Heterologous expression of termite cellulases in yeast

Patent metadata

Field	Value
Publication number	US-10214733-B2
Application number	US-201715825022-A
Country	US
Kind code	B2
Filing date	Nov 28, 2017
Priority date	Jul 7, 2008
Publication date	Feb 26, 2019
Grant date	Feb 26, 2019

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.

First claim

What is claimed is:

1. An isolated polynucleotide comprising a nucleic acid which encodes the mature endoglucanase of SEQ ID NO: 37, 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.

2. The isolated 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.

3. An isolated polynucleotide comprising a nucleic acid which encodes the mature endoglucanase of SEQ ID NO: 37, wherein said nucleic acid is codon-optimized for expression in a yeast 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.

4. An isolated polynucleotide comprising a nucleic acid which encodes the mature endoglucanase of SEQ ID NO: 37, 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.

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

6. The isolated polynucleotide of claim 1 comprising a nucleic acid coding sequence of SEQ ID NO: 17.

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

8. A host cell comprising a polynucleotide encoding the mature endoglucanase of SEQ ID NO: 37, 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.

9. A host cell comprising the polynucleotide of claim 1 .

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

11. A host cell comprising a polynucleotide encoding the mature endoglucanase of SEQ ID NO: 37, 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.

12. The host cell of claim 11 , wherein the cellulase is a cellobiohydrolase, a β-glucosidase, or a combination thereof.

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

14. The host cell of claim 8 , wherein the polynucleotide encoding the endoglucanase comprises the sequence of SEQ ID NO: 17.

15. A host cell comprising the polynucleotide of claim 6 .

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

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

18. A host cell comprising the polynucleotide of claim 3 .

19. The host cell of claim 11 having the ability to saccharify crystalline cellulose.

20. The host cell of claim 11 , wherein polynucleotide encoding the endoglucanase comprises the coding sequence of SEQ ID NO: 17.

Assignees

• Lallemand Hungary Liquidity Man Llc

Inventors

• Brevnova Elena E
• Rajgarhia Vineet
• Mellon Mark
• Warner Anne
• Mcbride John
• Gandhi Chhayal
• Wiswall Erin

Classifications

• C12N15/815

  for yeasts other than Saccharomyces · CPC title

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12P7/10

  substrate containing cellulosic material · CPC title

• C12N9/2437 Primary

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• C12Y302/01091

  Cellulose 1,4-beta-cellobiosidase (3.2.1.91) · CPC title