Elaiophylin biosynthetic gene cluster
AssigneeWyeth
InventorBradley Haltli
Life of this patent
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Abstract
The present invention relates especially to a DNA fragment that is obtainable from the gene cluster within the genome of streptomycete that is responsible for elaiophylin biosynthesis and that contains at least one gene or a part of a gene that codes for a polypeptide that is involved directly or indirectly in the biosynthesis of elaiophylin and to methods of preparing said DNA fragment. The present invention relates furthermore to recombinant DNA molecules containing one of the DNA fragments according to the invention and to the plasmids and vectors derived therefrom. Also included are host organisms transformed with the said plasmid or vector DNA.
Claims34
An isolated nucleic acid that comprises a sequence at least about 70% identical to the nucleotide sequence of an orf of the elaiophylin gene cluster of Streptomyces sp. NRRL 30748 having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or the complement thereof.
An isolated nucleic acid that comprises a sequence that encodes the amino acid sequence of an orf of the elaiophylin gene cluster of Streptomyces sp. NRRL 30748 having SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:48.
An isolated polypeptide comprising an amino acid sequence at least about 70% homologous to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, EQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:48.
An oligonucleotide that specifically hybridizes to a DNA molecule having the nucleotide sequence of SEQ ID NO:49 or the complement thereof.
A method of identifying a nucleic acid that encodes a polyketide synthase which comprises contacting the nucleic acid with an oligonucleotide that specifically binds to a nucleic acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and the complementary sequences thereof and detecting specific hybridization.
A method of inhibiting elaiophylin biosynthesis comprising disrupting an orf which encodes a polypeptide associated with elaiophylin biosynthesis.
A method of making an elaiophylin analog comprising substituting a polyketide synthase orf into an elaiophylin biosynthetic gene cluster.
A method of recombinantly substituting a nucleic acid into the elaiophylin biosynthetic cluster of a Streptomyces comprising:
- providing a host cell containing a plasmid which comprises the nucleic acid and can be conjugally transferred into the Streptomyces and selected,
- contacting the host cell with spores of the Streptomyces, and
- selecting the exconjugants.
A method of making elaiophylin comprising incubating Streptomyces sp. NRRL 30748 in a culture medium under conditions suitable for expression of elaiophylin and recovering elaiophylin from the culture medium.
A strain of Streptomyces sp. NRRL 30748 in which a polyketide synthase gene of the elaiophylin biosynthetic gene cluster is disrupted.
A method of catalyzing formation of a cyclic polyketide which comprises incorporating the thioesterase domain of the polyketide synthase of SEQ ID NO:22 into a heterologous polyketide biosynthetic gene cluster.
The method of catalyzing formation of a cyclic polyketide which comprises incorporating the polyketide synthase of SEQ ID NO:22 into a heterologous polyketide biosynthetic gene cluster.
Dependencies are inferred from the claim text.