阅读说明：本技术 蛋白质的制造方法 (Method for producing protein ) 是由 深田宽朗 河口礼佳 十仓充范 于 2018-10-09 设计创作，主要内容包括：本发明提供蛋白质的制造方法。通过培养基培养以使YscB蛋白质的活性降低的方式进行了修饰并且具有靶蛋白生产能力的Talaromyces cellulolyti cus,从而制造靶蛋白。(The present invention provides a method for producing a protein. Talaromyces cellulolyticus cus modified so as to reduce the activity of YscB protein and having the ability to produce a target protein is cultured in a medium to produce the target protein.)

1. A method for producing a target protein, comprising the steps of:

talaromyces cellulolyticus having an ability to produce a target protein is cultured in a medium,

the Talaromyces cellulolyticus is modified such that the activity of the YscB protein is reduced compared to a non-modified strain.

2. The method of claim 1, wherein,

the activity of the yscB protein is reduced by reducing the expression of the yscB gene or disrupting the yscB gene.

3. The method of claim 1 or 2,

the activity of the YscB protein is reduced by deletion of the yscB gene.

4. The method according to any one of claims 1 to 3,

the YscB protein is the protein described in the following (a), (b) or (c):

(a) a protein comprising the amino acid sequence represented by SEQ ID NO. 43;

(b) a protein having protease activity, which comprises an amino acid sequence comprising 1 to 10 substitutions, deletions, insertions, and/or additions of amino acid residues in the amino acid sequence represented by SEQ ID NO. 43;

(c) a protein which comprises an amino acid sequence having 90% or more homology with respect to the amino acid sequence represented by SEQ ID NO.43 and has a protease activity.

5. The method according to any one of claims 1 to 4,

the Talaromyces cellulolyticus is modified such that the activity of the C reA protein is reduced compared to a non-modified strain.

6. The method of claim 5, wherein,

the creA protein activity is decreased by decreasing the expression of the creA gene or disrupting the creA gene.

7. The method of claim 5 or 6,

the creA protein activity is decreased by deletion of the creA gene.

8. The method according to any one of claims 1 to 7,

the Talaromyces cellulolyticus is a modified strain derived from Talaromyces cellulolyticus S6-25 strain (NITE BP-01685).

9. The method according to any one of claims 1 to 8, further comprising the following steps:

recovering the target protein.

10. The method according to any one of claims 1 to 9,

the target protein is accumulated in the medium by the culture.

11. The method according to any one of claims 1 to 10,

the target protein is expressed as a fusion protein with a signal peptide that functions in Talaromyces cellulolyticus.

12. The method according to any one of claims 1 to 11,

the target protein is a heterologous protein.

13. The method of any one of claims 1 to 12, wherein,

the target protein is a protein derived from a human.

14. The method according to any one of claims 1 to 13,

the target protein is human serum albumin.

15. The method according to any one of claims 1 to 13,

the target protein is an antibody-related molecule.

16. The method according to any one of claims 1 to 13,

the target protein is a growth factor.

Technical Field

The present invention relates to a method for producing a protein.

Background

As a method for producing a protein, methods using various microorganisms such as coryneform bacteria, bacteria of the genus Bacillus, yeasts, and filamentous bacteria have been reported.

For example, non-patent document 1 discloses production of cellulase derived from a host using a filamentous fungus Talaromyces cellulolyticus (old name: Acremonium cellulolyticus). Patent document 1 discloses production of an antibody using a filamentous bacterium. Further, patent document 2 discloses production of a multimeric protein having an inner cavity using a filamentous bacterium such as Talaromycescellulolyticus.

Furthermore, patent documents 3 to 4 disclose production of a heterologous protein using a filamentous fungus in which the activity of an endogenous protease is attenuated. Patent document 5 discloses production of a heterologous protein using a filamentous fungus in which the activity of an endogenous alkaline protease is attenuated.

Further, patent document 6 discloses production of a heterologous protein using a yeast lacking carboxypeptidase ysc α activity. It is stated in the document that the yeast may also lack peptidase activities selected from the group consisting of yscA, yscB, yscY and y scS.

However, the relationship between the YscB protein in Talaromyces cellulolyticus and protein production is not known.

Disclosure of Invention

Technical problem to be solved by the invention

The purpose of the present invention is to provide a method for producing a protein.

Means for solving the problems

The present inventors have intensively studied to solve the above-mentioned problems, and as a result, have found that a protein production ability of Talaromyces cellulolyticus can be improved by modifying Talaromyces cellulolyticus so as to decrease an activity of YscB protein, and have completed the present invention.

That is, the present invention can be exemplified as follows.

1. A method for producing a target protein, comprising the steps of:

talaromyces cellulolyticus having an ability to produce a target protein is cultured in a medium,

the Talaromyces cellulolyticus is modified such that the activity of the Y scB protein is reduced compared to a non-modified strain.

2. The method, wherein,

the activity of the yscB protein is reduced by reducing the expression of the yscB gene or disrupting the yscB gene.

3. The method, wherein,

the activity of the YscB protein is reduced by deletion of the yscB gene.

4. The method, wherein,

the YscB protein is the protein described in the following (a), (b) or (c):

(a) a protein comprising the amino acid sequence represented by SEQ ID NO. 43;

(b) a protein having protease activity, which comprises an amino acid sequence comprising 1 to 10 substitutions, deletions, insertions, and/or additions of amino acid residues in the amino acid sequence represented by SEQ ID NO. 43;

(c) a protein which comprises an amino acid sequence having 90% or more homology with respect to the amino acid sequence represented by SEQ ID NO.43 and has a protease activity.

5. The method, wherein,

the Talaromyces cellulolyticus is modified such that the activity of the CreA protein is reduced compared to a non-modified strain.

6. The method, wherein,

the creA protein activity is decreased by decreasing the expression of the creA gene or disrupting the creA gene.

7. The method, wherein,

the creA protein activity is decreased by deletion of the creA gene.

8. The method, wherein,

the Talaromyces cellulolyticus is a modified strain derived from Talaromyces cellulolyticus S6-25 strain (NITE BP-01685).

9. The method further comprises the following steps:

recovering the target protein.

10. The method, wherein,

the target protein is accumulated in the medium by the culture.

11. The method, wherein,

the target protein is expressed as a fusion protein with a signal peptide that functions in Talaromyces cellulolyticus.

12. The method, wherein,

the target protein is a heterologous protein.

13. The method, wherein,

the target protein is a protein derived from a human.

14. The method, wherein,

the target protein is human serum albumin.

15. The method, wherein,

the target protein is an antibody-related molecule.

16. The method, wherein,

the target protein is a growth factor.

Drawings

Fig. 1 is a graph (photograph) showing the results of Human Serum Albumin (HSA) production based on t.cellulolyticus F09 strain and F09 Δ yscB strain.

Fig. 2 is a graph (photograph) showing the results of trastuzumab production based on t.cellulolyticus F09 strain and F09 Δ yscB strain.

FIG. 3 is a graph (photograph) showing the results of production of Keratinocyte growth factor (KGF-1) based on T.cellulyticus F09 strain and F09. delta. yscB strain.

FIG. 4 is a graph (photograph) showing the results of Vascular Endothelial Growth Factor (VEGF) production based on T.cellulolyticus F09 strain and F09. delta. yscB strain.

FIG. 5 is a photograph showing the results of evaluating the protease activity of a culture supernatant of T.cellulolyticus F09 strain.

FIG. 6 is a photograph showing the results of evaluating the protease activity of culture supernatants of T.cellulolyticus F09 strain and protease-deficient strain thereof.

Detailed Description

The present invention will be described in detail below.

The method of the present invention is a method for producing a target protein using Talaromyces cellulolyticus. Talaromyces cellulolyticus utilized in the process is also referred to as "microorganism of the invention".

<1> microorganism of the present invention

The microorganism of the present invention is Talaromyces cellulolyticus having a target protein-producing ability, which is modified so as to reduce the activity of YscB protein. In the description of the microorganism of the present invention, the microorganism of the present invention or Talaromyces c ellulolyticus used for constructing the microorganism is sometimes referred to as a "host".

