Commercial production of polysaccharide degrading enzymes in plants and methods of using same

• US 20060026715A1
• Filed: 09/02/2005
• Published: 02/02/2006
• Est. Priority Date: 12/06/2001
• Status: Active Grant

• Current Assignee: Applied Biotechnology Institute LLC
• Original Assignee: Applied Biotechnology Institute LLC
• Inventors: Hood, Elizabeth E., Howard, John A.
• Granted Patent: US 8,558,058 B2
• Time in Patent Office: Days
• US Class Current: 800/284
• 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 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon

Abstract

Expression of recombinant polysaccharide degrading enzymes in plants is described. In one embodiment, expression of the enzyme is preferentially directed to the seed of the plant. Expression may also be preferentially targeted to specific locations within the plant cell. Expression of cellulases in corn is shown. The result is the capacity to produce polysaccharide degrading enzymes in plants at commercially acceptable levels in a reliable manner. Methods of using same in production of ethanol is also described, including use of the plant-produced enzymes in the ethanol production process.

Claims

1. A corn plant comprising a heterologous nucleic acid molecule encoding a polysaccharide degrading enzyme.

2. The plant of claim 1, wherein the enzyme is expressed at levels of at least about 0.1% total soluble protein.

3. The plant of claim 1, wherein the enzyme is expressed at levels of at least about 1% total soluble protein.

4. The plant of claim 1, wherein the enzyme is expressed at levels of at least about 10% total soluble protein.

5. The plant of claim 1, wherein the enzyme is expressed at levels of at least about 20% total soluble protein.

6. The plant of claim 1, wherein the enzyme is cellobiohydrolase.

7. The plant of claim 1, wherein the enzyme is endo-1,4-β

-D-glucanase.

8. The plant of claim 6, wherein the enzyme is preferentially expressed to the seed of the plant.

9. The plant of claim 6, wherein the enzyme is secreted to the cell wall of the plant.

10. The plant of claim 6, wherein the enzyme is retained in the endoplasmic reticulum.

11. The plant of claim 7, wherein the enzyme is preferentially expressed to the seed of the plant.

12. The plant of claim 7, wherein the enzyme is secreted to the cell wall of the plant.

13. The plant of claim 7 wherein the enzyme is retained in the endoplasmic reticulum.

14. The plant of claim 7 wherein the enzyme is targeted to the vacuole.

15. Seed of the plant of claim 1.

16. Tissue of the plant of claim 1 selected from the group consisitng of leaf and stem.

17. A monocotyledenous plant comprising a heterologous nucleic acid molecule encoding a polysaccharide degrading enzyme.

18. The plant of claim 17, wherein the enzyme is selected from the group consisting of cellobiohydrolase and endo-1,4-β

-D-glucanase.

19. The plant of claim 17, wherein the enzyme is expressed at levels of at least about 0.1% total soluble protein.

20. Seed of the plant of claim 17.

21. Tissue of the plant of claim 17, selected from the group consisting of leaf and stem.

22. A method of increasing expression in a plant cell of a nucleic acid molecule encoding a cellulase enzyme, the cellulase enzyme selected from the group consisting of a cellobiohydrolase enzyme and an endo-1,4-β

-D-glucanase enzyme comprising secreting the enzyme to the cell wall.

23. A method of increasing expression in a plant cell of a nucleic acid molecule encoding a cellulase enzyme, the cellulase enzyme selected from the group consisting of a cellobiohydrolase enzyme and an endo-1,4-β

-D-glucanase enzyme comprising retaining the enzyme in the endoplasmic reticulum of the plant cell.

24. A method of increasing expression of a nucleic acid molecule encoding an endo-1,4-β

-D-glucanase enzyme in a plant cell comprising targeting expression of the enzyme to the vacuole of the plant cell.

25. A method of reducing costs of polysaccharide degradation of polysaccharides in plant biomass, comprising:

(a) transforming a polysaccharide-containing plant cell with a nucleic acid molecule expressing a polysaccharide-degrading enzyme;

(b) producing a plant expressing the enzyme from the plant cell;

(c) producing a biomass from the plant such that at least one of the plants of the biomass expresses the enzyme;

(d) harvesting the biomass; and

(e) contacting at least a portion of the biomass with the enzyme expressed in the plant under conditions such that the enzyme degrades the polysaccharides of at least a portion of the biomass.

26. A method of reducing costs of ethanol production from corn, comprising:

(a) transforming a corn plant cell with a nucleic acid molecule expressing a cellulase;

(b) producing from the plant cell a corn plant expressing the cellulase;

(c) producing a corn crop using the corn plant such that at least one of the plants in the crop expresses the cellulase;

(d) harvesting the corn;

(e) producing crop residue from the harvested corn;

(f) contacting the crop residue with the cellulase expressed in the corn plant under conditions such that the cellulase degrades cellulose of the crop residue; and

(g) producing ethanol from degradation of the cellulose of the crop residue.