# US 8,470,592 B2 · Isolation and characterization of Schizochytrium aggregatum cellobiohydrolase I (Cbh 1)

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- Number: US 8,470,592 B2
- Document ID: US8470592B2
- Status: Granted
- Kind: B2
- Application number: 13/003,199
- Filed: 2009-07-07
- Published: 2011-12-22
- Granted: 2013-06-25
- Earliest claimed: 2008-07-07
- Assignees: Mascoma Corporation
- Applicants: Elena E. Brevnova; Jim Flatt; Chhayal Gandhi; Vineet Rajgarhia; John McBride; Anne Warner
- Inventors: Elena E. Brevnova; Jim Flatt; Chhayal Gandhi; Vineet Rajgarhia; John McBride; Anne Warner
- Web page: https://patentgrep.com/patent/US8470592B2

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12Y 302/01091: Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention provides for the isolation and characterization of the cbh1 gene from Schizochytrium aggregatum. In particular, the present invention provides for the nucleic acid and amino acid sequences of Schizochytrium aggregatum cbh1, and domains, variants and derivatives thereof. The present invention further provides for the heterologous expression of Schizochytrium aggregatum Cbh1 in host cells, including yeast, e.g., Saccharomyces cerevisiae. Expression of Schizochytrium aggregatum Cbh1 in host cells will augment cellulose digestion and facilitate ethanol production by those host cells on cellulosic substrates. In certain embodiments, heterologous expression in Saccharomyces cerevisiae is in coordination with heterologous expression of other known, or newly identified saccharolytic enzymes. Therefore, the present invention also provides that the novel Schizochytrium aggregatum Cbh1 gene can utilized in a consolidated bioprocessing system.

## Claims

### Claim 1

An isolated polypeptide comprising an amino acid sequence having at least 95% identity to amino acids 20 to 456 of SEQ ID NO:3, wherein said polypeptide has cellobiohydrolase activity.

### Claim 2 (depends on 1)

The polypeptide of claim 1, said polypeptide comprising amino acids 20 to 456 of SEQ ID NO: 3.

### Claim 3 (depends on 1)

The polypeptide of claim 1, said polypeptide comprising amino acids 20-546 of SEQ ID NO: 3.

### Claim 4

An isolated polypeptide comprising an amino acid sequence having at least 95% identity to amino acids 457 to 546 of SEQ ID NO: 3 or to amino acids 507 to 546 of SEQ ID NO:3, wherein said polypeptide has cellulose binding activity.

### Claim 5 (depends on 4)

The polypeptide of claim 4, said polypeptide comprising amino acids 507 to 546 of SEQ ID NO: 3.

### Claim 6 (depends on 4)

The polypeptide of claim 4, said polypeptide comprising amino acids 457 to 546 of SEQ ID NO: 3.

### Claim 7

An isolated polypeptide comprising at least 50 contiguous amino acids within amino acids 20 to 456 of SEQ ID NO:3, wherein said polypeptide has cellobiohydrolase activity.

### Claim 8 (depends on 7)

A host cell comprising the polypeptide of claim 7.

### Claim 9 (depends on 8)

The host cell of claim 8, wherein the host cell is of a yeast organism.

### Claim 10 (depends on 8)

The host cell of claim 8, wherein the host cell is a thermophilic or mesophilic bacterial organism.

### Claim 11 (depends on 8)

The host cell of claim 8, said host cell further comprising at least one or more heterologously expressed endoglucanase polypeptides and/or at least one or more heterologously expressed β-glucosidase polypeptides and/or at least one or more heterologously expressed exoglucanase polypeptides.

### Claim 12 (depends on 11)

The host cell of claim 11, wherein said endoglucanase polypeptide is selected from the group consisting of Eg1, Eg2, Eg3, Eg4 and Eg5.

### Claim 13 (depends on 11)

The host cell of claim 11, wherein said μ-glucosidase polypeptide is selected from the group consisting of S. fibuligera Bgl1 and Bgl2.

### Claim 14 (depends on 11)

The host cell of claim 11, wherein said exogluconase polypeptide is selected from the group consisting of T. emersonii cellobiohydrolase I (Cbh1), T. reesei cellobiohydrolase I (Cbh1), T. reesei cellobiohydrolase II (Cbh2), Chaetomium thermophilum cellobiohydrolase I (Cbh1), and Neosartorya fischeri cellobiohydrolase I (Cbh1).

### Claim 15 (depends on 8)

A process for converting lignocellulosic biomass to ethanol, comprising contacting lignocellulosic biomass with the host cell of claim 8.

### Claim 16 (depends on 8)

A process for digesting as cellulosic substrate, comprising contacting said cellulosic substrate with the host cell of claim 8.

### Claim 17

An isolated polypeptide comprising at least 50 contiguous amino acids within amino acids 457 to 546 of SEQ ID NO: 3, wherein said polypeptide has cellulose binding activity.

### Claim 18

An isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having at least 95% identity to amino acids 20 to 456 of SEQ ID NO: 3, wherein said polypeptide has cellobiohydrolase activity.

### Claim 19 (depends on 18)

The polynucleotide of claim 18, wherein said nucleic acid sequence is codon optimized for expression in Saccharomyces cerevisiae and encodes for a polypeptide comprising amino acids 20 to 456 of SEQ ID NO:3.

### Claim 20 (depends on 18)

A vector comprising the polynucleotide of claim 18.

### Claim 21

An isolated polynucleotide comprising a nucleic acid sequence encoding amino acids 457 to 546 of SEQ ID NO:3 or amino acids 507 to 546 of SEQ ID NO:3, wherein said polypeptide has cellulose binding activity.