<1-1>Talaromyces cellulolyticus

The microorganism of the present invention is Talaromyces cellulolyticus. Talaromyces cellulolyticus is old under the name Acremonium cellulolyticus. That is, Acremonium cellulolyticus is reclassified as Talaromyces cellulolyticus (FEMS Microbiol. Lett.,2014,351:32-41) by revising the systematic classification. Specific examples of Talaromyces cellulolyticus include: the C1 strain (Japanese patent laid-open No. 2003-135052), the CF-2612 strain (Japanese patent laid-open No. 2008-271927), the TN strain (FERM BP-685), the S6-25 strain (NITEBP-01685), the Y-94 strain (FERM BP-5826, CBS 136886) and derivatives thereof. It should be noted that "Talaromyces cellulolyticus" is collectively referred to as: a fungus classified in talaromyces cellulolyticus at least at any point in time before, at and after application of the present application. That is, for example, a strain classified into Talaromyces cellulolyticus, such as the above-mentioned exemplified strain, is considered to belong to Talaromyces cellulolyticus even when the systematic classification is changed in the future.

The S6-25 strain was originally deposited at the NITE International patent organism depositary (Total Sickle 2-5-8122, Philippine 292-. The strain is obtained from TN strain (FERM BP-685), and has high cellulase production capacity. The Y-94 strain was originally deposited at 12.1.1983 at the institute of Industrial science and technology, Life engineering, and technology (NITE International patent organism depositary, postal code 292-.

For example, these strains may be initiated from a depository where each strain is deposited.

The microorganism of the present invention can be obtained by modifying Talaromyces cellulolyticus such as the above-mentioned strain. That is, for example, the microorganism of the present invention may be a modified strain derived from the exemplified strains. Specifically, for example, the microorganism of the present invention may be a modified strain derived from the S6-25 strain or the Y-94 strain. More specifically, for example, the microorganism of the present invention may be a modified strain derived from the S6-25 strain. The order of carrying out the modification for constructing the microorganism of the present invention is not particularly limited.

<1-2> target protein production ability

The microorganism of the present invention has a target protein-producing ability. "microorganism having the ability to produce a target protein" refers to a microorganism having the ability to produce a target protein. The "microorganism having the ability to produce a target protein" may specifically mean a microorganism having the ability to express a target protein and accumulate the target protein in a culture to such an extent that the target protein can be recovered when cultured in a medium. Specifically, the term "accumulation in a culture" may refer to accumulation in a culture medium, a cell surface layer, a cell body, or a combination thereof. The case where the target protein is accumulated outside the cell (for example, in a culture medium or on a cell surface layer) is referred to as "secretion" or "secretory production" of the target protein. That is, the microorganism of the present invention may have a secretory production ability of a target protein (an ability to secretly produce the target protein). The target protein can be accumulated in the medium. The amount of the target protein accumulated may be, for example, 10. mu.g/L or more, 1mg/L or more, 100mg/L or more, or 1g/L or more as the amount accumulated in the culture. The microorganism of the present invention may have the ability to produce 1 target protein, or may have the ability to produce 2 or more target proteins.

The microorganism of the present invention may be a substance having an ability to produce the target protein by itself, or a substance modified so as to have an ability to produce the target protein. Typically, the microorganism of the present invention may be a substance which inherently has cellulase producing ability (ability to produce cellulase). The microorganism of the present invention may be modified so as to enhance the ability to produce the target protein that the microorganism originally has. In the case of a microorganism having a target protein-producing ability, for example, it can be obtained by imparting a target protein-producing ability to Talaromyces cellulolyticus as described above, or by enhancing a target protein-producing ability of Talaromyces cellulolyticus as described above. The target protein production ability may be imparted or enhanced, for example, by introduction of a gene construct for expression of the target protein, introduction of other modification for improving the target protein production ability, or a combination thereof.

The microorganism of the present invention has a target protein-producing ability by virtue of having a gene construct for expression of at least a target protein. Specifically, the microorganism of the present invention may have a target protein-producing ability by having a gene construct for expression of a target protein, or by having a combination of a gene construct for expression of a target protein and other properties. That is, the microorganism of the present invention has a gene construct for expressing a target protein. The microorganism of the present invention may have 1 copy of a gene construct for expressing a target protein, or may have 2 or more copies of a gene construct for expressing a target protein. The microorganism of the present invention may have a gene construct for expression of 1 target protein, or may have a gene construct for expression of 2 or more target proteins. The copy number and the species number of the gene construct for expression of the target protein may be referred to as the copy number and the species number of the target protein gene, respectively.

In the microorganism of the present invention, the gene construct for expressing the target protein may be present on a vector autonomously replicating extrachromosomally such as a plasmid, or may be integrated into a chromosome. That is, the microorganism of the present invention may have, for example, 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. In addition, the microorganism of the present invention may have a gene construct for expression of a target protein on a chromosome, for example. In the case where the microorganism of the present invention has 2 or more gene constructs for expression of the target protein, these gene constructs may be retained in the microorganism of the present invention so long as the target protein can be produced. For example, these gene constructs may all be maintained on a single expression vector, or may all be maintained on the chromosome. Furthermore, these gene constructs may be maintained on a plurality of expression vectors, respectively, or may be maintained on a single or a plurality of expression vectors and on a chromosome, respectively.

The microorganism of the present invention may have a gene construct for expressing a target protein, or may have a modified gene construct for expressing a target protein. Typically, the microorganism of the present invention may be a substance originally having a gene construct for expression of cellulase. The microorganism of the present invention may be one into which a gene construct for expression of a target protein is introduced instead of an original gene construct for expression of a target protein, or one into which a gene construct for expression of a target protein is further introduced. The microorganism having the gene construct for expression of the target protein can be obtained by introducing the gene construct for expression of the target protein into Talaromyces cellulolyticus as described above.

The "gene construct for expressing a target protein" refers to a gene expression system configured to express the target protein. The gene construct for expression of the target protein is also referred to as "expression system of the target protein", "expression unit of the target protein", or "expression cassette of the target protein". The gene construct for expression of the target protein comprises a promoter sequence and a base sequence encoding the target protein in the 5 'to 3' direction. Promoter sequences are also referred to simply as "promoters". The base sequence encoding the amino acid sequence is also referred to as "gene". For example, a base sequence encoding a target protein is also referred to as "a gene encoding a target protein" or "a target protein gene". The gene of the target protein may be linked downstream of the promoter so as to be under the control of the promoter and express the target protein. In addition, the gene construct for expression of the target protein may have effective control sequences (operators, terminators, etc.) for expression of the target protein at appropriate positions so that they function. In the present invention, "expression of a target protein gene", "expression of a target protein", "production of a target protein", and "production of a target protein" may be used synonymously with each other, unless otherwise specified. The gene construct for expression of the target protein can be appropriately designed according to various conditions such as the type of the target protein.

The promoter is not particularly limited as long as it functions in Talaromyces cellulolyticus. The "promoter functional in Talaromyces cellulolyticus" refers to a promoter having a promoter activity in Talaromyces cellulolyticus, that is, a transcription activity of a gene.

The promoter may be a promoter derived from a host, or may be a promoter derived from a heterologous source. The promoter may be a promoter inherent to the target protein gene, or may be a promoter of another gene. The promoter may be an inducible promoter or a constitutive (constitutive) promoter. Examples of the promoter include promoters of cellulase genes of microorganisms. Specific examples of the promoter include a promoter of a cellulase gene of Talaromyces cellulolyticus. Examples of the cellulase gene include cbhI gene (also referred to as cbh1 gene) and cbhII gene (also referred to as cbh2 gene). That is, examples of the promoter include a cbhI gene promoter and a cbhII gene promoter. The promoter of the cbhI gene may also be referred to as the "cbhI promoter" or the "cbh 1 promoter". The promoter of the cbhII gene may also be referred to as the "cbhII promoter" or the "cbh 2 promoter". The base sequences of cbhI promoter and cbhII promoter of Talaromyces cellulolyticus are represented as SEQ ID Nos. 41 and 33, respectively. That is, the promoter may be, for example, one having the nucleotide sequence of the above-exemplified promoter (for example, the nucleotide sequence of SEQ ID NO.41 or 33). Further, the promoter may be a conservative variant of the exemplified promoters (for example, a promoter having the base sequence of SEQ ID NO.41 or 33). That is, for example, the above-mentioned exemplary promoter may be used as it is or after being appropriately modified. The terms "cbhI promoter" and "cbhI promoter" encompass conservative variants thereof in addition to the exemplary cbhI and cbhI promoters described. For conservative variants of the promoter, the description concerning conservative variants of the yscB gene described below can be applied. For example, the promoter may be a DNA having a nucleotide sequence having homology of 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more with respect to the nucleotide sequence of SEQ ID NO.41 or 33, if it retains its original function. The "original function" of the promoter refers to a function of expressing (e.g., inducing or constitutively expressing) a gene linked downstream. The function of the promoter can be confirmed, for example, by confirming the expression of the gene. For example, the expression of a gene can be confirmed using a reporter gene.

