Schizochytrium PKS genes

• US 20030101486A1
• Filed: 12/27/2002
• Published: 05/29/2003
• Est. Priority Date: 06/04/1997
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Boulder Corp. (DSM-Firmenich AG)
• Inventors: Lassner, Michael, Facciotti, Daniel, Metz, James George
• Granted Patent: US 7,214,853 B2
• Time in Patent Office: Days
• 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 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• 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 comprising:

a Vibrio marinus nucleotide sequence selected from the group consisting of ORF 6 (SEQ ID NO;

77), ORF 7 (SEQ ID NO;

78), ORF 8 (SEQ ID NO;

79), and ORF 9 (SEQ ID NO;

80), as shown in FIG. 6.

2. An isolated nucleic acid comprising:

a nucleotide sequence which encodes a polypeptide of a polyketide-like synthesis system, wherein said system produces a docosahexenoic acid when expressed in a host cell.

3. The isolated nucleic acid according to claim 2, wherein said nucleotide sequence is derived from a marine bacterium.

4. An isolated nucleic acid according to claim 2, wherein said nucleotide sequence is derived from Schizochytrium.

5. The isolated nucleic acid according to claim 2, wherein said nucleotide sequence is a Vibrio marinus ORF 8 (SEQ ID NO:

79), as shown in FIG. 6.

6. An isolated nucleic acid comprising a Schizochytrium nucleotide sequence comprising a sequence shown in a SEQ ID NO selected from the group consisting of SEQ ID NOS:

69, 71 and 76.

8. A recombinant microbial cell comprising at least one copy of an isolated nucleic acid according to claim 6.

9. The recombinant microbial cell according to claim 8, wherein said cell comprises each element of a polyketide-like synthesis system required to produce a long chain polyunsaturated fatty acid.

10. The recombinant microbial cell according to claim 9, wherein said cell is a eukaryotic cell.

11. The recombinant microbial cell according to claim 10, wherein said eukaryotic cell is a fungal cell, an algae cell or an animal cell.

12. The recombinant microbial cell according to claim 11, wherein said fungal cell is a yeast cell and said algae cell is a marine algae cell.

13. The recombinant microbial cell according to claim 8, wherein said cell is a prokaryotic cell.

14. The recombinant microbial cell according to claim 13, wherein said cell is a bacterial cell or a cyanobacterial cell.

15. A recombinant cell according to claim 14, wherein said bacterial cell is a lactobacillus cell.

16. The microbial cell according to claim 8, 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.

7. An isolated nucleic acid comprising:

a nucleotide sequence which is substantially identical to a sequence of at least 50 nucleotides of a Vibrio marinus nucleotide sequence selected from the group consisting of ORF 6 (SEQ ID NO;

77), ORF 7 (SEQ ID NO;

78), ORF 8 (SEQ ID NO;

79), and ORF 9 (SEQ ID NO;

80), as shown in FIG. 6.

17. A method for production of docosahexenoic acid in a microbial cell culture, said method comprising:

growing a microbial cell culture having a plurality of microbial cells, wherein said microbial cells or ancestors of said microbial cells were transformed with a vector comprising one or more nucleic acids having a nucleotide sequence which encodes a polypeptide of a polyketide synthesizing system, wherein said one or more nucleic acids are operably linked to a promoter, under conditions whereby said one or more nucleic acids are expressed and docosahexenoic acid is produced in said microbial cell culture.

19. The method according to claim 17 or claim 18 wherein said nucleotide sequence is shown in a SEQ ID NO selected from the group consisting of SEQ ID NOS:

69, 71 and 76.

21. The method according to claim 17, wherein said nucleotide sequence is selected from the group consisting of Vibrio marinus ORF 6 (SEQ ID NO:

77), ORF 7 (SEQ ID NO;

78), ORF 8 (SEQ ID NO;

79), and ORF 9 (SEQ ID NO;

80), as shown in FIG. 6 and Shewanella putrefaciens ORF 6 (SEQ ID NO;

83), ORF 7 (SEQ ID NO;

84), ORF 8 (SEQ ID NO;

85), ORF 9 (SEQ ID NO;

86), and ORF 3, which is complementary to SEQ ID NO;

4, as shown in FIG. 4.

18. 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 plant cells, wherein said plant cells or ancestors of said plant cells were transformed with a vector comprising one or more nucleic acids having a nucleotide sequence which encodes one or more polypeptides of a polyketide synthesizing system which produces a long chain polyunsaturated fatty acid, wherein each of said nucleic acids are operably linked to a promoter functional in a plant cell, under conditions whereby said polypeptides are expressed and a long chain polyunsaturated fatty acid is produced in said plant cells.

20. The method according to claim 18, wherein said long chain polyunsaturated fatty acid produced in said plant cells is a 20:

5 and 22;

6 fatty acid.

22. The method according to claim 18, wherein said nucleic acid constructs are derived from two or more polyketide synthesizing systems.

23. The method according to claim 18, wherein said long chain polyunsaturated fatty acid is eicosapentenoic acid.

24. The method according to claim 18, wherein said long chain polyunsaturated fatty acid is docosahexenoic acid.

25. A recombinant plant cell comprising:

one or more nucleic acids having a nucleotide sequence which encodes one or more polypeptides of a polyketide synthesizing system which produces a long chain polyunsaturated fatty acid, wherein each of said nucleic acids are operably linked to a promoter functional in said plant cell.

26. The recombinant plant cell according to claim 25, wherein said nucleotide sequence is shown in a SEQ ID NO selected from the group consisting of SEQ ID NOS:

69, 71 and 76.

27. The recombinant plant cell according to claim 26, wherein said recombinant plant cell is a recombinant seed cell.

28. The recombinant plant cell according to claim 27, wherein said recombinant seed cell is a recombinant embryo cell.

29. The recombinant plant cell according to claim 26, wherein said recombinant plant cell is from a plant selected from the group consisting of Brassica, soybean, safflower, and sunflower.

30. A plant oil produced by a recombinant plant cell according to claim 26.

31. The plant oil according to claim 30, wherein said plant oil comprises eicosapentenoic acid.

32. The plant oil according to claim 30, wherein said plant oil comprises docosahexenoic acid.

33. The plant oil according to claim 30, wherein said plant oil is encapsulated.

34. A dietary supplement comprising a plant oil according to claim 30.

35. A recombinant E. coli cell comprising:

one or more nucleic acids having a nucleotide sequence which encodes one or more polypeptides of a polyketide synthesizing system which produces a long chain polyunsaturated fatty acid, wherein each of said nucleic acids are operably linked to a promoter function in said E. coli cell.

36. The recombinant E. coli cell according to claim 35, wherein said long chain polyunsaturated fatty acid is docosahexenoic acid.

37. The recombinant E. coli cell according to claim 35, wherein said nucleotide sequence is shown in a SEQ ID NO selected from the group consisting of SEQ ID NOS:

69, 71 and 76.

38. A plant oil produced by a recombinant plant cell wherein said plant oil comprises a long chain polyunsaturated fatty acid exogenous to said plant oil, wherein said plant cell is produced according to a method comprising:

transforming said plant cell or an ancestor of said plant cell with a vector comprising one or more polypeptide of a polyketide synthesizing system which produces a long chain polyunsaturated fatty acid wherein each of said nucleic acids are operably linked to a promoter functional in said plant cell.

39. A plant oil according to claim 38, wherein said long chain polyunsaturated fatty acid is eicosapentenoic acid.

40. A plant oil according to claim 38, wherein said long chain polyunsaturated fatty acid is docosahexenoic acid.