# US 9,006,409 B2 · Methods of increasing secretion of polypeptides having biological activity

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- Number: US 9,006,409 B2
- Document ID: US9006409B2
- Status: Granted
- Kind: B2
- Application number: 14/524,793
- Filed: 2014-10-27
- Published: 2015-02-12
- Granted: 2015-04-14
- Earliest claimed: 2004-05-16
- Assignees: Novozymes, Inc.
- Applicants: Novozymes, Inc.
- Inventors: Sandra Merino
- Web page: https://patentgrep.com/patent/US9006409B2

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C07K 1/00: General methods for the preparation of peptides {, i.e. processes for the organic chemical preparation of peptides or proteins of any length}
- C12N 9/2434: {acting on beta-1,4-glucosidic bonds}
- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12N 15/80: for fungi
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- 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 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C07K 2319/00: Fusion polypeptide
- C07K 2319/036: targeting to the medium outside of the cell, e.g. type III secretion
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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 present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

## Claims

### Claim 1

A polynucleotide encoding a fusion protein comprising:
(a) a first amino acid sequence comprising a signal peptide;
(b) a second polynucleotide comprising a nucleotide sequence encoding at least a catalytic domain of a Cel45 endoglucanase or a portion thereof; and
(c) a third amino acid sequence comprising at least a catalytic domain of a polypeptide having biological activity or a portion thereof; wherein said fusion protein comprises SEQ ID NO: 74 or SEQ ID NO: 76.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the fusion protein comprises a carbohydrate binding module.

### Claim 3 (depends on 1)

The polynucleotide of claim 1, wherein the fusion protein comprises a linker located between the endoglucanase catalytic domain and the catalytic domain of the polypeptide having biological activity and/or the catalytic domain of a polypeptide having biological activity.

### Claim 4 (depends on 1)

The polynucleotide of claim 1, wherein the fusion protein comprises a fourth amino acid sequence comprising a second signal peptide.

### Claim 5 (depends on 4)

The polynucleotide of claim 4, wherein the second signal peptide is linked in frame to the N-terminus of the amino acid sequence comprising the catalytic domain of the polypeptide having biological activity or at least the catalytic domain of the endoglucanase.

### Claim 6 (depends on 1)

The polynucleotide of claim 1, comprising SEQ ID NO: 73 or SEQ ID NO: 75.

### Claim 7 (depends on 1)

The polynucleotide of claim 1, wherein the fusion protein has cellulolytic activity and/or hemicellulolytic activity.

### Claim 8 (depends on 1)

A fusion protein construct, comprising the polynucleotide of claim 1 and a promoter.

### Claim 9 (depends on 8)

The fusion protein construct of claim 8, wherein the promoter drives expression of the first, second, and third polynucleotides.

### Claim 10 (depends on 9)

The fusion protein construct of claim 9, further comprising a terminator.

### Claim 11 (depends on 10)

An expression vector, comprising the fusion protein construct of claim 10.

### Claim 12 (depends on 1)

A host cell transformed with the polynucleotide of claim 1.

### Claim 13 (depends on 12)

The host cell of claim 12, wherein the cell is a filamentous fungal host cell.

### Claim 14 (depends on 11)

A host cell transformed with the expression vector of claim 11.

### Claim 15 (depends on 14)

The host cell of claim 14, wherein the cell is a filamentous fungal host cell.

### Claim 16

An isolated polynucleotide encoding a fusion protein, wherein said fusion protein comprises SEQ ID NO: 74 or SEQ ID NO: 76.

### Claim 17 (depends on 16)

The isolated polynucleotide of claim 16, which further comprises a carbohydrate binding module.

### Claim 18 (depends on 16)

The isolated polynucleotide of claim 16, comprising SEQ ID NO: 73 or SEQ ID NO: 75.

### Claim 19 (depends on 16)

The isolated polynucleotide of claim 16, wherein the fusion protein has cellulolytic activity and/or hemicellulolytic activity.