# US 2019/0169238 A1 · Improved Filamentous Fungal Host

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0169238 A1
- Document ID: US20190169238A1
- Status: Published application
- Kind: A1
- Application number: 16/319,780
- Filed: 2017-07-19
- Published: 2019-06-06
- Earliest claimed: 2016-07-22
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Hiroshi Teramoto; Kaihei Kojima; Preethi Ramaiya
- Web page: https://patentgrep.com/patent/US20190169238A1

## CPC

- C07K 14/38: from Aspergillus
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 15/80: for fungi
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Y 302/01003: Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
- 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)}
- C12Y: ENZYMES

## Abstract

The instant invention relates to filamentous fungal host cells producing and secreting a heterologous polypeptide of interest, said mutant host cell comprising and expressing a mutated ireA gene or a homologue thereof encoding a modified IreA polypeptide or a homologue thereof, said modified IreA polypeptide or homologue thereof comprising amino acid substitutions in 5 positions corresponding to positions 81 and 84 in the Aspergillus niger IreA amino acid sequence shown in SEQ ID NO:16, as well as methods of producing a polypeptide of interest in said cells and methods for constructing said cells.

## Claims

### Claim 1

A filamentous fungal host cell producing and secreting a heterologous polypeptide of interest, said mutant host cell comprising and expressing a mutated ireA gene or a homologue thereof encoding a modified IreA polypeptide or a homologue thereof, said modified IreA polypeptide or homologue thereof comprising amino acid substitutions in positions corresponding to positions 81 and 84 in the Aspergillus niger IreA amino acid sequence shown in SEQ ID NO:16.

### Claim 2 (depends on 1)

The host cell of claim 1 which is of a genus selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma.

### Claim 3 (depends on 2)

The host cell of claim 2 which is an Aspergillus cell; preferably an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or an Aspergillus oryzae cell.

### Claim 4 (depends on 1)

The host cell of claim 1, wherein the secreted polypeptide of interest is an enzyme; preferably the enzyme is a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, or beta-xylosidase.

### Claim 5 (depends on 1)

The host cell of claim 1, wherein the nucleotide sequence of the mutated ireA gene or homologue thereof is at least 70% identical to the Aspergillus niger variant ireA nucleotide sequence shown in SEQ ID NO:17 or to its cDNA nucleotide sequence shown in SEQ ID NO:18.

### Claim 6 (depends on 1)

The host cell of claim 1, wherein the nucleotide sequence of the mutated ireA gene or homologue thereof encodes a variant IreA polypeptide or homologue thereof which comprises amino acid substitutions of alanine residues in positions corresponding to positions 81 and 84 in the Aspergillus niger IreA amino acid sequence shown in SEQ ID NO:16; preferably the alanine residues are substituted with threonine residues.

### Claim 7 (depends on 1)

The host cell of claim 1, wherein the modified IreA polypeptide or homologue thereof comprises amino acid substitutions of alanine residues in positions corresponding to positions 81 and 84 in the Aspergillus niger IreA amino acid sequence shown in SEQ ID NO:16; preferably the alanine residues are substituted with threonine residues.

### Claim 8 (depends on 1)

The host cell of claim 1, wherein the modified IreA polypeptide comprises an amino acid sequence at least 70% identical to SEQ ID NO:19.

### Claim 9 (depends on 1)

A method of producing a polypeptide of interest, said method comprising the steps of:
a) cultivating the filamentous fungal host cell of claim 1 under conditions suitable for the production and secretion of the polypeptide; and, optionally
b) recovering the polypeptide of interest.

### Claim 10

A method of improving the productivity or yield of a secreted polypeptide of interest in a filamentous fungal host cell, said method comprising the steps of:
a) providing a filamentous fungal host cell comprising and expressing an ireA gene or a homologue thereof encoding a IreA polypeptide or a homologue thereof; and
b) mutating the ireA gene or homologue thereof to provide a mutated ireA gene or homologue thereof that encodes a modified IreA polypeptide or a homologue thereof, said modified IreA polypeptide or homologue thereof comprising amino acid substitutions in positions corresponding to positions 81 and 84 in the Aspergillus niger IreA amino acid sequence shown in SEQ ID NO:16.

### Claim 11 (depends on 10)

The method of claim 10, wherein the filamentous fungal host cell is of a genus selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma.

### Claim 12 (depends on 11)

The method of claim 11, wherein the filamentous fungal host cell is an Aspergillus cell; preferably an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or an Aspergillus oryzae cell.

### Claim 13 (depends on 10)

The method of claim 10, wherein the secreted polypeptide of interest is an enzyme; preferably the enzyme is a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, or beta-xylosidase.

### Claim 14 (depends on 10)

The method of claim 10, wherein the nucleotide sequence of the mutated ireA gene or homologue thereof is at least 70% identical to the Aspergillus niger variant ireA nucleotide sequence shown in SEQ ID NO:17 or to its cDNA nucleotide sequence shown in SEQ ID NO:18.

### Claim 15 (depends on 10)

The method of claim 10, wherein the nucleotide sequence of the mutated ireA gene or homologue thereof encodes a variant IreA polypeptide or homologue thereof which comprises amino acid substitutions of alanine residues in positions corresponding to positions 81 and 84 in the Aspergillus niger IreA amino acid sequence shown in SEQ ID NO:16; preferably the alanine residues are substituted with threonine residues.

### Claim 16 (depends on 10)

The method of claim 10, wherein the modified IreA polypeptide or homologue thereof comprises amino acid substitutions of alanine residues in positions corresponding to positions 81 and 84 in the Aspergillus niger IreA amino acid sequence shown in SEQ ID NO:16; preferably the alanine residues are substituted with threonine residues.

### Claim 17 (depends on 10)

The method of claim 10, wherein the modified IreA polypeptide comprises an amino acid sequence at least 70% identical to SEQ ID NO:19.