Cellobiohydrolase I enzymes
View Patent ↗Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.
1. An isolated Penicillium funiculosum cellobiohydrolase I (Cel7A) polypeptide comprising a mutation at position 196 of SEQ ID NO:110 that creates a new N-linked glycosylation site at the asparagine residue at position 194 of SEQ ID NO:110, wherein the mutation results in a mutant polypeptide with improved cellulase activity relative to the wild type Cel7A polypeptide, and wherein the wild type Cel7A polypeptide has the amino acid sequence of SEQ ID NO:110.
2. The isolated polypeptide of claim 1 , wherein the polypeptide is produced by expressing the mutated polypeptide in a heterologous host cell.
3. The isolated polypeptide of claim 1 , further comprising the substitution of one or more amino acid residues in the linker domain with a serine or threonine residue or the addition of one or more serine or threonine residues to the linker domain, wherein the one or more substitutions or additions results in increased O-linked glycosylation of the linker domain relative to the linker domain of the wild type Cel7A polypeptide without the one or more substitutions or additions.
4. A composition comprising the isolated cellobiohydrolase I (Cel7A) polypeptide of claim 1 .
5. The composition of claim 4 , wherein the polypeptide is produced by expressing the mutated polypeptide in a heterologous host cell.
6. A method of making an active Penicillium funiculosum cellobiohydrolase I (Cel7A) polypeptide, comprising modifying a wild type Penicillium funiculosum Cel7A polypeptide to increase cellulase activity of the wild type Cel7A polypeptide, wherein the modification comprises introducing a mutation at position 196 of SEQ ID NO:110 to create a new N-linked glycosylation site at the asparagine residue at position 194 of SEQ ID NO:110, and wherein the wild type Cel7A polypeptide has the amino acid sequence of SEQ ID NO:110.
7. The method of claim 6 , wherein the modification is achieved using site-directed mutagenesis.
8. The method of claim 6 , wherein the Cel7A polypeptide is further modified by the substitution of one or more amino acid residues in the linker domain with a serine or threonine residue or the addition of one or more serine or threonine residues to the linker domain, wherein the one or more substitutions or additions results in increased O-linked glycosylation of the linker domain relative to the linker domain of the wild type Cel7A polypeptide without the one or more substitutions or additions.
9. The method of claim 6 , wherein the mutation is a substitution of the alanine residue at position 196 of SEQ ID NO:110 with a threonine residue.
10. The method of claim 6 , wherein the mutation is a substitution of the alanine residue at position 196 of SEQ ID NO:110 with a serine residue.
11. The method of claim 10 , wherein the Cel7A polypeptide is further modified by the substitution of one or more amino acid residues in the linker domain with a serine or threonine residue or the addition of one or more serine or threonine residues to the linker domain, wherein the one or more substitutions or additions results in increased O-linked glycosylation of the linker domain relative to the linker domain of the wild type Cel7A polypeptide without the one or more substitutions or additions.
12. The isolated polypeptide of claim 1 , wherein the mutation is a substitution of the alanine residue at position 196 of SEQ ID NO: 110 with a threonine residue.
13. The isolated polypeptide of claim 1 , wherein the mutation is a substitution of the alanine residue at position 196 of SEQ ID NO: 110 with a serine residue.
14. The isolated polypeptide of claim 13 , further comprising the substitution of one or more amino acid residues in the linker domain with a serine or threonine residue or the addition of one or more serine or threonine residues to the linker domain, wherein the one or more substitutions or additions results in increased O-linked glycosylation of the linker domain relative to the linker domain of the wild type Cel7A polypeptide without the one or more substitutions or additions.
15. The method of claim 9 , wherein the Cel7A polypeptide is further modified by the substitution of one or more amino acid residues in the linker domain with a serine or threonine residue or the addition of one or more serine or threonine residues to the linker domain, wherein the one or more substitutions or additions results in increased O-linked glycosylation of the linker domain relative to the linker domain of the wild type Cel7A polypeptide without the one or more substitutions or additions.
16. The isolated polypeptide of claim 12 , further comprising the substitution of one or more amino acid residues in the linker domain with a serine or threonine residue or the addition of one or more serine or threonine residues to the linker domain, wherein the one or more substitutions or additions results in increased O-linked glycosylation of the linker domain relative to the linker domain of the wild type Cel7A polypeptide without the one or more substitutions or additions.