Oils, lipids and fatty acids produced in transgenic Brassica plant

• US 10,301,638 B2
• Filed: 09/06/2016
• Issued: 05/28/2019
• Est. Priority Date: 02/21/2006
• Status: Active Grant

• Current Assignee: BASF Plant Science GmbH (BASF SE)
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Cirpus, Petra, Bauer, Jorg, Qiu, Xiao, Wu, Guohai, Cheng, Bifang, Truksa, Martin, Wetjen, Tom
• Primary Examiner(s): Robinson, Hope
• Application Number:
  US15/256,914
• Publication Number:
  US 20160369290A1
• Time in Patent Office: 994 Days
• Field of Search: None
• 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: 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: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 114/19 with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)

Abstract

The invention relates to a method for producing eicosapentaenoic acid, docosapentaenoic acid and/or docohexanoic acid in transgenic plants. According to said method, the plant is provided with at least one nucleic acid sequence coding for a polypeptide with a Δ6 desaturase activity, at least one nucleic acid sequence coding for a polypeptide with a Δ6 elongase activity, at least one nucleic acid sequence coding for a polypeptide with a Δ5 desaturase activity, and at least one nucleic acid sequence coding for a polypeptide with a Δ5 elongase activity, the nucleic acid sequence coding for a polypeptide with a Δ5 elongase activity being modified in relation to the nucleic acid sequence in the organism from which the sequence originates, such that it is adapted to the codon use in at least one type of plant. For the production of docosahexanoic acid, at least one nucleic acid sequence coding for a polypeptide with a Δ4 desaturase activity is also introduced into the plant.

Claims

1. Oils, lipids and/or fatty acids produced by a transgenic Brassica plant, wherein said oils, lipids and/or fatty acids comprise 60 to 85% by weight of polyunsaturated fatty acids based on the total fatty acids in the transgenic plant, wherein said polyunsaturated fatty acids comprise at least 20% by weight of eicosapentaenoic acid (EPA), at least 2% by weight of docosapentaenoic acid (DPA), and at least 4% by weight of docosahexaenoic acid (DHA) based on the total fatty acids in the transgenic plant in the form of triacylglycerides.

2. The oils, lipids and/or fatty acids of claim 1, wherein:

a) said polyunsaturated fatty acids comprise at least 20% by weight of EPA and at least 24% by weight of EPA is present in the sn-1, sn-2 or sn-3 position;

b) said polyunsaturated fatty acids comprise at least 2% by weight of DPA and at least 1.5% by weight of DPA is present in the sn-1, sn-2 or sn-3 position; and

c) said polyunsaturated fatty acids comprise at least 4% by weight of DHA and at least 3% by weight of DHA is present in the sn-1, sn-2 or sn-3 position.

3. The oils, lipids and/or fatty acids of claim 1, wherein:

a) said polyunsaturated fatty acids comprise at least 20% by weight of EPA and at least 24% by weight of EPA is present in the sn-2 position;

b) said polyunsaturated fatty acids comprise at least 2% by weight of DPA and at least 3% by weight of DPA is present in the sn-2 position; and

c) said polyunsaturated fatty acids comprise at least 4% by weight of DHA and at least 3% by weight of DHA is present in the sn-2 position.

4. The oils, lipids and/or fatty acids of claim 1, wherein said polyunsaturated fatty acids comprise:

a) at least 20% by weight of EPA and at least 2% by weight of DPA based on the total fatty acids in the transgenic plant in the form of triacylglycerides;

b) at least 20% by weight of EPA and at least 4% by weight of DHA based on the total fatty acids in the transgenic plant in the form of triacylglycerides;

or

c) at least 2% by weight of DPA and at least 4% by weight of DHA based on the total fatty acids in the transgenic plant in the form of triacylglycerides.

5. The oils, lipids and/or fatty acids of claim 1, wherein said polyunsaturated fatty acids comprise at least 30% long chain polyunsaturated fatty acids (LCPUFAs) based on the total fatty acids in the transgenic plant, and wherein the LCPUFAs are C20 and/or C22 fatty acid molecules having at least four double bonds.

6. The oils, lipids and/or fatty acids of claim 1, wherein said oils, lipids and/or fatty acids comprise about 25% by weight of EPA in the form of triacylglycerides based on the total fatty acids in the transgenic plant.

