PUFA polyketide synthase systems and uses thereof

• US 7,271,315 B2
• Filed: 06/12/2006
• Issued: 09/18/2007
• Est. Priority Date: 01/14/1999
• Status: Expired due to Fees

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Barclay, William R., Metz, James G., Kuner, Jerry M., Flatt, James H.
• Primary Examiner(s): Nashed; Nashaat T.
• Application Number:
  US11/452,138
• Publication Number:
  US 20070089199A1
• Time in Patent Office: 463 Days
• Field of Search: None
• US Class Current: 800/278
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• 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 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

Disclosed are the complete polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from Schizochytrium, and biologically active fragments and homologues thereof. More particularly, this invention relates to nucleic acids encoding such PUFA PKS systems, to proteins and domains thereof that comprise such PUFA PKS systems, to genetically modified organisms (plants and microorganisms) comprising such PUFA PKS systems, and to methods of making and using the PUFA PKS systems disclosed herein. This invention also relates to genetically modified plants and microorganisms and methods to efficiently produce lipids enriched in various polyunsaturated fatty acids (PUFAs) as well as other bioactive molecules by manipulation of a PUFA polyketide synthase (PKS) system.

Claims

1. A genetically modified plant or plant part, wherein cells of the plant are transfected with at least one recombinant nucleic acid molecule encoding a polyketide synthase (PKS) system for the production of polyunsaturated fatty acids (PUFAs) and a phosphopantetheine transferase, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

2. The genetically modified plant or plant part of claim 1, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 96% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 96% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 96% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

3. The genetically modified plant or plant part of claim 1, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 97% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 97% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 97% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

4. The genetically modified plant or plant part of claim 1, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

5. The genetically modified plant or plant part of claim 1, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 99% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 99% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 99% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

6. The genetically modified plant or plant part of claim 1, wherein said plant is a dicotyledonous plant.

7. The genetically modified plant or plant part of claim 1, wherein said plant is a monocotyledonous plant.

8. The genetically modified plant or plant part of claim 1, wherein said plant is selected from the group consisting of:

canola, soybean, rapeseed, linseed, corn, safflower, sunflower and tobacco.

9. The genetically modified plant or plant part of claim 1, wherein the plant is an oilseed plant and wherein the plant part is a mature oilseed.

10. The genetically modified plant or plant part of claim 1, wherein the total fatty acid profile in the plant or plant part comprises at least about 0.5% by weight of at least one PUFA selected from the group consisting of DHA (docosahexaenoic acid (C22:

6, n-3)) and DPA (docosapentaenoic acid (C22;

5, n-6), and wherein the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than about 10% of the total fatty acids produced by said plant or plant part.

11. The genetically modified plant or plant part of claim 10, wherein the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than 5% by weight of the total fatty acids produced by said plant or plant part.

12. The genetically modified plant or plant part of claim 10, wherein the fatty acids consisting of:

gamma-linolenic acid (GLA;

18;

3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds, comprise less than 5% by weight of the total fatty acids produced by said plant or plant part.

13. The genetically modified plant or plant part of claim 10, wherein gamma-linolenic acid (GLA;

18;

3, n-6) comprises less than 1% by weight of the total fatty acids produced by said plant or plant part.

14. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising growing the genetically modified plant or plant part of any one of claims 1 to 13, to produce at least one PUFA.

15. A method to produce a genetically modified plant that has a polyunsaturated fatty acid (PUFA) profile that differs from the naturally occurring plant and that produces at least one long chain polyunsaturated fatty acid (PUFA), comprising transfecting plant cells with at least one recombinant nucleic acid molecule encoding a polyketide synthase (PKS) system for the production of polyunsaturated fatty acids (PUFAs) and a phosphopantetheine transferase, and producing the plant from the plant cells, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

16. A recombinant plant cell, wherein the plant cell expresses at least one recombinant nucleic acid molecule encoding a polyketide synthase (PKS) system for the production of polyunsaturated fatty acids (PUFAs), wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

17. The recombinant plant cell of claim 16, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 96% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 96% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 96% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

18. The recombinant plant cell of claim 16, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 97% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 97% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 97% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

19. The recombinant plant cell of claim 16, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.

20. The recombinant plant cell of claim 16, wherein the PKS system comprises the following proteins:

a) a protein comprising an amino acid sequence that is at least 99% identical to SEQ ID NO;

2 and has β

-keto acyl-ACP synthase (KS) activity, malonyl-CoA;

ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO;

2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO;

2;

b) a protein comprising an amino acid sequence that is at least 990o identical to SEQ ID NO;

4 and has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO;

4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO;

4; and

c) a protein comprising an amino acid sequence that is at least 99% identical to SEQ ID NO;

6 and has FabA-like β

-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO;

6) at positions corresponding to amino acids 876-890 of SEQ ID NO;

6.