PUFA polyketide synthase systems and uses thereof

• US 20050100995A1
• Filed: 10/13/2004
• Published: 05/12/2005
• Est. Priority Date: 01/14/1999
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Weaver, Craig A., Zirkle, Ross, Metz, James G.
• Granted Patent: US 7,217,856 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C07K 14/195 from bacteria
• 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 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/62 Carboxylic acid esters
• 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 7/6472 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 the bacterial microorganisms Shewanella japonica and Shewanella olleyana, 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. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of;

SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

1, and SEQ ID NO;

12;

a) a nucleic acid sequence encoding a fragment of any of the amino acid sequences of (a) having at least one biological activity selected from the group consisting of enoyl-ACP reductase (ER) activity;

acyl carrier protein (ACP) activity;

β

-ketoacyl-ACP synthase (KS) activity;

acyltransferase (AT) activity;

β

-ketoacyl-ACP reductase (KR) activity;

FabA-like β

-hydroxyacyl-ACP dehydrase (DH) activity;

non-FabA-like dehydrase activity;

chain length factor (CLF) activity;

malonyl-CoA;

ACP acyltransferase (MAT) activity; and

4′

-phosphopantetheinyl transferase (PPTase) activity;

b) a nucleic acid sequence encoding an amino acid sequence that is at least about 65% identical to SEQ ID NO;

2 or SEQ ID NO;

8 and has at least one biological activity selected from the group consisting of;

KS activity, MAT activity, KR activity, ACP activity, and non-FabA-like dehydrase activity;

c) a nucleic acid sequence encoding an amino acid sequence that is at least about 60% identical to SEQ ID NO;

3 or SEQ ID NO;

9 and has AT biological activity;

d) a nucleic acid sequence encoding an amino acid sequence that is at least about 70% identical to SEQ ID NO;

4 or SEQ ID NO;

10 and has at least one biological activity selected from the group consisting of KS activity, CLF activity and DH activity;

e) a nucleic acid sequence encoding an amino acid sequence that is at least about 60% identical to SEQ ID NO;

6 or SEQ ID NO;

12 and has PPTase biological activity;

f) a nucleic acid sequence encoding an amino acid sequence that is at least about 85% identical to SEQ ID NO;

1 or at least about 95% identical to SEQ ID NO;

5 and has ER biological activity.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence is selected from the group consisting of:

a) a nucleic acid sequence encoding an amino acid sequence that is at least about 75% identical to SEQ ID NO;

2 or SEQ ID NO;

8 and has at least one biological activity selected from the group consisting of;

KS activity, MAT activity, KR activity, ACP activity, and non-FabA-like dehydrase activity;

b) a nucleic acid sequence encoding an amino acid sequence that is at least about 70% identical to SEQ ID NO;

3 or SEQ ID NO;

9 and has AT biological activity;

c) a nucleic acid sequence encoding an amino acid sequence that is at least about 80% identical to SEQ ID NO;

4 or SEQ ID NO;

10 and has at least one biological activity selected from the group consisting of KS activity, CLF activity and DH activity;

d) a nucleic acid sequence encoding an amino acid sequence that is at least about 70% identical to SEQ ID NO;

6 or SEQ ID NO;

12 and has PPTase biological activity;

e) a nucleic acid sequence encoding an amino acid sequence that is at least about 95% identical to SEQ ID NO;

11 or at least about 96% identical to SEQ ID NO;

5 and has ER biological activity.

3. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence is selected from the group consisting of:

a) a nucleic acid sequence encoding an amino acid sequence that is at least about 85% identical to SEQ ID NO;

2 or SEQ ID NO;

8 and has at least one biological activity selected from the group consisting of;

KS activity, MAT activity, KR activity, ACP activity, and non-FabA-like dehydrase activity;

b) a nucleic acid sequence encoding an amino acid sequence that is at least about 80% identical to SEQ ID NO;

3 or SEQ ID NO;

9 and has AT biological activity;

c) a nucleic acid sequence encoding an amino acid sequence that is at least about 90% identical to SEQ ID NO;

4 or SEQ ID NO;

10 and has at least one biological activity selected from the group consisting of KS activity, CLF activity and DH activity;

d) a nucleic acid sequence encoding an amino acid sequence that is at least about 80% identical to SEQ ID NO;

6 or SEQ ID NO;

12 and has PPTase biological activity;

e) a nucleic acid sequence encoding an amino acid sequence that is at least about 96% identical to SEQ ID NO;

11 or at least about 97% identical to SEQ ID NO;

5 and has ER biological activity.

4. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence is selected from the group consisting of:

a) a nucleic acid sequence encoding an amino acid sequence that is at least about 95% identical to SEQ ID NO;

2 or SEQ ID NO;

8 and has at least one biological activity selected from the group consisting of;

KS activity, MAT activity, KR activity, ACP activity, and non-FabA-like dehydrase activity;

b) a nucleic acid sequence encoding an amino acid sequence that is at least about 90% identical to SEQ ID NO;

3 or SEQ ID NO;

9 and has AT biological activity;

c) a nucleic acid sequence encoding an amino acid sequence that is at least about 95% identical to SEQ ID NO;

4 or SEQ ID NO;

10 and has at least one biological activity selected from the group consisting of KS activity, CLF activity and DH activity;

d) a nucleic acid sequence encoding an amino acid sequence that is at least about 90% identical to SEQ ID NO;

6 or SEQ ID NO;

12 and has PPTase biological activity;

e) a nucleic acid sequence encoding an amino acid sequence that is at least about 97% identical to SEQ ID NO;

11 or at least about 98% identical to SEQ ID NO;

5 and has ER biological activity.

5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of:

a) an amino acid sequence selected from the group consisting of;

SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12; and

b) a fragment of any of the amino acid sequences of (a) having at least one biological activity selected from the group consisting of enoyl-ACP reductase (ER) activity;

acyl carrier protein (ACP) activity;

β

-ketoacyl-ACP synthase (KS) activity;

acyltransferase (AT) activity;

β

-ketoacyl-ACP reductase (KR) activity;

FabA-like β

-hydroxyacyl-ACP dehydrase (DH) activity;

non-FabA-like dehydrase activity;

chain length factor (CLF) activity;

malonyl-CoA;

ACP acyltransferase (MAT) activity; and

4′

-phosphopantetheinyl transferase (PPTase) activity.

6. The isolated nucleic acid molecule of claim 1, wherein the fragment set forth in (b) is selected from the group consisting of:

a) a fragment of SEQ ID NO;

2 from about position 29 to about position 513 of SEQ ID NO;

2, wherein the domain has KS biological activity;

b) a fragment of SEQ ID NO;

2 from about position 625 to about position 943 of SEQ ID NO;

2, wherein the domain has MAT biological activity;

c) a fragment of SEQ ID NO;

2 from about position 1264 to about position 1889 of SEQ ID NO;

2, and subdomains thereof, wherein the domain or subdomain thereof has ACP biological activity;

d) a fragment of SEQ ID NO;

2 from about position 2264 to about position 2398 of SEQ ID NO;

2, wherein the domain has KR biological activity;

e) a fragment of SEQ ID NO;

2 comprising from about position 2504 to about position 2516 of SEQ ID NO;

2, wherein the fragment has non-FabA-like dehydrase biological activity;

f) a fragment of SEQ ID NO;

3 from about position 378 to about position 684 of SEQ ID NO;

3, wherein the domain has AT biological activity;

g) a fragment of SEQ ID NO;

4 from about position 5 to about position 483 of SEQ ID NO;

4, wherein the domain has KS biological activity;

h) a fragment of SEQ ID NO;

4 from about position 489 to about position 771 of SEQ ID NO;

4, wherein the domain has CLF biological activity;

i) a fragment of SEQ ID NO;

4 from about position 1428 to about position 1570 of SEQ ID NO;

4, wherein the domain has DH biological activity;

j) a fragment of SEQ ID NO;

4 from about position 1881 to about position 2019 of SEQ ID NO;

4, wherein the domain has DH biological activity;

k) a fragment of SEQ ID NO;

5 from about position 84 to about position 497 of SEQ ID NO;

5, wherein the domain has ER biological activity;

l) a fragment of SEQ ID NO;

6 from about position 40 to about position 186 of SEQ ID NO;

6, wherein the domain has PPTase biological activity;

m) a fragment of SEQ ID NO;

8 from about position 29 to about position 513 of SEQ ID NO;

8, wherein the domain has KS biological activity;

n) a fragment of SEQ ID NO;

8 from about position 625 to about position 943 of SEQ ID NO;

8, wherein the domain has MAT biological activity;

o) a fragment of SEQ ID NO;

8 from about position 1275 to about position 1872 of SEQ ID NO;

8, and subdomains thereof, wherein the domain or subdomain thereof has ACP biological activity;

p) a fragment of SEQ ID NO;

8 from about position 2240 to about position 2374 of SEQ ID NO;

8, wherein the domain has KR biological activity;

q) a fragment of SEQ ID NO;

8 comprising from about position 2480-2492 of SEQ ID NO;

8, wherein the fragment has non-FabA-like dehydrase activity;

r) a fragment of SEQ ID NO;

9 from about position 366 to about position 703 of SEQ ID NO;

9, wherein the domain has AT biological activity;

s) a fragment of SEQ ID NO;

10 from about position 10 to about position 488 of SEQ ID NO;

10, wherein the domain has KS biological activity;

t) a fragment of SEQ ID NO;

10 from about position 502 to about position 750 of SEQ ID NO;

10, wherein the domain has CLF biological activity;

u) a fragment of SEQ ID NO;

10 from about position 1431 to about position 1573 of SEQ ID NO;

10, wherein the domain has DH biological activity;

v) a fragment of SEQ ID NO;

10 from about position 1882 to about position 2020 of SEQ ID NO;

10, wherein the domain has DH biological activity;

w) a fragment of SEQ ID NO;

11 from about position 84 to about position 497 of SEQ ID NO;

1, wherein the domain has ER biological activity; and

x) a fragment of SEQ ID NO;

12 from about position 29 to about position 177 of SEQ ID NO;

12, wherein the domain has PPTase biological activity.

7. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of:

SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

1, and SEQ ID NO;

12.

8. An isolated nucleic acid molecule consisting essentially of a nucleic acid sequence that is fully complementary to the nucleic acid molecule of claim 1.

9. A recombinant nucleic acid molecule comprising the nucleic acid molecule of claim 1, operatively linked to at least one expression control sequence.

10. A recombinant cell transfected with the recombinant nucleic acid molecule of claim 9.

11. A genetically modified plant or a part of the plant, wherein the plant has been genetically modified to recombinantly express a PKS system comprising at least one biologically active protein or domain thereof of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system, wherein the protein or domain is encoded by a nucleic acid molecule of claim 1.

14. The genetically modified plant or part of the plant of claim 11, wherein the plant is genetically modified to recombinantly express at least one nucleic acid molecule encoding SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, and SEQ ID NO;

6.

16. The genetically modified plant or part of the plant of claim 11, wherein the plant is genetically modified to recombinantly express at least one nucleic acid molecule encoding SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12.

18. The genetically modified plant or part of a plant of claim 11, wherein the plant is additionally genetically modified to express at least one biologically active protein or domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system from a Thraustochytrid.

28. The genetically modified plant or part of a plant of claim 11, wherein the plant is a crop plant.

32. A genetically modified microorganism, wherein the microorganism has been genetically modified to recombinantly express an isolated nucleic acid molecule according to claim 1.

35. The genetically modified microorganism of claim 32, wherein the microorganism is genetically modified to recombinantly express at least one nucleic acid molecule encoding SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, and SEQ ID NO;

6.

37. The genetically modified microorganism of claim 32, wherein the microorganism is genetically modified to recombinantly express at least one nucleic acid molecule encoding SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12.

39. The genetically modified microorganism of claim 32, wherein the microorganism is additionally genetically modified to express at least one biologically active protein or domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system from a Thraustochytrid.

49. The genetically modified microorganism of claim 32, wherein the microorganism comprises an endogenous PUFA PKS system.

50. The genetically modified microorganism of claim 49, wherein the endogenous PUFA PKS system has been modified by substitution of another isolated nucleic acid molecule encoding at least one domain of a different PKS system for a nucleic acid sequence encoding at least one domain of the endogenous PUFA PKS system.

51. The genetically modified microorganism of claim 50, wherein the different PKS system is selected from the group consisting of a non-bacterial PUFA PKS system, a bacterial PUFA PKS system, a type I modular PKS system, a type I iterative PKS system, a type II PKS system, and a type III PKS system.

52. The genetically modified microorganism of claim 49, wherein the endogenous PUFA PKS system has been genetically modified by substitution of the isolated nucleic acid molecule according to claim 1 for a nucleic acid sequence encoding at least one domain of the endogenous PUFA PKS system.

53. The genetically modified microorganism of claim 49, wherein the microorganism has been genetically modified to recombinantly express a nucleic acid molecule encoding a chain length factor, or a chain length factor plus a β

-ketoacyl-ACP synthase (KS) domain, that directs the synthesis of C20 units.

54. The genetically modified microorganism of claim 49, wherein the endogenous PUFA PKS system has been modified in a domain or domains selected from the group consisting of a domain encoding FabA-like β

-hydroxy acyl-ACP dehydrase (DH) domain and a domain encoding β

-ketoacyl-ACP synthase (KS), wherein the modification alters the ratio of long chain fatty acids produced by the PUFA PKS system as compared to in the absence of the modification.

55. The genetically modified microorganism of claim 54, wherein the modification comprises substituting a DH domain that does not possess isomerization activity for a FabA-like β

-hydroxy acyl-ACP dehydrase (DH) in the endogenous PUFA PKS system.

57. The genetically modified microorganism of claim 49, wherein the endogenous PUFA PKS system has been modified in an enoyl-ACP reductase (ER) domain, wherein the modification results in the production of a different compound as compared to in the absence of the modification.

71. A method to produce a plant that has a polyunsaturated fatty acid (PUFA) profile that differs from the naturally occurring plant, comprising genetically modifying cells of the plant to express a PKS system comprising at least one recombinant nucleic acid molecule according to claim 9.

