# US 2019/0169239 A1 · Mutant Strain of Filamentous Fungus and Use Therefor

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- Number: US 2019/0169239 A1
- Document ID: US20190169239A1
- Status: Published application
- Kind: A1
- Application number: 16/321,101
- Filed: 2017-08-02
- Published: 2019-06-06
- Earliest claimed: 2016-08-02
- Assignees: Kao Corporation; National University Corporation Nagaoka University of Technology
- Applicants: Kao Corporation; National University Corporation Nagaoka University of Technology
- Inventors: Nozomu SHIBATA; Wataru OGASAWARA; Yosuke SHIDA
- Web page: https://patentgrep.com/patent/US20190169239A1

## CPC

- C07K 14/38: from Aspergillus
- C07K 14/37: from fungi
- C12N 15/09: Recombinant DNA-technology
- C12N 15/67: General methods for enhancing the expression
- C12N 15/80: for fungi
- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N 1/145: Fungi isolates
- C12R 2001/645: Fungi {; Processes using fungi}
- C12R 2001/885: Trichoderma
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12Y 302/01008: Endo-1,4-beta-xylanase (3.2.1.8)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12Y: ENZYMES

## Abstract

A filamentous fungus mutant strain showing improved secretory protein production and a method of producing a protein using the filamentous fungus are provided. The method of producing a protein comprises a step of culturing a filamentous fungus mutant strain in which a function of tubulin is reduced or lost and collecting a protein from a culture product.

## Claims

### Claim 1

A method of producing a protein, the method comprising a step of culturing a filamentous fungus mutant strain in which a function of tubulin is reduced or lost and collecting a protein from a culture product.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the reduction or loss of the function of tubulin is a reduction or loss of expression of tubulin compared to that in a parent strain.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the tubulin is α-tubulin and/or β-tubulin.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the reduction or loss of the function of tubulin is acquired by deleting or inactivating a gene encoding the tubulin.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the gene encoding the tubulin is a gene encoding α-tubulin and/or β-tubulin.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the gene encoding α-tubulin is represented by any of the following polynucleotides (a) to (f):
(a) a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1 or 3;
(b) a polynucleotide having a nucleotide sequence with an identity of 80% or more with the nucleotide sequence represented by SEQ ID NO: 1 or 3 and encoding a protein having a function as α-tubulin;
(c) a polynucleotide hybridizing to a complementary strand of the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and encoding a protein having a function as α-tubulin;
(d) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 or 4;
(e) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 or 4 in which one or several amino acids are deleted, substituted, added or inserted and having a function as α-tubulin; and
(f) a polynucleotide encoding a protein having an amino acid sequence with an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 2 or 4 and having a function as α-tubulin.

### Claim 7 (depends on 5)

The method according to claim 5, wherein the gene encoding β-tubulin is represented by any of the following polynucleotides (g) to (l):
(g) a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 5, 7, 9, 11, or 13;
(h) a polynucleotide having a nucleotide sequence with an identity of 80% or more with the nucleotide sequence represented by SEQ ID NO: 5, 7, 9, 11, or 13 and encoding a protein having a function as β-tubulin;
(i) a polynucleotide hybridizing to a complementary strand of the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 5, 7, 9, 11, or 13 under stringent conditions and encoding a protein having a function as β-tubulin;
(j) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, or 14;
(k) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, or 14 in which one or several amino acids are deleted, substituted, added or inserted and having a function as β-tubulin; and
(l) a polynucleotide encoding a protein having an amino acid sequence with an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, or 14 and having a function as β-tubulin.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the filamentous fungus is a filamentous fungus belonging to Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Myceliophthora, Neurospora, Penicillium, Piromyces, Talaromyces, Thermoascus, Thielavia, or Trichoderma.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the filamentous fungus is a Trichoderma filamentous fungus.

### Claim 10

(canceled)

### Claim 11 (depends on 1)

The method according to claim 1, wherein the protein is one or more selected from the group consisting of cellulase, xylanase, protease, lipase, exoglucanase, endoglucanase, β-glucosidase, mannase, arabinase, arabinofuranosidase, galactase, and amylase.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the protein is cellulase and/or xylanase.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the filamentous fungus mutant strain is cultured in the presence of a cellulase inducer selected from the group consisting of cellulose, sophorose, and a cellooligosaccharide, and cellulase or xylanase is collected from a culture product.

### Claim at position 14

-15. (canceled)

### Claim 16

A mutant strain of a Trichoderma filamentous fungus wherein a function of tubulin is reduced or lost, wherein the secretory protein productivity is improved compared to that of a parent fungal strain.

### Claim 17 (depends on 16)

The mutant strain according to claim 16, wherein the reduction or loss of the function of tubulin is a reduction or loss of expression of tubulin compared to that in a parent strain.

### Claim 18 (depends on 16)

The mutant strain according to claim 16, wherein the tubulin is α-tubulin and/or β-tubulin.

### Claim 19 (depends on 16)

The mutant strain according to claim 16, wherein the reduction or loss of the function of tubulin is acquired by deleting or inactivating a gene encoding the tubulin.

### Claim 20 (depends on 19)

The mutant strain according to claim 19, wherein the gene encoding the tubulin is a gene encoding α-tubulin and/or β-tubulin.

### Claim 21 (depends on 20)

The mutant strain according to claim 20, wherein the gene encoding α-tubulin is represented by any of the following polynucleotides (a) to (0:
(a) a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1 or 3;
(b) a polynucleotide having a nucleotide sequence with an identity of 80% or more with the nucleotide sequence represented by SEQ ID NO: 1 or 3 and encoding a protein having a function as α-tubulin;
(c) a polynucleotide hybridizing to a complementary strand of the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and encoding a protein having a function as α-tubulin;
(d) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 or 4;
(e) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 or 4 in which one or several amino acids are deleted, substituted, added or inserted and having a function as α-tubulin; and
(f) a polynucleotide encoding a protein having an amino acid sequence with an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 2 or 4 and having a function as α-tubulin.

### Claim 22 (depends on 20)

The mutant strain according to claim 20, wherein the gene encoding β-tubulin is represented by the following polynucleotides (g) to (l):
(g) a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 5, 7, 9, 11, or 13;
(h) a polynucleotide having a nucleotide sequence with an identity of 80% or more with the nucleotide sequence represented by SEQ ID NO: 5, 7, 9, 11, or 13 and encoding a protein having a function as β-tubulin;
(i) a polynucleotide hybridizing to a complementary strand of the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 5, 7, 9, 11, or 13 under stringent conditions and encoding a protein having a function as β-tubulin;
(j) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, or 14;
(k) a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, or 14 in which one or several amino acids are deleted, substituted, added or inserted and having a function as β-tubulin; and
(l) a polynucleotide encoding a protein having an amino acid sequence with an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, or 14 and having a function as β-tubulin.

### Claim 23 (depends on 16)

The mutant strain according to claim 16, wherein the filamentous fungus is Trichoderma reesei.

### Claim at position 23

-25. (canceled)