Carotenoid ketolase gene
AssigneeE.I. du Pont de Nemours and Company
InventorsQiong Cheng, Luan Tao
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Abstract
A ketolase gene has been isolated from Rhodococcus erythropolis AN12 strain encoding a carotenoid modification enzyme of the carotenoid biosynthetic pathway. The gene and gene product are the first isolated from a Rhodococcus strain. Six conserved amino acid motifs have been identified as the characteristic of this type of ketolase enzymes. The gene and gene product of the present invention may be used in a variety of ways for the production of keto-carotenoid compounds in a variety of organisms.
Claims35
An isolated nucleic acid molecule encoding a carotenoid ketolase enzyme, selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding an amino acid sequence containing all six conserved motifs as set forth in SEQ ID NOs:7,8,9,10, 11 and 12;
- (b) an isolated nucleic acid molecule encoding the amino acid sequence SEQ ID NO:2;
- (c) an isolated nucleic acid molecule that hybridizes with (a) or (b) under the following hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- an isolated nucleic acid molecule that is complementary to (a), or (b), wherein said isolated nucleic acid molecule is not SEQ ID NO 5 or SEQ ID NO:3.
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a carotenoid ketolase enzyme of at least 532 amino acids that has at least 70% 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 encoding a carotenoid ketolase enzyme, the enzyme having at least 70% identity based on the Smith-Waterman method of alignment to all of the amino acid sequences defining CrtO diagnostic motifs as set forth in SEQ ID NOs:7-12, provided the isolated nucleic acid molecule is not SEQ ID NO:5 or SEQ ID NO:3.
A method of obtaining a nucleic acid molecule encoding a carotenoid ketolase enzyme comprising:
- (a) synthesizing an at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3;
- and
- (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a);
- wherein the amplified insert encodes a carotenoid ketolase enzyme.
A method for obtaining a nucleic acid molecule encoding a carotenoid ketolase enzyme comprising:
- (a) providing nucleic acid probes encoding CrtO diagnostic motif sequences selected from the group consisting of SEQ ID NOs:7-12;
- (b) identifying a DNA clone that hybridizes with all of the probes of (a) under the following hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- and
- (c) sequencing the genomic fragment that comprises the clone identified in step (b),
- wherein the sequenced genomic fragment encodes a carotenoid ketolase enzyme.
A mutated gene encoding a carotenoid ketolase 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 carotenoid ketolase gene;
- b) a first population of nucleotide fragments which will hybridize to said native carotenoid ketolase gene;
- c) a second population of nucleotide fragments which will not hybridize to said native carotenoid ketoalse gene;
- 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 carotenoid ketoalse gene is produced encoding a protein having an altered biological activity.
Dependencies are inferred from the claim text.