Genes involved in the biosynthesis of isopentenyl diphosphate in Hevea brasiliensis latex
AssigneeE.I. du Pont de Nemours and Company
InventorsDavid L. Hallahan, Natalie M. Keiper-Hrynko
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Abstract
The instant invention relates to bioproduction of isopentenyl diphosphate within the latex of the rubber tree, Hevea brasiliensis. Isopentenyl diphosphate (IPP) synthesis from acetate is mediated by a set of enzymes encoded by six genes isolated as expressed sequence tag (EST) cDNAs from Hevea brasiliensis latex. In addition, a variant sequence of one of these enzymes, acetyl-coA acetyltransferase, has been found.
Claims28
An isolated nucleic acid molecule encoding an isopentenyl diphosphate pathway enzyme, selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13;
- (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).
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an acetyl-coA acetyltransferase enzyme that has at least 65% identity over length of 411 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:8 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 HMG-coA synthase enzyme that has at least 82% identity over length of 464 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:9 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a mevalonate kinase enzyme that has at least 68% identity over length of 386 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:11 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 phosphomevalonate kinase enzyme that has at least 73% identity over length of 503 amino acids based on the CLUSTALW 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 molecule comprising a first nucleotide sequence encoding a mevalonate diphosphate decarboxylase enzyme that has at least 77% identity over length of 415 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:13 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
A method of obtaining a nucleic acid molecule encoding an isopentenyl diphosphate pathway 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:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;
- 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 an isopentenyl diphosphate pathway enzyme.
A method for the production of a compound in the isopentenyl diphosphate pathway comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of a carbon substrate whereby a compound in the isopentenyl diphosphate pathway is produced, said transformed host cell comprising a set of nucleic acid molecules encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 under the control of suitable regulatory sequences.
A method of regulating isopentenyl diphosphate biosynthesis in an organism comprising, over-expressing at least one isopentenyl diphosphate gene selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in an organism such that isopentenyl diphosphate biosynthesis is altered in the organism.
A mutated gene encoding an isopentenyl diphosphate pathway 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 isopentenyl diphosphate gene;
- b) a first population of nucleotide fragments which will hybridize to said native isopentenyl diphosphate gene;
- c) a second population of nucleotide fragments which will not hybridize to said native isopentenyl diphosphate 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 isopentenyl diphosphate gene is produced encoding a protein having an altered biological activity.
An isolated nucleic acid molecule encoding an HMG-coA reductase enzyme having nucleotide sequence as set forth in SEQ ID NO:3.
Dependencies are inferred from the claim text.