Schizochytrium PKS genes

• US 6,566,583 B1
• Filed: 01/14/1999
• Issued: 05/20/2003
• Est. Priority Date: 06/04/1997
• Status: Expired due to Term

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Lassner, Michael, Facciotti, Daniel, Metz, James George
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US09/231,899
• Time in Patent Office: 1,587 Days
• Field of Search: 536/23.2, 435/419, 435/252.3, 435/320.1, 800/281
• US Class Current: 800/281
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/21 from Pseudomonadaceae (F)
• CPC Class Codes: C07K 14/28 from Vibrionaceae (F)
• CPC Class Codes: C07K 14/405 from algae
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

The present invention relates to compositions and methods for preparing poly-unsaturated long chain fatty acids in plants, plant parts and plant cells, such as leaves, roots, fruits and seeds. Nucleic acid sequences and constructs encoding PKS-like genes required for the poly-unsaturated long chain fatty acid production, including the genes responsible for eicosapentenoic acid production of Shewanella putrefaciens and novel genes associated with the production of docosahexenoic acid in Vibrio marinus are used to generate transgenic plants, plant parts and cells which contain and express one or more transgenes encoding one or more of the PKS-like genes associated with such long chain polyunsaturated fatty acid production. Expression of the PKS-like genes in the plant system permits the large scale production of poly-unsaturated long chain fatty acids such as eicosapentenoic acid and docosahexenoic acid for modification of the fatty acid profile of plants, plant parts and tissues. Manipulation of the fatty acid profiles allows for the production of commercial quantities of novel plant oils and products.

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence encoding SEQ ID NO;

73, or a portion thereof comprising at least one functional domain of SEQ ID NO;

73;

(b) a nucleic acid sequence encoding an amino acid sequence that is at least 60% identical to the amino acid sequence of (a), wherein said nucleic acid sequence encodes a protein having the biological activity of at least one functional domain of SEQ ID NO;

73;

(c) a nucleic acid sequence that is fully complementary to the nucleic acid sequence of (a) or (b).

2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence encoding at least about 20 contiguous amino acid residues of SEQ ID NO:

73.

3. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence encoding at least about 35 contiguous amino acid residues of SEQ ID NO:

73.

4. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence comprising at least about 50 contiguous nucleotides of SEQ ID NO:

76.

5. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence comprising at least about 60 contiguous nucleotides of SEQ ID NO:

76.

6. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence comprising at least about 75 contiguous nucleotides of SEQ ID NO:

76.

7. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence comprising at least about 110 contiguous nucleotides of SEQ ID NO:

76.

8. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO:

73.

9. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises SEQ ID NO:

76.

10. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule, when expressed by a host cell, modulates the production of a long chain polyunsaturated fatty acid.

11. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule, when expressed by a host cell, modulates the production of docosahexaenoic acid.

12. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule, when exressed by a host cell, modulates the production of eicosapentaenoic acid.

13. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes an amino acid sequence that is at least 80% identical to SEQ ID NO:

73, wherein said nucleic acid sequence encodes a protein having the biological activity of at least one functional domain of SEQ ID NO;

73.

14. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical to SEQ ID NO:

73, wherein said nucleic acid sequence encodes at least one protein having the biological activity of at least one functional domain of SEQ ID NO;

73.

15. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 95% identical to SEQ ID NO:

73, wherein said nucleic acid sequence encodes at least one protein having the biological activity of at least one functional domain of SEQ ID NO;

73.

16. A recombinant nucleic acid molecule comprising the isolated nucleic acid molecule of claim 1, operably linked to a promoter.

17. A recombinant microbial cell comprising at least one copy of an isolated nucleic acid molecule according to claim 1.

18. The recombinant microbial cell of claim 17, wherein said microbial cell comprises a nucleic acid molecule derived from two or more polyketide synthase systems.

19. The recombinant microbial cell of claim 17, wherein said microbial cell is a fungal cell.

20. The recombinant microbial cell of claim 17, wherein said fungal cell is a yeast cell.

21. The recombinant microbial cell of claim 17, wherein said microbial cell is an algal cell.

22. The recombinant microbial cell of claim 17, wherein said algal cell is a marine algal cell.

23. The recombinant microbial cell of claim 17, wherein said cell is a bacterial cell or a cyanobacterial cell.

24. The recombinant microbial cell of claim 17, wherein said bacterial cell is a Lactobacillus cell.

25. The recombinant microbial cell of claim 17, wherein said recombinant microbial cell is enriched for 22:

6 fatty acids as compared to a non-recombinant microbial cell which is devoid of said isolated nucleic acid.

26. A method for production of a long chain polyunsaturated fatty acid in a microbial cell culture, said method comprising growing a microbial cell culture having a plurality of recombinant microbial cells as set forth in claim 17, under conditions whereby a long chain polyunsaturated fatty acid is produced by said microbial cell culture.

27. A recombinant plant cell comprising at least one copy of an isolated nucleic acid molecule according to claim 1.

28. The recombinant plant cell of claim 27, wherein said recombinant plant cell is a recombinant seed cell.

29. The recombinant plant cell of claim 27, wherein said recombinant seed cell is a recombinant embryo cell.

30. The recombinant plant cell of claim 27, wherein said recombinant plant cell is from a plant selected from the group consisting of Brassica, soybean, safflower, Arabidopsis, corn and sunflower.

31. A method for production of a long chain polyunsaturated fatty acid in a plant cell, said method comprising growing a plant having a plurality of recombinant plant cells as set forth in claim 27, under conditions whereby a long chain polyunsaturated fatty acid is produced in said plant cells.

32. The method of claim 31, wherein said long chain polyunsaturated fatty acid produced in said plant cells is a 20:

5 and 22;

6 fatty acid.

33. The method of claim 31, therein said long chain polyunsaturated fatty acid is eicosapentaenoic acid.

34. The method of claim 31, wherein said long chain polyusaturated fatty acid is docosaheyaenoic acid.

35. The nucleic acid molecule of claim 1, wherein said biological activity is dehydrase activity.

36. An oligonucleotide probe comprising at least 50 nucleotides of SEQ ID NO:

76 or the complement thereof.