulase genes include the cbhI gene (also referred to as the cbh1 gene) and the cbhII gene (also referred to as the cbh2 gene). Specifically, examples of promoters include promoters of the cbhI gene and the cbhII gene. The cbhI gene promoter is also referred to as the "cbhI promoter" or "cbh1 promoter." The cbhII gene promoter is also referred to as the "cbhII promoter" or "cbh2 promoter." The nucleotide sequence of the cbhII promoter from Talaromyces cellulolyticus is shown in SEQ ID NO: 63. That is, the promoter may be, for example, a promoter having the nucleotide sequence of the above-exemplified promoter (e.g., the nucleotide sequence of SEQ ID NO: 63). The promoter may also be a conservative variant of the above-exemplified promoter (e.g., the promoter having the nucleotide sequence of SEQ ID NO: 63). That is, for example, the above-exemplified promoters can be used as is or after appropriate modification. The terms "cbhI promoter" and "cbhII promoter" encompass the above-exemplified cbhI promoter and cbhII promoter, as well as their conservative variants. The description of the conservative variant of the pep4 gene described below can be applied mutatis mutandis to conservative variants of promoters. For example, the promoter may be DNA having a nucleotide sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the nucleotide sequence of SEQ ID NO: 63, as long as the original function is maintained.The "original function" of a promoter refers to the function of expressing a gene linked immediately downstream (e.g., inducible or constitutive expression). The function of a promoter can be confirmed, for example, by confirming gene expression. Gene expression can be confirmed, for example, using a reporter gene.

[0029] The target protein is not particularly limited. It may be a protein derived from a host or a protein derived from a different species (heterologous protein). In the present invention, a "heterologous protein" refers to a protein that is exogenous to the Talaromyces cellulolyticus that produces the protein. The target protein may be, for example, a protein derived from a microorganism, a plant, an animal, a virus, or a protein with an artificially designed amino acid sequence. The target protein may particularly be a protein derived from a human. The target protein may be a monomeric protein or a multimeric protein. A multimeric protein refers to a protein that can exist as a multimer consisting of two or more subunits. In a multimer, the subunits may be linked by covalent bonds such as disulfide bonds, non-covalent bonds such as hydrogen bonds or hydrophobic interactions, or a combination thereof. It is preferable that the multimer contain one or more intermolecular disulfide bonds. The multimer may be a homomultimer consisting of a single type of subunit, or a heteromultimer consisting of two or more types of subunits. The phrase "the target protein is a heterologous protein" means that, in the case where the target protein is a heteromultimeric protein, at least one of the subunits constituting the multimer is a heterologous protein. That is, all of the subunits may be derived from a different species, or only some of the subunits may be derived from a different species. The target protein may be a secreted protein or a non-secreted protein. A secreted protein may be a protein that is secreted in nature, or a protein that is non-secreted in nature, but is preferably a protein that is secreted in nature. The term "protein" also encompasses what are called peptides, such as oligopeptides and polypeptides.

[0030] Examples of target proteins include enzymes, physiologically active proteins, receptor proteins, antigenic proteins, and any other proteins.

[0031] Examples of enzymes include cellulase, xylanase, transglutaminase, protein glutaminase, protein asparaginase, isomaltodextranase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, chitinase, γ-glutamylvaline synthetase, glutamic acid-cysteine ligase, and glutathione synthetase.

[0032] In the present invention, "cellulase" is a general term for enzymes that catalyze the hydrolysis of glycosidic bonds in cellulose. Examples of cellulases include endo-type cellulases (endoglucanases; EC 3.2.1.4), exo-type cellulases (cellobiohydrolases; EC 3.2.1.91), and cellobiases (β-glucosidases; EC 3.2.1.21). Depending on the substrate used to measure the activity, cellulases are also called avicelase, filter paper cellulases (FPases), carboxymethylcellulases (CMCases), and the like. Examples of cellulases include cellulases from fungi such as Trichoderma reesei and Talaromyces cellulolyticus, and bacteria such as Clostridium thermocellum.

[0033] Examples of transglutaminases include secretory transglutaminases from actinomycetes such as Streptoverticillium mobaraense IFO 13819 (WO 01 / 23591), Streptoverticillium cinnamoneum IFO 12852, Streptoverticillium griseocarneum IFO 12776, and Streptomyces lydicus (WO 9606931), and filamentous fungi such as Oomycetes (WO 9622366). Examples of protein glutaminases include protein glutaminase from Chryseobacterium proteolyticum (WO 2005 / 103278). Examples of isomaltodextranases include isomaltodextranase from Arthrobacter globiformis (WO 2005 / 103278).

[0034] Examples of physiologically active proteins include growth factors, hormones, cytokines, and antibody-related molecules.

[0035] Specific examples of growth factors include epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), acidic fibroblast growth factor (ACF), and erythropoietin (EPO). These include fibroblast growth factor (aFGF or FGF1), basic fibroblast growth factor (bFGF or FGF2), keratinocyte growth factor (KGF-1 or FGF7, KGF-2 or FGF10), hepatocyte growth factor (HGF), stem cell factor (SCF), and activin. Activins include activin A, C, and E.

[0036] Specific examples of hormones include insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), calcitonin, and exenatide.

[0037] Specific examples of cytokines include interleukins, interferons, and tumor necrosis factors (TNFs).

[0038] It is not necessary to strictly distinguish between growth factors, hormones, and cytokines. For example, a physiologically active protein may belong to any one group selected from growth factors, hormones, and cytokines, or may belong to multiple groups selected from these.

[0039] Furthermore, the physiologically active protein may be the entire protein or a portion thereof. Examples of the portion of the protein include a physiologically active portion. Specific examples of the physiologically active portion include teriparatide, a physiologically active peptide consisting of the N-terminal 34 amino acid residues of the mature form of parathyroid hormone (PTH).

[0040] An "antibody-related molecule" refers to a protein containing a molecular species consisting of a single domain selected from the domains constituting a complete antibody, or a combination of two or more domains. The domains constituting a complete antibody include the heavy chain domains VH, CH1, CH2, and CH3, and the light chain domains VL and CL. An antibody-related molecule may be a monomeric protein or a multimeric protein, so long as it contains the above-mentioned molecular species. When the antibody-related molecule is a multimeric protein, it may be a homomultimer consisting of a single type of subunit, or a heteromultimer consisting of two or more types of subunits. Specific examples of antibody-related molecules include complete antibodies, Fab, F(ab'), F(ab'), Fc, dimers consisting of heavy (H) and light (L) chains, Fc fusion proteins, heavy (H) and light (L) chains, single-chain Fvs (scFv), sc(Fv)2, disulfide-linked Fvs (sdFv), diabodies, and VHH fragments (nanobodies (registered trademark)). More specific examples of antibody-related molecules include trastuzumab, adalimumab, nivolumab, and the VHH antibody N15.

[0041] Receptor proteins include, for example, receptor proteins for physiologically active proteins and other physiologically active substances. Other physiologically active substances include, for example, neurotransmitters such as dopamine. Receptor proteins may also be orphan receptors for which the corresponding ligand is unknown.

[0042] The antigen protein is not particularly limited as long as it can induce an immune response. The antigen protein can be appropriately selected depending on, for example, the target of the expected immune response. The antigen protein can be used, for example, as a vaccine.

[0043] Other proteins include liver-type fatty acid-binding protein (LFABP), fluorescent proteins, immunoglobulin-binding proteins, albumin, fibroin-like proteins, and extracellular proteins. Fluorescent proteins include green fluorescent protein (GFP) and monomeric red fluorescent protein (mRFP). Immunoglobulin-binding proteins include protein A, protein G, and protein L. Albumins include human serum albumin. Fibroin-like proteins include those disclosed in WO2017 / 090665 and WO2017 / 171001.

[0044] Extracellular proteins include fibronectin, vitronectin, collagen, osteopontin, laminin, and their partial sequences. Laminin is a heterotrimeric protein consisting of an α chain, a β chain, and a γ chain. Examples of laminin include mammalian laminins. Mammals include primates such as humans, monkeys, and chimpanzees, rodents such as mice, rats, hamsters, and guinea pigs, and various other mammals such as rabbits, horses, cows, sheep, goats, pigs, dogs, and cats. Mammals, in particular, include humans. The subunit chains of laminin (i.e., α chains, β chains, and γ chains) include five α chains (α1-α5), three β chains (β1-β3), and three γ chains (γ1-γ3). Laminin forms various isoforms based on the combination of these subunit chains. Specific examples of laminins include laminin 111, laminin 121, laminin 211, laminin 213, laminin 221, laminin 311, laminin 321, laminin 332, laminin 411, laminin 421, laminin 423, laminin 511, laminin 521, and laminin 523. Examples of laminin partial sequences include laminin E8, which is the E8 fragment of laminin. Laminin E8 is a protein with a heterotrimeric structure consisting of an E8 fragment of the α chain (α chain E8), an E8 fragment of the β chain (β chain E8), and an E8 fragment of the γ chain (γ chain E8). The subunit chains of laminin E8 (i.e., α chain E8, β chain E8, and γ chain E8) are collectively referred to as the "E8 subunit chain." Examples of E8 subunit chains include E8 fragments of the laminin subunit chains listed above. Laminin E8 is composed of various isoforms depending on the combination of these E8 subunit chains. Specific examples of laminin E8 include laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, and laminin 521E8.

[0045] The target protein gene can be used as is or after appropriate modification. The target protein gene can be modified, for example, to obtain a desired activity. The description of conservative variants of the pep4 gene and Pep4 protein described below applies mutatis mutandis to target protein genes and target protein variants. For example, the target protein gene may be modified so that the amino acid sequence of the encoded target protein includes one or more amino acid substitutions, deletions, insertions, and / or additions. The protein identified by the biological species from which it is derived is not limited to the protein itself found in that biological species, but also includes proteins having the amino acid sequence of a protein found in that biological species and their variants. These variants may or may not be found in that biological species. For example, the term "human-derived protein" is not limited to the protein itself found in humans, but also includes proteins having the amino acid sequence of a protein found in humans and their variants. Furthermore, the target protein gene may have any codons substituted with equivalent codons. For example, the gene for a target protein may be modified to have optimal codons depending on the codon usage frequency of the host used.