The target protein is not particularly limited. The target protein may be a protein derived from a host, or may be a protein derived from a heterologous source (heterologous protein). In the present invention, a "heterologous protein" refers to a protein that is foreign (exogeno us) to Talaromyces cellulolyticus producing the protein. The target protein may be, for example, a protein derived from a microorganism, a protein derived from a plant, a protein derived from an animal, a protein derived from a virus, or a protein having an artificially designed amino acid sequence. In particular, the target protein may be a human protein. The target protein may be a monomeric protein or a multimeric protein. Multimeric proteins refer to proteins that exist as multimers comprising 2 or more subunits. In the multimer, the subunits may be linked by a covalent bond such as a disulfide bond, may be linked by a non-covalent bond such as a hydrogen bond or a hydrophobic interaction, or may be linked by a combination thereof. Preferably, the multimer contains 1 or more intermolecular disulfide bond. Multimers can be homomultimers comprising a single species of subunit, or heteromultimers comprising 2 or more species of subunit. In the case where the target protein is a heteromultimeric protein, the phrase "the target protein is a heteromultimeric protein" means that at least 1 subunit of the subunits constituting the heteromultimer should be a heteromultimeric protein. That is, all subunits may be heterologous-derived, or only a portion of the subunits may be heterologous-derived. The target protein may be a secretory protein or a non-secretory protein. The secretory protein may be a naturally secretory protein or a naturally non-secretory protein, and is preferably a naturally secretory protein. The term "protein" also includes substances called peptides, such as oligopeptides and polypeptides.

Examples of the target protein include: enzymes, bioactive proteins, receptor proteins, antigenic proteins for use as vaccines, and any other proteins.

Examples of the enzyme include: cellulase, glutamine transaminase, protein glutaminase (Pr oeinglutinase), isomaltose glucanase (Isomaltoglucanase), protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, chitinase, and the like.

In the present invention, "cellulase" is a generic name of enzymes that catalyze a reaction of hydrolyzing a glycosidic bond contained in cellulose. Examples of the cellulase include: endo-cellulases (endoglucanases; EC 3.2.1.4), exo-cellulases (cellobiohydrolases; EC 3.2.1.91), cellobiases (beta-glucosidases; EC 3.2.1.21). In addition, cellulases are called microcrystalline cellulase (avicel), filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and the like, depending on the substrate used for the activity measurement. Examples of the cellulase include: cellulases derived from fungi such as Trichoderma reesei (Trichoderma reesei) and Talaromyces cellulolyticus, and bacteria such as Clostridium thermocellum (Clostridium thermocellum).

Examples of glutaminyl aminotransferase include: secretory glutamyl transaminase of filamentous bacteria such as actinomycetes such as Streptomyces mobaraense IFO13819(WO01/23591), Streptomyces cinnnamoneum IFO 12852, Streptomyces griseocorneum IFO 12776, Streptomyces lydicus (WO9606931) and microorganisms such as microorganisms (WO 9622366). Examples of the protein glutaminase include protein glutaminase of Chryseobacterium proteolyticum (WO 2005/103278). Examples of the isomaltose glucanase include isomaltose glucanase from Arthrobact globiformis (WO 2005/103278).

Examples of the biologically active protein include: growth factors (Growth factor), hormones, cytokines, antibody-related molecules.

Specific examples of the Growth factor (Growth factor) include: epidermal Growth Factor (EGF), Insulin-like growth factor-1 (Insulin-like growth factor-1; IGF-1), Transforming Growth Factor (TGF), Nerve Growth Factor (NGF), Brain-derived neurotrophic factor (Brain-derived neurotrophic factor; BDNF), Vascular Endothelial Growth Factor (VEGF), Granulocyte-colony stimulating factor (G-CSF), Granulocyte-colony stimulating factor (GM-CSF), Platelet-derived growth factor (PDGF-CSF; PDGF), Thrombopoietin (TPO), fibroblast growth factor (EPO), fibroblast growth factor (FGF-84), fibroblast growth factor (FGF-FGF), fibroblast growth factor (FGF-84), fibroblast growth factor (FGF-growth factor; FGF-growth factor (FGF-83; FGF-growth factor, FGF-growth factor (FGF-growth factor; FGF-growth factor, NGF, VEGF-growth factor, VEGF, Granulocyte-colony stimulating factor, G-macrophage-stimulating factor, Granulocyte colony stimulating factor (PDGF), Thrombopoietin, PDGF, VEGF, fibroblast growth factor (EPO, FGF-growth factor, fibroblast growth factor, FGF-growth factor (EPO, fibroblast growth factor, fibroblast growth, Keratinocyte growth factor (KGF-1 or FGF7, KGF-2 or FGF10), Hepatocyte Growth Factor (HGF).

Specific examples of the hormone include: insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), calcitonin (calcein), exenatide (exenatide).

Specific examples of the cytokine include: interleukins, interferons, Tumor Necrosis Factor (TNF).

It should be noted that Growth factors (Growth factors), hormones and cytokines may not be strictly distinguished from each other. For example, the biologically active protein may belong to any one group selected from Growth factors (Growth factors), hormones, and cytokines, or may belong to a plurality of groups selected from these.

In addition, the biologically active protein may be the whole protein or a part thereof. Examples of the protein include a physiologically active portion. Specific examples of the physiologically active moiety include a physiologically active peptide Teriparatide (Teriparatide) containing 34 amino acid residues at the N-terminus of a mature form of parathyroid hormone (PTH).

"antibody-related molecule" refers to a protein comprising a molecular species selected from a single domain or a combination of 2 or more domains constituting the domain of a complete antibody. As the domains constituting the whole antibody, there can be mentioned: VH, CH as the Domain of the heavy chain1. CH2 and CH3, and VL and CL as domains of a light chain. The antibody-related molecule may be a monomeric protein or a multimeric protein, as long as it contains the above-mentioned molecular species. In the case where the antibody-related molecule is a multimeric protein, it may be a homomultimer comprising a single kind of subunit, or a heteromultimer comprising 2 or more kinds of subunits. Specific examples of the antibody-related molecule include: intact antibody, Fab, F (ab')₂Fc, a dimer comprising a heavy chain (H chain) and a light chain (L chain), an Fc fusion protein, a heavy chain (H chain), a light chain (L chain), a single-chain fv (scFv), an sc (fv)₂Disulfide bond fv (sdfv), diabody (diabody), and VHH fragment (nanobody (registered trademark)). More specifically, examples of the antibody-related molecule include trastuzumab and nivolumab.

The receptor protein is not particularly limited, and may be, for example, a receptor protein for a biologically active protein or other physiologically active substance. Examples of the other physiologically active substance include neurotransmitters such as dopamine. In addition, the receptor protein may also be an orphan receptor for which the corresponding ligand is unknown.

The antigen protein used for the vaccine is not particularly limited as long as it can elicit an immune response, and may be appropriately selected according to the subject of the presumed immune response.

In addition, as other proteins, there may be mentioned: liver-type fatty acid binding protein Liver-type fatty acid-binding protein (LFABP), fluorescent protein, immunoglobulin binding protein, albumin and extracellular protein. The Fluorescent Protein may be Green Fluorescent Protein (GFP). Examples of the immunoglobulin-binding Protein include Protein A, Protein G and Protein L. Examples of albumin include human serum albumin.

Examples of extracellular proteins include: fibronectin, vitronectin, collagen, osteopontin, laminin, partial sequences thereof. Laminins are proteins having a heterotrimeric structure that contains an alpha chain, a beta chain, and a gamma chain. As the laminin, mammalian laminin can be mentioned. Examples of the mammal 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. The mammal is particularly a human. The subunit chains of laminin (i.e., alpha, beta, and gamma chains) include: 5 alpha chains (alpha 1-alpha 5), 3 beta chains (beta 1-beta 3) and 3 gamma chains (gamma 1-gamma 3). Laminins constitute various isoforms through 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, laminin 523. As a partial sequence of laminin, laminin E8, which is a fragment E8 of laminin, may be mentioned. Specifically, laminin E8 is a protein having a heterotrimeric structure comprising 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., alpha chain E8, beta chain E8, and gamma chain E8) are also collectively referred to as "E8 subunit chains". Examples of the subunit chain of E8 include the E8 fragment of the subunit chain of laminin mentioned above. Laminin E8 constitutes various isoforms through 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, laminin 521E 8.

