Genes involved in isoprenoid compound production
AssigneeE.I. du Pont de Nemours and Company
InventorsMichael G. Bramucci, Patricia C. Brzostowicz, Qiong Cheng, Kristy N. Kostichka, Pierre E. Rouviere, Vasantha Nagarajan, Luan Tao, Stuart M. Thomas
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Abstract
Genes have been isolated from Rhodococcus erythropolis AN12 strain encoding the isoprenoid biosynthetic pathway. The genes and gene products are the first isolated from a Rhodococcus strain. The genes and gene products of the present invention may be used in a variety of ways for the production of isoprenoid compounds in a variety of organisms.
Claims34
An isolated nucleic acid molecule selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding an isoprenoid biosynthetic enzyme, having an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18 and 20;
- (b) an isolated nucleic acid molecule encoding an isoprenoid biosynthetic enzyme, that hybridizes with (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;
- or
- an isolated nucleic acid molecule that is complementary to (a), or (b).
An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 648 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 comprising a first nucleotide sequence encoding a polypeptide of at least 385 amino acids that has at least 71% 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 a polypeptide of at least 232 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:6 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 polypeptide of at least 311 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: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 a polypeptide of at least 158 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: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 a polypeptide of at least 344 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: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 polypeptide of at least 378 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:14 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 polypeptide of at least 314 amino acids of 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:16 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 polypeptide of at least 530 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:18 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 polypeptide of at least 376 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:20 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
A method of obtaining a nucleic acid molecule encoding an isoprenoid biosynthetic 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 NOs:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19;
- 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 isoprenoid biosynthetic enzyme.
A method for the production of isoprenoid compounds comprising:
- contacting a transformed host cell under suitable growth conditions with an effective amount of a fermentable carbon substrate whereby an isoprenoid compound is produced, said transformed host cell comprising a set of nucleic acid molecules encoding SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 under the control of suitable regulatory sequences.
A method of regulating isoprenoid biosynthesis in an organism comprising, over-expressing at least one isoprenoid gene selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 in an organism such that the isoprenoid biosynthesis is altered in the organism.
A mutated gene encoding a isoprenoid 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 isoprenoid gene;
- b) a first population of nucleotide fragments which will hybridize to said native isoprenoid gene;
- c) a second population of nucleotide fragments which will not hybridize to said native isoprenoid gene;
- wherein a mixture of restriction fragments is 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 isoprenoid gene is produced encoding a protein having an altered biological activity.
Dependencies are inferred from the claim text.