Methods for generating cellulases
View Patent ↗The invention relates to carboxymethyl cellulases and to polynucleotides encoding the carboxymethyl cellulases. In addition methods of designing new carboxymethyl cellulases and method of use thereof are also provided. The carboxymethyl cellulases have increased activity and stability at increased pH and temperature.
1. A method of generating a nucleic acid encoding a cellulase comprising:
(a) providing a template nucleic acid encoding a polypeptide having cellulase activity comprising (i) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, (ii) a nucleic acid sequence encoding a polypeptide, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:2, (iii) a nucleic acid that hybridizes under high stringency conditions to the complement of the nucleic acid of SEQ ID NO:1 (iv) a nucleic acid that hybridizes under high stringency conditions to the complement of a nucleic acid encoding a polypeptide, wherein the polypeptide has the sequence of SEQ ID NO:2, or, (v) sequences complementary to (i), (ii), (iii), or (iv);
(b) varying the template nucleic acid of (a) by modifying one or more nucleotides in said sequence to another nucleotide, deleting one or more nucleotides in said sequence, or adding one or more nucleotides to said sequence, thereby generating a variant nucleic acid encoding a cellulase; and
(c) selecting a variant nucleic acid that encodes a cellulase, thereby generating a nucleic acid encoding a cellulase.
2. The method of claim 1 , wherein the modifications, deletions or additions are introduced by a method selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM) and any combination thereof.
3. The method of claim 1 , wherein the modifications, deletions or additions are introduced by error-prone PCR.
4. The method of claim 1 , wherein the modifications, deletions or additions are introduced by shuffling.
5. The method of claim 1 , wherein the modifications, deletions or additions are introduced by oligonucleotide-directed mutagenesis.
6. The method of claim 1 , wherein the modifications, deletions or additions are introduced by assembly PCR.
7. The method of claim 1 , wherein the modifications, deletions or additions are introduced by sexual PCR mutagenesis.
8. The method of claim 1 , wherein the modifications, deletions or additions are introduced by in vivo mutagenesis.
9. The method of claim 1 , wherein the modifications, deletions or additions are introduced by cassette mutagenesis.
10. The method of claim 1 , wherein the modifications, deletions or additions are introduced by recursive ensemble mutagenesis.
11. The method of claim 1 , wherein the modifications, deletions or additions are introduced by exponential ensemble mutagenesis.
12. The method of claim 1 , wherein the modifications, deletions or additions are introduced by site-specific mutagenesis.
13. The method of claim 1 , wherein the modifications, deletions or additions are introduced by gene reassembly.
14. The method of claim 1 , wherein the modifications, deletions or additions are introduced by Gene Site Saturation Mutagenesis (GSSM).
15. The method of claim 1 , wherein the sequence identity is at least 95% sequence identity.
16. The method of claim 1 , wherein the sequence identity is at least 97% sequence identity.
17. The method of claim 16 , wherein the nucleic acid has the sequence of SEQ ID NO:1 or encodes a polypeptide having the sequence of SEQ ID NO:2.
18. A method of generating a cellulase comprising:
(a) providing a template nucleic acid encoding a polypeptide having cellulase activity comprising (i) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, (ii) a nucleic acid sequence encoding a polypeptide, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:2, (iii) a nucleic acid that hybridizes under high stringency conditions to the complement of the nucleic acid (iv) a nucleic acid that hybridizes under high stringency conditions to the complement of a nucleic acid encoding a polypeptide, wherein the polypeptide has the sequence of SEQ ID NO:2, or, (v) sequences complementary to (i), (ii), (iii), or (iv);
(b) varying the template nucleic acid of (a) by modifying one or more nucleotides in the sequence to another nucleotide, deleting one or more nucleotides in the sequence, or adding one or more nucleotides to the sequence of (a), thereby generating a variant nucleic acid encoding a cellulase; and
(c) expressing the variant nucleic acid and testing the expressed polypeptide for cellulase activity, thereby generating a nucleic acid encoding a cellulase.
19. A method of making a variant of a nucleic acid encoding a cellulase comprising:
(a) providing a template nucleic acid encoding a polypeptide having cellulase activity comprising (i) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, (ii) a nucleic acid sequence encoding a polypeptide, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:2, (iii) a nucleic acid that hybridizes under high stringency conditions to the complement of the nucleic acid of SEQ ID NO: 1 (iv) a nucleic acid that hybridizes under high stringency conditions to the complement of a nucleic acid encoding a polypeptide, wherein the polypeptide has the sequence of SEQ ID NO:2, or, (v) sequences complementary to (i), (ii), (iii), or (iv); and
(b) making a variant sequence of (a) by modifying one or more nucleotides in the sequence to another nucleotide, deleting one or more nucleotides in the sequence, or adding one or more nucleotides to the sequence of (a).
20. The method of claim 18 , wherein the modifications, deletions or additions are introduced by a method selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM) and any combination thereof.
21. The method of claim 19 , wherein the modifications, deletions or additions are introduced by a method selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM) and any combination thereof.
22. The method of claim 1 , wherein the method is iteratively repeated.
23. The method of claim 18 , wherein the method is iteratively repeated.
24. The method of claim 19 , wherein the method is iteratively repeated.
25. The method of claim 1 , wherein the polypeptide encoded by the variant nucleic acid has cellulase activity at a temperature, pH or salinity level that is different from that of the polypeptide encoded by the template nucleic acid.
26. The method of claim 18 , wherein the polypeptide encoded by the variant nucleic acid has cellulase activity at a temperature, pH or salinity level that is different from that of the polypeptide encoded by the template nucleic acid.
27. The method of claim 19 , wherein the polypeptide encoded by the variant nucleic acid has cellulase activity at a temperature, pH or salinity level that is different from that of the polypeptide encoded by the template nucleic acid.
28. The method of claim 1 , wherein the polypeptide encoded by the variant nucleic acid has a cellulase activity that has a different stereo-, regio- or chemo-selectivity from that of the polypeptide encoded by the template nucleic acid.
29. The method of claim 18 , wherein the polypeptide encoded by the variant nucleic acid has a cellulase activity at a temperature, pH or salinity level that is different from that of the polypeptide encoded by the template nucleic acid.
30. The method of claim 19 , wherein the polypeptide encoded by the variant nucleic acid has a cellulase activity at a temperature, pH or salinity level that is different from that of the polypeptide encoded by the template nucleic acid.