The target protein gene may be used as it is or after appropriate modification. The target protein gene may be modified, for example, to obtain a desired activity. For the target protein gene and the variant of the target protein, the descriptions of conservative variants of the below-described yscB gene and yscB protein can be applied. For example, the target protein gene may be modified in such a manner that substitution, deletion, insertion and/or addition of 1 or several amino acids are contained in the amino acid sequence of the encoded target protein. It should be noted that the specific protein derived from a biological species is not limited to the protein itself found in the biological species, and includes proteins having the amino acid sequence of the protein found in the biological species and variants thereof. These variants may or may not be found in the species. That is, for example, the term "human protein" is not limited to proteins found in the human body, but includes proteins having amino acid sequences of proteins found in the human body and variants thereof. In addition, the target protein gene may have any codon substituted by a codon equivalent thereto. For example, with respect to the target protein gene, modification may be made so as to have an optimum codon according to the codon usage frequency of the host used.

In the case of the target protein, other amino acid sequences may be contained in addition to the amino acid sequences of the exemplified target proteins. That is, the target protein may be a fusion protein with other amino acid sequences. The "other amino acid sequence" is not particularly limited as long as a target protein having desired properties can be obtained. The "other amino acid sequence" can be appropriately selected depending on various conditions such as the purpose of use thereof. Examples of the "other amino acid sequence" include: signal peptide (also referred to as signal sequence), peptide tag, recognition sequence for protease. "other amino acid sequences", for example, may be linked to the N-terminus or C-terminus, or both, of the target protein. As the "other amino acid sequences", 1 kind of amino acid sequence may be used, or 2 or more kinds of amino acid sequences may be used in combination.

The signal peptide can be used, for example, in 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, the gene construct for expression of the target protein may include a promoter sequence, a base sequence encoding a signal peptide, and a base sequence encoding the target protein in the 5 'to 3' direction. In this case, a nucleic acid sequence encoding the target protein may be ligated 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 phrase "the target protein is expressed as a fusion protein with the signal peptide" is not limited to the case where the target protein is adjacent to the signal peptide and is expressed as a fusion protein with 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 target protein is produced by secretion using a signal peptide, the signal peptide is usually cleaved at the time of secretion, and the target protein without the signal peptide can be secreted extracellularly. That is, the phrase "the target protein is expressed as a fusion protein with the signal peptide" or "the target protein includes the signal peptide" means that the target protein is not required to constitute a fusion protein with the signal peptide at the time of expression, and the finally obtained target protein is not required to constitute a fusion protein with the signal peptide.

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

The signal peptide may be a signal peptide derived from a host or a signal peptide derived from a heterologous source. The signal peptide may be a signal peptide inherent to the target protein, or may be a signal peptide of another protein. The signal peptide includes a signal peptide of a secretory cellulase of a microorganism. Specific examples of the signal peptide include a signal peptide of a secretory cellulase of Talaromyces cellulolyticus. Examples of the secretory cellulase include: the cbhI protein encoded by the cbhI gene (also referred to as Cbh1 protein), the cbhI protein encoded by the cbhI gene (also referred to as Cbh2 protein). That is, examples of the signal peptide include: the signal peptide of CbhI protein, the signal peptide of CbhII protein. The signal peptide of the CbhI protein is also referred to as the "CbhI signal peptide" or "Cbh 1 signal peptide". The signal peptide of the CbhII protein is also referred to as the "CbhII signal peptide" or "Cbh 2 signal peptide". The amino acid sequence of the CbhI signal peptide of Talaromyces cellulolyticus is shown in SEQ ID NO. 42. That is, the signal peptide may be, for example, a signal peptide having the amino acid sequence of the exemplified signal peptide (e.g., the amino acid sequence of SEQ ID NO. 42). Furthermore, the signal peptide may be a conservative variant of the exemplified signal peptide (e.g., a signal peptide having the amino acid sequence of SEQ ID NO. 42). That is, for example, the signal peptide can be used as it is or after suitable modification. The terms "CbhI signal peptide" and "CbhI signal peptide" encompass conservative variants thereof in addition to the exemplary CbhI signal peptide and CbhI signal peptide. For conservative variants of the signal peptide, the description concerning conservative variants of the YscB protein described later can be applied. For example, the signal peptide may be a peptide having an amino acid sequence in which 1 or several amino acids at 1 or several positions are substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO.42, if it retains its original function. In addition, the "1 or several" in the signal peptide variants means, specifically, preferably 1 to 7, more preferably 1 to 5, and near some preferably 1 to 3, and particularly preferably 1 to 2. For example, the signal peptide may have an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more homology with the amino acid sequence of SEQ ID NO.42, if it retains its original function. The "original function" of the signal peptide is a function that can cause secretion of the target protein when the signal peptide is linked to the N-terminus of the target protein. The function of the signal peptide can be confirmed, for example, by confirming secretion of the protein by linking to the N-terminus of the protein.

Specific examples of the peptide tag include: his tag, FLAG tag, GST tag, Myc tag, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (thioredoxin), GFP (green fluorescent protein), HRP (Horseradish peroxidase), ALP (Alkalinephosphopeptide), Fc region of antibody. The peptide tag can be used, for example, for detection and purification of a target protein obtained by expression.

Specific examples of the recognition sequence of the protease include: HRV3C protease recognition sequence, Factor Xa protease recognition sequence, and protEV protease recognition sequence. The recognition sequence of the protease can be used, for example, for cleavage of a target protein obtained by expression. Specifically, for example, when a target protein is expressed as a fusion protein with a peptide tag, a recognition sequence of a protease is introduced into a junction between the target protein and the peptide tag, so that the peptide tag can be cleaved from the expressed target protein by the protease to obtain a target protein without the peptide tag.

The resulting N-terminal region of the target protein may be the same as or different from that of the natural protein. For example, the N-terminal region of the resulting target protein may have 1 or several additional amino acids added or deleted compared to the native protein. The "1 or more" means that the number varies depending on the total length, structure, etc. of the target protein of interest, and specifically, 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.

The target protein may be expressed as a protein to which a precursor structural moiety is added (precursor protein). When the target protein is expressed as a precursor protein, the target protein to be finally obtained may or may not be a precursor protein. That is, the precursor protein can be cleaved from the precursor structure to become a mature protein. In the case of cleavage, for example, cleavage can be performed by a protease. When a protease is used, the precursor protein is generally cleaved at substantially the same position as the native protein, and more preferably cleaved at substantially the same position as the native protein to obtain a mature protein identical to the native protein, from the viewpoint of the activity of the finally obtained protein. Thus, in general, a specific protease that cleaves a precursor protein at a position that produces the same protein as the naturally occurring mature protein is most preferred. However, as described above, the N-terminal region of the finally obtained target protein may be different from that of the natural protein. For example, a protein having an N-terminus 1 to several amino acids longer or shorter than that of a natural protein may have a more suitable activity depending on the type of target protein to be produced, the intended use, and the like. The protease to be used in the present invention includes a protease obtained from a culture solution of a microorganism, for example, a culture solution of actinomycetes, in addition to a commercially available protease such as Dispase (manufactured by BOEHRINGER MANNHEIM). Such a protease may be used in an unpurified state, or may be used after being purified to an appropriate purity as required.

The target protein gene can be obtained, for example, by cloning. For cloning, for example, a nucleic acid such as genomic DNA or cDNA containing a gene of a target protein can be used. Furthermore, the target protein Gene can be obtained, for example, by total synthesis based on the base sequence thereof (Gene,60(1),115-127 (1987)). The obtained target protein gene may be used as it is or after appropriate modification. That is, a target protein gene can be modified to obtain a variant thereof. Modification of the gene can be carried out by a known method. For example, a desired mutation can be introduced into a desired site of DNA by a site-specific mutation method. Examples of the site-specific mutation method include: methods using PCR (Higuchi, R.61, in PCR technology, Erlich, H.A.Eds., Stockton press (1989); Carter, P., meth.in enzymol.,154,382(1987)), methods using phages (Kramer, W.and Frits, H.J., meth.in enzymol.,154,350 (1987); Kunkel, T.A.et., meth.in enzymol.,154,367 (1987)). Alternatively, variants of the target protein gene may be synthesized entirely. Furthermore, the obtained target protein gene may be modified as appropriate by introduction of a promoter sequence, to obtain a gene construct for expression of the target protein. Other components (e.g., promoter sequences) of the gene construct for expressing the target protein and the gene construct for expressing the target protein can be obtained in the same manner as the target protein gene.

