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

• US 7,049,485 B2
• Filed: 10/18/2001
• Issued: 05/23/2006
• 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: Dale, Bruce E., Sticklen, Masomeh B., Maqbool, Shahina B.
• Primary Examiner(s): KALLIS, RUSSELL
• Application Number:
  US09/981,900
• Publication Number:
  US 20020138878A1
• Time in Patent Office: 1,678 Days
• Field of Search: 800/278, 800/284, 800/298, 800/287, 800/288, 800/312, 800/317.2, 800/317.3, 800/320, 800/320.1, 800/320.2, 800/320.3, 536/23.1, 536/23.2, 536/23.7
• 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 herbaceous transgenic plant which degrades lignocellulose when the transgenic plant is ground to produce a plant material comprising:

(a) the DNA of SEQ ID NO;

4 encoding a cellulase which is operably linked a nucleotide sequence encoding a signal peptide that directs the cellulase to a plastid or apoplast of the transgenic plant; and

(b) the DNA of SEQ ID NO;

11 encoding a ligninase comprising a lignin peroxidase which is operably linked to a nucleotide sequence encoding a signal peptide that directs the ligninase to the plastid or apoplast of the transgenic plant,

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 and the DNA encoding the ligninase are each operably linked to a leaf-specific promoter.

3. The transgenic plant of claim 2 wherein the leaf-specific promoter is a rubisco small subunit (rbcS) promoter for a rbcS coding sequence.

4. The transgenic plant of claim 1 wherein the nucleotide sequence encoding the signal peptide is a rubisco small subunit (rbcS) signal peptide coding sequence.

5. The transgenic plant of claim 3 or claim 4 wherein the rbcS comprises the nucleotide sequence of SEQ ID NO:

1.

6. 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.

7. 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.

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

9. The transgenic plant of claim 8 wherein the DNA encoding the selectable marker provides the transgenic plant with resistance to an antibiotic, an herbicide, or to environmental stress.

10. The transgenic plant of claim 9 wherein the DNA encoding resistance to the herbicide is a DNA encoding phosphinothricin acetyl transferase which confers resistance to the herbicide phosphinothricin.

11. The transgenic plant of claim 1 wherein the plastid of the transgenic plant is the chloroplast.

36. The transgenic plant of claim 1 wherein the lignocellulose is degrade to fermentable sugars.

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

(a) providing a first transgenic plant which includes the DNA of SEQ ID NO;

4 encoding a cellulase, which is operably linked to a nucleotide sequence encoding a signal peptide that directs the cellulose to a plastid or apoplast of the transgenic plant, and a second transgenic plant which includes the DNA of SEQ ID NO;

11 encoding a ligninase comprising a lignin peroxidase which is operably linked to a nucleotide sequence encoding a signal peptide that directs the ligninase to the plastid or apoplast of the transgenic plant; and

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

wherein the transgenic plant which includes the DNA encoding the cellulase and the DNA encoding the ligninase degrades the lignocellulose when ground to produce the plant material.

13. The method of claim 12 wherein the DNA encoding the cellulase and the DNA encoding the ligninase are each operably linked to a leaf-specific promoter.

14. The method of claim 13 wherein the leaf-specific promoter is a rubisco small subunit (rbcS) promoter for a rbcS coding sequence.

15. The method of claim 12 wherein the nucleotide sequence encoding the signal peptide is a rubisco small subunit (rbcS) signal peptide coding sequence.

16. The method of claim 14 or claim 15 wherein the rbcS comprises the nucleotide sequence set forth in SEQ ID NO:

1.

17. The method of claim 12 wherein the plant is selected from the group consisting of maize, wheat, barley, rye, hops, hemp, rice, potato, soybean, sorghum, sugarcane, clover, tobacco, alfalfa, arabidopsis.

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

19. The method of claim 12 wherein the first, second, or both transgenic plants further includes a DNA encoding a selectable marker operably linked to a constitutive promoter.

20. The method of claim 19 wherein the DNA encoding the selectable marker provides the transgenic plant with resistance to an antibiotic, an herbicide, or to environmental stress.

21. The method of claim 20 wherein the DNA encoding resistance to the herbicide is a DNA encoding phosphinothricin acetyl transferase which confers resistance to the herbicide phosphinothricin.

22. The method of claim 12 wherein the plastid of the transgenic plant is the chloroplast.

23. The method of claim 12 wherein transformed progeny of the third transgenic plant which includes the DNA encoding the cellulase and the DNA encoding the ligninase are mated by sexual fertilization to a transgenic plant selected from the group consisting of the first, second, and third transgenic plant to produce a transgenic plant comprising multiple copies of the DNA encoding the cellulase and multiple copies of the DNA encoding the ligninase.

37. The method of claim 12 wherein the lignocellulose is degraded to fermentable sugars.

24. A method for converting lignocellulose in a herbaceous transgenic plant material to fermentable sugars comprising:

(a) providing a herbaceous transgenic plant which includes the DNA of SEQ ID NO;

4 encoding a cellulase, which is operably linked to a nucleotide sequence encoding a signal peptide that directs the cellulase to a plastid or apoplast of the transgenic plant and the DNA of SEQ ID NO;

11 encoding a ligninase comprising a lignin peroxidase which is operably linked to a nucleotide sequence encoding a signal peptide wherein the signal peptide directs the ligninase to the plastid or apoplast of the transgenic plant;

(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 plastid or apoplast of the transgenic plant;

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

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

(e) incubating the herbaceous transgenic 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 herbaceous transgenic plant material; and

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

25. The method of claim 24 wherein DNA encoding the cellulase and the DNA encoding the ligninase are each operably linked to a leaf-specific promoter.

26. The transgenic plant of claim 25 wherein the leaf-specific promoter is a promoter for rbcS.

27. The method of claim 24 wherein the nucleotide sequence encoding the signal peptide encodes a signal peptide of rbcS.

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

1.

29. The method of claim 24 wherein the plant is selected from the group consisting of maize, wheat, barley, rye, hops, hemp, rice, potato, soybean, sorghum, sugarcane, clover, tobacco, alfalfa, and arabidopsis.

30. The method of claim 24 wherein the cellulase and ligninase DNAs are stably integrated into nuclear or plastid DNA of the transgenic plant.

31. The method of claim 24 wherein transgenic plant further includes a DNA encoding a selectable marker operably linked to a constitutive promoter.

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

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

34. The method of claim 24 wherein the plastid of the transgenic plant is a chloroplast.

35. The method claim of 24 further comprising adding to (d) a plant material from a non-transgenic plant.

38. The method of claim 24 further comprising:

(g) fermenting the sugars to ethanol.