[0046] The target protein may contain other amino acid sequences in addition to the amino acid sequence of the target protein exemplified above. That is, the target protein may be a fusion protein with other amino acid sequences. The "other amino acid sequences" are not particularly limited as long as a target protein with desired properties is obtained. The "other amino acid sequences" can be appropriately selected depending on various conditions such as the intended use. Examples of "other amino acid sequences" include signal peptides (also called signal sequences), peptide tags, and protease recognition sequences. The "other amino acid sequences" may be linked, for example, to the N-terminus, C-terminus, or both of the target protein. The "other amino acid sequences" may be one type of amino acid sequence, or a combination of two or more types of amino acid sequences.

[0047] Signal peptides can be used, for example, for the secretory production of a target protein. The signal peptide may be linked to the N-terminus of the target protein. That is, in one embodiment, a gene construct for expressing a target protein may contain, from 5' to 3', a promoter sequence, a nucleotide sequence encoding a signal peptide, and a nucleotide sequence encoding the target protein. In this case, the nucleic acid sequence encoding the target protein may be linked downstream of the nucleic acid sequence encoding the signal peptide so that the target protein is expressed as a fusion protein with the signal peptide. In such a fusion protein, the signal peptide and the target protein may or may not be adjacent to each other. That is, "the target protein is expressed as a fusion protein with a signal peptide" does not necessarily mean that the target protein is expressed as a fusion protein with the signal peptide adjacent to the signal peptide, but also includes the case where the target protein is expressed as a fusion protein with the signal peptide via another amino acid sequence. When a signal peptide is used for secretory production of a target protein, the signal peptide is usually cleaved during secretion, and the target protein without the signal peptide is secreted outside the bacterial cell. In other words, "the target protein is expressed as a fusion protein with a signal peptide" or "the target protein contains a signal peptide" means that the target protein forms a fusion protein with the signal peptide upon expression, and it is not necessary that the target protein finally obtained forms a fusion protein with the signal peptide.

[0048] The signal peptide is not particularly limited as long as it functions in Talaromyces cellulolyticus. A "signal peptide that functions in Talaromyces cellulolyticus" refers to a peptide that, when linked to the N-terminus of a target protein, causes secretion of the target protein in Talaromyces cellulolyticus.

[0049] The signal peptide may be derived from the host or from a heterologous source. The signal peptide may be the native signal peptide of the target protein or a signal peptide of another protein. Examples of signal peptides include signal peptides of secreted cellulases from microorganisms. Specific examples of signal peptides include signal peptides of secreted cellulases from Talaromyces cellulolyticus. Secreted cellulases include the CbhI protein (also known as Cbh1 protein) encoded by the cbhI gene and the CbhII protein (also known as Cbh2 protein) encoded by the cbhII gene. Specifically, signal peptides include the signal peptides of CbhI proteins and CbhII proteins. The signal peptide of the CbhI protein is also referred to as the "CbhI signal peptide" or "Cbh1 signal peptide." The signal peptide of the CbhII protein is also referred to as the "CbhII signal peptide" or "Cbh2 signal peptide." The amino acid sequence of the CbhI signal peptide of Talaromyces cellulolyticus is shown in SEQ ID NO: 72. That is, the signal peptide may be, for example, a signal peptide having the amino acid sequence of the above-exemplified signal peptide (e.g., the amino acid sequence of SEQ ID NO: 72). The signal peptide may also be a conservative variant of the above-exemplified signal peptide (e.g., the signal peptide having the amino acid sequence of SEQ ID NO: 72). That is, for example, the above-exemplified signal peptides can be used as is or after appropriate modification. The terms "CbhI signal peptide" and "CbhII signal peptide" encompass not only the above-exemplified CbhI signal peptide and CbhII signal peptide, but also their conservative variants. The description of conservative variants of the Pep4 protein described below can be applied mutatis mutandis to conservative variants of signal peptides.For example, the signal peptide may be a peptide having an amino acid sequence in which one or several amino acids at one or several positions in the amino acid sequence of SEQ ID NO: 72 have been substituted, deleted, inserted, and / or added, so long as the original function is maintained. Note that the above-mentioned "one or several" in the signal peptide variant specifically means preferably 1 to 7, more preferably 1 to 5, even more preferably 1 to 3, and particularly preferably 1 to 2. Furthermore, for example, the signal peptide may be a peptide having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 72, so long as the original function is maintained. Note that the "original function" of a signal peptide may be the function of causing secretion of a target protein when linked to the N-terminus of the target protein. The function of a signal peptide can be confirmed, for example, by confirming secretion of the protein upon linkage to the N-terminus of the protein.

[0050] Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, maltose binding protein (MBP), cellulose binding protein (CBP), thioredoxin (TRX), green fluorescent protein (GFP), horseradish peroxidase (HRP), alkaline phosphatase (ALP), and antibody Fc regions. Peptide tags can be used, for example, to detect and purify expressed proteins of interest.

[0051] Specific examples of protease recognition sequences include the HRV3C protease recognition sequence, the Factor Xa protease recognition sequence, and the proTEV protease recognition sequence. Protease recognition sequences can be used, for example, to cleave an expressed target protein. Specifically, when a target protein is expressed as a fusion protein with a peptide tag, a protease recognition sequence can be introduced at the junction between the target protein and the peptide tag, allowing the peptide tag to be cleaved from the expressed target protein using a protease, thereby obtaining a target protein without the peptide tag.

[0052] The N-terminal region of the target protein obtained in the end may be identical to that of the naturally occurring protein, or may not be identical to that of the naturally occurring protein. For example, the N-terminal region of the target protein obtained in the end may have one or several extra amino acids added or deleted compared to that of the naturally occurring protein. Note that the term "one or several" varies depending on the full length and structure of the target protein, but specifically means preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3 amino acids.

[0053] Alternatively, the target protein may be expressed as a protein with a pro-structure (proprotein). When the target protein is expressed as a proprotein, the final target protein may or may not be a proprotein. That is, the proprotein may be cleaved to form a mature protein by cleaving the pro-structure. Cleavage can be carried out, for example, by a protease. When a protease is used, from the viewpoint of the activity of the final protein, it is generally preferable that the proprotein be cleaved at approximately the same position as the native protein, and more preferably, it is cleaved at exactly the same position as the native protein to obtain a mature protein identical to the native protein. Therefore, in general, a specific protease that cleaves the proprotein at a position that produces a protein identical to the naturally occurring mature protein is most preferred. However, as mentioned above, the N-terminal region of the final target protein does not have to be identical to that of the native protein. For example, depending on the type of target protein to be produced and the intended use, a protein with an N-terminus that is one to several amino acids longer or shorter than the native protein may have more appropriate activity. Proteases that can be used in the present invention include commercially available ones such as Dispase (Boehringer Mannheim), as well as ones obtained from microbial culture media, for example, actinomycete culture media, etc. Such proteases can be used in an unpurified state, or may be purified to an appropriate purity as needed.

[0054] The target protein gene can be obtained, for example, by cloning. For cloning, for example, nucleic acids such as genomic DNA or cDNA containing the target protein gene can be used. The target protein gene can also be obtained, for example, by total synthesis based on its nucleotide sequence (Gene, 60(1), 115-127 (1987)). The obtained target protein gene can be used as is or after appropriate modification. In other words, by modifying the target protein gene, its variant can be obtained. Gene modification can be carried out by known techniques. For example, a target mutation can be introduced into a target site in DNA by site-directed mutagenesis. Examples of site-directed mutagenesis include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, a variant of the target protein gene may be totally synthesized. Furthermore, a gene construct for expressing the target protein can be obtained by appropriately modifying the obtained target protein gene, such as by introducing a promoter sequence. Other components of the gene construct for expressing the target protein (e.g., promoter sequence) and the gene construct for expressing the target protein can also be obtained in the same manner as the target protein gene.

[0055] Genetic modification can be carried out by known techniques. For example, a desired mutation can be introduced into a target site in DNA by site-directed mutagenesis. Examples of site-directed mutagenesis include PCR-based methods (Higuchi, R., 61, in PCR Technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0056] The method for introducing a gene construct for expressing a target protein into Talaromyces cellulolyticus is not particularly limited. "Introduction of a gene construct for expressing a target protein" refers to maintaining the gene construct for expressing a target protein in a host. Specifically, it may refer to introducing the target protein gene into the host so that it can be expressed. Unless otherwise specified, "introduction of a gene construct for expressing a target protein" does not necessarily mean introducing a pre-constructed gene construct for expressing a target protein into the host all at once, but also includes introducing a portion of the gene construct for expressing a target protein into the host and constructing the gene construct for expressing the target protein within the host. For example, a gene construct for expressing a target protein may be constructed on a chromosome by introducing the target protein gene downstream of a promoter native to the host.

[0057] A gene construct for expressing a target protein can be introduced into a host using, for example, a vector containing the gene construct for expressing a target protein. A vector containing the gene construct for expressing a target protein is also referred to as a "target protein expression vector." A target protein expression vector can be constructed, for example, by ligating the gene construct for expressing a target protein to a vector. Alternatively, for example, if the vector contains a promoter, the target protein expression vector can also be constructed by ligating the target protein gene downstream of the promoter. Transforming a host with the target protein expression vector results in a transformant incorporating the vector, i.e., the target protein expression gene construct can be introduced into the host. The vector is not particularly limited as long as it is capable of autonomous replication within host cells. The vector may be a single-copy vector, a low-copy vector, or a multi-copy vector. The vector may contain a marker gene for selecting a transformant. The vector may also contain a promoter or terminator for expressing the target protein gene.

