Factors for the production and accumulation of polyunsaturated fatty acids (pufas) derived from pufa synthases

Patent metadata

Field	Value
Publication number	US-2016348083-A1
Application number	US-201515112885-A
Country	US
Kind code	A1
Filing date	Jan 28, 2015
Priority date	Jan 28, 2014
Publication date	Dec 1, 2016
Grant date	—

Abstract

Factors For the Production and Accumulation of Polyunsaturated Fatty Acids (PUFAs) Derived from PUFA Synthases Abstract Disclosed are novel enhancing factor proteins of the PUFA synthase systems, nucleic acid molecules encoding the same, recombinant nucleic acid molecules and recombinant host cells comprising such nucleic acid molecules, genetically modified microorganisms comprising the same, and methods of making and using the same. Also disclosed are genetically modified microorganisms that have been genetically modified to express a PUFA synthase system for the production of PUFAs, wherein the microorganisms have been modified to express the novel enhancing factor proteins of the PUFA synthase system.

First claim

What is claimed is:

1 . A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide that is at least 90% identical to an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

2 . The recombinant nucleic acid molecule of claim 1 , wherein the polypeptide is at least 95% identical to an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

3 . The recombinant nucleic acid molecule of claim 1 , wherein the polypeptide enhances the enzymatic activity of a PUFA synthase.

4 . The recombinant nucleic acid molecule of claim 3 , wherein said recombinant nucleic acid molecule comprises a nucleic acid sequence which is an enzymatically active fragment of SEQ ID NO:1 or SEQ ID NO:3.

5 . The recombinant nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encodes a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

6 . The recombinant nucleic acid molecule of claim 1 , wherein the nucleic acid sequence is SEQ ID NO:5 or SEQ ID NO:7.

7 . A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide that is at least 90% identical to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

8 . The recombinant nucleic acid molecule of claim 7 , wherein the polypeptide is at least 95% identical to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

9 . The recombinant nucleic acid molecule of claim 7 , wherein the polypeptide enhances the enzymatic activity of a PUFA synthase.

10 . The recombinant nucleic acid molecule of claim 9 , wherein said recombinant nucleic acid molecule comprises a nucleic acid sequence which is an enzymatically active fragment of SEQ ID NO:2 or SEQ ID NO:4.

11 . The recombinant nucleic acid molecule of claim 7 , wherein the nucleic acid sequence encodes a polypeptide having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

12 . The recombinant nucleic acid molecule of claim 7 , wherein the nucleic acid sequence is SEQ ID NO:6 or SEQ ID NO:8.

13 . An isolated protein encoded by the nucleic acid molecule of any one of claims 1 to 12 .

14 . A recombinant nucleic acid molecule, comprising the nucleic acid molecule according to any one of claims 1 to 12 , operatively linked to an expression control sequence.

15 . A recombinant host cell comprising the recombinant nucleic acid molecule of claim claim 14 .

16 . The recombinant host cell of claim 15 , wherein the host cell is a microorganism.

17 . A genetically modified microorganism, wherein the microorganism has been genetically modified to express the recombinant nucleic acid molecule of any one of claims 1 to 12 .

18 . A genetically modified microorganism, wherein the microorganism has been genetically modified to express at least one recombinant nucleic acid molecule of any one of claims 1 to 6 and at least one recombinant nucleic acid molecule of any one of claims 7 to 12 .

19 . The genetically modified microorganism of any one of claims 17 to 18 , wherein the organism endogenously expresses a PUFA synthase system, a phosphopantetheinyl transferase (PPTase), and/or an acyl-CoA synthetase (ACS).

20 . The genetically modified microorganism of claim 19 , wherein the microorganism is a Thraustochytriales microorganism.

21 . The genetically modified microorganism of claim 20 , wherein the microorganism is a Schizochytrium.

22 . The genetically modified microorganism of claim 19 , wherein the microorganism is a bacterium.

23 . The genetically modified microorganism of any one of claims 17 to 18 , wherein the organism has been further genetically modified to exogenously express a PUFA synthase system, a phosphopantetheinyl transferase (PPTase), and/or an acyl-CoA synthetase (ACS).

24 . The genetically modified microorganism of claim 23 , wherein the PUFA synthase system comprises at least one functional domain from a PUFA synthase system from a Thraustochytriales microorganism.

25 . The genetically modified microorganism of claim 24 , wherein the PUFA synthase system comprises at least one functional domain from a PUFA synthase system from a Schizochytrium.

26 . The genetically modified microorganism of claim 23 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from a microorganism selected from the group consisting of Schizochytrium sp. American Type Culture Collection (ATCC) No. 20888 , Schizochytrium sp. American Type Culture Collection (ATCC) No. PTA-9695 , Thraustochytrium 23B American Type Culture Collection (ATCC) No. 20892, and a mutant of any of said microorganisms.

27 . The genetically modified microorganism of claim 23 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from a marine bacterium.

28 . The genetically modified microorganism of claim 27 , wherein the microorganism is a microalga, a yeast, or a bacterium.

29 . The genetically modified microorganism of any one of claims 23 to 28 , wherein one or more nucleic acid sequences encoding the PUFA synthase has been optimized to improve the expression of the PUFA synthase in the microorganism.

30 . The genetically modified microorganism of any one of claims 19 to 29 , wherein the microorganism comprises at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of: DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), EPA (C20:5, n-3), ARA (C20:4, n-6), GLA (C18:3, n-6), and/or SDA (C18:4, n-3).

31 . The genetically modified microorganism of any one of claims 19 to 29 , wherein the genetically modified microorganism comprises DHA, DPAn-6 and/or EPA.

32 . The genetically modified microorganism of claim 18 , wherein the amount of DHA, DPAn-6 and/or EPA produced in said genetically modified microorganism is higher than that is produced in the counterpart microorganism which none of the recombinant nucleic acid molecule of claims 1 to 12 is expressed.

33 . The genetically modified microorganism of claim 18 , wherein the ratio of DHA:DPAn-6 produced in said genetically modified microorganism is higher than that produced in the counterpart microorganism which none of the recombinant nucleic acid molecule of claims 1 to 12 is expressed.

34 . The genetically modified microorganism of claim 32 or claim 33 , wherein the microorganism is a microalga, a yeast, or a bacterium.

35 . A genetically modified microorganism, wherein the microorganism has been genetically modified to delete or inactivate the nucleic acid molecule of any one of claims 1 to 12 expressed by the microorganism.

36 . The genetically modified microorganism of claim 35 , wherein the microorganism is a Thraustochytriales microorganism.

37 . The genetically modified microorganism of claim 3 , wherein the microorganism is a Schizochytrium.

38 . An oil obtained from the genetically modified microorganism of any one of claims 17 to 37 .

39 . A method to produce an oil comprising at least one polyunsaturated fatty acid (PUFA), comprising growing the microorganism of any one of claims 17 to 37 .

40 . An oil produced by the method of claim 39 .

Assignees

• Dsm Ip Assets Bv

Inventors

• Metz James George
• Kuner Jerry M
• Mccaskill David Glen
• Foster Mendy Louise

Classifications

• C12N15/79

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• A61K31/202

  having three or more double bonds, e.g. linolenic (eicosanoids, e.g. leukotrienes A61K31/557) · CPC title

• C12P7/6427

  Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone · CPC title

• C12Y301/01

  Carboxylic ester hydrolases (3.1.1) · CPC title