Variant CBH I polypeptides with reduced product inhibition
View Patent ↗The present disclosure relates to variant CBH I polypeptides that have reduced product inhibition, and compositions, e.g., cellulase compositions, comprising variant CBH I polypeptides. The variant CBH I polypeptides and related compositions can be used in variety of agricultural and industrial applications. The present disclosure further relates to nucleic acids encoding variant CBH I polypeptides and host cells that recombinantly express the variant CBH I polypeptides.
1. A polypeptide comprising a variant cellobiohydrolase I (CBH I) catalytic domain as compared to a reference CBH I catalytic domain, wherein the variant CBH I catalytic domain comprises an amino acid sequence having at least 90% sequence identity to amino acids 26-455 of SEQ ID NO:1, the polypeptide comprising:
(a) a substitution at the amino acid position corresponding to R268 of T. reesei CBH I of SEQ ID NO:2(R268 substitution);
(b) a substitution at the amino acid position corresponding to R411 of T. reesei CBH I of SEQ ID NO: 2 (R411 substitution); or
(c) both an R268 substitution and an R411 substitution,
wherein substitution (a), (b) or (c) decreases product inhibition as compared to the reference CBH I catalytic domain.
2. The polypeptide of claim 1 , which comprises an R268 substitution.
3. The polypeptide of claim 2 , wherein the R268 substituent is a lysine.
4. The polypeptide of claim 2 , wherein the R268 substituent is an alanine.
5. The polypeptide of claim 1 , which comprises an R411 substitution.
6. The polypeptide of claim 5 , wherein the R411 substituent is a lysine.
7. The polypeptide of claim 5 , wherein the R411 substituent is an alanine.
8. The polypeptide of claim 1 , wherein the variant CBH I catalytic domain comprises an amino acid sequence having at least 95% sequence identity to amino acids 26-455 of SEQ ID NO:1.
9. The polypeptide of claim 8 , wherein, other than said R268 and/or R411 substitutions, the variant CBH I catalytic domain comprises the sequence of amino acids 26-455 of SEQ ID NO:1.
10. The polypeptide of claim 1 , wherein the variant CBH I catalytic domain comprises one of the following amino acid substitutions or pairs of amino acid substitutions as compared to a protein of SEQ ID NO:1:
(a) R273K and R422K;
(b) R273K and R422A;
(c) R273A and R422K;
(d) R273A and R422A;
(e) R273A;
(f) R273K;
(g) R422A; and
(h) R422K.
11. The polypeptide of claim 1 , wherein the variant CBH I catalytic domain comprises the amino acid substitutions R273K and R422K as compared to a protein of SEQ ID NO:1.
12. The polypeptide of claim 1 , wherein the variant CBH I catalytic domain does not comprise both R273K and R422K substitutions as compared to a protein of SEQ ID NO:1.
13. The polypeptide of claim 1 in which the variant CBH I catalytic domain is linked to a cellulose binding domain.
14. The polypeptide of claim 13 in which the catalytic domain is linked to a cellulose binding domain via a linker.
15. The polypeptide of claim 13 in which the cellulose binding domain is C-terminal to the catalytic domain.
16. The polypeptide of claim 13 in which the cellulose binding domain is N-terminal to the catalytic domain.
17. The polypeptide of claim 1 which further comprises a signal sequence.
18. The polypeptide of claim 1 towards which cellobiose has an IC 50 that is at least 2-fold the IC 50 of a reference CBH I lacking said R268 substitution and/or R411 substitution.
19. The polypeptide of claim 1 which CBH I activity that is at least 50% the CBH I activity of a reference CBH I lacking said R268 substitution and/or R411 substitution.
20. A composition comprising a polypeptide according to claim 1 .
21. The composition of claim 20 in which said polypeptide represents at least 1% of all polypeptides in said composition.
22. The composition of claim 21 in which said polypeptide represents at least 5% of all polypeptide in said composition.
23. The composition of claim 22 in which said polypeptide represents at least 25% of all polypeptide in said composition.
