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

• US 8,350,123 B2
• Filed: 10/19/2011
• Issued: 01/08/2013
• 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.
• Primary Examiner(s): Page, Brent T
• Application Number:
  US13/276,662
• Publication Number:
  US 20120090231A1
• Time in Patent Office: 447 Days
• Field of Search: None
• US Class Current: 800/284
• 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 9/0004 Oxidoreductases (1.)
• 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: 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 111/01014 Lignin peroxidase (1.11.1.14)
• 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: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention provides transgenic cover crop plants which after harvest degrade the hemicellulose and cellulose therein to fermentable sugars which can further be fermented to ethanol or other products. In particular, the transgenic plants comprise cellulase, hemicellulase and optionally ligninase 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 hemicellulase and cellulase which then degrade the hemicellulose and cellulose of the transgenic plants to produce the fermentable sugars.

Claims

1. A method for providing transgenic plant material from a cover crop with low amounts of lignin, the method comprising:

(a) providing a transgenic monocot plant which is the low-lignin cover crop which includes (i) at least one DNA encoding a cellulase and hemicellulase which is operably linked to a nucleotide sequence encoding a signal peptide wherein the signal peptide directs the cellulase to an organelle of the transgenic monocot plant and (ii) at least one DNA encoding a ligninase which is operably linked to a nucleotide sequence encoding a signal peptide wherein the signal peptide directs the ligninase to the organelle of the transgenic monocot plant;

(b) growing the transgenic monocot plant for a time sufficient for the transgenic monocot plant to accumulate a sufficient amount of the cellulase, the hemicellulase, the ligninase in sub-cellular compartments of the transgenic monocot plant; and

(c) harvesting the transgenic monocot plant which has accumulated the cellulase, the hemicellulase, the ligninase in the sub-cellular compartments of the transgenic monocot plant.

2. The method of claim 1, wherein the transgenic monocot plant is selected from the group consisting of rice, wheat, barley, oat, triticale, and rye.

3. The method of claim 1, wherein the sub-cellular compartment of the transgenic monocot plant is selected from the group consisting of nucleus, microbody, endoplasmic reticulum, endosome, vacuole, mitochondria, chloroplast, and plastid.

4. The method of claim 1, wherein the sub-cellular compartment of the transgenic monocot plant is selected from the group consisting of chloroplast, apoplast, endoplasmic reticulum, mitochondria, and vacuole.

5. The method of claim 1, wherein the DNA encoding the cellulase and hemicellulase and the DNA encoding the ligninase are each operably linked to a leaf-specific promoter.

6. The method of claim 1, wherein the DNA encoding the cellulase and hemicellulase and the DNA encoding the ligninase are stably integrated into nuclear or plastid DNA of the transgenic monocot plant.

7. The method of claim 1, wherein:

(i) 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;

(ii) the DNA encoding the hemicellulase is from Cochliobolus carbonum; and

(iii) the DNA encoding the ligninase is from Phanerochaete chrysosporium.

8. The method of claim 1, wherein:

(i) the DNA encoding the cellulase is selected from the group consisting of an e1 gene comprising the nucleotide sequence set forth in SEQ ID NO;

4 from Acidothermus cellulyticus, a cbh1 gene comprising the nucleotide sequence set forth in SEQ ID NO;

10 from Trichoderma reesei, a dextranase gene comprising the nucleotide sequence set forth in SEQ ID NO;

8 from Streptococcus salivarius, and a beta-glucosidase gene comprising the nucleotide sequence set forth in SEQ ID NO;

6 from Actinomyces naeslundi;

(ii) the DNA encoding the hemicellulase is a Xyl1 gene encoding a xylanase; and

(iii) the DNA encoding the ligninase is selected from the group consisting of a ckg4 gene comprising the nucleotide sequence set forth in SEQ ID NO;

11, and a ckg5 gene comprising the nucleotide sequence set forth in SEQ ID NO;

13.

9. A method for providing transgenic plant material from a cover crop with low amounts of lignin, the method comprising:

(a) growing and harvesting a first cash crop on a plot of land;

(b) providing a transgenic monocot plant which is the low-lignin cover crop which includes at (i) least one DNA encoding a cellulase and hemicellulase which is operably linked to a nucleotide sequence encoding a signal peptide wherein the signal peptide directs the cellulase to an organelle of the transgenic monocot plant and (ii) optionally at least one DNA encoding a ligninase which is operably linked to a nucleotide sequence encoding a signal peptide wherein the signal peptide directs the ligninase to the organelle of the transgenic monocot plant;

(c) growing the transgenic monocot plant on the plot (i) during a winter season, (ii) subsequent to the harvesting of the first cash crop from the plot, and (iii) for a time sufficient for the transgenic monocot plant to accumulate a sufficient amount of the cellulase, the hemicellulase, and the ligninase if present in sub-cellular compartments of the transgenic monocot plant; and

(d) harvesting the transgenic monocot plant which has accumulated the cellulase, the hemicellulase, and the ligninase if present in the sub-cellular compartments of the transgenic monocot plant.

10. The method of claim 9, wherein the first cash crop is corn.

11. The method of claim 9, wherein the first cash crop is soybean.

12. The method of claim 9, wherein the transgenic monocot plant is selected from the group consisting of rice, wheat, barley, oat, triticale, and rye.

13. The method of claim 9, wherein the sub-cellular compartment of the transgenic monocot plant is selected from the group consisting of nucleus, microbody, endoplasmic reticulum, endosome, vacuole, mitochondria, chloroplast, and plastid.

14. The method of claim 9, wherein the sub-cellular compartment of the transgenic monocot plant is selected from the group consisting of chloroplast, apoplast, endoplasmic reticulum, mitochondria, and vacuole.

15. The method of claim 9, wherein the DNA encoding the cellulase and hemicellulase is stably integrated into nuclear or plastid DNA of the transgenic monocot plant.

16. The method of claim 9, wherein the transgenic monocot plant includes the at least one DNA encoding the ligninase.

17. The method of claim 9, wherein:

(i) 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; and

(ii) the DNA encoding the hemicellulase is from Cochliobolus carbonum.

18. The method of claim 17, wherein the transgenic monocot plant includes the at least one DNA encoding the ligninase, and the DNA encoding the ligninase is from Phanerochaete chlysosporium.

19. The method of claim 9, wherein:

(i) the DNA encoding the cellulase is selected from the group consisting of an e1 gene comprising the nucleotide sequence set forth in SEQ ID NO;

4 from Acidothermus cellulyticus, a cbh1 gene comprising the nucleotide sequence set forth in SEQ ID NO;

10 from Trichoderma reesei, a dextranase gene comprising the nucleotide sequence set forth in SEQ ID NO;

8 from Streptococcus salivarius, and a beta-glucosidase gene comprising the nucleotide sequence set forth in SEQ ID NO;

6 from Actinomyces naeslundi; and

(ii) the DNA encoding the hemicellulase is a Xyl1 gene encoding a xylanase.

20. The method of claim 19, wherein the transgenic monocot plant includes the at least one DNA encoding the ligninase, and the DNA encoding the ligninase is selected from the group consisting of a ckg4 gene comprising the nucleotide sequence set forth in SEQ ID NO:

11, and a ckg5 gene comprising the nucleotide sequence set forth in SEQ ID NO;

13.