Transgenic plants containing ligninase and cellulase which degrade lignin and cellulose to fermentable sugars

• US 7,696,411 B2
• Filed: 11/20/2007
• Issued: 04/13/2010
• Est. Priority Date: 10/20/2000
• Status: Expired due to Fees

• Current Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Original Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Inventors: Sticklen, Masomeh B., Maqbool, Shahina B., Dale, Bruce E.
• Primary Examiner(s): Kallis; Russell
• Application Number:
  US11/986,073
• Publication Number:
  US 20080104724A1
• Time in Patent Office: 875 Days
• Field of Search: None
• US Class Current: 800/288
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8242 with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
• CPC Class Codes: C12N 15/8246 Non-starch polysaccharides, e.g. cellulose, fructans, levans
• CPC Class Codes: C12N 15/8255 involving lignin biosynthesis
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01011 Dextranase (3.2.1.11)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention provides transgenic plants which after harvest degrade the lignin and cellulose therein to fermentable sugars which can further be fermented to ethanol or other products. In particular, the transgenic plants comprise ligninase and cellulase genes from microbes operably linked to a DNA encoding a signal peptide which targets the fusion polypeptide produced therefrom to an organelle of the plant, in particular the chloroplasts. When the transgenic plants are harvested, the plants are ground to release the ligninase and cellulase which then degrade the lignin and cellulose of the transgenic plants to produce the fermentable sugars.

Claims

1. A transgenic herbaceous plant which degrades lignocellulose when the transgenic plant is ground to produce a plant material comprising:

(a) at least one DNA encoding a microbial cellulase which is operably linked to a nucleotide sequence encoding a signal peptide as a first fusion protein wherein the signal peptide directs the cellulase to a first organelle of the transgenic plant wherein the fusion protein protects the transgenic plant from degradation while it is growing; and

(b) at least one DNA encoding a fungal ligninase which is operably linked to a nucleotide sequence encoding a signal peptide as a second fusion protein wherein the signal peptide directs the ligninase to a second different organelle of the transgenic plant, wherein the fusion protein protects the transgenic plant fusion from degradation while growing,

wherein the transgenic plant degrades the lignocellulose when ground to produce the plant material.

2. The transgenic plant of claim 1 wherein the DNA encoding the cellulase is from an organism selected from the group consisting of Trichoderma reesei, Acidothermus cellulyticus, Streptococcus salivarius, Actinomyces naeslundi, and Thermomonospora fusca.

3. The transgenic plant of claim 1 wherein the DNA encoding the cellulase is selected from the group consisting of an e1 gene from Acidothermus cellulyticus, a cbh1 gene from Trichoderma reesei, a dextranase gene from Streptococcus salivarius, and a beta-glucosidase gene from Actinomyces naeslundi.

4. The transgenic plant of claim 3 wherein the e1 gene comprises the nucleotide sequence set forth in SEQ ID NO:

4, the cbh1 gene comprises the nucleotide sequence set forth in SEQ ID NO;

10, the dextranase gene comprises the nucleotide sequence set forth in SEQ ID NO;

8, and the beta-glucosidase gene comprises the nucleotide sequence set forth in SEQ ID NO;

6.

5. The transgenic plant of claim 1 wherein the DNA encoding the ligninase is from Phanerochaete chrysosporium.

6. The transgenic plant of claim 5 wherein the ligninase is ckg4 comprising the nucleotide sequence set forth in SEQ ID NO:

11 or ckg5 comprising the nucleotide sequence set forth in SEQ ID NO;

13.

7. The transgenic plant of claim 1 wherein the DNA encoding the cellulase and the DNA encoding the ligninase are each operably linked to a leaf-specific promoter.

8. The transgenic plant of claim 7 wherein the leaf-specific promoter is a promoter for rbcS.

9. The transgenic plant of claim 1 wherein the nucleotide sequence encoding the signal peptide encodes a signal peptide of rbcS.

10. The transgenic plant of claim 8 or 9 wherein the rbcS comprises the nucleotide sequence set forth in SEQ ID NO:

1.

11. The transgenic plant of claim 1 selected from the group consisting of maize, wheat, barley, rye, hops, hemp, rice, potato, soybean, sorghum, sugarcane, clover, tobacco, alfalfa and arabidopsis.

12. The transgenic plant of claim 1 wherein the DNA encoding the cellulase and the DNA encoding the ligninase are stably integrated into nuclear or plastid DNA of the transgenic plant.

13. The transgenic plant of claim 1 wherein the transgenic plant further includes a DNA encoding a selectable marker operably linked to a constitutive promoter.

14. The transgenic plant of claim 13 wherein the DNA encoding the selectable marker provides the transgenic plant with resistance to an antibiotic, an herbicide, or to environmental stress.

15. The transgenic plant of claim 14 wherein the DNA encoding resistance to the herbicide is a DNA encoding phosphinothricin acetyl transferase which confers resistance to the herbicide phosphinothricin.