[0058] Alternatively, a gene construct for expressing a target protein may be introduced into a host chromosome. Introduction of a gene into a chromosome can be achieved by homologous recombination. Specifically, a host can be transformed with recombinant DNA containing the gene construct for expressing a target protein, and homologous recombination can occur with the target site on the host chromosome, thereby introducing the gene construct for expressing a target protein into the host chromosome. The structure of the recombinant DNA used for homologous recombination is not particularly limited, as long as it allows homologous recombination to occur in the desired manner. For example, a host can be transformed with linear DNA containing the gene construct for expressing a target protein, with sequences upstream and downstream of the target site on the chromosome at both ends of the gene construct for expressing a target protein. This allows homologous recombination to occur upstream and downstream of the target site, thereby replacing the target site with the gene construct for expressing a target protein. The recombinant DNA used for homologous recombination may contain a marker gene for selecting transformants. Introduction of a portion of the gene construct for expressing a target protein, such as the target protein gene or promoter, into a chromosome can be achieved in the same manner as introduction of the entire gene construct for expressing a target protein into a chromosome.

[0059] The marker gene can be selected appropriately depending on the traits, such as auxotrophy, of the host. For example, if the host exhibits uracil auxotrophy due to a mutation in the pyrF gene or pyrG gene, the pyrF gene or pyrG gene can be used as a marker gene to select a strain into which the desired modification has been introduced, using complementation of the uracil auxotrophy (i.e., uracil non-auxotrophy) as an indicator. Furthermore, a drug resistance gene, such as a hygromycin resistance gene, can be used as a marker gene.

[0060] Transformation can be carried out by techniques commonly used for transforming eukaryotic microorganisms such as molds and yeasts, including the protoplast method.

[0061] <1-3>Decreased activity of Pep4 protein The microorganisms of the present invention have been modified to reduce the activity of the Pep4 protein. Specifically, the microorganisms of the present invention have been modified to reduce the activity of the Pep4 protein compared to a non-modified strain. More specifically, the microorganisms of the present invention may be modified, for example, to reduce expression of the pep4 gene or to disrupt the pep4 gene. By modifying Talaromyces cellulolyticus to reduce the activity of the Pep4 protein, the ability of the microorganism to produce a target protein can be improved, i.e., the production of a target protein by the microorganism can be increased.

[0062] The Pep4 protein and the pep4 gene that encodes it are described below.

[0063] The Pep4 protein is a protease. The term "protease" refers to a protein that has the activity of catalyzing a reaction that hydrolyzes a protein. This activity is also referred to as "protease activity."

[0064] The pep4 gene (including introns) of the Talaromyces cellulolyticus Y-94 strain (FERM BP-5826, CBS 136886) corresponds to the complementary sequence of positions 2810881 to 2812244 of the genome sequence registered with NCBI under NCBI ACCESSION DF933830.1. The Pep4 protein of the Talaromyces cellulolyticus Y-94 strain has been registered with NCBI under NCBI ACCESSION GAM39722.1. The nucleotide sequence of the pep4 gene (including introns) of the Talaromyces cellulolyticus Y-94 strain and the amino acid sequence of the Pep4 protein encoded by the gene are shown in SEQ ID NOs: 70 and 71, respectively. That is, the pep4 gene may be, for example, a gene having the nucleotide sequence shown in SEQ ID NO: 70. The Pep4 protein may also be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 71. Unless otherwise specified, the expression "a gene or protein has a base sequence or an amino acid sequence" may mean that the gene or protein contains the base sequence or amino acid sequence, and may also include cases where the gene or protein consists of the base sequence or amino acid sequence.

[0065] The pep4 gene may be a variant of the above-exemplified pep4 gene (e.g., the gene having the nucleotide sequence shown in SEQ ID NO: 70), so long as the original function is maintained. Similarly, the Pep4 protein may be a variant of the above-exemplified Pep4 protein (e.g., the protein having the amino acid sequence shown in SEQ ID NO: 71), so long as the original function is maintained. Such variants that maintain the original function may be referred to as "conservative variants." In the present invention, the term "pep4 gene" is not limited to the above-exemplified pep4 genes and encompasses conservative variants thereof. Similarly, the term "Pep4 protein" is not limited to the above-exemplified Pep4 proteins and encompasses conservative variants thereof. Examples of conservative variants include homologs and artificially modified forms of the above-exemplified pep4 genes and Pep4 proteins.

[0066] "Maintaining the original function" means that a gene or protein variant has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. In other words, in the case of the pep4 gene, "maintaining the original function" means that the gene variant encodes a protein that maintains the original function. In addition, in the case of the Pep4 protein, "maintaining the original function" means that the protein variant has protease activity.

[0067] Protease activity can be measured by incubating an enzyme with a substrate (protein) and measuring the enzyme-dependent degradation of the substrate. Alternatively, protease activity can be measured using a commercially available protease activity measurement kit.

[0068] Examples of conservative variants are shown below.

[0069] Homologues of the pep4 gene or Pep4 protein can be easily obtained from public databases by, for example, BLAST or FASTA searches using the nucleotide sequence of the pep4 gene or the amino acid sequence of the Pep4 protein as a query sequence. Homologues of the pep4 gene can also be obtained by PCR using, for example, the chromosome of Talaromyces cellulolyticus as a template and oligonucleotides prepared based on the nucleotide sequence of these known pep4 genes as primers.

[0070] As long as the original function is maintained, the pep4 gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions in the amino acid sequence of the Pep4 protein exemplified above (for example, the amino acid sequence shown in SEQ ID NO: 71) have been substituted, deleted, inserted, and / or added. Note that the term "one or several" varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0071] The above-mentioned substitution, deletion, insertion, and / or addition of one or several amino acids is a conservative mutation that maintains normal protein function. A typical conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted with each other when the substitution site is an aromatic amino acid; Leu, Ile, and Val are substituted with each other when the substitution site is a hydrophobic amino acid; Gln and Asn are substituted with each other when the substitution site is a polar amino acid; Lys, Arg, and His are substituted with each other when the substitution site is a basic amino acid; Asp and Glu are substituted with each other when the substitution site is an acidic amino acid; and Ser and Thr are substituted with each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Il Examples of such substitutions include substitutions of Lys with Leu, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, or additions also include those resulting from naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the gene is derived.

[0072] Furthermore, the pep4 gene may be a gene encoding a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence of the Pep4 protein exemplified above (e.g., the amino acid sequence shown in SEQ ID NO: 71), as long as the original function is maintained.

[0073] Furthermore, the pep4 gene may be a DNA that hybridizes under stringent conditions with a complementary sequence of the nucleotide sequence of the pep4 gene (e.g., the nucleotide sequence shown in SEQ ID NO: 70) or a probe that can be prepared from the complementary sequence, as long as the original function is maintained. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. One example of such conditions is a condition under which DNAs with high identity, for example, DNAs with an identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, will hybridize with each other, while DNAs with lower identity will not hybridize with each other; or a condition under which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0074] The probe may be, for example, a portion of the complementary sequence of a gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on the base sequence of a known gene as primers and a DNA fragment containing this base sequence as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. In such cases, washing conditions for hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0075] Furthermore, the pep4 gene may be one in which any codon has been replaced with an equivalent codon, i.e., the pep4 gene may be a variant of the above-exemplified pep4 gene due to codon degeneracy.

[0076] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using the default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using the default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0077] The above descriptions regarding gene and protein variants can also be applied mutatis mutandis to any proteins, such as target proteins, and the genes encoding them.

[0078] <1-4>Other properties The microorganism of the present invention may have other desired properties (e.g., modifications) as long as the ability to produce a target protein is not impaired. Examples of modifications include those that improve the ability of Talaromyces cellulolyticus to produce a target protein. Specific examples of modifications include those that reduce the activity of the YscB protein and those that reduce the activity of the CreA protein. These properties and modifications can be used alone or in appropriate combinations.

[0079] That is, the microorganism of the present invention may be modified, for example, so that the activity of the YscB protein is reduced. Specifically, the microorganism of the present invention may be modified so that the activity of the YscB protein is reduced compared to a non-modified strain. More specifically, the microorganism of the present invention may be modified, for example, so that the expression of the yscB gene is reduced, or so that the yscB gene is disrupted. The YscB protein is a protease.

[0080] The nucleotide sequence of the yscB gene (including introns) of the Talaromyces cellulolyticus S6-25 strain and the amino acid sequence of the YscB protein encoded by the gene are shown in SEQ ID NOs: 60 and 73, respectively. That is, the yscB gene may be, for example, a gene having the nucleotide sequence shown in SEQ ID NO: 60. The YscB protein may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 73. The yscB gene and YscB protein may be conservative variants of the yscB gene and YscB protein exemplified above, respectively. The same descriptions regarding conservative variants of the pep4 gene and Pep4 protein can be applied mutatis mutandis to conservative variants of the yscB gene and YscB protein. Note that, in the case of the YscB protein, "maintaining the original function" may mean that the protein variant has protease activity. Protease activity can be measured, for example, as described above.

[0081] Furthermore, the microorganism of the present invention may be modified, for example, to reduce the activity of the CreA protein. Specifically, the microorganism of the present invention may be modified so that the activity of the CreA protein is reduced compared to an unmodified strain. More specifically, the microorganism of the present invention may be modified, for example, to reduce the expression of the creA gene, or may be modified so that the creA gene is disrupted. The creA gene encodes a transcription factor involved in catabolite repression. The creA gene is known to be involved in the expression of cellulase in filamentous fungi (Mol Gen Genet. 1996 Jun 24;251(4):451-60, Biosci Biotechnol Biochem. 1998 Dec;62(12):2364-70).

[0082] The nucleotide sequence of the creA gene of Talaromyces cellulolyticus strain S6-25 is shown in SEQ ID NO: 74. That is, the creA gene may be, for example, a gene having the nucleotide sequence shown in SEQ ID NO: 74. Furthermore, the CreA protein may be, for example, a protein having an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 74. The creA gene and CreA protein may be conservative variants of the creA gene and CreA protein exemplified above, respectively. The same descriptions regarding conservative variants of the pep4 gene and Pep4 protein can be applied mutatis mutandis to conservative variants of the creA gene and CreA protein. Note that, in the case of the CreA protein, "maintaining the original function" may mean that the protein variant has the function as a transcription factor involved in catabolite repression.

[0083] <1-5> Methods for reducing protein activity Below, methods for reducing the activity of proteins such as the Pep4 protein, the YscB protein, and the CreA protein will be described.