7. The oils, lipids and/or fatty acids of claim 1, wherein said oils, lipids and/or fatty acids comprise a total amount of about 30% by weight of EPA and DHA in the form of triacylglycerides based on the total fatty acids in the transgenic plant.

8. The oils, lipids and/or fatty acids of claim 1, wherein said oils, lipids and/or fatty acids comprise a total amount of at least 54% by weight of polyunsaturated ω

3-fatty acids based on the total fatty acids in the transgenic plant.

17. The oils, lipids and/or fatty acids of claim 1, comprising:

a) at least 4% by weight of DHA and at least 2% by weight of DPA based on the total fatty acids in the transgenic plant;

b) at least 4% by weight of DHA and at least 20% by weight of EPA based on the total fatty acids in the transgenic plant;

or

c) at least 4% by weight of DHA, at least 2% by weight of DPA, and at least 20% by weight of EPA based on the total fatty acids in the transgenic plant.

18. A method for producing oils, lipids and/or fatty acids of claim 1, comprising expressing in a Brassica plant a nucleic acid encoding a Δ

6-desaturase, a nucleic acid encoding a Δ

5-desaturase, a nucleic acid encoding a Δ

6-elongase, a nucleic acid encoding a ω

3-desaturase, a nucleic acid encoding a Δ

5-elongase, and a nucleic acid encoding a Δ

4-desaturase, wherein said nucleic acid encoding a Δ

5-elongase is codon-optimized by adapting to the codon usage of Brassica.

19. The method of claim 18, wherein said nucleic acid encoding a Δ

5-elongase comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO;

64, and wherein said nucleotide sequence is obtained by adapting at least 30% of the nucleotide sequence of SEQ ID NO;

109 to the codon usage of Brassica.

20. The method of claim 19, wherein said nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO:

64, or wherein said nucleotide sequence encodes a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO;

65.

21. The method of claim 19, wherein said nucleotide sequence is adapted based on the natural frequency of individual codons.

22. The method of claim 19, wherein said nucleotide sequence is obtained by adapting at least 40% of the nucleotide sequence of SEQ ID NO:

109 to the codon usage of Brassica.

23. The method of claim 18, wherein said nucleic acid encoding a Δ

5-elongase comprises a translated section coding for a protein having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO;

65, and wherein the translated section comprises a nucleotide sequence obtained by adapting at least 30% or 40% of the nucleotide sequence of SEQ ID NO;

109 to the codon usage of Brassica.

9. Oils, lipids and/or fatty acids produced by a transgenic Brassica plant, wherein said oils, lipids and/or fatty acids comprise a total amount of at least 54% by weight of polyunsaturated ω

3-fatty acids based on the total fatty acids in the transgenic plant.

10. The oils, lipids and/or fatty acids of claim 9, wherein said oils, lipids and/or fatty acids comprise 60 to 85% by weight of polyunsaturated fatty acids based on the total fatty acids in the transgenic plant.

11. The oils, lipids and/or fatty acids of claim 9, wherein said polyunsaturated fatty acids comprise at least 20% by weight of EPA based on the total fatty acids in the transgenic plant in the form of triacylglycerides.

12. The oils, lipids and/or fatty acids of claim 9, wherein said polyunsaturated fatty acids comprise at least 2% by weight of DPA based on the total fatty acids in the transgenic plant in the form of triacylglycerides.

13. The oils, lipids and/or fatty acids of claim 9, wherein said polyunsaturated fatty acids comprise at least 4% by weight of DHA based on the total fatty acids in the transgenic plant in the form of triacylglycerides.

14. The oils, lipids and/or fatty acids of claim 9, wherein said polyunsaturated fatty acids comprise at least 30% long chain polyunsaturated fatty acids (LCPUFAs) based on the total fatty acids in the transgenic plant, and wherein the LCPUFAs are C20 and/or C22 fatty acid molecules having at least four double bonds.

15. The oils, lipids and/or fatty acids of claim 9, wherein said oils, lipids and/or fatty acids comprise about 25% by weight of EPA in the form of triacylglycerides based on the total fatty acids in the transgenic plant.

16. The oils, lipids and/or fatty acids of claim 9, wherein said oils, lipids and/or fatty acids comprise a total amount of about 30% by weight of EPA and DHA in the form of triacylglycerides based on the total fatty acids in the transgenic plant.