Modification of the gene can be carried out by a known method. For example, a desired mutation can be introduced into a desired site of DNA by a site-specific mutation method. Examples of the site-specific mutation method include: methods using PCR (Higuchi, R.61, in PCR technology, Erlich, H.A.Eds., Stockton press (1989); Carter, P., meth.in enzymol.,154,382(1987)), methods using phages (Kramer, W.and Frits, H.J., meth.in enzymol.,154,350 (1987); Kunkel, T.A.et., meth.in enzymol.,154,367 (1987)).

The method for introducing a gene construct for expressing a target protein into Talaromyces cellulolyticus is not particularly limited. The "introduction of a gene construct for expression of a target protein" is sufficient if the gene construct for expression of the target protein is held in a host, and specifically, the target protein gene is introduced into the host so as to be expressed. The "introduction of a gene construct for expression of a target protein" is not limited to the case of introducing a gene construct for expression of a target protein, which is constructed in advance, into a host together, unless otherwise specified, and also includes the case of introducing a part of a gene construct for expression of a target protein into a host and constructing a gene construct for expression of a target protein in the host. For example, a gene construct for expressing a target protein can be constructed on a chromosome by introducing the target protein gene downstream of a promoter originally possessed by the host.

The gene construct for expression of the target protein can be introduced into a host using, for example, a vector containing the gene construct for expression of the target protein. A vector containing a gene construct for expression of a target protein is also referred to as "expression vector for a target protein". The expression vector for the target protein can be constructed, for example, by linking a gene construct for expression of the target protein to the vector. In addition, for example, in the case where the vector includes a promoter, an expression vector for the target protein can be constructed by ligating the gene for the target protein to the downstream of the promoter. The host is transformed with an expression vector for the target protein to obtain a transformant into which the vector has been introduced, that is, a gene construct for expression of the target protein can be introduced into the host. The vector is not particularly limited as long as it can autonomously replicate in the host cell. The vector may be a 1-copy vector, a low-copy vector or a multi-copy vector. The vector may be provided with a marker gene for selecting transformants. The vector may also include a promoter and a terminator for expressing the target protein gene.

In addition, a gene construct for expression of the target protein may be introduced into the chromosome of the host. Introduction of a gene into a chromosome can be performed by homologous recombination. Specifically, the host is transformed with a recombinant DNA containing a gene construct for expression of the target protein, and homologous recombination occurs with a target site on the chromosome of the host, so that the gene construct for expression of the target protein can be introduced into the chromosome of the host. The structure of the recombinant DNA used in the homologous recombination is not particularly limited as long as the homologous recombination occurs in a desired manner. For example, a linear DNA comprising a gene construct for expression of a target protein may be used, and the gene construct for expression of a target protein may be substituted with the gene construct for expression of a target protein by transforming a host with linear DNAs each having sequences upstream and downstream of the site to be substituted on the chromosome at both ends of the gene construct for expression of a target protein and causing homologous recombination upstream and downstream of the site to be substituted. The recombinant DNA used for homologous recombination may have a marker gene for selecting transformants. The introduction of the target protein gene, promoter, etc. into a part of the chromosome of the gene construct for expression of the target protein may be performed in the same manner as the introduction into the chromosome of the entire gene construct for expression of the target protein.

The marker gene may be appropriately selected depending on the properties such as auxotrophy of the host. For example, when a host shows Uracil (Uracil) deficiency due to mutation of the pyrF gene or the pyrG gene, a strain into which a desired modification has been introduced can be selected using the pyrF gene or the pyrG gene as a marker gene and complementation of Uracil (Uracil) deficiency (i.e., non-Uracil (Uracil) deficiency) as an indicator. In addition, as the marker gene, a drug resistance gene such as a hygromycin resistance gene can be used.

Transformation can be carried out by a technique generally used for transformation of eukaryotic microorganisms such as molds and yeasts, for example. Such a method includes a protoplast method.

<1-3> reduction of Activity of YscB protein

The microorganism of the present invention is modified so that the activity of the YscB protein is decreased. Specifically, the microorganism of the present invention is modified so that the activity of the YscB protein is reduced as compared with a non-modified strain. More specifically, for example, the microorganism of the present invention may be modified so that the expression of the yscB gene is reduced or so that the yscB gene is disrupted. By modifying Talaromyces cellulolyticus so as to decrease the activity of the YscB protein, the target protein production ability of the microorganism can be improved, that is, the production of the target protein by the microorganism can be increased.

The YscB protein and the yscB gene encoding the same are described below.

The YscB protein is a protease. "protease" refers to a protein having an activity of catalyzing a reaction of hydrolyzing a protein. In addition, the activity is also referred to as "protease activity".

The base sequence of the yscB gene (containing an intron) of Talaromyces cellulolyticus S6-25 strain and the amino acid sequence of the YscB protein encoded by the gene are shown in SEQ ID Nos. 32 and 43, respectively. That is, the yscB gene may be, for example, a gene having the base sequence shown in SEQ ID NO. 32. Further, the YscB protein may be, for example, a protein having the amino acid sequence shown by SEQ ID NO. 43. The expression "having a (amino acid or base) sequence" includes the case of "including a (amino acid or base) sequence" and the case of "consisting of a (amino acid or base) sequence".

The yscB gene may be a variant of the above-mentioned exemplary yscB gene (for example, a gene having the base sequence shown in SEQ id No.32) as long as it retains its original function. Similarly, the YscB protein may be a variant of the exemplified YscB protein (for example, a protein having the amino acid sequence shown in SEQ ID NO. 43) as long as it retains the original function. Such variants that retain the original function are also sometimes referred to as "conservative variants". In the present invention, the term "yscB gene" is not limited to the exemplified yscB gene, but also encompasses conservative variants thereof. Likewise, the term "YscB protein" is not limited to the exemplified YscB proteins, but also encompasses conservative variants thereof. Examples of conservative variants include: the exemplified yscB gene, homologues of yscB protein, artificial modifications.

The phrase "retains the original function" means that the variant of the gene or protein has a function (activity, property) corresponding to the function (activity, property) of the original gene or protein. That is, "retains the original function" means that, in the case of the yscB gene, a variant of the gene encodes a protein that retains the original function. The phrase "retains the original function" means that, in the case of the YscB protein, the variant of the protein has a protease activity.

Protease activity can be measured by incubating the enzyme with a substrate (protein) (Incubate) and measuring the enzyme-dependent degradation of the substrate. The protease activity can be measured using a commercially available protease activity measurement kit.

Hereinafter, conservative variants will be exemplified.

Homologs of the yscB gene or homologs of the yscB protein can be easily obtained from public databases by, for example, BLAST search or FASTA search using the exemplified base sequence of the yscB gene or the exemplified amino acid sequence of the yscB protein as a query sequence. Furthermore, homologues of the yscB gene can be obtained, for example, by PCR using an oligonucleotide prepared based on the base sequence of the known yscB gene as a primer, using a chromosome of Talaromyces cellulolyticus as a template.

The yscB gene may be a gene encoding a protein having an amino acid sequence in which 1 or several amino acids at 1 or several positions are substituted, deleted, inserted and/or added in the amino acid sequence of the above-exemplified yscB protein (for example, the amino acid sequence shown in SEQ ID No. 43) as long as the gene retains its original function. The "1 or more" may vary depending on the position in the three-dimensional structure of the protein of the amino acid residue and the type of the amino acid residue, and specifically, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably 1 to 3.

The substitution, deletion, insertion and/or addition of 1 or several amino acids is a conservative variation that normally maintains the function of the protein. Representative conservative variations are conservative substitutions. Conservative substitution is a substitution between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid, between Gln and Asn when the substitution site is a polar amino acid, between Lys, Arg, and His when the substitution site is a basic amino acid, between Asp and Glu when the substitution site is an acidic amino acid, and between Ser and Thr when the substitution site is an amino acid having a hydroxyl group. Specific examples of the substitution considered to be conservative substitution include: substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, substitution from Asp to Asn, Glu or Gln, substitution from Cys to Ser or Ala, substitution from Gln to Asn, Glu, Lys, His, Asp or Arg, substitution from Glu to Gly, Asn, Gln, Lys or Asp, substitution from Gly to Pro, substitution from His to Asn, Lys, Gln, Arg or Tyr, substitution from Ile to Leu, Met, Val or Phe, substitution from Leu to Ile, Met, Val or Phe, substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, substitution from Ser to Thr or Ala, substitution from Thr to Ala, substitution from Trp to Phe, substitution from Tyr, Val to Phe or Phe, and substitution from Leu to Met, Ser to Phe, Ser, His or Phe. In addition, the substitution, deletion, insertion or addition of such amino acids includes a case where the amino acids are caused by naturally occurring variation (mutant or variant) based on the individual difference, species difference or the like of the gene-derived organism.