[0084] "Decreased protein activity" means that the activity of the protein is reduced compared to a non-modified strain. Specifically, "decreased protein activity" means that the activity of the protein per cell is reduced compared to a non-modified strain. Here, "non-modified strain" refers to a control strain that has not been modified to reduce the activity of the target protein. Examples of non-modified strains include wild-type strains and parent strains. Specific examples of non-modified strains include the strains exemplified in the description of Talaromyces cellulolyticus. In other words, in one embodiment, the activity of the protein may be reduced compared to the Talaromyces cellulolyticus S6-25 strain. Note that "decreased protein activity" also includes cases where the activity of the protein is completely lost. More specifically, "decreased protein activity" may mean that the number of molecules of the protein per cell and / or the function per molecule of the protein are reduced compared to a non-modified strain. In other words, the "activity" in the phrase "decreased protein activity" is not limited to the catalytic activity of the protein, but may also refer to the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. The "number of protein molecules per cell" may refer to the average number of molecules of the protein per cell. Note that "decreased number of protein molecules per cell" also includes cases where the protein is completely absent. Furthermore, "decreased function per protein molecule" also includes cases where the function per protein molecule is completely lost. The degree of reduction in protein activity is not particularly limited, as long as the protein activity is reduced compared to that of an unmodified strain. The protein activity may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0085] Modifications that reduce the activity of a protein can be achieved, for example, by reducing the expression of the gene encoding the protein. "Reduced gene expression" means that the expression of the gene is reduced compared to an unmodified strain. "Reduced gene expression" specifically means that the expression level of the gene per cell is reduced compared to an unmodified strain. "Expression level of the gene per cell" may refer to the average expression level of the gene per cell. "Reduced gene expression" may more specifically mean a decrease in the transcription level (mRNA level) of the gene and / or a decrease in the translation level (protein level) of the gene. "Reduced gene expression" includes cases where the gene is not expressed at all. "Reduced gene expression" is also referred to as "attenuated gene expression." Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0086] Decreased gene expression may be due to, for example, decreased transcription efficiency, decreased translation efficiency, or a combination thereof. Decreased gene expression can be achieved, for example, by modifying the expression regulatory sequence of a gene. "Expression regulatory sequence" is a general term for a site that affects gene expression, such as a promoter. Expression regulatory sequences can be determined, for example, using a promoter search vector or genetic analysis software such as GENETYX. When modifying an expression regulatory sequence, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases are modified in the expression regulatory sequence. Decreased gene transcription efficiency can be achieved, for example, by replacing the promoter of a gene on a chromosome with a weaker promoter. A "weaker promoter" refers to a promoter that weakens gene transcription compared to the native wild-type promoter. An example of a weaker promoter is an inducible promoter. In other words, an inducible promoter can function as a weaker promoter under non-inducing conditions (e.g., in the absence of an inducer). Alternatively, a partial or entire region of the expression regulatory sequence may be deleted (deleted). Reduction of gene expression can also be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Reduction of gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that reduces gene expression. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. Furthermore, gene expression itself can be reduced, for example, by gene disruption as described below.

[0087] Furthermore, a modification that reduces the activity of a protein can be achieved, for example, by disrupting the gene that encodes the protein. "Disrupting a gene" means that the gene is modified so that it does not produce a protein that functions normally. "Not producing a protein that functions normally" includes cases where no protein is produced from the gene at all, and cases where the gene produces a protein with reduced or lost function (activity or properties) per molecule.

[0088] Gene disruption can be achieved, for example, by deleting (deleting) the gene on a chromosome. "Gene deletion" refers to the deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on the chromosome. The sequences before and after the coding region of the gene may include, for example, a gene expression regulatory sequence. As long as a reduction in protein activity can be achieved, the region to be deleted may be any region, such as the N-terminal region (the region encoding the N-terminal side of the protein), an internal region, or a C-terminal region (the region encoding the C-terminal side of the protein). Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. The region to be deleted may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the entire coding region of the gene. Furthermore, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. A mismatch in the reading frame can cause a frameshift downstream of the region to be deleted. Specifically, in the case of the creA gene, the gene can be disrupted by deleting the portion corresponding to positions 3262 to 4509 of SEQ ID NO: 74 (JP 2016-131533 A).

[0089] Gene disruption can also be achieved by, for example, introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting one or two bases (frameshift mutation) (Journal of Biological Chemistry 272:8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

[0090] Gene disruption can also be achieved, for example, by inserting another base sequence into the coding region of the gene on a chromosome. The insertion site may be anywhere in the gene, but the longer the inserted base sequence, the more reliably the gene can be inactivated. Furthermore, it is preferable that the sequences before and after the insertion site do not match in reading frame. A mismatch in reading frame can cause a frameshift downstream of the insertion site. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples include marker genes and genes useful for producing a target protein.

[0091] Gene disruption may be carried out, in particular, to delete (delete) the amino acid sequence of the encoded protein. In other words, modification that reduces the activity of a protein can be achieved, for example, by deleting the amino acid sequence of the protein, specifically by modifying the gene to encode a protein from which the amino acid sequence has been deleted. The term "deletion of the amino acid sequence of a protein" refers to the deletion of a portion or the entire region of the amino acid sequence of a protein. The term "deletion of the amino acid sequence of a protein" also refers to the absence of the original amino acid sequence in the protein, and also encompasses cases in which the original amino acid sequence is changed to a different amino acid sequence. For example, a region that has been changed to a different amino acid sequence due to frameshifting may be considered a deleted region. While deletion of the amino acid sequence of a protein typically shortens the overall length of the protein, it may also result in the overall length of the protein remaining unchanged or being extended. For example, deletion of a portion or the entire coding region of a gene can delete the region encoded by the deleted region in the amino acid sequence of the encoded protein. For example, by introducing a stop codon into the coding region of a gene, the region coded for by the region downstream of the introduction site in the amino acid sequence of the encoded protein can be deleted. For example, by causing a frameshift in the coding region of a gene, the region coded for by the frameshift site can be deleted. The position and length of the region to be deleted in the deletion of an amino acid sequence can be determined mutatis mutandis from the explanation of the position and length of the region to be deleted in the deletion of a gene.

[0092] The above-described modification of a gene on a chromosome can be achieved, for example, by creating a disrupted gene modified so that it does not produce a normally functioning protein, transforming a host with recombinant DNA containing the disrupted gene, and inducing homologous recombination between the disrupted gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the disrupted gene. In this case, the recombinant DNA can be easily manipulated by incorporating a marker gene according to the host's traits, such as its nutritional requirements. Examples of disrupted genes include genes lacking part or all of the coding region of a gene, genes with missense mutations, genes with nonsense mutations, genes with frameshift mutations, and genes with insertion sequences such as transposons or marker genes. Even if a protein encoded by a disrupted gene is produced, it will have a different three-dimensional structure from the wild-type protein, resulting in reduced or lost function.

[0093] The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as it allows homologous recombination to occur in the desired manner. For example, a host can be transformed with linear DNA containing an arbitrary sequence, with sequences upstream and downstream of the target site on the chromosome at both ends of the arbitrary sequence, and homologous recombination can be caused upstream and downstream of the target site, thereby replacing the target site with the arbitrary sequence in a single step. For example, a sequence containing a marker gene can be used as the arbitrary sequence.

[0094] Marker genes can be selected appropriately depending on the traits of the host, such as its nutritional requirements. For example, if a host exhibits uracil auxotrophy due to a mutation in the pyrF gene or pyrG gene, the pyrF gene or pyrG gene can be used as a marker gene to select a strain into which a desired modification has been introduced, using complementation of uracil auxotrophy (i.e., uracil non-auxotrophy) as an indicator. Furthermore, if a host exhibits methionine auxotrophy due to a mutation in the sC gene (sulfate permiase gene), the sC gene can be used as a marker gene to select a strain into which a desired modification has been introduced, using complementation of methionine auxotrophy (i.e., methionine non-auxotrophy). Furthermore, drug resistance genes such as the hygromycin resistance gene can be used as marker genes.

[0095] Modifications that reduce the activity of proteins may also be performed by, for example, mutation treatments, such as X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0096] The decrease in protein activity can be confirmed by measuring the activity of the protein. The activities of the Pep4 protein and the YscB protein can be measured, for example, as described above. The activity of the CreA protein can be measured, for example, by measuring the degree of catabolite repression. The degree of catabolite repression can be measured, for example, by measuring cellulase production under culture conditions containing glucose as a carbon source. That is, the decrease in CreA protein activity can be confirmed, specifically, for example, by using the improvement in cellulase production under culture conditions containing glucose as a carbon source as an indicator.

[0097] A decrease in protein activity can also be confirmed by confirming a decrease in expression of the gene encoding the protein. A decrease in gene expression can be confirmed by confirming a decrease in the transcription level of the gene or a decrease in the amount of protein expressed from the gene.

[0098] The reduction in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain. Methods for assessing the amount of mRNA include Northern hybridization and RT-PCR (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA (e.g., the number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.

[0099] The reduction in the protein amount can be confirmed by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The protein amount (e.g., number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0100] Gene disruption can be confirmed by determining the nucleotide sequence, restriction enzyme map, or full length of a part or all of the gene, depending on the means used for disruption.

[0101] Transformation can be carried out by techniques commonly used for transforming eukaryotic microorganisms such as molds and yeasts, including the protoplast method.

[0102] <2> Methods of the Invention A target protein can be produced using the microorganism of the present invention. Specifically, the target protein can be produced by culturing the microorganism of the present invention. That is, the method of the present invention may specifically be a method for producing a target protein, which comprises culturing the microorganism of the present invention in a medium.

[0103] The medium used is not particularly limited as long as it allows the microorganism of the present invention to grow and the target protein to be produced. For example, a medium containing components selected from a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic and inorganic components as needed can be used. The types and concentrations of medium components can be appropriately determined by those skilled in the art. For specific medium compositions, reference can be made to the medium compositions described in previous reports on Talaromyces cellulolyticus (e.g., Japanese Patent Application Laid-Open Nos. 2003-135052, 2008-271826, and 2008-271927), as well as medium compositions for various other cellulase-producing microorganisms such as Trichoderma reesei.

