Polyhydroxyalkanoate synthesis in plants

• US 6,103,956 A
• Filed: 03/31/1998
• Issued: 08/15/2000
• Est. Priority Date: 03/31/1998
• Status: Expired due to Fees

• Current Assignee: University of Minnesota
• Original Assignee: Regents of The University of Minnesota (University of Minnesota)
• Inventors: Hahn, J. J., Eschenlauer, Arthur C., Srienc, Friedrich, Somers, David A.
• Primary Examiner(s): Smith, Lynette R. F.
• Assistant Examiner(s): Nelson, Amy J.
• Application Number:
  US09/052,607
• Time in Patent Office: 868 Days
• Field of Search: 536/23.2, 536/23.7, 435/69.1, 435/320.1, 435/419, 435/468, 435/135, 800/278, 800/287, 800/298
• US Class Current: 800/298
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Abstract

Novel transgenic plants and plant cells are capable of biosynthesis of polyhydroxyalkanoate (PHA). Heterologous enzymes involved in PHA biosynthesis, particularly PHA polymerase, are targeted to the peroxisome of a transgenic plant. Transgenic plant materials that biosynthesize short chain length monomer PHAs in the absence of heterologous β-ketothiolase and acetoacetyl-CoA reductase are also disclosed.

Claims

1. A transgenic plant comprising recombinant DNA comprising a heterologous nucleotide sequence encoding a polyhydroxyalkanoate (PHA) polymerase operably linked to a peroxisome-targeting sequence.

2. The transgenic plant of claim 1 wherein the recombinant DNA further comprises a promoter operably linked to the heterologous nucleotide sequence encoding the PHA polymerase.

3. The transgenic plant of claim 2 wherein the PHA polymerase is targeted to a glyoxysome.

4. The transgenic plant of claim 1 wherein the PHA polymerase comprises a PHA_SCL polymerase.

5. The transgenic plant of claim 4 wherein the recombinant DNA further comprises a heterologous nucleotide sequence encoding an acetoacetyl-CoA reductase operably linked to a peroxisome-targeting sequence.

6. The transgenic plant of claim 4 wherein the recombinant DNA further comprises a heterologous nucleotide sequence encoding a β

-ketothiolase operably linked to a peroxisome-targeting sequence.

7. The transgenic plant of claim 6 wherein the recombinant DNA further comprises a heterologous nucleotide sequence encoding an acetoacetyl-CoA reductase operably linked to a peroxisome-targeting sequence.

8. The transgenic plant of claim 1 wherein the PHA polymerase comprises a PHA_MCL polymerase.

9. The transgenic plant of claim 1 which produces polyhydroxyalkanoate.

10. A transgenic plant comprising recombinant DNA comprising a heterologous nucleotide sequence encoding a polyhydroxyalkanoate (PHA) polymerase operably linked to a peroxisome-targeting sequence, wherein expression of the heterologous nucleotide sequence leads to the production of polyhydroxyalkanoate in the plant.

11. The transgenic plant of claim 10 wherein the PHA polymerase comprises a PHA_SCL polymerase.

12. The transgenic plant of claim 11 wherein the polyhydroxyalkanoate is produced in a plant tissue selected from the group consisting of a seed, a root, a stem, a tuber, a rhizome, a leaf, a fruit and a bud.

13. The transgenic plant of claim 11 wherein the recombinant DNA further comprises each of a heterologous nucleotide sequence encoding a β

-kethothiolase operably linked to a peroxisome-targeting sequence and a heterologous nucleotide sequence encoding a acetoacetyl-CoA reductase operably linked to a peroxisome-targeting sequence.

14. The transgenic plant of claim 11 wherein the PHA polymerase comprises a PHA_MCL polymerase.

15. The transgenic plant of claim 10 wherein the polyhydroxyalkanoate is produced in a plant tissue selected from the group consisting of a seed, a root, a stem, a tuber, a rhizome, a leaf, a fruit and a bud.

29. A method for making polyhydroxyalkanoate comprising isolating polyhydroxyalkanoate from at least one tissue of the transgenic plant of claim 10.

30. The method of claim 29 wherein the at least one tissue of the transgenic plant is selected from the group consisting of a seed, a root, a stem, a tuber, a leaf, a fruit and a bud.

16. A transgenic differentiated plant cell comprising recombinant DNA comprising a heterologous nucleotide sequence encoding a PHA_SCL polymerase operably linked to a peroxisome-targeting sequence, wherein expression of the heterologous nucleotide sequence encoding the PHA_SCL polymerase in the transgenic plant cell in the absence of functional heterologous β

-kethothiolase and functional heterologous acetoacetyl-CoA reductase leads to production of polyhydroxyalkanoate.

17. A transgenic plant comprising the transgenic differentiated plant cell of claim 16.

18. A transgenic differentiated plant cell comprising recombinant DNA comprising a heterologous nucleotide sequence encoding a polyhydroxyalkanoate (PHA) polymerase operably linked to a peroxisome-targeting sequence.

19. The transgenic differentiated plant cell of claim 18 wherein the PHA polymerase comprises a PHA_SCL polymerase.

20. The transgenic differentiated plant cell of claim 18 wherein the PHA polymerase comprises a PHA_MCL polymerase.

21. A transgenic differentiated plant cell comprising recombinant DNA comprising a heterologous nucleotide sequence encoding a PHA_MCL polymerase operably linked to a peroxisome-targeting sequence.

22. A transgenic plant comprising the differentiated plant cell of claim 21.

23. A transgenic seed comprising recombinant DNA comprising a heterologous nucleotide sequence encoding a polyhydroxyalkanoate (PHA) polymerase operably linked to a peroxisome-targeting sequence.

24. A method for making a transgenic plant comprising:

(a) transforming a plant cell with recombinant DNA comprising a heterologous nucleotide sequence encoding a polyhydroxyalkanoate (PHA) polymerase operably linked to a peroxisome-targeting sequence, to yield a transgenic plant cell; and

(b) regenerating the transgenic plant cell into a transgenic plant.

25. The method of claim 24 wherein expression of the heterologous nucleotide sequence encoding the PHA polymerase leads to the production of polyhydroxyalkanoate in the transgenic plant.

26. The method of claim 24 wherein the PHA polymerase comprises a PHA_SCL polymerase, and wherein expression of the heterologous nucleotide sequence encoding the PHA_SCL polymerase in the transgenic plant cell in the absence of functional heterologous β

-ketothiolase and functional heterologous acetoacetyl-CoA reductase leads to production of polyhydroxyalkanoate.

27. The method of claim 24 wherein the PHA polymerase comprises a PHA_MCL polymerase.

28. A method for making polyhydroxyalkanoate comprising:

(a) transforming a plant cell with recombinant DNA comprising a heterologous nucleotide sequence encoding a polyhydroxyalkanoate (PHA) polymerase operably linked to a peroxisome-targeting sequence to yield a transgenic plant cell, wherein expression of the heterologous nucleotide sequence encoding the PHA polymerase in the transgenic plant cell leads to production of polyhydroxyalkanoate; and

(b) isolating the polyhydroxyalkanoate.