Construction of protrophic/celluloytic yeast strains expressing tethered and secreted cellulases
View Patent ↗The present invention is directed to the construction of prototrophic, cellulo lytic strains of Saccharomyces cerevisiae with tethered and secreted cellulases and selection-based improvement of growth on cellulose by these strains. In some embodiments, host cells of the invention are able to produce ethanol using crystalline cellulose as a sole carbon source.
1. A transformed host cell comprising:
(a) at least one heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase which when expressed is tethered to the cell surface;
(b) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase I which when expressed is tethered to the cell surface;
(c) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase II which when expressed is tethered to the cell surface;
(d) at least one heterologous polynucleotide comprising a nucleic acid which encodes a β-glucosidase which when expressed is tethered to the cell surface; and
(e) at least one additional heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase which when expressed, is secreted by the cell;
wherein the host cell is capable of producing ethanol from microcrystalline cellulose.
2. The host cell of claim 1 , wherein the host cell is an organism selected from the group consisting of Saccharomyces cerevisiae ( S. cerevisiae ), Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans , and Schwanniomyces occidentalis.
3. The host cell of claim 1 , wherein the at least one tethered endoglucanase is an endoglucanase I.
4. The host cell of claim 3 , wherein the polynucleotide encoding the endoglucanase comprises a nucleic acid sequence that encodes endoglucanase I from Trichoderma reesei ( T. reesei ).
5. The host cell of claim 4 , wherein the polynucleotide encoding the endoglucanase further comprises a linker sequence.
6. The host cell of claim 5 , wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 20.
7. The host cell of claim 1 , wherein the polynucleotide encoding cellobiohydrolase I comprises a nucleic acid sequence that encodes cellobiohydrolase I from T. reesei.
8. The host cell of claim 7 , wherein the polynucleotide further comprises a linker sequence.
9. The host cell of claim 7 , wherein the polynucleotide further comprises a cell wall anchoring sequence.
10. The host cell of claim 9 , wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 21.
11. The host cell of claim 1 , wherein the polynucleotide encoding the cellobiohydrolase II comprises a nucleic acid sequence that encodes cellobiohydrolase II from T. reesei.
12. The host cell of claim 11 , wherein the polynucleotide encoding the cellobiohydrolase II further comprises a linker sequence.
13. The host cell of claim 12 , wherein the polynucleotide encoding the cellobiohydrolase II comprises the nucleotide sequence of SEQ ID NO: 22.
14. The host cell of claim 1 , wherein the at least one tethered β-glucosidase is a β-glucosidase I (Bgl1) from Saccharomycopsis fibuligera ( S. fibuligera ).
15. The host cell of claim 1 , wherein the nucleic acid which encodes the cellobiohydrolase II is from Talaromyces emersonii ( T. emersonii ).
16. The host cell of claim 1 , wherein the secreted enzyme is a cellobiohydrolase I.
17. The host cell of claim 16 , wherein the polynucleotide encoding the secreted cellobiohydrolase I comprises a nucleic acid sequence that encodes T. emersonii cellobiohydrolase I.
18. The host cell of claim 17 , wherein the polynucleotide encoding the secreted cellobiohydrolase I comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 9, or SEQ ID NO: 93.
19. The host cell of claim 16 , wherein the polynucleotide encoding the secreted cellobiohydrolase I comprises a nucleic acid sequence that encodes a fusion protein comprising T. emersonii Cbh1.
20. The host cell of claim 19 , wherein the fusion protein comprises T. emersonii Cbh1 and the cellulose binding module (CBM) of T. reesei Cbh2.
21. The host cell of claim 19 , wherein the fusion protein comprises T. emersonii Cbh1 and the cellulose binding module (CBM) of T. reesei Cbh1.
22. The host cell of claim 20 , wherein the CBM is fused to the T. emersonii Cbh1 via a linker sequence.
23. The host cell of claim 21 , wherein the CBM is fused to the T. emersonii Cbh1 via a linker sequence.
24. The host cell of claim 1 , wherein the at least one heterologous polynucleotide encoding a secreted cellobiohydrolase comprises a nucleotide sequence selected from the group consisting of SED ID NOs: 1-10.
25. The host cell of claim 2 , wherein the host cell is S. cerevisiae.
26. The host cell of claim 1 , wherein the secreted cellobiohydrolase is a cellobiohydrolase II.
27. The host cell of claim 26 , wherein the polynucleotide encoding the secreted cellobiohydrolase II comprises a nucleic acid sequence that encodes T. emersonii cellobiohydrolase II.
28. The host cell of claim 27 , wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10.
29. The host cell of claim 1 , wherein the nucleic acid which encodes a secreted cellobiohydrolase is a cellobiohydrolase I comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4 or 7-9.
30. The host cell of claim 1 , wherein the cellobiohydrolase I comprises the nucleotide sequence of SEQ ID NO: 9.
31. The host cell of claim 1 , wherein the tethered endoglucanase is T. reesei Eg 1, the tethered cellobiohydrolase I and cellobiohydrolase II are T. reesei Cbh1 and Cbh2, said tethered β-glucosidase is S. fibuligera Bgl1, and the secreted cellobiohydrolase is T. emersonii Cbh1 or a fusion protein comprising T. emersonii Cbh1.
32. The host cell of claim 31 , wherein the fusion protein comprises T. emersonii Cbh1 and the cellulose binding module (CBM) of T. reesei Cbh2.
33. The host cell of claim 31 , wherein the fusion protein comprises T. emersonii Cbh1 and the cellulose binding module (CBM) of T. reesei Cbh1.
34. The host cell of claim 32 wherein the CBM is fused to the T. emersonii Cbh1 via a linker sequence.
35. The host cell of claim 33 , wherein the CBM is fused to the T. emersonii Cbh1 via a linker sequence.
36. The host cell of claim 7 , wherein the cellobiohydrolase I comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from any of the cellobiohydrolases of Table 3 or Table 4.
37. The host cell of claim 1 , wherein the host cell has the ability to saccharify crystalline cellulose.
38. The host cell of claim 37 , wherein the cell has the ability to ferment the crystalline cellulose.
39. A method of fermenting cellulose using the host cell of claim 1 , the method comprising culturing the transformed host cell in medium that contains crystalline cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose.
40. The method of claim 39 , wherein the host cell produces ethanol.
41. The host cell of claim 17 , wherein the polynucleotide encoding the secreted cellobiohydrolase I comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 9, or SEQ ID NO: 93.
42. The host cell of claim 17 , wherein the polynucleotide encoding the secreted cellobiohydrolase I comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 9, or SEQ ID NO: 93.