Cellobiohydrolase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-11053489-B2
Application number	US-202016917505-A
Country	US
Kind code	B2
Filing date	Jun 30, 2020
Priority date	Mar 2, 2016
Publication date	Jul 6, 2021
Grant date	Jul 6, 2021

Abstract

The present invention relates to cellobiohydrolase variants, polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing and using the variants.

First claim

What is claimed is:

1. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide variant having cellobiohydrolase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host, and wherein the polypeptide variant having cellobiohydrolase activity comprises a substitution at one or more positions corresponding to positions 201, 243, 286, and 343 of the polypeptide of SEQ ID NO: 1, wherein the substitution at a position corresponding to position 201 is with Asp, the substitution at a position corresponding to position 243 is with Glu, Lys, or Val, the substitution at a position corresponding to position 286 is with Ala, and the substitution at a position corresponding to position 343 is with Glu or Gly, and wherein the variant has at least 80%, but less than 100%, sequence identity to SEQ ID NO: 1, 2, 5, 6, 7, 8, or 9 or at least 95% sequence identity, but less than 100% sequence identity, to SEQ ID NO: 3.

2. The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide variant having cellobiohydrolase activity comprises one or more substitutions selected from the group consisting of S201D, L243E,K,V, V286A, and S343E,G of SEQ ID NO: 1.

3. The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide variant having cellobiohydrolase activity further comprises a substitution at one or more positions corresponding to positions 101, 143, 186, 217, 236, 245, 250, 251, 289, 295, 311, 321, 327, 333, 365, 374, 421, and 441 of SEQ ID NO: 1.

4. A recombinant host cell transformed with the nucleic acid construct or expression vector of claim 1 .

5. A method of producing a cellobiohydrolase variant, comprising:
a. cultivating the recombinant host cell of claim 4 under conditions conducive for production of the variant; and optionally
b. recovering the variant.

6. A transgenic plant, plant part or plant cell transformed with the nucleic acid construct or expression vector of claim 1 .

7. A method of producing a cellobiohydrolase variant, comprising:
a. cultivating a transgenic plant, plant part or a plant cell of claim 6 under conditions conducive for production of the variant; and optionally
b. recovering the variant.

8. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide variant having cellobiohydrolase activity, wherein the poly nucleotide is operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host, and wherein the polypeptide variant having cellobiohydrolase activity comprises a substitution at one or more positions corresponding to positions 201, 243, 286, and 343 of the polypeptide of SEQ ID NO: 1, wherein the substitution at a position corresponding to position 201 is with Asp, the substitution at a position corresponding to position 243 is with Glu, Lys, or Val, the substitution at a position corresponding to position 286 is with Ala, and the substitution at a position corresponding to position 343 is with Glu or Gly and wherein the variant has at least 85%, but less than 100%, sequence identity to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, or 9.

9. The nucleic acid construct or expression vector of claim 1 , wherein the variant has at least 96%, but less than 100%, sequence identity to SEQ ID NO: 3.

10. The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide variant having cellobiohydrolase activity further comprises a substitution at one or more positions corresponding to positions E101H, S186Y, A236S, C245L, T251K, N289D, D321N, Q327K, L333F, G365E, G374C, T429Q, and N441C of SEQ ID NO: 1.

Assignees

• Novozymes As

Inventors

• Wogulis Mark
• Demars Leslie
• Jones Aubrey
• Ding Hanshu
• Osborn David

Classifications

• 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

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y302/01004

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• C12N9/2437 Primary

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