Method for production of asymmetric carotenoids
AssigneeE. I. Du Pont de Nemours and Company
InventorsQiong Cheng, Luan Tao
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Abstract
Genes have been isolated from Rhodococcus and Deinococcus which encode a specific lycopene β-cyclase capable of converting acyclic carotenoids with at least one ψ-end group to the corresponding asymmetric carotenoid containing a single β-ionone ring end group. The genes are new. Transformed host cells expressing the present genes and methods for the bio-conversion of acylic carotenoid substrates to corresponding asymmetric carotenoid are also provided.
Claims20
A method for the production of asymmetric carotenoid compounds comprising:
- a) providing a host cell which produces acyclic carotenoids having at least one ψ-end group wherein said host cell expresses a lycopene β-cyclase selected from the group consisting of:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4;
- (ii) an isolated nucleic acid molecule that hybridizes with (i) 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
- (iii) an isolated nucleic acid molecule that encodes a polypeptide having at least 70% identity to the polypeptides selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4, based on the Smith-Waterman method of alignment;
- b) growing the host cell of (a) under conditions whereby asymmetric carotenoids are produced;
- and
- c) Optionally recovering said asymmetric carotenoids.
A method for the production of asymmetric carotenoid compounds comprising:
- a) providing a host cell wherein said host cell expresses a lycopene β-cyclase selected from the group consisting of:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4;
- (ii) an isolated nucleic acid molecule that hybridizes with (i) 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
- (iii) an isolated nucleic acid molecule that encodes a polypeptide having at least 70% identity to the polypeptides selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4, based on the Smith-Waterman method of alignment;
- b) contacting the host cell of (a) with an acyclic carotenoid comprising least one ψ-end group;
- c) growing the host cell of (b) under conditions whereby asymmetric carotenoids are produced;
- and
- d) optionally recovering said asymmetric carotenoids.
A method of regulating asymmetric carotenoid biosynthesis in an organism comprising:
- (a) providing a host cell which produces acyclic carotenoids with at least one ψ-end group;
- (b) introducing into the host cell of (a) a lycopene β-cyclase gene selected from the group consisting of:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NO:
- and SEQ ID NO:
- 4;
- (ii) an isolated nucleic acid molecule that hybridizes with (i) 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
- (iii) an isolated nucleic acid molecule that encodes a polypeptide having at least 70% identity to the polypeptides selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4, based on the Smith-Waterman method of alignment;
- (c) growing the host cell of (b) under conditions whereby the lycopene β-cyclase gene is expressed and asymmetric carotenoid biosynthesis is regulated.
A method for the identification of a gene encoding a lycopene β-cyclase which catalyzes the production of asymmetric carotenoids from acyclic carotenoids having at least one ψ-end group comprising:
- (a) probing a genomic library with a portion of a nucleic acid molecule selected from the group consisting of:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO:
- 4;
- (ii) an isolated nucleic acid molecule that hybridizes with (i) 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
- (iii) an isolated nucleic acid molecule that encodes a polypeptide having at least 70% identity to the polypeptides selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4, based on the Smith-Waterman method of alignment;
- (b) identifying a DNA clone that hybridizes with the isolated nucleic acid molecule of (a);
- and
- (c) sequencing the genomic fragment that comprises the clone identified in step (b),
- wherein the sequenced genomic fragment encodes a carotenoid biosynthetic enzyme.
A method for the identification of a gene encoding a lycopene β-cyclase which catalyzes the production of asymmetric carotenoids from acyclic carotenoids having at least one ψ-end group comprising:
- (a) synthesizing an at least one oligonucleotide primer corresponding to a portion of a sequence selected from the group consisting of:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4;
- (ii) an isolated nucleic acid molecule that hybridizes with (i) 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
- (iii) an isolated nucleic acid molecule that encodes a polypeptide having at least 70% identity to the polypeptides selected from the group consisting of SEQ ID NO:
- 2 and SEQ ID NO:
- 4, based on the Smith-Waterman method of alignment;
- 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 carotenoid biosynthetic enzyme.
Dependencies are inferred from the claim text.