16. The transgenic plant of claim 1 wherein the organelle of the transgenic plant is selected from the group consisting of nucleus, microbody, endoplasmic reticulum, endosome, vacuole, mitochondria, chloroplast, or plastid.

17. The transgenic plant of claim 16 wherein the organelle of the transgenic plant is the chloroplast.

54. The transgenic plant of claim 1 wherein the lignocellulose is degraded to fermentable sugars.

57. The transgenic plant of claim 1 wherein the cellulase is encoded by SEQ ID NO:

4 and the ligninase is encoded by SEQ ID NO;

13.

58. The transgenic plant of claim 1 wherein the second different organelle is a peroxisome.

59. The transgenic plant of claim 1 wherein the first organelle is a chloroplast.

60. The transgenic plant of claim 1 wherein the signal peptide for the cellulase is encoded by SEQ ID NO:

2.

18. A method for producing a transgenic herbaceous plant which degrades lignocellulose when the transgenic plant is ground to produce a plant material comprising:

(a) providing a first transgenic plant which includes a DNA encoding a microbial cellulase which is operably linked to a nucleotide sequence encoding a signal peptide as a first fusion protein wherein the signal peptide directs the cellulase to a first organelle of the transgenic plant, wherein the fusion protein protects the transgenic plant from degradation while it is growing and a second transgenic plant which includes a DNA encoding a fungal ligninase which is operably linked to a nucleotide sequence encoding a signal peptide as a second fusion protein wherein the signal peptide directs the ligninase to a second different organelle of the transgenic plant, wherein the fusion protein protects the transgenic plant fusion from degradation while growing; and

(b) mating by sexual fertilization the first and the second transgenic plants to produce a third transgenic plant which includes the first DNA encoding the cellulase and the second DNA encoding the ligninase,

wherein the transgenic plant degrades the lignocellulose when ground to produce the plant material.

19. The method of claim 18 wherein the DNA encoding the cellulase is from an organism selected from the group consisting of Trichoderma reesei, Acidothermus cellulyticus, Streptococcus salivarius, Actinomyces naeslundi, and Thermomonospora fusca.

20. The method of claim 18 wherein the DNA encoding the cellulase is selected from the group consisting of an e1 gene from Acidothermus cellulyticus, a cbh1 gene from Trichoderma reesei, a dextranase gene from Streptococcus salivarius, and a beta-glucosidase gene from Actinomyces naeslundi.

21. The method of claim 20 wherein the e1 gene comprises the nucleotide sequence set forth in SEQ ID NO:

4, the cbh1 gene comprises the nucleotide sequence set forth in SEQ ID NO;

10, the dextranase gene comprises the nucleotide sequence set forth in SEQ ID NO;

8, and the beta-glucosidase gene comprises the nucleotide sequence set forth in SEQ ID NO;

6.

22. The method of claim 18 wherein the DNA encoding the ligninase is from Phanerochaete chrysosporium.

23. The method of claim 22 wherein the ligninase is ckg4 comprising the nucleotide sequence set forth in SEQ ID NO:

11 or ckg5 comprising the nucleotide sequence set forth in SEQ ID NO;

13.

24. The method of claim 18 wherein the DNA encoding the cellulase and the DNA encoding the ligninase are each operably linked to a leaf-specific promoter such as a promoter for rbcS.

25. The method of claim 24 wherein the leaf-specific promoter is a promoter for rbcS.

26. The method of claim 18 wherein the nucleotide sequence encoding the signal peptide encodes a signal peptide of rbcS.

27. The method of claim 25 or 26 wherein the rbcS comprises the nucleotide sequence set forth in SEQ ID NO:

1.

28. The method of claim 18 selected from the group consisting of maize, wheat, barley, rye, hops, hemp, rice, potato, soybean, sorghum, sugarcane, clover, tobacco, alfalfa and arabidopsis.

29. The method of claim 18 wherein the DNA encoding the cellulase and the DNA encoding the ligninase are stably integrated into nuclear or plastid DNA of the transgenic plant.

30. The method of claim 18 wherein the first, second, or both transgenic plants further includes a DNA encoding a selectable marker operably linked to a constitutive promoter.

31. The method of claim 30 wherein the DNA encoding the selectable marker provides the transgenic plant with resistance to an antibiotic, an herbicide, or to environmental stress.

32. The method of claim 31 wherein the DNA encoding resistance to the herbicide is a DNA encoding phosphinothricin acetyl transferase which confers resistance to the herbicide phosphinothricin.

33. The method of claim 18 wherein the organelle of the transgenic plant is selected from the group consisting of nucleus, microbody, endoplasmic reticulum, endosome, vacuole, mitochondria, chloroplast, or plastid.

