PUFA polyketide synthase systems and uses thereof
First Claim
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 Assignments
0 Petitions
Accused Products
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.
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Citations
86 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 16, 18, 28, 32, 35, 37, 39, 49, 50, 51, 52, 53, 54, 55, 57, 71, 72, 73, 75)
- a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of;
- 12. (canceled)
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13. (canceled)
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15. (canceled)
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17. (canceled)
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19-27. -27. (canceled)
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29-31. -31. (canceled)
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33. (canceled)
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34. (canceled)
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36. (canceled)
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38. (canceled)
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40-48. -48. (canceled)
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56. (canceled)
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58-61. -61. (canceled)
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64-70. -70. (canceled)
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74. (canceled)
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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:
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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. - View Dependent Claims (77, 78)
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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.
- C., and wherein the bacterial PUFA PKS system comprises;
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80-85. -85. (canceled)
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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.
- C., and wherein the bacterial PUFA PKS system comprises;
Specification