[0104] The carbon source is not particularly limited as long as it can be assimilated by the microorganism of the present invention to produce the target protein. Examples of carbon sources include sugars and cellulosic substrates. Specific examples of sugars include glucose, fructose, galactose, xylose, arabinose, sucrose, lactose, cellobiose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates. Specific examples of cellulosic substrates include microcrystalline cellulose (Avicel), filter paper, waste paper, pulp, wood, rice straw, wheat straw, rice husks, rice bran, wheat bran, sugarcane bagasse, coffee grounds, and tea leaves. Cellulosic substrates may be used as carbon sources after pretreatment such as hydrothermal decomposition, acid treatment, alkali treatment, steaming, explosion, or pulverization. A commercially available suitable cellulosic substrate is Solkafloc (International Fiber Corp, North Tonawanda, NY, USA). As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0105] Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, corn steep liquor, and soy protein hydrolysate, ammonia, and urea. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0106] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0107] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0108] Specific examples of other various organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acids, yeast extract, and soy protein hydrolysate. As other various organic and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

[0109] The culture conditions are not particularly limited as long as the microorganism of the present invention can grow and the target protein can be produced. Culture can be performed under standard conditions used for culturing microorganisms such as filamentous fungi. For specific culture conditions, reference can be made to the culture conditions described in previous reports on Talaromyces cellulolyticus (e.g., Japanese Patent Application Laid-Open Nos. 2003-135052, 2008-271826, and 2008-271927), as well as the culture conditions for various other cellulase-producing microorganisms such as Trichoderma reesei.

[0110] Culturing can be carried out under aerobic conditions, for example, using a liquid medium. Specifically, culturing under aerobic conditions can be carried out by aerobic culture, shaking culture, agitation culture, or a combination thereof. The culture temperature may be, for example, 15 to 43°C, particularly about 30°C. The culture period may be, for example, 2 hours to 20 days. Culturing can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The medium at the start of culturing is also called the "initial medium." The medium supplied to the culture system (fermentor) in fed-batch culture or continuous culture is also called the "fed-batch medium." Supplying a fed-batch medium to the culture system in fed-batch culture or continuous culture is also called "fed-batch." Culturing can also be carried out in two stages: preculture and main culture. For example, preculture can be carried out on a solid medium such as an agar medium, and main culture can be carried out in a liquid medium. Cultivation may be continued, for example, until the carbon source in the medium is consumed or until the activity of the microorganism of the present invention disappears.

[0111] In the present invention, each medium component may be contained in the initial medium, the feed medium, or both. The types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium. Furthermore, the concentrations of each component contained in the initial medium may or may not be the same as the concentrations of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feeds are performed intermittently, the types and / or concentrations of components contained in the feed medium for each feed may or may not be the same.

[0112] The concentrations of various components can be measured by gas chromatography (Hashimoto, K. et al. 1996. Biosci. Biotechnol. Biochem. 70:22-30) or HPLC (Lin, JT et al. 1998. J. Chromatogr. A. 808: 43-49).

[0113] By culturing the microorganism of the present invention as described above, the target protein is expressed, and a culture containing the target protein is obtained. Specifically, the target protein may accumulate in the medium, on the cell surface, within the cell, or a combination thereof. The target protein may particularly accumulate in the medium.

[0114] Production of a target protein can be confirmed by known methods used for detecting or identifying proteins. Such methods include, for example, SDS-PAGE, Western blotting, mass spectrometry, N-terminal amino acid sequence analysis, and enzyme activity measurement. These methods may be used alone or in combination of two or more.

[0115] The produced target protein can be collected as appropriate. That is, the method for producing a target protein of the present invention may include collecting the produced target protein. Specifically, the target protein can be collected as an appropriate fraction containing the target protein. Examples of such fractions include cultures, culture supernatants, bacterial cells, treated bacterial cells (disrupted cells, lysates, and extracts (cell-free extracts)). The bacterial cells may be provided in the form of immobilized bacterial cells immobilized on a carrier such as acrylamide or carrageenan.

[0116] The target protein may be separated and purified to a desired extent. The target protein may be provided in a free state or in the form of an immobilized enzyme immobilized on a solid phase such as a resin.

[0117] When the target protein accumulates in the medium, the target protein can be separated and purified from the supernatant after removing solid matter such as bacterial cells from the culture by centrifugation or the like.

[0118] When a target protein accumulates within bacterial cells, the target protein can be separated and purified from the treated product, for example, by subjecting the bacterial cells to treatment such as disruption, lysis, or extraction. The bacterial cells can be recovered from the culture by centrifugation or the like. Treatment such as cell disruption, lysis, or extraction can be carried out by known methods. Examples of such methods include ultrasonic disruption, the Dynomill method, bead disruption, French press disruption, and lysozyme treatment. These methods may be used alone or in appropriate combination of two or more.

[0119] When a target protein accumulates on the bacterial cell surface, the target protein can be solubilized and then separated and purified from the solubilized product. Solubilization can be performed by known methods. Examples of such methods include increasing the salt concentration and using a surfactant. These methods may be used alone or in combination of two or more.

[0120] Purification of the target protein (e.g., from the supernatant, treated product, or solubilized product as described above) can be carried out by known methods used for protein purification. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation. These methods may be used alone or in combination of two or more.

[0121] In addition to the target protein, other enzymes, such as cellulase and hemicellulases such as xylanase, xylobiase (β-xylosidase), and arabinofuranosidase, may also be produced and accumulated in the culture. The target protein may be recovered as a mixture with such other enzymes, or may be recovered separately from such other enzymes.

[0122] The recovered target protein may be formulated as appropriate. The dosage form is not particularly limited and can be appropriately selected depending on various conditions, such as the intended use of the target protein. Examples of dosage forms include liquids, suspensions, powders, tablets, pills, and capsules. Pharmacologically acceptable additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, odorants, fragrances, diluents, and surfactants can be used in the formulation. [Example]

[0123] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0124] (1) Construction of a yscB gene-deficient strain derived from Talaromyces cellulolyticus F09 Using the Talaromyces cellulolyticus F09 strain (JP 2016-131533 A) as a parent strain, the sC gene (SEQ ID NO: 59) was disrupted by the following procedure to construct the T. cellulolyticus F09 ΔsC strain so that the sC gene could be used as a recombination marker. The F09 strain was derived from the T. cellulolyticus S6-25 strain (NITE BP-01685) as a parent strain and contains a mutation (single nucleotide substitution) in the pyrF gene. The F09 strain exhibits uracil auxotrophy due to the mutation in the pyrF gene.

[0125] First, a DNA fragment for disrupting the sC gene, containing the nucleotide sequence of the upstream region of the T. cellulolyticus sC gene and the downstream region of the T. cellulolyticus sC gene, was prepared as follows. The upstream region of the sC gene was amplified by PCR using primers (SEQ ID NOs: 1 and 2) and primers (SEQ ID NOs: 3 and 4) with the genomic DNA of T. cellulolyticus Y-94 (FERM BP-5826, CBS 136886) as a template. The downstream region of the sC gene was amplified by PCR using primers (SEQ ID NOs: 3 and 4). The PCR product was purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The purified PCR product was then ligated into the pUC plasmid provided with the In-Fusion HD Cloning Kit (Takara Bio). The reaction mixture was transformed into Escherichia coli JM109, and colonies were formed by overnight incubation at 37°C on LB agar medium (containing 100 mg / L ampicillin). The pUC-ΔsC plasmid, which contained the DNA fragment for disrupting sC, was isolated from the resulting transformants using the Wizard Plus Miniprep System (Promega). The DNA fragment for disrupting sC was amplified by PCR using the pUC-ΔsC plasmid as a template and primers (SEQ ID NOs: 1 and 4), and then concentrated and purified by ethanol precipitation.

[0126] Next, the F09 strain was inoculated into a medium containing 12 g / L Potato Dextrose Broth (Difco) and 20 g / L Bacto Agar (Difco) and cultured at 30°C. A single agar disk was obtained by punching out the edge of a colony formed on the agar medium with a straw. The resulting agar disk was inoculated into a medium containing 30 g / L Glucose, 20 g / L Yeast Extract (Becton, Dickinson and Company), 1 g / L Uracil, and 1 g / L Uridine and cultured at 30°C and 120 rpm for 2 days. After culturing with shaking at 30°C and 120 rpm for 2 days, 2 ml of the preculture was inoculated into a medium containing 24 g / L Potato Dextrose Broth and cultured at 30°C and 220 rpm for 1 day. The cells were harvested by centrifugation (5,000 rpm, 5 min). 30 mL of an aqueous solution containing 10 g / L Yatalase (Takara Bio), 10 mM KH2PO4, and 0.8 M NaCl (pH 6.0) was added and incubated with shaking at 30°C for 2 hours to digest the cell walls and generate protoplasts. After removing debris using a glass filter, the protoplasts were harvested by centrifugation (2,000 rpm, 10 min) and suspended in 1 mL of Tris-HCl buffer (pH 7.5) containing 1.2 M sorbitol and 10 mM CaCl2 to prepare a protoplast solution. To 200 μL of the protoplast solution, 10 μg of the purified sC disruption DNA fragment and 50 μL of Tris-HCl buffer (pH 7.5) containing 400 g / L PEG4000 and 10 mM CaCl2 were added and left on ice for 30 minutes. After that, 1 mL of Tris-HCl buffer (pH 7.5) containing 400 g / L PEG4000 and 10 mM CaCl2 was added, mixed, and left at room temperature for 15 minutes to allow transformation. Protoplasts were collected by centrifugation (2,000 rpm, 10 minutes). The protoplasts were then cultured in minimal medium (10 g / L glucose, 10 mM NH4Cl, 10 mM KH2PO4, 7 mM KCl, 2 mM MgSO4, 0.06 mg / L H3BO3, 0.26 mg / L (NH4)6Mo7O) containing 1 M sucrose, 1 mM sodium selenate, 30 mg / L methionine, 1 g / L uracil, and 1 g / L uridine. 24The strains were plated on a medium containing 1 mM sodium selenate, 30 mg / L methionine, 1 g / L uracil, and 1 g / L uridine. After incubation at 30°C for 4 days, selenate-tolerant strains were selected. The sC gene-disrupted strain exhibited selenate tolerance and methionine auxotrophy, allowing selection on selenate-containing medium containing methionine. The resulting colonies were inoculated onto minimal medium containing 1 mM sodium selenate, 30 mg / L methionine, 1 g / L uracil, and 1 g / L uridine. After incubation at 30°C for 4 days, the sC gene was confirmed to be deleted, yielding the F09-derived sC-disrupted strain (F09 ΔsC strain).