The yscB gene may be a gene encoding a protein having an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more homology to the entire amino acid sequence of the exemplary yscB protein (for example, the amino acid sequence shown in SEQ id No. 43) as long as the gene retains its original function. In the present specification, "homology" means "identity".

The yscB gene may be a DNA that hybridizes under stringent conditions to a complementary sequence of the nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID No.32) of the above-mentioned exemplary yscB gene or to a probe prepared from the complementary sequence, as long as the original function is maintained. "stringent conditions" means conditions under which so-called specific hybridization is formed and non-specific hybridization is not formed. If an example is shown, mention may be made of: the conditions under which DNAs having high homology hybridize with each other, for example, DNAs having homology of 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more hybridize with each other and DNAs having low homology do not hybridize with each other, or the conditions under which washing is performed 1 time, preferably 2 to 3 times at a salt concentration and temperature equivalent to 60 ℃,1 XSSC, 0.1% SDS, preferably 60 ℃, 0.1 XSSC, 0.1% SDS, more preferably 68 ℃, 0.1 XSSC, 0.1% SDS, which are conditions for ordinary Southern hybridization.

The probe may be, for example, a part of a complementary sequence of a gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on the base sequences of known genes as primers and DNA fragments containing these base sequences as templates. As the probe, for example, a DNA fragment of about 300bp in length can be used. In such a case, the conditions for washing the hybridization include: 50 ℃,2 × SSC, 0.1% SDS.

The yscB gene may be obtained by substituting an arbitrary codon with a codon equivalent thereto. That is, the yscB gene may be a variant of the exemplified yscB gene based on codon degeneracy.

The percentage of sequence homology between 2 sequences can be determined, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms include: myers and Miller (1988) CABIOS 4:11-17, Smith et al (1981) adv.Appl.Math.2:482, Needleman and Wunsch (1970) J.mol.biol.48: 443) the same alignment algorithm, a method to search for the similarity of Pearson and Lipman (1988) Proc.Natl.Acad.Sci.85: 2444-.

Sequence comparisons (calibrations) to determine sequence homology can be performed using programs based on these mathematical algorithms. The program may suitably be carried out by a computer. Such a procedure is not particularly limited, and examples thereof include: CLUSTAL (available from Intelligenetics, Mountain View, Cali f.), ALIGN program (Version2.0), and GAP, BESTFIT, BLAST, FASTA, and TFASTA, of Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG),575Science Drive, Madis on, Wis., USA). Calibration using these procedures can be performed using, for example, initial parameters. The CLUSTAL program is well described in Higgins et al (1988) Gene 73:237-244, Higgins et al (1989) CABIOS 5:151-153, Corpet et al (1988) Nucleic Acids Res.16:10881-90, Huang et al (1992) CABIOS8:155-65 and Pearson et al (1994) Metal.mol.biol.24: 307-331.

To obtain a nucleotide sequence having homology with the nucleotide sequence of the protein to be encoded, a BLAST nucleotide search can be performed, for example, by a BLAST program under a score of 100 and a word length of 12. To obtain an amino acid sequence having homology with the target protein, a BLAST protein search can be performed, for example, by a BLASTX program with a score of 50 and a word length of 3. For BLAST nucleotide searches, BLAST protein searches, see http:// www.ncbi.nlm.nih.gov. In addition, Gapped BLAST (BLAST 2.0) can be used to obtain alignment with gaps added for comparison. In addition, PSI-BLAST (BLAST 2.0) can be used to examine the segregation relationships between the sequenced columns and to perform repeated searches. For Gapped BLAST and PSI-BLAST, refer to Altschul et al (1997) nucleic acids Res.25: 3389. In the case of using BLAST, Gapp ed BLAST, or PSI-BLAST, for example, the initial parameters of each program (e.g., BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. The calibration may be performed manually.

The sequence homology among 2 sequences was calculated as the ratio of residues that were identical among 2 sequences when the 2 sequences were aligned so as to make the maximum agreement. The term "homology" between amino acid sequences means, specifically, unless otherwise specified, a homology between amino acid sequences calculated using "designing Parameters" (Matrix: BLOSUM 62; Gap Costs: Existence ═ 11, Extension ═ 1; composition Adjustments: composition score adjusted by blastp default). The term "homology" between base sequence sequences means, specifically, homology between base sequence sequences calculated using "cloning Parameters" (Match/Mismatch Scores ═ 1, -2; Gap Costs ═ Linear) set by default by blastn unless otherwise specified.

The description relating to the gene and the protein variant can also be applied to any protein such as a target protein and a gene encoding the protein.

<1-4> other Properties

The microorganism of the present invention may have other desired properties (e.g., modification) as long as the target protein-producing ability is not impaired. The modification may be a modification that increases the production ability of a target protein of Talaromyces cellulolyticus. Specific examples of the modification include a modification for reducing the activity of CreA protein. These properties and modifications may be used alone or in appropriate combinations.

That is, for example, the microorganism of the present invention may be modified such that the activity of CreA protein is decreased. Specifically, the microorganism of the present invention may be modified such that the activity of CreA protein is reduced as compared to a non-modified strain. More specifically, for example, the microorganism of the present invention may be modified such that the expression of the creA gene is reduced, or such that the creA gene is disrupted. The creA gene is a gene encoding a transcription factor involved in catabolite repression. The creA gene is known to be involved in cellulase expression in filamentous fungi (Mol Gen Genet.1996Jun 24; 251(4):451-60, Biosci Biotechnol biochem.1998Dec; 62(12): 2364-70).

The base sequence of the creA gene of Talaromyces cellulolyticus S6-25 strain is shown in SEQ ID NO. 44. That is, for example, the creA gene may be a gene having the base sequence shown by SEQ ID NO. 44. Further, for example, the creA protein may be a protein having an amino acid sequence encoded by the base sequence shown in SEQ ID NO. 44. The creA gene and creA protein may be conservative variants of the exemplary creA gene and creA protein, respectively. For conservative variants of the creA gene and the creA protein, the statements on conservative variants of the yscB gene and the YscB protein can be applied. In the case of CreA protein, the term "retains the original function" means that the protein variant has a function as a transcription factor involved in catabolite repression.

<1-5> method for reducing protein activity

Hereinafter, a method for reducing the activity of a protein such as YscB protein or CreA protein will be described.

"reduced activity of a protein" means that the activity of the protein is reduced as compared with that of a non-modified strain. Specifically, "activity of a protein is decreased" means that the activity of the protein is decreased in each cell as compared with a non-modified strain. The term "non-modified strain" as used herein means a control strain which has not been modified in such a manner that the activity of a target protein is reduced. Examples of the non-modified strain include a wild strain and a parent strain. Specific examples of the non-modified strain include those exemplified in the description of Talaromycescellulolyticus. That is, in one embodiment, the activity of the protein may be reduced as compared to the Talaromyces cellulolyticus S6-25 strain. The phrase "activity of a protein is decreased" also includes a case where the activity of the protein is completely lost. More specifically, "the activity of the protein is decreased" may mean that the number of molecules of the protein per cell is decreased, and/or the function of each molecule of the protein is decreased, as compared to a non-modified strain. That is, the "activity" in the case of "activity of protein is decreased" is not limited to the catalytic activity of protein, and may be a transcription amount (mRNA amount) or a translation amount (protein amount) of a gene encoding protein. The phrase "the number of molecules of the protein per cell is reduced" also includes the case where the protein is not present at all. The phrase "the function of each molecule of the protein is reduced" also includes a case where the function of each molecule of the protein is completely lost. The degree of reduction of the activity of the protein is not particularly limited if the activity of the protein is reduced as compared with that of a non-modified strain. The activity of the protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the activity of the unmodified strain.

