HIGH LEVEL EXPRESSION AND ACCUMULATION OF HETEROLOGOUS POLYPEPTIDE IN PLANT TISSUE

• US 20090193541A1
• Filed: 01/26/2009
• Published: 07/30/2009
• Est. Priority Date: 01/30/2008
• Status: Active Grant

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Miles, Stacy
• Granted Patent: US 8,168,863 B2
• Time in Patent Office: Days
• US Class Current: 800/287
• CPC Class Codes: C12N 15/8221 Transit peptides
• 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
• 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: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Abstract

Compositions and methods for increasing the expression and/or accumulation of cellobiohydrolase enzyme in the vacuoles of plant cells are provided. The method involves targeting the enzyme to the vacuoles through the use of a barley polyamine oxidase (BPAO) vacuole sorting signal peptide. Plants transformed with an expression construct encoding the vacuole sorting signal peptide operably linked to the cellobiohydrolase enzyme direct expression of the polypeptide to the vacuoles of the plant cells. Transgenic plants, seeds, and plant tissues, and plant parts are provided. Downstream uses of transgenic plants or plant material expressing the constructs of the invention include agronomical and industrial uses, for example, human food, animal feed, biofuel, industrial alcohol, fermentation feedstocks, and the like.

Claims

1. An isolated nucleic acid molecule comprising a protein storage vacuole targeting sequence operably linked to a nucleic acid encoding a heterologous polypeptide which is operatively linked to a plant green tissue promoter.

2. The isolated nucleic acid of claim 1, wherein the protein storage vacuole targeting sequence is encoded by SEQ ID NO:

2.

3. The isolated nucleic acid of claim 1, wherein the heterologous polypeptide is a protein secreted by a fungus.

4. The isolated nucleic acid of claim 3, wherein the heterologous polypeptide is an enzyme.

5. The isolated nucleic acid of claim 4, wherein the enzyme is a cellulase.

6. The isolated nucleic acid of claim 5, wherein the cellulase is selected from the group consisting of CBH1 and CBH2.

7. The isolated nucleic acid of claim 6, wherein the CBH1 is encoded by a polypeptide selected from the group consisting ofSEQ ID NOs:

11, 15, 19 and 21.

8. The isolated nucleic acid of claim 6, wherein the CBH2 is encoded by a polypeptide selected from the group consisting of SEQ ID NOs:

13 and 17.

9. A method for accumulating a heterologous protein in a transgenic plant comprising the steps of:

a) operatively linking a plant green tissue promoter, a protein storage vacuole targeting sequence and a nucleic acid encoding the heterologous polypeptide; and

b) generating a transgenic plant wherein the heterologous polypeptide is targeted to the vacuole.

10. The method of claim 9, wherein the protein storage vacuole targeting sequence is encoded by SEQ ID NO:

2.

11. The method of claim 9, wherein the heterologous polypeptide is a protein secreted by a fungus.

12. The method of claim 11, wherein the heterologous polypeptide is an enzyme.

13. The method of claim 12, wherein the enzyme is a cellulase.

14. The method of claim 13, wherein the cellulase is selected from the group consisting of CBH1 and CBH2.

15. The method of claim 14, wherein the CBH1 is encoded by a polypeptide selected from the group consisting of SEQ ID NOs:

11, 15, 19 and 21.

16. The method of claim 14, wherein the CBH2 is encoded by a polypeptide selected from the group consisting of SEQ ID NOs:

13 and 17.

17. A transgenic plant cell comprising a nucleic acid molecule comprising a protein storage vacuole targeting sequence operably linked to a nucleic acid encoding a heterologous polypeptide which is operatively linked to a plant green tissue promoter.

18. The transgenic plant cell of claim 17, wherein the plant is a monocot.

19. The transgenic plant cell of claim 17, wherein the plant is a dicot.

20. The transgenic plant cell of claim 17, wherein the protein storage vacuole targeting sequence is encoded by SEQ ID NO:

2.

21. The transgenic plant cell of claim 17, wherein the heterologous polypeptide is a protein secreted by a fungus.

22. The transgenic plant cell of claim 18, wherein the monocot is maize.

23. The transgenic plant cell of claim 21, wherein the heterologous polypeptide is an enzyme.

24. The transgenic plant cell of claim 23, wherein the enzyme is a cellulase.

25. The transgenic plant cell of claim 24, wherein the cellulase is selected from the group consisting of CBH1 and CBH2.

26. The transgenic plant cell of claim 25, wherein the CBH1 is encoded by a polypeptide selected from the group consisting of SEQ ID NOs:

11, 15, 19 and 21.

27. The transgenic plant cell of claim 25, wherein the CBH2 is encoded by a polypeptide selected from the group consisting of SEQ ID NOs:

13 and 17.

28. A method for accumulating a heterologous polypeptide in plant stover comprising the steps of

a) introducing into said plant cell a nucleic acid construct comprising a protein storage vacuole sorting signal sequence operably linked to a nucleotide sequence encoding a heterologous polypeptide;

and

b) allowing the plant cell to senesce to form stover.

29. The method of claim 28, wherein the heterologous polypeptide accumulates at least 3 fold more heterologous polypeptide in the stover than in the green tissue wherein the heterologous polypeptide is measured as an amount of heterologous polypeptide per unit of gram of tissue.

30. The method of claim 28, wherein the stover is maize.

31. The method of claim 28, wherein the protein storage vacuole sorting sequence is encoded by SEQ ID NO:

2.

32. The method of claim 28, wherein the heterologous polypeptide is a protein secreted by a fungus.

33. The method of claim 32, wherein the heterologous polypeptide is an enzyme.

34. The method of claim 33, wherein the enzyme is a cellulase.

35. The method of claim 34, wherein the cellulase is selected from the group consisting of CBH1 and CBH2.

36. The method of claim 35, wherein the CBH1 is encoded by a polypeptide selected from the group consisting of SEQ ID NOs:

11, 15, 19 and 21.

37. The method of claim 35, wherein the CBH2 is encoded by a polypeptide selected from the group consisting of SEQ ID NOs:

13 and 17.

41. The method of any one of claims 28 to 37, wherein the polypeptide is stable in senesced tissue for at least 2 to 3 months, at least 3 to 6 months, at least 6 to 9 months, at least 9 months to 1 year or more than 1 year.

38. A method of expressing a cellobiohydrolase close to the predicted size comprising the steps of:

a) operably linking a plant promoter, an ER retention sequence and a cellobiohydrolase;

b) isolating a transgenic plant expressing said cellobiohydrolase wherein said cellobiohydrolase, by Western blot, migrates close to the predicted size.

39. The method of claim 38, wherein the plant promoter is selected from the group consisting of a green tissue promoter and a seed preferred promoter.

40. The method of claim 38, wherein the transgenic plant is a soybean.