Δ-12 desaturase gene suitable for altering levels of polyunsaturated fatty acids in oleaginous yeasts

• US 7,214,491 B2
• Filed: 05/06/2004
• Issued: 05/08/2007
• Est. Priority Date: 05/07/2003
• Status: Active Grant

• Current Assignee: Dupont US Holding, LLC (International Flavors & Fragrances Incorporated)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Yadav, Narendra S., Zhang, Hongxiang
• Primary Examiner(s): Guzo; David
• Assistant Examiner(s): JOIKE, MICHELE K
• Application Number:
  US10/840,325
• Publication Number:
  US 20050043527A1
• Time in Patent Office: 1,097 Days
• Field of Search: None
• US Class Current: 435/134
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)

Abstract

The present invention relates to a Δ12 fatty acid desaturase able to catalyze the conversion of oleic acid to linoleic acid (LA; 18:2). Nucleic acid sequences encoding the desaturase, nucleic acid sequences that hybridize thereto, DNA constructs comprising the desaturase gene, and recombinant host microorganisms expressing increased levels of the desaturase are described. Methods of increasing production of specific ω-3 and/or ω-6 fatty acids are described by overexpression of the Δ12 fatty acid desaturase or by disruption of the native gene.

Claims

1. An isolated nucleic acid molecule encoding a Yarrowia Δ

12 desaturase enzyme, selected from the group consisting of;

(a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO;

24;

(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;

0.1X SSC, 0.1% SDS, 65°

C. and washed with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS;

or

an isolated nucleic acid molecule that is complementary to (a) or (b).

2. The isolated nucleic acid molecule of claim 1 as set forth in SEQ ID NO:

23.

3. A chimeric gene comprising the isolated nucleic acid molecule of any of claims 1–

2 operably linked to suitable regulatory sequences.

4. A transformed host cell comprising the chimeric gene of claim 3.

5. A transformed host cell according to claim 4 selected from the group consisting of plants, algae, bacteria, yeast and fungi.

6. A transformed host cell according to claim 5 wherein the yeast is an oleaginous yeast.

7. A transformed host cell according to claim 6 wherein the oleaginous yeast is selected from the group consisting of Yarrowia, Mortierella, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

8. A transformed host cell according to claim 7 wherein the oleaginous yeast is Yarrowia sp.

9. A method for modulating the biosynthesis of ω

-3 or ω

-6 fatty acids in a Yarrowia host cell comprising;

a) providing a Yarrowia host cell comprising a functional ω

-3/ω

-6 fatty acid biosynthetic pathway;

b) over-expressing a Δ

12 desaturase gene encoding the Δ

12 desaturase enzyme as set forth in SEQ ID NO;

24 in the host cell of (a);

whereby the biosynthesis of ω

-3 or ω

-6 fatty acids is modulated.

10. A method of obtaining a nucleic acid molecule encoding a Δ

12 desaturase enzyme comprising;

(a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence as set forth in SEQ ID NOs;

23; and

(b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a);

wherein the amplified insert encodes a portion of an amino acid sequence encoding a Δ

12 desaturase enzyme.