[0127] Next, using the T. cellulolyticus F09 ΔsC strain as a parent strain, the yscB gene (SEQ ID NO: 60) was disrupted by the following procedure to construct the T. cellulolyticus F09 ΔyscB strain.

[0128] First, a DNA fragment for disrupting the yscB gene was prepared according to the following procedure. The fragment contained the upstream region of the T. cellulolyticus yscB gene, the T. cellulolyticus sC gene marker (SEQ ID NO: 61), and the downstream region of the T. cellulolyticus yscB gene. The upstream region of the yscB gene was amplified by PCR using primers (SEQ ID NOs: 5 and 6) and the downstream region of the yscB gene was amplified by PCR using primers (SEQ ID NOs: 7 and 8). The sC gene marker was amplified by PCR using primers (SEQ ID NOs: 9 and 10). The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System. The purified PCR products were then ligated into the pUC plasmid provided with the In-Fusion HD Cloning Kit. The reaction mixture was transformed into E. coli JM109 strain and cultured overnight at 37°C on LB agar medium (containing 100 mg / L ampicillin) to allow colonies to form. The pUC-yscB::sC plasmid, incorporating the DNA fragment for disrupting the yscB gene, was isolated from the resulting transformants using the Wizard Plus Miniprep System. The DNA fragment for disrupting the yscB gene was amplified by PCR using the pUC-yscB::sC plasmid as a template and primers (SEQ ID NOs: 5 and 8), and then concentrated and purified by ethanol precipitation.

[0129] Next, the F09 ΔsC strain was inoculated into a medium containing 12 g / L potato dextrose broth and 20 g / L Bacto agar and cultured at 30°C. A single agar disk was obtained by punching out the edge of a colony formed on the agar medium with a straw. The resulting disk was inoculated into a medium containing 30 g / L glucose, 20 g / L yeast extract, 1 g / L uracil, and 1 g / L uridine and cultured at 30°C with shaking at 120 rpm for 2 days. Afterward, 2 ml of the preculture was inoculated into a medium containing 24 g / L potato dextrose broth and cultured at 30°C with shaking at 220 rpm for 1 day. The cells were harvested by centrifugation (5,000 rpm, 5 min). 30 mL of an aqueous solution containing 10 g / L Yatalase, 10 mM KH2PO4, and 0.8 M NaCl (pH 6.0) was added and incubated with shaking at 30°C for 2 hours to digest the cell walls and generate protoplasts. After removing debris using a glass filter, the protoplasts were harvested by centrifugation (2,000 rpm, 10 min) and suspended in 1 mL of Tris-HCl buffer (pH 7.5) containing 1.2 M sorbitol and 10 mM CaCl2 to prepare a protoplast solution. To 200 μL of the protoplast solution, 10 μg of the purified yscB gene disruption DNA fragment and 50 μL of Tris-HCl buffer (pH 7.5) containing 400 g / L PEG4000 and 10 mM CaCl2 were added and left on ice for 30 minutes. After that, 1 mL of Tris-HCl buffer (pH 7.5) containing 400 g / L PEG4000 and 10 mM CaCl2 was added, mixed, and left at room temperature for 15 minutes to allow transformation. Protoplasts were collected by centrifugation (2,000 rpm, 10 minutes). They were then cultured in minimal medium (10 g / L glucose, 10 mM NH4Cl, 10 mM KH2PO4, 7 mM KCl, 2 mM MgSO4, 0.06 mg / L H3BO3, 0.26 mg / L (NH4)6Mo7O) containing 1 M sucrose, 1 g / L uracil, and 1 g / L uridine. 24The strains were plated on a medium containing 0.08 mg / L FeCl₃·4H₂O, 1 mg / L FeCl₃·6H₂O, 0.4 mg / L CuSO₄·5H₂O, 0.08 mg / L MnCl₂, 2 mg / L ZnCl₂, and 20 g / L Bacto Agar and cultured at 30°C for 7 days to select strains with complemented methionine auxotrophy. The resulting colonies were inoculated onto minimal medium containing 1 g / L uracil and 1 g / L uridine and cultured at 30°C for 4 days. After confirming that the yscB gene had been replaced with the sC gene, the yscB gene-disrupted strain derived from the F09 strain (F09 ΔyscB strain) was obtained.

[0130] (2) Construction and cultivation of a pep4 gene-deficient strain derived from T. cellulolyticus F09 Using the T. cellulolyticus F09 ΔyscB strain as a parent strain, a pep4 gene disruptant encoding Pep4 protease (GenBank accession number: GAM39722.1) was constructed by the following procedure.

[0131] First, a DNA fragment for disrupting the pep4 gene was prepared using the following procedure: the upstream region of the T. cellulolyticus pep4 gene, the pyrF gene marker (SEQ ID NO: 62), and the downstream region of the T. cellulolyticus pep4 gene were linked in this order. Using genomic DNA from T. cellulolyticus Y-94 (FERM BP-5826) as a template, the upstream region of the pep4 gene was amplified by PCR using primers (SEQ ID NOs: 27 and 28), the downstream region of the pep4 gene was amplified by PCR using primers (SEQ ID NOs: 29 and 30), and the pyrF gene marker was amplified by PCR using primers (SEQ ID NOs: 31 and 32). The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System. The purified PCR products were cloned into the pUC plasmid provided with the In-Fusion HD Cloning Kit in combination with the upstream and downstream regions of the pep4 gene and the pyrF marker gene. The reaction mixture was transformed into E. coli JM109 strain and cultured overnight at 37°C on LB agar medium (containing 100 mg / L ampicillin) to allow colonies to form. The pUC-pep4::pyrF plasmid, incorporating the DNA fragment for disrupting the pep4 gene, was isolated from the resulting transformants using the Wizard Plus Miniprep System. The DNA fragment for disrupting the Pep4 protease gene was amplified by PCR using primers (SEQ ID NOs: 33 and 34) and the pUC-pep4::pyrF plasmid as a template, followed by concentration and purification by ethanol precipitation.

[0132] Next, the F09 ΔyscB strain was cultured and protoplasted using the same method as in (1), and transformed with the purified DNA fragment for the pep4 gene disruption in the same manner as in (1). Protoplasts were recovered by centrifugation (2,000 rpm, 10 minutes) and plated on minimal medium containing 1 M sucrose. After culturing at 30°C for 7 days, strains with complemented uracil auxotrophy were selected. The resulting colonies were inoculated onto minimal medium and cultured at 30°C for 4 days. After confirming that the pep4 gene region had been replaced with the pyrF gene marker, a pep4 gene disruptant derived from the F09 ΔyscB strain (hereafter referred to as the "Δpep4 strain") was obtained.

[0133] In addition, to provide a control with consistent auxotrophy, a yscB gene-disrupted strain was constructed using the F09 strain as a parent strain by the following procedure.

[0134] First, a DNA fragment for disrupting the yscB gene was prepared according to the following procedure. The fragment contained the upstream region of the T. cellulolyticus yscB gene, the pyrF gene marker (SEQ ID NO: 62), and the downstream region of the T. cellulolyticus yscB gene. The upstream region of the yscB gene was amplified by PCR using primers (SEQ ID NOs: 5 and 35) and the downstream region of the yscB gene was amplified by PCR using primers (SEQ ID NOs: 36 and 8). The pyrF gene marker was amplified by PCR using primers (SEQ ID NOs: 31 and 32). The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System. The purified PCR products were cloned into the pUC plasmid provided with the In-Fusion HD Cloning Kit. The reaction mixture was transformed into E. coli JM109 strain and cultured overnight at 37°C on LB agar medium (containing 100 mg / L ampicillin) to allow colonies to form. The pUC-yscB::pyrF plasmid, incorporating the DNA fragment for disrupting the yscB gene, was isolated from the resulting transformants using the Wizard Plus Miniprep System. The DNA fragment for disrupting the yscB gene was amplified by PCR using primers (SEQ ID NOs: 5 and 8) and the pUC-yscB::pyrF plasmid as a template, and then concentrated and purified by ethanol precipitation.

[0135] Next, the F09 strain was cultured and protoplasted using the same method as in (1), and transformed with the purified DNA fragment for disrupting the yscB gene in the same manner as in (1). Protoplasts were recovered by centrifugation (2,000 rpm, 10 minutes), plated on minimal medium containing 1 M sucrose, and cultured at 30°C for 7 days to select strains that had complemented uracil auxotrophy. The resulting colonies were inoculated onto minimal medium and cultured at 30°C for 4 days, after which it was confirmed that the yscB gene region had been replaced with the pyrF gene marker. This gave a yscB gene-disrupted strain derived from the F09 strain (hereinafter also referred to as the "control strain") for use as a control for protease activity evaluation.

[0136] The control strain and the Δpep4 strain were inoculated into a medium containing 12 g / L potato dextrose broth and 20 g / L Bacto agar and cultured at 30°C. A colony formed on the agar medium was punched near the edge with a straw to obtain an agar disk, which was then crushed and transferred to a 14 ml polypropylene round tube (Corning). 2 mL of liquid medium containing 5 g / L potato dextrose broth was added, and the mixture was cultured at 30°C with reciprocal shaking at 120 rpm for 2 days. The entire culture was inoculated into 20 mL of liquid medium containing 40 g / L Solka Floc® (International Fiber Corporation), 1 g / L corn steep liquor (Sigma-Aldrich, Lot number: MKBN0183V), 24 g / L KH2PO4, 5 g / L (NH4)2SO4, 4 g / L urea, 1 g / L Tween 80, 1.2 g / L MgSO4·7H2O, 0.01 g / L ZnSO4·7H2O, 0.01 g / L MnSO4·5H2O, and 0.01 g / L CuSO4·5H2O. The culture was then cultured in a 300 mL Erlenmeyer flask with gyratory shaking (30°C, 220 rpm) for 10 days. The resulting culture was centrifuged at 15,000 rpm for 5 minutes and then filtered through a 0.22 μm filter to obtain the culture supernatant.

