US4935340A
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Method of isolating antibiotic biosynthetic genes
Assignee: Eli Lilly and Co
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Filing: 1985-06-07
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Granted: 1990-06-19
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Expires: 2005-06-07
Abstract
Abstract The present invention is a method for isolating antibiotic biosynthetic genes. To practice the method, an antibiotic resistance-conferring DNA segment is labelled and used as a probe to find, via DNA hydridization, homologous DNA in a genetic library which comprises chromosomal and plasmid DNA of an antibiotic-producing organism. Individual vectors of the genetic library which hybridize to the antibiotic resistance-conferring gene, and which comprise ˜1-45 kb of contiguous DNA from the antibiotic-producing organism, which also comprise an antibiotic biosynthetic gene. The present method is exemplified by using the erythromycin resistance-conferring gene of Streptomyces erythreus to clone the erythromycin biosynthetic pathway from the same organism. The erythromycin biosynthetic pathway isolated with the present method synthesizes erythromycin when introduced into S. lividans TK23.
Inventors
- 👤 Richard H. Baltz
- 👤 R. Nagaraja Rao
- 👤 Richard K. Stanzak
- 👤 Patti J. Treadway
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 16:50
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/76; C12N15/11; C12N15/52
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
51 words
A method for identifying and isolating an antibiotic biosynthetic gene linked to an antibiotic resistance-conferring gene, said method comprising identifying an antibiotic biosynthetic gene-containing DNA sequence, said DNA sequence not previously isolated, by hybridizing a labelled antibiotic resistance conferring-gene to said antibiotic biosynthetic gene-containing DNA and isolating said DNA thus identified.
Claim 2
Independent Claim
51 words
A method for identifying and isolating an antibiotic biosynthetic gene linked to an antibiotic resistance-conferring gene, said method comprising identifying an antibiotic biosynthetic gene-containing DNA sequence, said DNA sequence not previously isolated, by hybridizing a labelled antibiotic resistance conferring-gene to said antibiotic biosynthetic gene-containing DNA and isolating said DNA thus identified.
Claim 3
Dependent Claim
82 words
The method of claim 1, which comprises: (1) isolating chromosomal and plasmid DNA from an antibiotic-producing organism; (2) preparing a genetic library of said chromosomal and plasmid DNA, such that the recombinant vectors of said library comprise a fragment of said DNA of a size ranging from ˜1-45 kb; (3) hybridizing a labelled antibiotic resistance conferring gene to said library; (4) identifying which recombinant vectors of said library hybridize to said antibiotic resistance-conferring gene; (5) isolating said hybridizing vectors from said library.
Claim 4
Dependent Claim
20 words
The method of claim 1, wherein said DNA comprises the genes which encode the enzymes comprising an antibiotic biosynthetic pathway.
Claim 5
Dependent Claim
37 words
The method of claim 1, wherein said DNA is isolated from a species of a genus selected from the group of genera consisting of Actinomadura, Actinoplanes, Bacillus, Cephalosporium, Corynebacterium, Dactylosporangium, Micromonospora, Nocardia, Penicillium, Saccharopolyspora, Streptomyces, and Streptoverticillium.
Claim 6
Dependent Claim
42 words
The method of claim 4, wherein said genus is Streptomyces and said species is selected from the group consisting of albireticuli, ambofaciens, antibioticus, aureofaciens, cattleya, chartreusis, cinnamonensis, clavuligerus, erythreus, fradiae, hygroscopicus, kanamyceticus, lavendulae, macrosporeus, narbonensis, olivaceus, rimosus, tenebrarius, thermotolerans, toyocoensis, and virginiae.
Claim 7
Dependent Claim
10 words
The method of claim 5, wherein said species is erythreus.
Claim 8
Dependent Claim
10 words
The method of claim 5, wherein said species is fradiae.
Claim 9
Dependent Claim
35 words
The method of claim 1, wherein said antibiotic resistance-conferring gene confers resistance to an antibiotic selected from the group consisting of antibiotics apramycin, β-lactams, erythromycin, hygromycin, kanamycin, methelenomycin, neomycin, spiramycin, tobramycin, thiostrepton, tylosin, and viomycin.
Claim 10
Dependent Claim
30 words
The method of claim 1, wherein said antibiotic resistance-conferring gene encodes an enzymatic activity selected from the group consisting of acetyltransferase activity, β-lactamase activity, phosphotransferase activity, and rRNA methyltransferase activity.
Claim 11
Dependent Claim
14 words
The method of claim 8, wherein said antibiotic resistance-conferring gene confers resistance to erythromycin.
Claim 12
Dependent Claim
14 words
The method of claim 8, wherein said antibiotic resistance-conferring gene confers resistance to tylosin.
Claim 13
Dependent Claim
14 words
The method of claim 9, wherein said antibiotic resistance-conferring gene encodes rRNA methyltransferase activity.
Claim 14
Dependent Claim
8 words
The ˜34 kb Bg1II fragment of plasmid pKC488.
Claim 15
Dependent Claim
8 words
The ˜34 kb Bg1II fragment of plasmid pKC488.
Claim 16
Dependent Claim
8 words
A Streptomyces host cell transformed with plasmid pKC488.
Claim 17
Dependent Claim
8 words
A Streptomyces host cell transformed with plasmid pKC488.
Claim 18
Dependent Claim
10 words
The host cell of claim 14 that is Streptomyces lividans/pKC488.
Claim 19
Dependent Claim
20 words
A DNA sequence which encodes an enzyme in the erythromycin biosynthetic pathway, said DNA sequence being comprised on plasmid pK488.
Claim 20
Dependent Claim
20 words
A DNA sequence which encodes an enzyme in the erythromycin biosynthetic pathway, said DNA sequence being comprised on plasmid pK488.
References Cited (Backward Citations)
No citation records indexed.
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