Oxidation of a cyclohexanone derivative using a brevibacterium cyclohexanone monooxygenase
AssigneeE. I. Du Pont de Nemours and Company
InventorsPatricia C. Brzostowicz, Pierre E. Rouviere
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Abstract
Two gene clusters have been isolated from an Brevibacterium sp HCU that encode the enzymes expected to convert cyclohexanol to adipic acid. Individual open reading frames (ORF's) on each gene cluster are useful for the production of intermediates in the adipic acid biosynthetic pathway or of related molecules. All the ORF's have been sequenced. Identification of gene function has been made on the basis of sequence comparison and biochemical analysis.
Claims21
An isolated nucleic acid fragment encoding an adipic acid synthesizing protein selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24;
- (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).
A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding an adipic acid synthesizing protein comprising:
- (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23;
- (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a),
- wherein the amplified insert encodes a portion of an amino acid sequence encoding an adipic acid synthesizing enzyme.
A method for the production of adipic acid comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of substrate selected from the group consisting of, cyclohexanol, cyclohexanone, 1-caprolactone, 6-hydroxy hexanoic acid, 6-aldehyde and hexanoic acid, whereby adipic acid is produced, said transformed host cell containing the nucleic acid fragments as set forth in SEQ ID NO:15 and SEQ ID NO:16.
A method for the production of cyclohexanone comprising:
- contacting a transformed cell with an effective amount of cyclohexanol whereby cyclohexanone is produced, the transformed cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of, SEQ ID NO:12 and SEQ ID NO:14 operably linked to suitable regulatory sequences.
A method for the production of ε-caprolactone comprising:
- contacting a transformed cell with an effective amount of cyclohexanone whereby ε-caprolactone is produced, the transformed cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of, SEQ ID NO:6 and SEQ ID NO:22 operably linked to suitable regulatory sequences.
A method for the production of 6-hydroxy hexanoic acid comprising:
- contacting a transformed cell with an effective amount of ε-caprolactone whereby 6-hydroxy hexanoic acid is produced, the transformed cell comprising a nucleic acid fragment encoding the polypeptide as set forth in, SEQ ID NO:4 operably linked to suitable regulatory sequences.
A method for the production of 6-aldehyde hexanoic acid comprising:
- contacting a transformed cell with an effective amount of 6-hydroxy hexanoic acid whereby 6-aldehyde hexanoic acid is produced, the transformed cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of SEQ ID NO:18 and SEQ ID NO:8, operably linked to suitable regulatory sequences.
A method for the oxidation of a cyclohexanone derivative comprising:
- contacting a transformed cell with an effective amount of a cyclohexanone derivative whereby oxidation of the derivative occurs, the transformed cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:22 operably linked to suitable regulatory sequences.
An isolated nucleic acid fragment encoding adipic acid synthesizing proteins selected from the group consisting of:
- (a) an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:16;
- (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).
A Brevibacterium sp. comprising the 16s rDNA sequence as set forth in SEQ ID NO:49.
Dependencies are inferred from the claim text.