72. A method to produce a recombinant microbe, comprising genetically modifying microbial cells to express at least one recombinant nucleic acid molecule according to claim 9.

73. A method to modify an endproduct to contain at least one fatty acid, comprising adding to the endproduct an oil produced by a recombinant host cell that expresses at least one recombinant nucleic acid molecule according to claim 9.

75. A method to produce a humanized animal milk, comprising genetically modifying milk-producing cells of a milk-producing animal with at least one recombinant nucleic acid molecule according to claim 9.

12. (canceled)

62. A method to produce a bioactive molecule that is produced by a polyketide synthase system, comprising growing under conditions effective to produce the bioactive molecule, a genetically modified plant according to claim 12.

13. (canceled)

15. (canceled)

17. (canceled)

19-27. -27. (canceled)

29-31. -31. (canceled)

33. (canceled)

34. (canceled)

36. (canceled)

38. (canceled)

40-48. -48. (canceled)

56. (canceled)

58-61. -61. (canceled)

64-70. -70. (canceled)

74. (canceled)

76. A recombinant host cell which has been modified to express a recombinant bacterial polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system, wherein the PUFA PKS catalyzes both iterative and non-iterative enzymatic reactions, and wherein the PUFA PKS system comprises:

a) at least one enoyl ACP-reductase (ER) domain;

b) at least six acyl carrier protein (ACP) domains;

c) at least two β

-keto acyl-ACP synthase (KS) domains;

d) at least one acyltransferase (AT) domain;

e) at least one ketoreductase (KR) domain;

f) at least two FabA-like β

-hydroxy acyl-ACP dehydrase (DH) domains;

g) at least one chain length factor (CLF) domain;

h) at least one malonyl-CoA;

ACP acyltransferase (MAT) domain; and

i) at least one 4′

-phosphopantetheinyl transferase (PPTase) domain; and

wherein the PUFA PKS system produces PUFAs at temperatures of at least about 25°

C.

77. The recombinant host cell of claim 76, wherein the PUFA PKS system comprises:

a) one enoyl ACP-reductase (ER) domain;

b) six acyl carrier protein (ACP) domains;

c) two β

-keto acyl-ACP synthase (KS) domains;

d) one acyltransferase (AT) domain;

e) one ketoreductase (KR) domain;

f) two FabA-like β

-hydroxy acyl-ACP dehydrase (DH) domains;

g) one chain length factor (CLF) domain;

h) one malonyl-CoA;

ACP acyltransferase (MAT) domain; and

i) one 4′

-phosphopantetheinyl transferase (PPTase) domain.

78. The recombinant host cell of claim 76, wherein the PUFA PKS system is a PUFA PKS system from a marine bacterium selected from the group consisting of Shewanella japonica and Shewanella olleyana.

79. A genetically modified organism comprising at least one protein or domain of a bacterial polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system, wherein the bacterial PUFA PKS system catalyzes both iterative and non-iterative enzymatic reactions, wherein the bacterial PUFA PKS system produces PUFAs at temperatures of at least about 25°

C., and wherein the bacterial PUFA PKS system comprises;

a) at least one enoyl ACP-reductase (ER) domain;

b) at least six acyl carrier protein (ACP) domains;

c) at least two β

-keto acyl-ACP synthase (KS) domains;

d) at least one acyltransferase (AT) domain;

e) at least one ketoreductase (KR) domain;

f) at least two FabA-like β

-hydroxy acyl-ACP dehydrase (DH) domains;

g) at least one chain length factor (CLF) domain;

h) at least one malonyl-CoA;

ACP acyltransferase (MAT) domain; and

i) at least one 4′

-phosphopantetheinyl transferase (PPTase) domain; and

wherein the genetic modification affects the activity of the PUFA PKS system.

80-85. -85. (canceled)

86. An isolated recombinant nucleic acid molecule encoding at least one protein or functional domain of a bacterial (PUFA) polyketide synthase (PKS) system, wherein the bacterial PUFA PKS system catalyzes both iterative and non-iterative enzymatic reactions, wherein the bacterial PUFA PKS system produces PUFAs at temperatures of at least about 25°

C., and wherein the bacterial PUFA PKS system comprises;

a) at least one enoyl ACP-reductase (ER) domain;

b) at least six acyl carrier protein (ACP) domains;

c) at least two β

-keto acyl-ACP synthase (KS) domains;

d) at least one acyltransferase (AT) domain;

e) at least one ketoreductase (KR) domain;

f) at least two FabA-like β

-hydroxy acyl-ACP dehydrase (DH) domains;

g) at least one chain length factor (CLF) domain;

h) at least one malonyl-CoA;

ACP acyltransferase (MAT) domain; and

i) at least one 4′

-phosphopantetheinyl transferase (PPTase) domain.