Such modification to reduce the activity of a protein can be achieved, for example, by reducing the expression of a gene encoding the protein. By "reduced expression of a gene" is meant reduced expression of the gene as compared to a non-modified strain. The "expression of a gene is reduced" means, specifically, that the expression amount of the gene per cell is reduced as compared with a non-modified strain. The "expression of a gene is decreased" may mean, more specifically, that the amount of transcription (amount of mRNA) of the gene is decreased, and/or that the amount of translation (amount of protein) of the gene is decreased. "the expression of a gene is reduced" includes the case where the gene is not expressed at all. The "reduction in the expression of a gene" is also referred to as "attenuation of the expression of a gene". The expression of the gene may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

The reduction of the expression of the gene may be based on, for example, a reduction in transcription efficiency, a reduction in translation efficiency, or a combination thereof. The reduction of the expression of a gene can be achieved, for example, by modifying an expression regulatory sequence of the gene. The "expression control sequence" is a general term for a site that affects the expression of a gene such as a promoter. The expression control sequence can be determined, for example, using a gene analysis software such as a promoter search vector or GENETYX. When the expression control sequence is modified, the expression control sequence is preferably modified at 1 base or more, more preferably at 2 bases or more, and particularly preferably at 3 bases or more. The reduction in the transcription efficiency of a gene can be achieved, for example, by replacing the promoter of the gene on a chromosome with a weaker promoter. "weaker promoter" means a promoter in which transcription of a gene is weaker than the wild-type promoter originally present. Examples of the weaker promoter include inducible promoters. That is, an inducible promoter can function as a weaker promoter under non-inducible conditions (e.g., in the absence of an inducing substance). In addition, a region of a part or all of the expression regulatory sequence may be deleted (defective). In addition, the reduction of expression of a gene can be achieved, for example, by manipulating factors involved in expression control. Examples of factors involved in expression control include: low molecules (inducing substances, inhibiting substances, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.) involved in the control of transcription and translation. In addition, the reduction of the expression of a gene can be achieved by, for example, introducing a mutation that reduces the expression of a gene into a coding region of the gene. For example, the expression of a gene can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. In addition, for example, the expression of the gene itself can be reduced by disruption of the gene as described below.

Such modification to reduce the activity of the protein can be achieved, for example, by disrupting a gene encoding the protein. "disrupting a gene" means that the gene is modified so that normally functioning proteins are not produced. "not producing a protein that normally functions" includes a case where no protein is produced from the gene, and a case where a protein in which the function (activity, property) of each molecule is reduced or eliminated is produced from the gene.

Disruption of a gene can be achieved, for example, by deleting (defective) a gene on a chromosome. "deletion of a gene" refers to deletion of a part or all of a coding region of a gene. Further, the entire gene including sequences before and after the coding region of the gene on the chromosome may be deleted. The sequence around the coding region of the gene may include, for example, an expression control sequence of the gene. The deleted region may be any of the N-terminal region (region on the N-terminal side of the encoded protein), the internal region, the C-terminal region (region on the C-terminal side of the encoded protein), and the like, as long as the activity of the protein can be reduced. In general, when the deleted region is long, the gene can be reliably inactivated. The deleted region may be, for example, a region having a length of 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 length of the coding region of the gene. In addition, it is preferable that the sequences before and after the deleted region are not identical in reading frame. Frame shifting can occur downstream of the deleted region by virtue of the inconsistency in the reading frames. In the case of the creA gene, specifically, the gene can be disrupted by deleting a portion corresponding to positions 3262 to 4509 of SEQ ID NO.44, for example.

In addition, disruption of the gene can be achieved, for example, by introducing amino acid substitution (missense mutation), introduction of a stop codon (nonsense mutation), introduction of addition or deletion of 1 to 2 bases (frameshift variation), etc. into the coding region of the gene on the chromosome (Journal of Biological Chemistry 272:8611-8617(1997), Proceedings of the national Academy of Sciences, USA 955511-5515 (1998), Journal of Biological Chemistry 26116, 20833-20839 (1991)).

In addition, disruption of the gene can be achieved, for example, by inserting another base sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, and if the inserted nucleotide sequence is long, the gene can be surely inactivated. Furthermore, it is preferred that the sequences before and after the insertion site are not in reading frame. Frame shifting can occur downstream of the insertion site by virtue of the disparity in the reading frames. The other nucleotide sequence is not particularly limited as long as the activity of the encoded protein is reduced or eliminated, and examples thereof include a marker gene and a gene useful for producing a target protein.

In particular, disruption of the gene can be carried out such that the amino acid sequence of the encoded protein is deleted (defective). In other words, such modification to reduce the activity of the protein can be achieved by, for example, deleting the amino acid sequence of the protein, and specifically, by modifying a gene so as to encode the protein with the deleted amino acid sequence. The term "deletion of an amino acid sequence of a protein" refers to deletion of a part or all of a region of the amino acid sequence of the protein. The phrase "deletion of an amino acid sequence of a protein" means that the original amino acid sequence is not present in the protein, and includes a case where the original amino acid sequence is changed to another amino acid sequence. That is, for example, a region that is changed to another amino acid sequence by a frame shift can be considered to be a deleted region. The entire length of a protein is typically shortened by deletion of the amino acid sequence of the protein, but the entire length of the protein may be unchanged or extended. For example, a region encoded by a region in which a part or all of the coding region of a gene is deleted can be deleted in the amino acid sequence of the encoded protein. In addition, for example, by introducing a stop codon into a coding region of a gene, a region encoded in a region downstream of the introduction site can be deleted in the amino acid sequence of the encoded protein. In addition, for example, the region encoded by the frameshift site can be deleted by frameshifting in the coding region of the gene. For the position and length of the deleted region in the deletion of the amino acid sequence, the description of the position and length of the deleted region in the deletion of the gene can be applied.

In the case where the gene on the chromosome is modified as described above, this can be achieved, for example, by: a disrupted gene modified so that it does not produce a protein that normally functions is prepared, a host is transformed with a recombinant DNA comprising the disrupted gene, and homologous recombination occurs by disrupting the disrupted gene and a wild-type gene on a chromosome, thereby replacing the wild-type gene on the chromosome with the disrupted gene. In this case, the recombinant DNA can be easily manipulated if it contains a marker gene depending on the properties of the host such as auxotrophy. Examples of the disrupted gene include: a gene lacking a part or all of the coding region of the gene, a gene into which a missense mutation is introduced, a gene into which a nonsense mutation is introduced, a gene into which a frameshift mutation is introduced, and a gene into which an insertion sequence such as a transposon or a marker gene is introduced. Even when produced, the protein encoded by the disrupted gene has a different steric structure from the wild-type protein, and the function thereof is reduced or eliminated.

The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as homologous recombination occurs in a desired manner. For example, a linear DNA comprising an arbitrary sequence, a host is transformed with a linear DNA having sequences upstream and downstream of a site to be replaced on a chromosome at both ends of the arbitrary sequence, and homologous recombination is performed upstream and downstream of the site to be replaced, respectively, whereby the site to be replaced can be replaced with the arbitrary sequence in step 1. As the arbitrary sequence, for example, a sequence including a marker gene can be used.

The marker gene may be appropriately selected depending on the properties such as auxotrophy of the host. For example, when a host shows Uracil (Uracil) deficiency due to mutation of pyrF gene or pyrG gene, a strain to be introduced with a desired modification can be selected by using the pyrF gene or pyrG gene as a marker gene and using complementation of Uracil (Uracil) deficiency (i.e., non-Uracil (Uracil) deficiency) as an indicator. In addition, for example, when methionine deficiency is expressed by mutation of sC gene (sulfate perminase gene), a strain to be purposefully modified can be selected by using the sC gene as a marker gene to indicate the complementation of methionine deficiency (i.e., methionine non-deficiency). In addition, as the marker gene, a drug resistance gene such as a hygromycin resistance gene can be used.

Such modification for reducing the activity of the protein may be performed, for example, by mutation treatment. Examples of the mutation treatment include: x-ray irradiation, ultraviolet irradiation, and treatment with a mutagen such as N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), Ethyl Methanesulfonate (EMS), and Methyl Methanesulfonate (MMS).

The activity of the protein can be measured to confirm the decrease in the activity of the protein. The activity of the YscB protein can be determined, for example, in the manner described above. For example, the activity of CreA protein can be determined by measuring the degree of catabolite repression. For example, the degree of catabolite repression can be determined by measuring cellulase production under culture conditions comprising glucose as a carbon source. Specifically, for example, the decrease in CreA protein activity can be confirmed by using, as an index, an increase in cellulase production under culture conditions containing glucose as a carbon source.

A decrease in the activity of a protein can also be confirmed by confirming a decrease in the expression of a gene encoding the protein. The reduced expression of a gene can also be determined by: by confirming a decrease in the amount of transcription of the gene or by confirming a decrease in the amount of protein expressed from the gene.

The confirmation of the decrease in the amount of transcription of the gene can be performed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain. Examples of the method for evaluating the amount of mRNA include: northern hybridization, RT-PCR, etc. (molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA (e.g., the number of molecules per cell) can be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain. .

