Biological method for the production of adipic acid and intermediates
First Claim
Patent Images
1. An isolated nucleic acid fragment encoding an adipic acid synthesizing enzyme selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO;
12, SEQ ID NO;
20, SEQ ID NO;
24, and SEQ ID NO;
26, or an enzymatically active fragment thereof;
(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;
0.1×
SSC, 0.1% SDS at 65°
C.; and
washed with 2×
SSC, 0.1% SDS followed by 0.1×
SSC, 0.1% SDS; and
(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).
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Abstract
A gene cluster has been isolated from an Acinetobacter sp. that encodes the enzymes expected to convert cyclohexanol to adipic acid. The entire gene cluster has been cloned and all open reading frames have been sequenced. Cosmid clones have been identified containing the gene cluster. Demonstration of conversion of cyclohexanol to adipic acid has been made with the recombinant E. coli host strain containing the cosmids.
36 Citations
20 Claims
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1. An isolated nucleic acid fragment encoding an adipic acid synthesizing enzyme selected from the group consisting of:
-
(a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO;
12, SEQ ID NO;
20, SEQ ID NO;
24, and SEQ ID NO;
26, or an enzymatically active fragment thereof;
(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;
0.1×
SSC, 0.1% SDS at 65°
C.; and
washed with 2×
SSC, 0.1% SDS followed by 0.1×
SSC, 0.1% SDS; and
(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 18)
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10. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding an adipic acid synthesizing enzyme comprising:
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(a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO;
11, SEQ ID NO;
19, SEQ ID NO;
23, and SEQ ID NO;
25; and
(b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified insert encodes a portion of an amino acid sequence encoding an adipic acid synthesizing enzyme. - View Dependent Claims (11)
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12. A method for the production of adipic acid comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of cyclohexanol whereby adipic acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
20, SEQ ID NO;
24, and SEQ ID NO;
26 under the control of suitable regulatory sequences. - View Dependent Claims (13)
- contacting a transformed host cell under suitable growth conditions with an effective amount of cyclohexanol whereby adipic acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO;
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14. A method for the production of cyclohexanone comprising:
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(a) transforming a host cell with a nucleic acid fragment encoding the polypeptide as set forth in SEQ ID NO;
20 under the control of suitable regulatory sequences; and
(b) contacting said transformed cell of (a) with an effective amount of cyclohexanol whereby cyclohexanone is produced.
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15. A method for the production of 6-alcohol hexanoic acid comprising:
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(a) transforming a host cell with a nucleic acid fragment encoding the polypeptide as set forth in SEQ ID NO;
26 under the control of suitable regulatory sequences; and
(b) contacting said transformed cell of (a) with an effective amount of ε
-caprolactone whereby 6-alcohol hexanoic acid is produced.
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16. A method for the production of 6-aldehyde hexanoic acid comprising:
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(a) transforming a host cell with a nucleic acid fragment encoding the polypeptide as set forth in SEQ ID NO;
24 under the control of suitable regulatory sequences; and
(b) contacting said transformed cell of (a) with an effective amount of 6-alcohol hexanoic acid where by 6-aldehyde hexanoic acid is produced.
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17. A method for the production of adipic acid comprising:
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(a) transforming a host cell with a nucleic acid fragment encoding the polypeptide as set forth in SEQ ID NO;
12 under the control of suitable regulatory sequences; and
(b) contacting said transformed cell of (a) with an effective amount of 6-aldehyde hexanoic acid whereby adipic acid is produced.
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19. An isolated nucleic acid fragment encoding adipic acid synthesizing enzymes selected from the group consisting of:
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(a) an isolated nucleic acid molecule as set forth in SEQ ID NO;
27;
(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;
0.1×
SSC, 0.1% SDS at 65°
C.; and
washed with 2×
SSC, 0.1% SDS followed by 0.1×
SSC, 0.1% SDS;
(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).
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20. An Acinetobacter sp. comprising endogenous genes encoding the proteins as set forth by SEQ ID NO:
- 10, SEQ ID NO;
12, SEQ ID NO;
20, SEQ ID NO;
24, and SEQ ID NO;
26, said Acinetobacter sp. having the ability to convert cyclohexanol to adipic acid under suitable growth conditions.
- 10, SEQ ID NO;
Specification