Fad4, Fad5, Fad5-2, and Fad6, novel fatty acid desaturase family members and uses thereof

• US 20060117414A1
• Filed: 01/30/2006
• Published: 06/01/2006
• Est. Priority Date: 09/28/2000
• Status: Active Grant

• Current Assignee: Bioriginal Food & Science Corp. (Omega Protein Corporation)
• Original Assignee: Bioriginal Food & Science Corp. (Omega Protein Corporation)
• Inventors: Hong, Haiping, Qiu, Xiao
• Granted Patent: US 7,671,252 B2
• Time in Patent Office: Days
• US Class Current: 800/281
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A61K 31/202 having three or more double bonds, e.g. linolenic eicosanoids, e.g. leukotrienes A61K31/557
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C12N 15/8247 : involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0071 : acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/0083 : Miscellaneous (1.14.99)
• 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 7/6431 : Linoleic acids [18:2[n-6]]
• 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
• CPC Class Codes: C12Y 114/19003 : Linoleoyl-CoA desaturase (1.14.19.3)
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

The invention provides isolated nucleic acid molecules which encode novel fatty acid desaturase family members. The invention also provides recombinant expression vectors containing desaturase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for large-scale production of long chain polyunsaturated fatty acids (LCPUFAs), e.g., DHA.

Claims

1. An isolated nucleic acid molecule selected from the group consisting of

a) an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:

7;

b) an isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO;

8;

c) an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO;

7 and wherein the nucleic acid molecule encodes a Δ

6 fatty acid desaturase molecule capable of converting linoleic acid (18;

2) to gamma-linolenic acid;

d) an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO;

7 and wherein the nucleic acid molecule encodes a Δ

6 fatty acid desaturase molecule capable of converting linolenic acid (18;

3) to stearidonic acid;

e) an isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO;

8 and wherein the polypeptide is a Δ

6 fatty acid desaturase molecule capable of converting linoleic acid (18;

2) to gamma-linolenic acid;

f) an isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO;

8 and wherein the polypeptide is a Δ

6 fatty acid desaturase molecule capable of converting linolenic acid (18;

3) to stearidonic acid;

g) an isolated nucleic acid molecule encoding a Δ

6 fatty acid desaturase molecule capable of converting linoleic acid (18;

2) to gamma-linolenic acid, wherein the desaturase molecule comprises an amino acid sequence which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO;

8 and further comprises a heme-binding motif and a histidine-rich motif;

h) an isolated nucleic acid molecule encoding a Δ

6 fatty acid desaturase molecule capable of converting linolenic acid (18;

3) to stearidonic acid, wherein the desaturase molecule comprises an amino acid sequence which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO;

8 and further comprises a heme-binding motif and a histidine-rich motif; and

i) an isolated nucleic acid molecule comprising a nucleotide sequence of at least 12 contiguous nucleotides of SEQ ID NO;

7.

2. The nucleic acid molecule of claim 1, wherein the desaturase molecule comprises at least one motif represented by the amino acid residues selected from the group consisting of residues 42-47, residues 178-183, residues 215-220, and residues 400-405 of SEQ ID NO:

11.

3. The nucleic acid molecule of claim 1, wherein the desaturase molecule comprises at least two conservative histidine motifs.

4. The nucleic acid molecule of claim 1 comprising amino acid residues 42-47, 178-183, 215-220, and 400-405 of SEQ ID NO:

8.

5. A vector comprising the nucleic acid molecule of claim 1.

6. The vector of claim 5, wherein the vector is an expression vector.

7. A host cell comprising the expression vector of claim 6.

8. The host cell of claim 7, wherein the cell is selected from the group consisting of a plant cell, a microbial cell, and an animal cell.

9. The host cell of claim 8, wherein the plant cell is a cell obtained from an oilseed crop selected from the group consisting of flax (Linum sp.), Brassica napus, Brassica juncea, rapeseed (Brassica sp.), soybean (Glycine and Soja sp.), sunflower (Helianthus sp.), corron (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobroma cacoa), and peanut (Arachis sp.).

10. The host cell of claim 8, wherein the microbial cell is selected from the group consisting of Thraustochytrium, Pythium irregulare, Schizochytrium, and Crythecodinium.

11. A method of producing an unsaturated fatty acid comprising culturing or cultivating the cell of claim 7 under conditions such that the unsaturated fatty acid is produced.

12. The method of claim 11, wherein the unsaturated fatty acid is gamma-linolenic acid (GLA).

13. The method of claim 11, wherein the unsaturated fatty acid is stearidonic acid (SDA).

14. The method of claim 11, further comprising recovering the unsaturated fatty acid.