24. The composition of claim 20 which is a whole cellulase.
25. The composition of claim 24 , wherein the whole cellulase is produced by a host cell that recombinantly expresses said polypeptide.
26. The composition of claim 20 which is filamentous fungal whole cellulase.
27. A fermentation broth comprising a polypeptide according to claim 1 .
28. The fermentation broth of claim 27 , which is a filamentous fungal fermentation broth.
29. The fermentation broth of claim 27 , which is a cell-free fermentation broth.
30. A method for saccharifying biomass, comprising: treating biomass with a composition according to claim 20 .
31. The method of claim 30 , further comprising recovering monosaccharides.
32. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of claim 1 .
33. A vector comprising the nucleic acid of claim 32 .
34. The vector of claim 33 which further comprises an origin of replication.
35. The vector of claim 33 which further comprises a promoter sequence operably linked to said nucleotide sequence.
36. The vector of claim 35 , wherein the promoter sequence is operable in yeast.
37. The vector of claim 35 , wherein the promoter sequence is operable in filamentous fungi.
38. A recombinant cell engineered to express the nucleic acid of claim 32 .
39. The recombinant cell of claim 38 which is a eukaryotic cell.
40. The recombinant cell of claim 39 which is a filamentous fungal cell.
41. The recombinant cell of claim 40 , wherein the filamentous fungal cell is of the genus Aspergillus, Penicillium, Rhizopus, Chrysosporium, Myceliophthora, Trichoderma, Humicola, Acremonium or Fusarium.
42. The recombinant cell of claim 40 , wherein the filamentous fungal cell is of the species Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum, Myceliophthora thermophila , or Rhizopus oryzae.
43. The recombinant cell of claim 39 which is a yeast cell.
44. The recombinant cell of claim 43 which is a yeast cell of the genus Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Klockera, Schwanniomyces or Yarrowia.
45. The recombinant cell of claim 44 , wherein the yeast cell is of the species S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S uvarum, S. diastaticus, K lactis, K. marxianus or K. fragilis.
46. The recombinant cell of claim 45 , which is a S. cerevisiae cell.
47. A host cell transformed with the vector of claim 33 .
48. The host cell of claim 47 which is a prokaryotic cell.
49. The host cell of claim 48 which is a bacterial cell.
50. The host cell of claim 47 which is a eukaryotic cell.
51. A method of producing a polypeptide according to claim 1 , comprising culturing the recombinant cell engineered to express said polypeptide under conditions in which the polypeptide is expressed.
52. The method of claim 51 , wherein the polypeptide comprises a signal sequence and wherein the recombinant cell is cultured under conditions in which the polypeptide is secreted from the recombinant cell.
53. The method of claim 52 , further comprising recovering the polypeptide from the cell culture.
54. The method of claim 53 , wherein recovering the polypeptide comprises a step of centrifuging away cells and/or cellular debris.
55. The method of claim 53 , wherein recovering the polypeptide comprises a step of filtering away cells and/or cellular debris.
56. A method for generating a product tolerant variant CBH I polypeptide, wherein the variant CBH I catalytic domain comprises an amino acid sequence having at least 90% sequence identity to amino acids 26-455 of SEQ ID NO:1 comprising:
(a) modifying the nucleotide sequence of a CBH I-encoding nucleic acid so that the nucleic acid encodes a variant CBH I polypeptide, wherein said variant CBH I polypeptide comprises:
(i) an R273 substitution of SEQ ID NO: 1;
(ii) an R422 substitution of SEQ ID NO: 1; or
(iii) both an R273 substitution and an R422 substitution; and
(b) expressing said variant CBH I polypeptide, thereby generating a product tolerant variant CBH I polypeptide.
57. A method for producing ethanol, comprising:
(a) treating biomass with a fermentation broth according to claim 27 , thereby producing monosaccharides; and
(b) culturing a fermenting microorganism in the presence of the monosaccharides produced in step (a) under fermentation conditions, thereby producing ethanol.