Mutant strain of filamentous fungus and use therefor

Patent metadata

Field	Value
Publication number	US-10844100-B2
Application number	US-201716321101-A
Country	US
Kind code	B2
Filing date	Aug 2, 2017
Priority date	Aug 2, 2016
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

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.

First claim

The invention claimed is:

1. A method of producing a protein, the method comprising culturing a filamentous fungus mutant strain under conditions in which the protein is produced, wherein, in the mutant strain, a function of tubulin is reduced or lost, wherein the function of tubulin that is reduced or lost is (i) expression of α-tubulin or β-tubulin or both, and (ii) the formation of α-tubulin and β-tubulin heterodimers, as compared to that in the mutant's parent strain, and
collecting the protein from a culture product of the culturing.

2. The method according to claim 1 , wherein the expression of α-tubulin is reduced or lost.

3. The method according to claim 1 , wherein the expression of β-tubulin is reduced or lost.

4. The method according to claim 1 , wherein the reduced or lost function of tubulin is the result of deleting or inactivating a gene encoding α-tubulin.

5. The method according to claim 1 , wherein the reduced or lost function of tubulin is the result of deleting or inactivating a gene encoding β-tubulin.

6. The method according to claim 4 , wherein the gene encoding α-tubulin comprises any of the following polynucleotides (a) to (f):
(a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 3;
(b) a polynucleotide having a nucleotide sequence with an identity of 80% or more with the nucleotide sequence of SEQ ID NO: 1 or 3 and encoding a protein that forms microtubules with β-tubulin in the mutant's parent strain;
(c) a polynucleotide that hybridizes to a complementary strand of the polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 3 under stringent conditions that require the polynucleotide that hybridizes to the complementary strand have 80% or more identity to SEQ ID NO: 1 or 3, and wherein the polynucleotide encodes a protein that forms microtubules with β-tubulin in the mutant's parent strain;
(d) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 2 or 4;
(e) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 2 or 4 in which one or several amino acids are deleted, substituted, added or inserted and that forms microtubules with β-tubulin in the mutant's parent strain; and
(f) a polynucleotide encoding a protein having an amino acid sequence with an identity of 80% or more with the amino acid sequence of SEQ ID NO: 2 or 4 and that forms microtubules with β-tubulin in the mutant's parent strain.

7. The method according to claim 5 , wherein the gene encoding β-tubulin is comprises any of the following polynucleotides (g) to (l):
(g) a polynucleotide having the nucleotide sequence of 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 of SEQ ID NO: 5, 7, 9, 11, or 13 and encoding a protein that forms microtubules with α-tubulin in the mutant's parent strain;
(i) a polynucleotide that hybridizes to a complementary strand of the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, 7, 9, 11, or 13 under stringent conditions that require the polynucleotide that hybridizes to the complementary strand have 80% or more identity to SEQ ID NO: 5, 7, 9, 11 or 13, and wherein the polynucleotide encodes a protein that forms microtubules with α-tubulin in the mutant's parent strain;
(j) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, or 14;
(k) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, or 14 in which one or several amino acids are deleted, substituted, added or inserted and that forms microtubules with α-tubulin in the mutant's parent strain; and
(l) a polynucleotide encoding a protein having an amino acid sequence with an identity of 80% or more with the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, or 14 and that forms microtubules with α-tubulin in the mutant's parent strain.

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

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

10. 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.

11. The method according to claim 10 , wherein the protein is cellulase and/or xylanase.

12. The method according to claim 11 , 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.

13. The method of claim 4 , wherein the protein encoded by the α-tubulin gene has the amino acid sequence of SEQ ID NO:2.

14. The method of claim 5 , wherein the protein encoded by the β-tubulin gene has the amino acid sequence of SEQ ID NO: 6.

Assignees

• Kao Corp

Inventors

• Shibata Nozomu
• Ogasawara Wataru
• SHIDA Yosuke

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12R2001/885

  Trichoderma · CPC title

• C12R2001/645

  Fungi {; Processes using fungi} · CPC title

• C12N1/145

  Fungi isolates · CPC title

• C12N15/67

  General methods for enhancing the expression · CPC title