[0137] (3) Measurement of protease activity in T. cellulolyticus culture supernatant in the presence and absence of pepstatin A The protein concentration of the culture supernatant of each strain was measured using Protein Assay CBB (Nacalai Tesque). To confirm protease activity against casein, protease activity was measured using the Amplite™ Universal Fluorimetric Protease Activity Assay Kit *Green (AAT Bioquest, Inc.). Simultaneously, protease activity was measured under conditions in which the aspartic acid protease inhibitor pepstatin A was added.

[0138] Fifty μL of the kit's 2x assay buffer containing 0.5 μL of the kit's protease substrate (green fluorescent casein substrate) was dispensed into a 96-well plate (Greiner, product number 655090), and 50 μL of culture supernatant from the control strain and the Δpep4 strain was added to each well. For the pepstatin A-containing conditions, pepstatin A (Sigma-Aldrich, product number P2032) was added to a final concentration of 10 μM. Protease activity was measured every 2 minutes at 37°C using a SpectraMax® M2 microplate reader (Molecular Devices) by measuring relative fluorescence units (RFU, excitation / emission = 490 / 525 nm). The specific protease activity per total protein in the culture supernatant was calculated by dividing the RFU by the total protein amount used and the reaction time.

[0139] The results are shown in Table 1. In the table, "Relative activity" indicates the ratio of the protease specific activity of each sample to the protease specific activity of the control strain in the absence of pepstatin A. In the absence of pepstatin A, the protease specific activity of the culture supernatant of the Δpep4 strain decreased to approximately 36% of that of the control strain. Furthermore, most of the protease specific activity of the culture supernatant of the control strain (approximately 88%) was inhibited by pepstatin A. In contrast, the protease specific activity of the culture supernatant of the Δpep4 strain showed almost no change in either the presence or absence of pepstatin A. This indicates that the majority of the extracellular protease activity of T. cellulolyticus is derived from aspartic proteases, and that the majority of the protease activity derived from aspartic proteases is derived from Pep4 protease. These results suggest that the majority of the protease activity in the culture supernatant of T. cellulolyticus is derived from Pep4 protease, and that Pep4 protease is an important protease that should be deleted when expressing heterologous proteins using T. cellulolyticus as a host.

[0140] [Table 1]

[0141] (4) Evaluation of human IgG (Trastuzumab) degradation activity by T. cellulolyticus culture supernatant To confirm the trastuzumab-degrading activity of proteases contained in the culture supernatants of the control and Δpep4 strains, 0.5 g / L Herceptin® (Chugai Pharmaceutical, Trastuzumab (recombinant)) was dissolved in a solution (pH 4.3) simulating the medium used for actual liquid culture, containing 24 g / L KH2PO4, 5 g / L (NH4)2SO4, 2 g / L urea, 1 g / L Tween 80, 1.2 g / L MgSO4·7H2O, 0.01 g / L ZnSO4·7H2O, 0.01 g / L MnSO4·5H2O, and 0.01 g / L CuSO4·5H2O. To 90 μL of this Herceptin solution, 10 μL of culture supernatant from the control or Δpep4 strains diluted with water to a total protein concentration of 0.2 g / L or water was added. Furthermore, 10 μL of the culture supernatant (total protein concentration: 0.2 g / L) of the control strain and the Δpep4 strain, whose protease activity had been inactivated by treatment at 95°C for 5 minutes, was added to 90 μL of the Herceptin solution. Each mixture was incubated at 37°C for 3 days.

[0142] Western blotting was then performed on each of the incubated mixtures. 3 μL of each sample, diluted 20-fold with water, was added to 2 μL of water and 5 μL of Laemmli buffer. After heating at 70°C for 10 minutes, the entire mixture was loaded onto an Any kD™ Mini-PROTEAN™ TGX™ Precast Gel (Bio-Rad) and electrophoresed at 200 V for 40 minutes with a protein molecular weight marker, the A Precision Plus Protein Dual Color Standard (Bio-Rad). Proteins in the gel were transferred to a PVDF membrane (Invitrogen) using an iBind Western Device (ThermoFisher). The gel was probed with anti-human IgG F(ab′)2, F(ab′)2 fragment, highly cross-absorbed-peroxidase antibody produced in goat (Sigma-Aldrich, Product No. SAB3701242), diluted 1:20,000 in 5-fold diluted iBind™ FD Solution (Invitrogen). ECL Prime Western Blotting Detection Reagent (GE Healthcare) was added to the PVDF membrane to perform the ECL (Enhanced Chemiluminescence) reaction, and the luminescence was detected using an Amersham Imager 600 (GE Healthcare).

[0143] The results are shown in Figure 1. In samples incubated with water or protease-inactivated culture supernatant and Herceptin®, only a band representing the full-length Trastuzumab was detected. In contrast, in samples incubated with Herceptin® and the culture supernatant of the control strain, a band representing a Trastuzumab degradation product was detected at a lower molecular weight (around 150 kDa), suggesting that some Trastuzumab had been degraded. In samples incubated with Herceptin® and the culture supernatant of the Δpep4 strain, almost no band representing a Trastuzumab degradation product was detected. This indicates that the Pep4 protease contained in the culture supernatant of the control strain was responsible for the majority of the enzyme-dependent degradation of Trastuzumab. Therefore, Pep4 protease is considered to be an important protease that should be deleted when secreting and expressing heterologous proteins (e.g., IgG such as Trastuzumab) using T. cellulolyticus as a host.

[0144] (5) Evaluation of the effect of Pep4 protease gene disruption in Trastuzumab-producing T. cellulolyticus strains Using the T. cellulolyticus F09 ΔyscB strain as a parent strain, a trastuzumab-expressing strain was constructed by the following procedure.

[0145] First, a DNA fragment for expressing Trastuzumab having a nucleotide sequence in which the upstream region of the T. cellulolyticus creA gene, the upstream region of the T. cellulolyticus cbh2 gene (promoter; SEQ ID NO: 63), the T. cellulolyticus CBH1 secretion signal sequence (SEQ ID NO: 64), the Trastuzumab heavy chain gene (SEQ ID NO: 65), the downstream region of the T. cellulolyticus cbh1 gene (terminator; SEQ ID NO: 66), the T. cellulolyticus pyrF gene marker (SEQ ID NO: 62), the upstream region of the T. cellulolyticus cbh2 gene (promoter; SEQ ID NO: 63), the T. cellulolyticus CBH1 secretion signal sequence (SEQ ID NO: 64), the Trastuzumab light chain gene (SEQ ID NO: 67), the downstream region of the T. cellulolyticus cbh2 gene (terminator; SEQ ID NO: 68), and the downstream region of the T. cellulolyticus creA gene were linked in this order was prepared according to the following procedure. Using the genomic DNA of T. cellulolyticus Y-94 strain (FERM BP-5826) as a template, the upstream region of the creA gene was amplified by PCR using primers (SEQ ID NOs: 37 and 38), the upstream region of the cbh2 gene was amplified by PCR using primers (SEQ ID NOs: 39 and 40), the CBH1 secretion signal sequence was amplified by PCR using primers (SEQ ID NOs: 41 and 42), the downstream sequence of the cbh1 gene was amplified by PCR using primers (SEQ ID NOs: 43 and 44), the pyrF gene marker was amplified by PCR using primers (SEQ ID NOs: 31 and 32), the upstream region of the cbh2 gene was amplified by PCR using primers (SEQ ID NOs: 45 and 40), the CBH1 secretion signal sequence was amplified by PCR using primers (SEQ ID NOs: 41 and 46), the downstream sequence of the cbh2 gene was amplified by PCR using primers (SEQ ID NOs: 47 and 48), and the downstream region of the creA gene was amplified by PCR using primers (SEQ ID NOs: 49 and 50).Additionally, the Trastuzumab heavy chain gene was amplified by PCR using primers (SEQ ID NOs: 51 and 52) and primers (SEQ ID NOs: 53 and 54) with a fully synthetic gene purchased from Eurofins as a template. The Trastuzumab light chain gene was amplified by PCR using primers (SEQ ID NOs: 53 and 54). The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System. Two of the purified PCR products were mixed and used as templates for repeated PCR and ligation. The products were then integrated into the pUC plasmid provided with the In-Fusion HD Cloning Kit using the In-Fusion HD Cloning Kit. The reaction mixture was transformed into E. coli JM109 and grown overnight at 37°C on LB agar medium (containing 100 mg / L ampicillin) to form colonies. The resulting transformants were then isolated using the Wizard Plus Miniprep System to obtain the pUC-creA::Pcbh2-Her_H-pyrF-Pcbh2-Her_L plasmid, incorporating the Trastuzumab expression DNA fragment. The DNA fragment for Trastuzumab expression was amplified by PCR using the pUC-creA::Pcbh2-Her_H-pyrF-Pcbh2-Her_L plasmid as a template and primers (SEQ ID NOs: 37 and 50), and then concentrated and purified by ethanol precipitation. Note that by ligating the upstream and downstream sequences of the creA gene to both ends of the Trastuzumab expression sequence, it was possible to target the Trastuzumab expression sequence to the creA gene region rather than to a random location on the genome.

[0146] Next, the F09 ΔyscB strain was cultured and protoplasted using the same method as in (1), and then transformed with the purified Trastuzumab expression DNA fragment in the same manner as in (1). Protoplasts were recovered by centrifugation (2,000 rpm, 10 minutes) and plated on minimal medium containing 1 M sucrose. After culturing at 30°C for 7 days, strains with complemented uracil auxotrophy were selected. The resulting colonies were inoculated onto minimal medium and cultured at 30°C for 4 days. After confirming that the creA gene region had been replaced with the Trastuzumab expression sequence, a Trastuzumab-expressing strain derived from the F09 ΔyscB strain was obtained.

