# US 8,785,159 B2 · Extracellular secretion of recombinant proteins

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- Number: US 8,785,159 B2
- Document ID: US8785159B2
- Status: Granted
- Kind: B2
- Application number: 12/950,442
- Filed: 2010-11-19
- Published: 2011-05-26
- Granted: 2014-07-22
- Earliest claimed: 2009-11-25
- Assignees: Alliance for Sustainable Energy, LLC
- Applicants: Jeffrey G. Linger; Aldis Darzins
- Inventors: Jeffrey G. Linger; Aldis Darzins
- Web page: https://patentgrep.com/patent/US8785159B2

## CPC

- C07K 14/195: from bacteria
- C12N 1/22: Processes using, or culture media containing, cellulose or hydrolysates thereof
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- 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}
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C07K 2319/034: containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
- C07K 2319/036: targeting to the medium outside of the cell, e.g. type III secretion
- 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

Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

## Claims

### Claim 1

A recombinant vector comprising an isolated nucleic acid molecule encoding a polypeptide that functions as a secretion signal in Zymomonas mobilis, wherein the polypeptide has an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:2.

### Claim 2 (depends on 1)

The recombinant vector of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO:2.

### Claim 3 (depends on 1)

The recombinant vector of claim 1, wherein the nucleic acid molecule has the nucleotide sequence of SEQ ID NO:1.

### Claim 4 (depends on 1)

The recombinant vector of claim 1, further comprising a nucleic acid sequence encoding a polypeptide with cellulase activity.

### Claim 5 (depends on 4)

The recombinant vector of claim 4, wherein the polypeptide with cellulase activity is an endoglucanase.

### Claim 6 (depends on 4)

The recombinant vector of claim 4, wherein the polypeptide with cellulase activity is endoglucanase E1 or endoglucanase GH12 from Acidothermus cellulolyticus.

### Claim 7 (depends on 4)

The recombinant vector of claim 4, wherein the nucleic acid molecule has the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:9, or wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:10.

### Claim 8 (depends on 1)

The recombinant vector of claim 1, wherein the recombinant vector is an expression vector.

### Claim 9 (depends on 1)

An isolated host cell comprising the recombinant vector of claim 1.

### Claim 10 (depends on 9)

The host cell of claim 9, wherein the cell is a microbial cell.

### Claim 11 (depends on 10)

The host cell of claim 10, wherein the cell is a Zymomonas cell.

### Claim 12 (depends on 8)

A method for producing a recombinant polypeptide, comprising:
a) culturing an isolated host cell comprising the expression vector of claim 8, wherein the host cell expresses a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:2; and
b) isolating the recombinant polypeptide.

### Claim 13 (depends on 12)

The method of claim 12, wherein the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO:2 fused to a protein with cellulase activity.

### Claim 14 (depends on 12)

The method of claim 12, wherein the host cell is a bacterial cell and wherein the recombinant polypeptide is secreted from the host cell and isolated from the cell culture.

### Claim 15 (depends on 9)

A method for degrading lignocellulosic biomass, comprising culturing the host cell of claim 9 with the lignocellulosic biomass.

### Claim 16 (depends on 12)

The method of claim 12, wherein the host cell is a Zymomonas cell.

### Claim 17 (depends on 12)

The method of claim 12, wherein the host cell is a Zymomonas mobilis cell.

### Claim 18 (depends on 1)

The recombinant vector of claim 1, wherein the polypeptide has an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:2.