Confirmation of the decrease in the amount of protein can be performed by western blotting using an antibody (molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein (e.g., the number of molecules per cell) can be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Gene disruption can be confirmed by: the nucleotide sequence, restriction map, or full length of a part or all of the gene is determined by the method used for disruption.

Transformation can be carried out by a method generally used for transformation of eukaryotic microorganisms such as molds and yeasts. Such a method includes a protoplast method.

<2> method of the present invention

The target protein can be produced by using the microorganism of the present invention. Specifically, the target protein can be produced by culturing the microorganism of the present invention. Specifically, the method of the present invention may be a method for producing a target protein comprising culturing the microorganism of the present invention in a medium.

The medium to be used is not particularly limited as long as the microorganism of the present invention can proliferate and produce the target protein. As the medium, for example, a medium containing a component selected from a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, other various organic components and inorganic components as required can be used. Those skilled in the art can appropriately set the kind and concentration of the medium components. As the specific medium composition, for example, the medium composition described in the report on Talaromyces cellulolyticus (Japanese patent laid-open Nos. 2003-135052, 2008-271826, 2008-271927 and the like), the medium composition for other various cellulase-producing microorganisms such as Trichoderma reesei and the like can be referred to.

The carbon source is not particularly limited as long as it can be assimilated by the microorganism of the present invention to produce a target protein. Examples of the carbon source include: sugars, cellulose-based substrates. Specific examples of the saccharide include: glucose, fructose, galactose, xylose, arabinose, sucrose, lactose, cellobiose, molasses, starch hydrolysate, biomass hydrolysate. Specific examples of the cellulose-based substrate include: microcrystalline cellulose (Avicel), filter paper, waste paper, pulp, wood, Straw (richetraw), wheat Straw (Straw), rice hull, rice bran, wheat bran, bagasse, coffee grounds, tea grounds. The cellulose-based substrate may be used as a carbon source after being subjected to pretreatment such as hydrothermal decomposition treatment, acid treatment, alkali treatment, cooking, crushing, pulverization, or the like. As a suitable cellulose-based substrate that is commercially available, there may be mentioned SOLKA FLOC (International Fiber Corp, North Tonawanda, NY, U.S. A). As the carbon source, 1 kind of carbon source may be used, or 2 or more kinds of carbon sources may be used in combination.

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 soybean protein decomposition product, ammonia and urea. As the nitrogen source, can use 1 kind of nitrogen source, can also be combined with 2 or more than 2 kinds of nitrogen source.

Specific examples of the phosphoric acid source include: phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphoric acid polymers such as pyrophosphoric acid. As the phosphoric acid source, 1 kind of phosphoric acid source may be used, or 2 or more kinds of phosphoric acid sources may be used in combination.

Specific examples of the sulfur source include: inorganic sulfur compounds such as sulfates, thiosulfates, sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, 1 kind of sulfur source may be used, or 2 or more kinds of sulfur sources may be used in combination.

Specific examples of the other various organic components 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 amide, and vitamin B12; amino acids; nucleic acids; organic components including peptone, casamino acid, yeast extract, soybean protein decomposition product and the like. As other various organic components and inorganic components, can use 1 kind of components, also can be combined with 2 or more than 2 kinds of components.

The culture conditions are not particularly limited as long as the microorganism of the present invention can proliferate and produce the target protein. The culture can be performed under the usual conditions used for culturing microorganisms such as filamentous fungi. As specific culture conditions, for example, the culture conditions described in reports relating to Talaromyces cell ulolyyticus (Japanese patent laid-open publication No. 2003-135052, Japanese patent laid-open publication No. 2008-271826, Japanese patent laid-open publication No. 2008-271927, etc.), and the culture conditions for other various cellulase-producing microorganisms such as Trichoderma reesei can be referred to.

For the culture, for example, a liquid medium can be used and the culture can be carried out under aerobic conditions. The culture under aerobic conditions may be specifically carried out by aeration culture, shaking culture, agitation culture or a combination thereof. The culture temperature may be, for example, 15 to 43 ℃ and particularly about 30 ℃. The culture period may be, for example, 2 hours to 20 days. The culture may be performed by batch culture (b culture), Fed-batch culture (Fed-batch culture), continuous culture (continuous culture), or a combination thereof. The medium at the start of the culture is also referred to as "initial medium". Further, a medium supplied to a culture system (fermentation tank) in the fed-batch culture or continuous culture is also referred to as "fed-batch medium". Further, feeding a feeding medium to a culture system in a feeding culture or a continuous culture is also referred to as "feeding". The culture can be performed by dividing into a preculture and a main culture. For example, the preculture may be performed in a solid medium such as an agar medium, and the main culture may be performed in a liquid medium. The 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 is lost.

In the present invention, each medium component may be contained in the initial medium, the fed medium, or both. The kind of the components contained in the initial medium may be the same as or different from the kind of the components contained in the fed-batch medium. The concentrations of the respective components contained in the initial medium may be the same as or different from the concentrations of the respective components contained in the fed-batch medium. In addition, can use the containing component types and/or concentrations of different 2 or more than 2 kinds of fed-batch culture medium. For example, in the case of intermittently performing the feeding a plurality of times, the kinds and/or concentrations of the components contained in the fed-batch medium of each time may be the same or different.

The concentrations of the various constituents can be determined by gas chromatography (Hashimoto, K.et al 1996.biosci. Biotechnol. biochem.70:22-30), HPLC (Lin, J.T.et al 1998. J.Chromatog. A.808:43-49)

By culturing the microorganism of the present invention as described above, the target protein is expressed to obtain a culture containing the target protein. Specifically, the target protein may be accumulated in a medium, a cell surface layer, a cell body, or a combination thereof. The target protein can be accumulated in the medium.

The production of the target protein can be confirmed by a known method used for detection or identification of the protein. Examples of such a method include: SDS-PAGE, Western blotting, mass spectrometry, N-terminal amino acid sequence analysis, and enzyme activity assay. These methods may be used alone in 1 kind, or may be used in combination of 2 or more than 2 kinds.

The resulting target protein can be recovered as appropriate. That is, the method for producing a target protein of the present invention may comprise recovering the produced target protein. Specifically, the target protein can be recovered as an appropriate fraction containing the target protein. Examples of such a moiety include: culture, culture supernatant, cell treated product (disrupted product, lysate, extract (cell-free extract)). The cells can be provided as immobilized cells immobilized with a carrier such as acrylamide or carrageenan, for example.

In addition, the target protein can be isolated and purified to a desired degree. The target protein may be supplied in a free state, or may be supplied in a state of an immobilized enzyme immobilized on a solid phase such as a resin.

When the target protein is accumulated in the medium, the target protein may be separated and purified from the supernatant after removing solid components such as cells from the culture by centrifugation or the like, for example.

When the target protein is present in the cell body, the target protein may be separated and purified from the treated product after, for example, the cell body is subjected to a treatment such as disruption, dissolution or extraction. The cells can be collected from the culture by centrifugation or the like. The treatment such as cell disruption, lysis or extraction can be performed by a known method. Examples of such a method include: ultrasonication, Dyno-mill, bead disruption, French press disruption (French press disruption), and lysozyme treatment. These methods may be used alone in 1 kind, or may be used in combination of 2 or more than 2 kinds.

When the target protein accumulates on the surface layer of the cell body, the target protein may be solubilized, and then separated and purified from the solubilized product. The solubilization can be carried out by a known method. Examples of such a method include: increase in salt concentration, use of a surfactant. These methods may be used alone in 1 kind, or may be used in combination of 2 or more than 2 kinds.

Purification of the target protein (for example, purification from the supernatant, the treated product, or the soluble product as described above) can be carried out by a known method used for purification of proteins. Examples of such a method include: ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation. These methods may be used alone in 1 kind, or may be used in combination of 2 or more than 2 kinds.

In addition, other enzymes, for example, hemicellulases such as cellulase, xylanase, xylosidase (β -xylosidase), arabinofuranosidase (Arabi nonfuranosidase), and the like, may be produced together with the target protein in the culture. The target protein may be recovered as a mixture with these other enzymes, or may be recovered by separating from these other enzymes.

The recovered target protein may be appropriately formulated. The dosage form is not particularly limited, and can be appropriately set according to various conditions such as the use application of the target protein. Examples of the agent include: liquid, suspension, powder, tablet, pill, and capsule. In the formulation, for example, there may be used: pharmacologically acceptable additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, deodorizing agents, perfumes, diluents, and surfactants.