Desaturase genes and uses thereof

• US 7,067,285 B2
• Filed: 01/22/2002
• Issued: 06/27/2006
• Est. Priority Date: 01/25/2001
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Das, Tapas, Mukerji, Pradip, Huang, Yung-Sheng, Thurmond, Jennifer, Pereira, Suzette L., Leonard, Amanda Eun-Yeong
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): Sullivan, Daniel M.
• Application Number:
  US10/054,534
• Publication Number:
  US 20030167525A1
• Time in Patent Office: 1,617 Days
• Field of Search: 536/23.1, 536/23.2, 800/8, 435/325, 435/320.1, 435/254.11, 435/71.1, 435/189, 424/93.21
• US Class Current: 435/71.1
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• CPC Class Codes: A61P 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/02 : Nutrients, e.g. vitamins, minerals
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• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
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• 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
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• 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 9/0083 : 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

Abstract

The subject invention relates to the identification of genes involved in the desaturation of polyunsaturated fatty acids at carbon 5 (i.e., “Δ5-desaturase”) and at carbon 6 (i.e., “Δ6-desaturase”) and to uses thereof. In particular, Δ5-desaturase may be utilized, for example, in the conversion of dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA) and in the conversion of 20:4n-3 to eicosapentaenoic acid (EPA). Delta-6 desaturase may be used, for example, in the conversion of linoleic (LA) to γ-linolenic acid (GLA). AA or polyunsaturated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

Claims

1. An isolated nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to SEQ ID NO:

14, wherein said nucleic acid sequence encodes a functionally active Δ

6-desaturase.

2. The isolated nucleic acid sequence of claim 1 wherein said Δ

6-desaturase utilizes a polyunsaturated fatty acid as a substrate.

3. The isolated nucleic acid sequence of claim 1 wherein said sequence is isolated from Saprolegnia diclina.

4. A method of producing a desaturase in a host cell in vitro comprising the steps of:

a) isolating a nucleotide sequence comprising SEQ ID NO;

13;

b) constructing a vector comprising;

i) said isolated nucleotide sequence operably linked to ii) a regulatory sequence;

c) introducing said vector into a host cell in vitro for a time and under conditions sufficient for expression of said desaturase.

5. A vector comprising:

a) a nucleotide sequence comprising SEQ ID NO;

13, operably linked to b) a regulatory sequence.

6. An isolated host cell comprising said vector of claim 5.

7. An isolated mammalian cell comprising said vector of claim 5, wherein expression of said nucleotide sequence of said vector results in production of altered levels of arachidonic acid (AA), eicosapentaenoic acid (EPA), gamma-linolenic acid (GLA) or stearidonic acid (STA), when said cell is grown in a culture media comprising at least one fatty acid selected from the group consisting of linoleic acid (LA), alpha-linolenic acid (ALA), dihomo-gamma-linolenic acid (DGLA) and eicosapentaenoic acid (EPA).

8. An isolated plant cell or plant tissue comprising said vector of claim 5, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid by said plant cell, plant or plant tissue.

9. The isolated plant cell or plant tissue of claim 5 wherein said polyunsaturated fatty acid is selected from the group consisting of AA, EPA, GLA and STA.

10. A method for producing a polyunsaturated fatty acid comprising the steps of:

a) isolating a nucleotide sequence comprising SEQ ID NO;

13;

b) constructing a vector comprising said isolated nucleotide sequence;

c) introducing said vector into a host cell in vitro for a time and under conditions sufficient for expression of Δ

6-desaturase enzyme; and

d) exposing said expressed Δ

6-desaturase enzyme to a substrate polyunsaturated fatty acid in order to convert said substrate to a product polyunsaturated fatty acid.

11. The method according to claim 10, wherein said substrate polyunsaturated fatty acid is LA or ALA and said product polyunsaturated fatty acid is GLA or STA, respectively.

12. The method according to claim 10 further comprising the step of exposing said product polyunsaturated fatty acid to an elongase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid.

13. The method according to claim 12 wherein said product polyunsaturated fatty acid is GLA or STA and said another polyunsaturated fatty acid is DGLA or ETA, respectively.

14. The method of claim 12 further comprising the step of exposing said another polyunsaturated fatty acid to an additional desaturase in order to convert said another polyunsaturated fatty acid to a final polyunsaturated fatty acid.

15. The method of claim 14 wherein said final polyunsaturated fatty acid is AA or EPA.

16. An isolated nucleic acid sequence encoding a polypeptide wherein said polypeptide:

1) has at least 70% sequence identity to SEQ ID NO;

14;

     2) has a third histidine box motif consisting essentially of QXXHH; and

     3) encodes a functionally active Δ

6-desaturase.