34. The method of claim 33 wherein the organelle of the transgenic plant is the chloroplast.

35. The method of claim 18 wherein progeny of the third transgenic plant are mated by sexual fertilization to a transgenic plant selected from the group consisting of the first, second, and third transgenic plants to produce a transgenic plant comprising multiples of genes encoding cellulases and ligninases.

36. A method for converting lignocellulose in a plant material to fermentable sugars comprising:

(a) providing a transgenic herbaceous plant which includes at least one DNA encoding a microbial cellulase which is operably linked to a nucleotide sequence encoding a signal peptide as a first fusion protein wherein the signal peptide directs the cellulase to a first organelle of the transgenic plant, wherein the fusion protein protects the transgenic plant from degradation while it is growing and at least one DNA encoding a fungal ligninase which is operably linked to a nucleotide sequence encoding a signal peptide as a second fusion protein wherein the signal peptide directs the ligninase to a second different organelle of the transgenic plant, wherein the fusion protein protects the transgenic plant fusion from degradation while growing;

(b) growing the transgenic plant for a time sufficient for the transgenic plant to accumulate a sufficient amount of the cellulase and the ligninase in the organelle of the transgenic plant;

(c) harvesting the transgenic plant which has accumulated the cellulase and ligninase in the organelle of the transgenic plant;

(d) grinding the transgenic plant for a time sufficient to produce the plant material wherein the cellulase and ligninase produced by the transgenic plant are released from the organelle of the transgenic plant;

(e) incubating the plant material for a time sufficient for the cellulase and ligninase in the plant material to produce the fermentable sugars from the lignocellulose in the plant material; and

(f) extracting the fermentable sugars produced from the lignocellulose by the cellulase and the ligninase from the plant material.

37. The method of claim 36 wherein the DNA encoding the cellulase is from an organism selected from the group consisting of Trichoderma reesei, Acidothermus cellulyticus, Streptococcus salivarius, Actinomyces naeslundi, and Thermomonospora fusca.

38. The method of claim 36 wherein the DNA encoding the cellulase is selected from the group consisting of an e1 gene from Acidothermus cellulyticus, a cbh1 gene from Trichoderma reesei, a dextranase gene from Streptococcus salivarius, and a beta-glucosidase gene from Actinomyces naeslundi.

39. The method of claim 38 wherein the e1 gene comprises the nucleotide sequence set forth in SEQ ID NO:

4, the cbh1 gene comprises the nucleotide sequence set forth in SEQ ID NO;

10, the dextranase gene comprises the nucleotide sequence set forth in SEQ ID NO;

8, and the beta-glucosidase gene comprises the nucleotide sequence set forth in SEQ ID NO;

6.

40. The method of claim 36 wherein the DNA encoding the ligninase is from Phanerochaete chrysosporium.

41. The method of claim 40 wherein the ligninase is ckg4 comprising the nucleotide sequence set forth in SEQ ID NO:

11 or ckg5 comprising the nucleotide sequence set forth in SEQ ID NO;

13.

42. The method of claim 36 wherein DNA encoding the cellulase and the DNA encoding the ligninase are each operably linked to a leaf-specific promoter.

43. The transgenic plant of claim 42 wherein the leaf-specific promoter is a promoter for rbcS.

44. The method of claim 36 wherein the nucleotide sequence encoding the signal peptide encodes a signal peptide of rbcS.

45. The method of claim 43 or 44 wherein the rbcS comprises the nucleotide sequence set forth in SEQ ID NO:

1.

46. The method of claim 36 selected from the group consisting of maize, wheat, barley, rye, hops, hemp, rice, potato, soybean, sorghum, sugarcane, clover, tobacco, alfalfa and arabidopsis.

47. The method of claim 36 wherein the first and second DNAs are stably integrated into nuclear or plastid DNA of the transgenic plant.

48. The method of claim 36 wherein the transgenic plant further includes a DNA encoding a selectable marker operably linked to a constitutive promoter.

49. The method of claim 48 wherein the DNA encoding the selectable marker provides the transgenic plant with resistance to an antibiotic, an herbicide, or to environmental stress.

50. The method of claim 49 wherein the DNA encoding resistance to the herbicide is a DNA encoding phosphinothricin acetyl transferase which confers resistance to the herbicide phosphinothricin.

51. The method of claim 36 wherein the organelle of the transgenic plant is selected from the group consisting of nucleus, microbody, endoplasmic reticulum, endosome, vacuole, mitochondria, chloroplast, or plastid.

52. The method of claim 51 wherein the organelle of the transgenic plant is the chloroplast.

53. The method of claim 36 wherein the plant material further includes a plant material made from a non-transgenic plant.

55. The method of claim 48 wherein the lignocellulose is degraded to fermentable sugars.

56. The method of claim 36 wherein the fermentable sugars are fermented to ethanol.

61. The transgenic plant wherein the signal peptide for the ligninase is SEQ ID NO:

3.