Genes involved in cyclododecanone degradation pathway
AssigneeE. I. Du Pont de Nemours and Company
InventorsMario W. Chen, Qiong Cheng, Katharine Janet Gibson, Kristy Nan-Shan Kostichka, Stuart M. Thomas, Vasantha Nagarajan
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Abstract
A 10 kb gene cluster has been isolated from Rhodococcus ruber SC1 comprising genes encoding enzymes useful for the synthesis of dodecanoic diacid from cyclododecanone and other cyclic intermediates. The six specific open reading frames have been identified that are associated with dodecanoic diacid biosynthesis. In addition to the expected substrates the enzymes of the instant invention have moderate specificity for C11-C15 compounds.
Claims33
An isolated nucleic acid fragment encoding a dodecanoic diacid synthesizing enzyme selected from the group consisting of:
- (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 10, and SEQ ID NO:
- 12;
- (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 10, and SEQ ID NO:
- 12;
- (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions:
- 6×SSC (1 M NaCl), 50% formamide, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. ;and
- (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c).
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an enzymatically active cyclododecanone monooxygenase of at least 603 amino acids that has at least 30% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 2 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an enzymatically active lauryl lactone esterase of at least 386 amino acids that has at least 41% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 4 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an enzymatically active 12-hydroxylauric acid dehydrogenase of at least 374 amino acids that has at least 49% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 10 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an enzymatically active 12-oxo lauric acid dehydrogenase of at least 474 amino acids that has at least 50% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 12 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
An isolated nucleic acid fragment having 80% identity to the nucleic acid fragment selected from the group consisting of SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 26, SEQ ID NO:
- 27, SEQ ID NO:
- 28, SEQ ID NO:
- 29, SEQ ID NO:
- 30, SEQ ID NO:
- 31, SEQ ID NO:
- 32, SEQ ID NO:
- 33, SEQ ID NO:
- 34, SEQ ID NO:
- 35, SEQ ID NO:
- 36, and SEQ ID NO:
- 37.
An isolated nucleic acid fragment having 90% identity to the nucleic acid fragment selected from the group consisting of SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 26, SEQ ID NO:
- 27, SEQ ID NO:
- 28, SEQ ID NO:
- 29, SEQ ID NO:
- 30, SEQ ID NO:
- 31, SEQ ID NO:
- 32, SEQ ID NO:
- 33, SEQ ID NO:
- 34, SEQ ID NO:
- 35, SEQ ID NO:
- 36, and SEQ ID NO:
- 37.
A method of obtaining a nucleic acid fragment encoding all or a substantial portion a dodecanoic diacid synthesizing enzyme 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:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 26, SEQ ID NO:
- 27, SEQ ID NO:
- 28, SEQ ID NO:
- 29, SEQ ID NO:
- 30, SEQ ID NO:
- 31, SEQ ID NO:
- 32, SEQ ID NO:
- 33, SEQ ID NO:
- 34, SEQ ID NO:
- 35, SEQ ID NO:
- 36, and SEQ ID NO:
- 37;
- and
- (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 a dodecanoic diacid synthesizing enzyme.
A method for the production of dodecanedioic acid comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of cyclododecanone whereby dodecanedioic acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 10, and SEQ ID NO:
- 12 under the control of suitable regulatory sequences.
A method for the production of lauryl lactone comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of cyclododecanone whereby lauryl lactone is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:
- 2, under the control of suitable regulatory sequences.
A method for the production of 12-hydroxy lauric acid comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of lauryl lactone whereby 12-hydroxy lauric acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:
- 4, under the control of suitable regulatory sequences.
A method for the production of 12-oxo lauric acid comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount 12-hydroxy lauric acid whereby 12-oxo lauric acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:
- 10, under the control of suitable regulatory sequences.
A method for the production of dodecanedioic acid comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount 12-oxo lauric acid whereby dodecanedioic acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:
- 12, under the control of suitable regulatory sequences.
An isolated nucleic acid fragment encoding a dodecanedioic acid synthesizing enzyme selected from the group consisting of:
- (a) an isolated nucleic acid molecule as set forth in SEQ ID NO:
- 13;
- (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions:
- 6×SSC (1 M NaCl), 50% formamide, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.;
- and
- (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).
A Rhodococcus ruber comprising endogenous genes encoding the proteins as set forth by SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 10, and SEQ ID NO:
- 12, said Rhodococcus ruber having the ability to convert cyclododecanone to dodecanedioic acid under suitable growth conditions.
A method for the production of hydroxy acids comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of a cyclic ketone having from six to eighteen carbon atoms, whereby the corresponding hydroxy acid is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:
- 2, and SEQ ID NO:
- 4 under the control of suitable regulatory sequences.
A method for the production of macrolactones having at least ten carbon atoms comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of a cyclic ketone having at least ten carbon atoms whereby the corresponding macrolactone is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:
- 2 under the control of suitable regulatory sequences.
A mutated gene encoding a dodecanedioic acid synthesizing enzyme having an altered biological activity produced by a method comprising the steps of:
- (i) digesting a mixture of nucleotide sequences with restriction endonucleases wherein said mixture comprises:
- (a) a native sequence encoding a dodecanedioic acid synthesizing enzyme selected from the group consisting of SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 10, and SEQ ID NO:
- 12;
- (b) a first population of nucleotide fragments which will hybridize to said native sequence;
- c) a second population of nucleotide fragments which will not hybridize to said native sequence;
- wherein a mixture of restriction fragments are produced;
- (ii) denaturing said mixture of restriction fragments;
- (iii) incubating the denatured said mixture of restriction fragments of step (ii) with a polymerase;
- (iv) repeating steps (ii) and (iii) wherein a mutated native sequence is produced encoding a dodecanedioic acid synthesizing enzyme having an altered biological activity.
Dependencies are inferred from the claim text.