# US 8,658,398 B2 · Heterologous expression of termite cellulases yeast

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- Number: US 8,658,398 B2
- Document ID: US8658398B2
- Status: Granted
- Kind: B2
- Application number: 13/003,200
- Filed: 2009-07-07
- Published: 2012-01-05
- Granted: 2014-02-25
- Earliest claimed: 2008-07-07
- Assignees: Mascoma Corporation
- Applicants: Elena E. Brevnova; Vineet Rajgarhia; Mark Mellon; Anne Warner; John McBride; Chhayal Gandhi; Erin Wiswall
- Inventors: Elena E. Brevnova; Vineet Rajgarhia; Mark Mellon; Anne Warner; John McBride; Chhayal Gandhi; Erin Wiswall
- Web page: https://patentgrep.com/patent/US8658398B2

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 15/81: for yeasts
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12P 7/10: substrate containing cellulosic material
- 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 302/01091: 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)
- 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

The present invention provides for heterologous expression of termite and termite-associated symbiont cellulases. The cellulases can, for example, be codon-optimized and expressed in yeast host cells, such as the yeast Saccharomyces cerevisiae. The cellulases can also be co-expressed in host cells with other cellulases. The expression in such host cells of the termite and termite-associated symbiont cellulases, and variants and combinations thereof, result in yeast with improved cellulosic activity. Thus, such genes and expression systems are useful for efficient and cost-effective consolidated bioprocessing systems.

## Claims

### Claim 1

An isolated polynucleotide comprising a nucleic acid which encodes the mature endoglucanase of SEQ ID NO:36, wherein said nucleic acid is codon-optimized for expression in a yeast strain wherein at least one nucleotide within a sequence of 4, 5, 6, 7, 8, 9, or 10 consecutive A, T, C or G nucleotides is replaced with a different nucleotide, wherein the nucleotide replacement does not alter the amino acid sequence encoded by the polynucleotide and wherein the nucleotide replacement creates a codon that is the second most frequently used codon to encode an amino acid in the yeast strain.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluveromyces lactus, Kluyveromyces marxianus, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis.

### Claim 3

An isolated polynucleotide comprising a nucleic acid which encodes the mature endoglucanase of SEQ ID NO:36, wherein said nucleic acid is codon-optimized for expression in a s east strain, wherein at least one restriction enzyme site within the polynucleotide is removed by replacing at least one nucleotide within the restriction enzyme site with a different nucleotide, wherein the nucleotide replacement does not alter the amino acid sequence encoded by the polynucleotide and wherein the nucleotide replacement creates a codon that is the second most frequently used codon to encode an amino acid in the yeast strain.

### Claim 4

An isolated polynucleotide comprising a nucleic acid which encodes the mature endoglucanase of SEQ ID NO:36, wherein said nucleic acid is codon-optimized for expression in a yeast strain, wherein one or more direct repeats, inverted repeats and mirror repeats with lengths of 10 bases or longer within said polynucleotide is altered by replacing at least one nucleotide within the repeat with a different nucleotide, wherein the nucleotide replacement does not alter the amino acid sequence encoded by the polynucleotide and wherein the nucleotide replacement creates a codon that is the second most frequently used codon to encode an amino acid in the yeast strain.

### Claim 5 (depends on 1)

The polynucleotide of claim 1, wherein said polynucleotide is operably associated with a heterologous nucleic acid.

### Claim 6

An isolated polynucleotide comprising a nucleic acid, wherein the nucleic acid comprises the a nucleic acid coding sequence of SEQ ID NO:16.

### Claim 7 (depends on 1)

A vector comprising a first polynucleotide, wherein said first polynucleotide is the polynucleotide of claim 1.

### Claim 8

A host cell comprising a polynucleotide encoding the mature endoglucanase of SEQ ID NO:36 wherein the host cell is a yeast cell, wherein the polynucleotide is codon-optimized for expression in yeast and wherein the endoglucanase is expressed, wherein the host cell is co-cultured with one or more additional host cells, wherein each of the one or more additional host cells expresses one or more endoglucanases, cellobiohydrolases and/or β-glucosidases.

### Claim 9 (depends on 1)

A host cell comprising the polynucleotide of claim 1.

### Claim 10 (depends on 8)

The host cell of claim 8, wherein the host cell in the co-culture has the ability to saccharify crystalline cellulose.

### Claim 11

A host cell comprising a polynucleotide encoding the mature endoglucanase of SEQ ID NO: 6, wherein the host cell is a yeast cell, wherein the polynucleotide is codon-optimized for expression in yeast and wherein the endoglucanase is expressed, and wherein the host cell further comprises a polynucleotide encoding a cellulase.

### Claim 12 (depends on 1)

The host cell of claim 1, wherein the cellulose is a cellobiohydrolase, a β-glucosidase, or a combination thereof.

### Claim 13 (depends on 12)

The host cell of claim 12, wherein the cellulase is a T. emersonii cellobiohydrolase I.

### Claim 14 (depends on 8)

The host cell of claim 8, wherein polynucleotide encoding the endoglucanase comprises the sequence of SEQ ID NO:16.

### Claim 15 (depends on 6)

A host cell comprising the polynucleotide of claim 6.

### Claim 16 (depends on 3)

The polynucleotide of claim 3, wherein said polynucleotide is operably associated with a heterologous nucleic acid.

### Claim 17 (depends on 3)

A vector comprising a first polynucleotide, wherein said first polynucleotide is the polynucleotide of claim 3.

### Claim 18 (depends on 3)

A host cell comprising the polynucleotide of claim 3.

### Claim 19 (depends on 11)

The host cell of claim 11, wherein the host cell has the ability to saccharify crystalline cellulose.

### Claim 20 (depends on 11)

The host cell of claim 11, wherein polynucleotide encoding the endoglucanase comprises the coding sequence of SEQ ID NO:16.