Transgenic plants expressing enzymes involved in fatty acid biosynthesis

• US 20050164192A1
• Filed: 03/17/2003
• Published: 07/28/2005
• Est. Priority Date: 03/16/2002
• Status: Active Grant

• Current Assignee: University of York
• Original Assignee: University of York
• Inventors: Graham, Ian Alexander, Tonon, Thierry
• Granted Patent: US 7,705,202 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/93 Ligases (6)

Abstract

This invention relates to nucleic acid molecules which comprise nucleic acid sequences which encode enzymes involved in the biosynthesis of n-3 fatty acides, particularly docosahexanoic acid (DHA), or variants thereof; polypeptides encoded by said nucleic acids; cells transfected with said nucleic acid sequences and products comprising said nucleic acid sequences, polypeptides and/or cells.

Claims

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

a) the DNA sequence as represented in SEQ ID NO;

3, 5, 8, 9, 12, 14, 15. or 17;

b) DNA sequences which hybridise to the sequences identified in a) and

c) DNA sequences that are degenerate as a result of the genetic code to the DNA sequence defined in a) and b).

2. The isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid molecule anneals under stringent hybridisation conditions to the DNA sequence of claim 1.

3. The isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid molecule is isolated from an algal species.

4. The isolated nucleic acid molecule according to claim 3 wherein said algal species is Amphidinium carterae, Amphiphora hyalina, Amphiphora sp., Chaetoceros gracilis, Coscinodiscus sp., Crypthecodinium coohnii, Cryptomonas sp., Cylindrotheca fusiformis, Haslea ostrearia, Isochrysis galbana, Nannochloropsis oculata, Navicula sp., Nitzschia closterium, Pavlova lutheri, Phaeodactylum tricornutum, Prorocentrum minimum, Rhizosolenia setigera, Skeletonema costatum, Skeletonema sp., Tetraselmis tetrathele, Thalassiosira nitzschioides, Thalassiosira heterophorma, Thalassiosira pseudonana, or Thalassiosira stellaris.

5. A polypeptide encoded by the isolated nucleic acid molecule claim 1.

6. The polypeptide according to claim 5, wherein the peptide comprises a variant of the amino acid sequence represented in SEQ ID NO:

4, 6, 7, 10, 11, 13, 16, 18 or 19 wherein the sequence has been modified by deletion, addition or substitution of at least one amino acid residue, and wherein said modification enhances an enzyme activity of said polypeptide.

7. The polypeptide according to claim 5 wherein said polypeptide comprises the amino acid sequence represented in SEQ ID NO:

4, 6, 7, 10, 11, 13, 16, 18 or 19.

8. The polypeptide of claim 7 wherein said polypeptide consists of the amino acid sequence represented in SEQ ID NO:

4, 6, 7, 10, 11, 13, 16, 18 or 19.

9. A vector comprising the isolated nucleic acid of claim 1.

10. The vector according to claim 9, wherein said isolated nucleic acid is operably linked to a promoter.

11. The vector according to claim 10, wherein the promoter is an inducible promoter or a developmentally regulated promoter.

12. A cell transfected or transformed with at least one isolated nucleic acid molecule of claim 1.

13. The cell according to claim 12, wherein the at least one isolated nucleic acid molecules encodes a peptide having elongase activity, desaturase activity, acyl-CoA synthetase activity, and diacylglycerol acyltransferase activity, wherein the a cell has at least part of a 3-n fatty acid biosynthetic pathway.

14. A cell transfected or transformed with the vector of claim 9.

15. The cell according to claim 12, wherein said cell is transfected or transformed with nucleic acid molecules which comprise the sequences as represented by SEQ ID NO:

3, 5, 8, 9, 12, 14, 15, or 17.

16. The cell of claim 12, wherein the cell is a mammalian cell;

yeast cell;

an algal cell;

or a plant cell.

17. The cell according to claim 16, wherein the cell is a plant cell.

18. A plant comprising the cell according to claim 17.

19. The plant according to claim 18, wherein said plant is selected from the group consisting of:

corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.),flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Iopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

20. A seed comprising the cell according to claim 17.

21. The cell according to claim 16, wherein a the yeast cell is a member of the genus Saccharomyces spp.

22. The cell according to claim 21, wherein said yeast cell is Saccharomyces cerevisiae.

23. A foodstuff product comprising the yeast cell according to claim 21.

24. The foodstuff product according to claim 23, wherein said product is wine;

beer;

a baking products;

a dairy products;

or a vegetable extracts.

25. The foodstuff product according to claim 24, wherein said wine or beer is non-alcoholic.

27. A fermentation process comprising

providing a vessel containing the cell of claim 16 and constituents required for fermentation and fatty acid biosynthesis, thereby producing a liquid composition;

and

providing conditions conducive to fermentation of the liquid composition contained in said vessel.

28. An animal feed product comprising the cell of claim 12.

29. A method of modulating a level of fatty acids, or variants thereof, in a plant cell comprising;

providing the plant cell according to claim 17;

regenerating the plant cell into a plant; and

monitoring fatty acid production by said plant.

30. A method for producing fatty acids or variants thereof, comprising:

providing the cell claim 12;

and

providing conditions conducive to growth of said cell, wherein the cell thereby produces fatty acids or variants thereof.

31. A method for producing fatty acids, or variants thereof, comprising:

providing the plant cell of claim 17;

regenerating said cell into a plant; and

extracting fatty acids, or variants thereof, from said plant, thereby producing fatty acids or variants thereof.

32. Fatty acids, or variants thereof, obtained by the method according to claim 30.

33. A pharmaceutical composition comprising the fatty acids, or variants thereof, according to claim 32.

34. A method of treating a condition in a subject that would benefit from administration of fatty acids, or variants thereof, comprising administration of the pharmaceutical composition of claim 33 to the subject.

35. The method of claim 34, wherein said condition is cardiac arrhythmia;

rheumatoid arthritis;

Crohn'"'"'s disease;

schizophrenia;

cancer;

foetal alcohol syndrome;

attention deficient hyperactivity disorder;

cystic fibrosis;

phenylketonuria;

unipolar depression;

aggressive hostility;

adrenoleukodystophy;

coronary heart disease, hypertension, type II diabetes, or ocular disease.

36. A non-human transgenic animal comprising the isolated nucleic acid molecule of claim 1.

37. A reaction vessel comprising the isolated polypeptide of claim 5, and fatty acid substrates, wherein the vessel is adapted for the conversion of fatty acids substrates contained therein.

38. The reaction vessel comprising the cell of claim 12 and fatty acid substrates, wherein the vessel is adapted for the conversion of fatty acids substrates contained therein.

39. A reaction vessel according to claim 37 wherein said vessel is a bioreactor.

40. The method of claim 30, further comprising extracting fatty acids or variants thereof from the cell

26. (canceled)