[0147] Next, a pep4 gene disruptant was constructed using the Trastuzumab-expressing strain derived from the F09 ΔyscB strain as a parent strain by the following procedure.

[0148] First, a DNA fragment for disrupting the pep4 gene was prepared using the following procedure. The fragment contained the upstream region of the T. cellulolyticus pep4 gene, the hygromycin resistance gene marker (SEQ ID NO: 69), and the downstream region of the T. cellulolyticus pep4 gene. The upstream region of the pep4 gene was amplified by PCR using primers (SEQ ID NOs: 27 and 55) and primers (SEQ ID NOs: 56 and 30) with the genomic DNA of T. cellulolyticus Y-94 strain (FERM BP-5826) as a template. The downstream region of the pep4 gene was amplified by PCR using primers (SEQ ID NOs: 57 and 58) with the hygromycin resistance gene-containing vector pcDNA3.1 / Hygro(+) (Life Technologies) as a template. The hygromycin resistance gene (including the promoter and terminator) was amplified by PCR using primers (SEQ ID NOs: 57 and 58). The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System. The purified PCR product was ligated into the pUC plasmid provided with the In-Fusion HD Cloning Kit. E. coli JM109 was transformed with the reaction mixture and cultured overnight at 37°C on LB agar medium (containing 100 mg / L ampicillin) to allow colonies to form. The pUC-pep4::hyg plasmid, incorporating the DNA fragment for disrupting the pep4 gene, was isolated from the resulting transformants using the Wizard Plus Miniprep System. The DNA fragment for disrupting the pep4 gene was amplified by PCR using primers (SEQ ID NOs: 27 and 30) with the pUC-pep4::hyg plasmid as a template, and then concentrated and purified by ethanol precipitation.

[0149] Next, the F09 ΔyscB-derived trastuzumab-expressing strain was cultured and protoplasted as described in (1), and transformed with the purified pep4 gene-disrupting DNA fragment as described in (1). Protoplasts were harvested by centrifugation (2,000 rpm, 10 min). They were then plated on minimal medium containing 1 M sucrose and cultured at 30°C for 1 day. Hygromycin-resistant strains were selected by overlaying a medium containing 0.5 g / L hygromycin B, 24 g / L potato dextrose broth, and 7 g / L Bacto agar and culturing at 30°C for another 3 days. The resulting colonies were inoculated onto minimal medium containing 0.5 g / L hygromycin B and cultured at 30°C for 4 days. Replacement of the pep4 gene with the hygromycin resistance gene was confirmed, yielding a pep4 gene-disrupted trastuzumab-expressing strain derived from the F09 ΔyscB strain.

[0150] The Trastuzumab-expressing strain derived from the F09 ΔyscB strain and its pep4 gene-disrupted strain were inoculated into a medium containing 12 g / L Potato Dextrose Broth (Difco) and 20 g / L Bacto Agar (Difco) and cultured at 30°C. One agar disk obtained by punching out the edge of a colony formed on the agar medium with a straw was inoculated into a medium containing 30 g / L glucose, 20 g / L yeast extract, 1 g / L uracil, and 1 g / L uridine and cultured at 30°C for 2 days with shaking at 120 rpm. After that, 2 ml of the preculture was added to 20 mL of 40 g / L Solka Floc, 10 g / L Pharmamedia (Archer Daniels Midland Company), 24 g / L KH2PO4, 5 g / L (NH4)2SO4, 4 g / L urea, 4.7 g / L potassium sodium (+)-tartrate tetrahydrate, 1 g / L Tween 80, 1.2 g / L MgSO4 7H2O, 0.01 g / L ZnSO4 7H2O, 0.01 g / L MnSO4 5H2O, and 0.01 g / L The bacteria were inoculated into a liquid medium containing CuSO4·5H2O and cultured at 30°C and 220 rpm for 7 days. The resulting culture medium was passed through a 0.22 μm filter to obtain the culture supernatant.

[0151] To confirm the secretory production amounts of Trastuzumab and Trastuzumab degradation products in the Trastuzumab-expressing strain derived from the F09 ΔyscB strain and its pep4 gene-disrupted strain, Western blotting was performed using the same method as in (4).

[0152] The results are shown in Figure 2. Compared to the F09 ΔyscB-derived Trastuzumab-expressing strain, the ratio of the amount of Trastuzumab degradation products to the amount of full-length Trastuzumab was reduced in the pep4 gene-disrupted strain of the F09 ΔyscB-derived Trastuzumab-expressing strain. This suggests that disruption of the pep4 gene suppresses the degradation of Trastuzumab. These results demonstrate that the Pep4 protease is an important protease that should be deleted when secreting and expressing heterologous proteins (e.g., IgG such as Trastuzumab) using T. cellulolyticus as a host. [Industrial Applicability]

[0153] According to the present invention, proteins can be produced efficiently.

[0154] <Description of Sequence Listing> SEQ ID NOs: 1 to 58: Primers SEQ ID NO: 59: Nucleotide sequence of the sC gene of Talaromyces cellulolyticus S6-25 strain SEQ ID NO: 60: Nucleotide sequence of the yscB gene of Talaromyces cellulolyticus S6-25 strain SEQ ID NO: 61: Nucleotide sequence of sC gene marker SEQ ID NO: 62: Nucleotide sequence of pyrF gene marker SEQ ID NO: 63: Nucleotide sequence of the cbh2 promoter of Talaromyces cellulolyticus SEQ ID NO: 64: Nucleotide sequence encoding the Cbh1 signal peptide of Talaromyces cellulolyticus SEQ ID NO: 65: Nucleotide sequence of trastuzumab heavy chain gene SEQ ID NO: 66: Nucleotide sequence of cbh1 terminator SEQ ID NO: 67: Nucleotide sequence of trastuzumab light chain gene SEQ ID NO: 68: Base sequence of cbh2 terminator SEQ ID NO: 69: Nucleotide sequence of hygromycin resistance gene marker SEQ ID NO: 70: Nucleotide sequence of the pep4 gene of Talaromyces cellulolyticus Y-94 strain SEQ ID NO: 71: Amino acid sequence of the Pep4 protein of Talaromyces cellulolyticus strain Y-94 SEQ ID NO: 72: Amino acid sequence of the Cbh1 signal peptide of Talaromyces cellulolyticus SEQ ID NO: 73: Amino acid sequence of the YscB protein of Talaromyces cellulolyticus strain S6-25 SEQ ID NO: 74: Nucleotide sequence of the creA gene of Talaromyces cellulolyticus S6-25 strain

Claims

1. A method for producing a target protein, comprising: Cultivating Talaromyces cellulolyticus capable of producing a target protein in a medium; Including, The Talaromyces cellulolyticus has been modified so that the activity of the Pep4 protein is reduced compared to that of an unmodified strain, The activity of the Pep4 protein is reduced by reducing the expression of the pep4 gene, by disrupting the pep4 gene, or by deleting the pep4 gene; The Pep4 protein is a protein described in the following (A), (B), or (C): (A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 71; (B) a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 71, and having protease activity; (C) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 71 and having protease activity; A method, wherein the pep4 gene is any one of the following genes (a) to (d): (a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 70; (b) a gene encoding the amino acid sequence shown in SEQ ID NO: 71; (c) a gene encoding a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 71 and having protease activity; (d) A gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 71 and having protease activity.

2. The method according to claim 1, wherein the Talaromyces cellulolyticus has been further modified to reduce the activity of the YscB protein and / or the CreA protein compared to a non-modified strain, The activity of the YscB protein and / or the CreA protein reduces the expression of the yscB gene and / or the creA gene, thereby or by deletion of the yscB gene and / or the creA gene, The YscB protein is a protein described in (A-1), (B-1), or (C-1) below: (A-1) a protein comprising the amino acid sequence shown in SEQ ID NO: 73; (B-1) a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 73, and having protease activity; (C-1) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 73 and having protease activity; The CreA protein is a protein described in the following (A-2), (B-2), or (C-2): (A-2) a protein comprising an amino acid sequence encoded by the base sequence shown in SEQ ID NO: 74; (B-2) A protein comprising an amino acid sequence encoded by the base sequence shown in SEQ ID NO: 74, but containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and having protease activity; (C-2) A protein having an amino acid sequence having 90% or more identity to the amino acid sequence encoded by the base sequence shown in SEQ ID NO: 74 and having protease activity; The yscB gene is a gene described in the following (a-1), (b-1), or (c-1): (a-1) a gene comprising the nucleotide sequence shown in SEQ ID NO: 60; (b-1) a gene encoding a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 73 and having protease activity; (c-1) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 73 and having protease activity; A method in which the creA gene is a gene described in the following (a-2), (b-2), or (c-2): (a-2) a gene comprising the nucleotide sequence shown in SEQ ID NO: 74; (b-2) a gene encoding a protein having a protease activity, which contains an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence encoded by a gene containing the base sequence shown in SEQ ID NO: 74; (c-2) A gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence encoded by a gene comprising the base sequence shown in SEQ ID NO: 74 and having protease activity.

3. The Talaromyces cellulolyticus is Talaromyces cellulolyticus S6-25 The method according to claim 1 or 2, wherein the modified strain is derived from the strain (NITE BP-01685).

4. The method according to any one of claims 1 to 3, further comprising recovering the target protein.

5. The method according to any one of claims 1 to 4, wherein the target protein accumulates in the medium by the culture.

6. The method according to any one of claims 1 to 5, wherein the target protein is expressed as a fusion protein with a signal peptide that functions in Talaromyces cellulolyticus.

7. The method according to any one of claims 1 to 6, wherein the target protein is a heterologous protein.

8. The method according to any one of claims 1 to 7, wherein the target protein is a human-derived protein.

9. The method according to any one of claims 1 to 8, wherein the target protein is an antibody-related molecule.

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