Cloning genes from Streptomyces avermitilis for avermectin biosynthesis and the methods for their use
AssigneeMerck & Co., Inc.
InventorsKeith M. Gewain, Douglas J. MacNeil, Tanya MacNeil, Philip S. Paress, Carolyn L. Ruby, Stanley L. Streicher
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Abstract
There are disclosed plasmids containing DNA isolated from Streptomyces avermitilis, the microorganism which is used to prepare avermectin compounds, identified as pAT1, pVE650 pVE855, pVE859, pVE1446, pVE923, and pVE924 which contain the genetic information for the biosynthesis of the avermectins. Methods for the isolation of such plasmid and for the manipulation of the plasmids to alter the formation of the avermectin compound are also disclosed.
Claims46
Plasmid pAT1 (44.05 kb), pVE650, pVE923, pVE924, pVE855 and pVE859, and pVE1446.
The plasmid pVE1446.
The DNA responsible for avermectin biosynthesis contained on plasmid pAT1 (44.05 kb), pVE650, pVE923, pVE924, pVE885, pVE859, or pVE1446.
A method for improving the yields of avermectin compounds from fermentation broths containing a microorganism capable of producing avermectin compounds which comprises incorporating one or more of the plasmids pAT1, (44.05 kb) pVE650, pVE923, pVE924, pVE855, pVE859, or pVE1446 or BamHi restriction fragments from said plasmids into such microorganism.
A process for the isolation of DNA from microorganisms comprising:
- a) constructing a cosmid library of DNA from a microorganism in Escherichia coli;
- b) preparing filters which contain DNA from said cosmid library;
- c) incorporating .sup.32 P into a purified Bam HI restriction DNA fragment comprising a portion of the DNA responsible for avermectin biosynthesis wherein said Bam HI fragment is contained on a plasmid selected from the group consisting of pAT1 (44.05 kb), pVE650, pVE923, pVE924, pVE855, pVE859, and pVE1446;
- d) using the .sup.32 P containing DNA of step c) as a probe in DNA--DNA hybridization with the filter from step b);
- and
- e) isolating the cosmid DNA from a replica of the colony which hybridized to the .sup.32 P containing DNA of step c).
A process for the isolation of avermectin genes from Streptomyces avermitilis which comprises the complementation of Streptomyces avermitilis mutants with cloned Streptomyces avermitilis DNA wherein the cloned Streptomyces avermitilis DNA is contained on a plasmid selected from the group consisting of pVE650, pAT1 (44.05 kb), pVE923, pVE924, pVE855, pVE859, and pVE1446.
The microbiological strain Streptomyces lividans containing plasmid pAT1 (44.05 kb).
The microbiological strain Streptomyces lividans containing plasmid pVE650.
A microbiological strain Escherichia coli containing plasmid pVE923.
The microbiological strain Escherichia coli containing plasmid pVE924.
The microbiological strain Escherichia coli containing plasmid pVE855.
A microbiological strain Escherichia coli containing the plasmid pVE859.
A microbiological strain Escherichia coli containing the plasmid pVE1446.
A Bam HI restriction fragment comprising a portion of the DNA responsible for avermectin biosynthesis wherein said Bam HI fragment is contained on pAT1 (44.05 kb).
A Bam HI restriction fragment comprising a portion of the DNA responsible for avermectin biosynthesis wherein said Bam HI fragment is contained on plasmid pVE650.
A Bam HI restriction fragment comprising a portion of the DNA responsible for avermectin biosynthesis wherein said Bam HI fragment is contained on a plasmid selected from the group consisting of pVE923, pVE924, pVE855, and pVE859.
A Bam HI restriction fragment comprising a portion of the DNA responsible for avermectin biosynthesis wherein said Bam HI fragment is contained on plasmid pVE1446.
Dependencies are inferred from the claim text.