# US 9,751,917 B2 · Polynucleotide for cell surface layer expression

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- Number: US 9,751,917 B2
- Document ID: US9751917B2
- Status: Granted
- Kind: B2
- Application number: 14/778,250
- Filed: 2014-03-25
- Published: 2016-11-10
- Granted: 2017-09-05
- Earliest claimed: 2013-03-25
- Assignees: National University Corporation Kobe University
- Applicants: National University Corporation Kobe University
- Inventors: Akihiko Kondo; Tomohisa Hasunuma; Kentaro Inokuma
- Web page: https://patentgrep.com/patent/US9751917B2

## CPC

- C07K 14/39: from yeasts
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12P 7/10: substrate containing cellulosic material
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C07K 2319/035: containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

Disclosed is a polynucleotide for cell surface expression. The polynucleotide of the invention comprises a promoter, a secretion signal sequence, a sequence encoding an intended protein, and a sequence encoding a cell surface-localized protein or a cell membrane-binding domain thereof, wherein the promoter is a promoter of a gene encoding the cell surface-localized protein. Provided is a polynucleotide for cell surface expression allowing the production of yeast displaying an enzyme on the cell surface with a high activity.

## Claims

### Claim 1

A polynucleotide for cell surface expression, comprising various heterologous elements: a) an SED1 promoter of SEQ ID NO: 5, b) a sequence encoding a secretion signal peptide of Rhizopus oryzae-derived glucoamylase or Saccharomyces cerevisiae α factor, c) a sequence encoding an intended protein, and d) a sequence encoding a cell surface-localized protein SED1 of SEQ ID NO: 2 or a cell membrane-binding domain thereof including positions 110 to 338 of SEQ ID NO: 2, said intended protein being a protein selected from the group consisting of enzyme, antibody, ligand and fluorescent protein.

### Claim 2 (depends on 1)

An expression vector, comprising the polynucleotide for cell surface expression of claim 1.

### Claim 3 (depends on 1)

A recombinant yeast into which the polynucleotide for cell surface expression of claim 1 has been introduced.

### Claim 4 (depends on 3)

The recombinant yeast of claim 3, obtained from a host yeast in which at least one selected from the group consisting of SED1 and SSD1 is deficient.

### Claim 5 (depends on 4)

The recombinant yeast of claim 4, obtained from a host yeast in which SED1 and SSD1 are deficient.

### Claim 6 (depends on 3)

The recombinant yeast of claim 3, displaying at least one enzyme selected from the group consisting of cellulose-degrading enzymes and starch-degrading enzymes on the cell surface.

### Claim 7 (depends on 6)

A method for producing ethanol, comprising:
performing fermentation culture of the recombinant yeast of claim 6.

### Claim 8 (depends on 2)

A recombinant yeast into which the expression vector of claim 2 has been introduced.

### Claim 9 (depends on 8)

The recombinant yeast of claim 8, obtained from a host yeast in which at least one selected from the group consisting of SED1 and SSD1 is deficient.

### Claim 10 (depends on 9)

The recombinant yeast of claim 9, obtained from a host yeast in which SED1 and SSD1 are deficient.

### Claim 11 (depends on 8)

The recombinant yeast of claim 8, displaying at least one enzyme selected from the group consisting of cellulose-degrading enzymes and starch-degrading enzymes on the cell surface.