US5824485A
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Methods for generating and screening novel metabolic pathways
Assignee: Cubist Pharmaceuticals LLC
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Filing: 1996-04-24
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Granted: 1998-10-20
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Expires: 2016-04-24
Abstract
Abstract The present invention relates to a novel drug discovery system for generating and screening molecular diversity. The system provides methods for mixing and cloning genetic materials from a plurality of species of organisms in combinatorial gene expression libraries to generate novel metabolic pathways and classes of compounds. The system also involves methods for pre-screening or identifying for host organisms containing a library that are capable of generating such novel pathways and compounds. The host organisms may be useful in drug screening for particular diseases, and in commercial production of compounds of interest. The methods of the invention are also useful in preserving the genomes of organisms that are known or prospective sources of drugs.
Inventors
- 👤 Katie A. Thompson
- 👤 Lyndon M. Foster
- 👤 Todd C. Peterson
- 👤 Nicole Marie Nasby
- 👤 Paul Brian
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 19:09
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1093; C12Q; C12Q1/00; C12Q1/68; C12Q1/6811; C12N15/102; C12N15/1027; C12N15/1086; C12N15/63; C12N15/66
Claims Analysis
Total Claims: 51
Claim 1
Independent Claim
85 words
A combinatorial gene expression library, comprising a pool of expression constructs, each expression construct containing one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are derived from a plurality of species of donor organisms, and wherein the cDNA or genomic DNA fragments in each expression construct are operably-associated each with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism.
Claim 2
Dependent Claim
85 words
A combinatorial gene expression library, comprising a pool of expression constructs, each expression construct containing one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are derived from a plurality of species of donor organisms, and wherein the cDNA or genomic DNA fragments in each expression construct are operably-associated each with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism.
Claim 3
Dependent Claim
69 words
A combinatorial chimeric pathway gene expression library, comprising a pool of expression constructs, each expression construct containing randomly concatenated cDNA or genomic DNA fragments derived from one or more species of donor organisms, in which the concatenated cDNA or genomic DNA fragments are operably-associated with one or more regulatory regions that drive expression of genes encoded by the concatenated cDNA or genomic DNA fragments in an appropriate host organism.
Claim 4
Dependent Claim
69 words
A combinatorial chimeric pathway gene expression library, comprising a pool of expression constructs, each expression construct containing randomly concatenated cDNA or genomic DNA fragments derived from one or more species of donor organisms, in which the concatenated cDNA or genomic DNA fragments are operably-associated with one or more regulatory regions that drive expression of genes encoded by the concatenated cDNA or genomic DNA fragments in an appropriate host organism.
Claim 5
Dependent Claim
68 words
A biased combinatorial gene expression library, comprising a pool of expression constructs, each expression construct containing cDNA or genomic DNA fragments preselected from a plurality of species of donor organisms for a specific property, in which the cDNA or genomic DNA fragments are operably-associated with one or more regulatory regions that drive expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism.
Claim 6
Dependent Claim
68 words
A biased combinatorial gene expression library, comprising a pool of expression constructs, each expression construct containing cDNA or genomic DNA fragments preselected from a plurality of species of donor organisms for a specific property, in which the cDNA or genomic DNA fragments are operably-associated with one or more regulatory regions that drive expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism.
Claim 7
Dependent Claim
32 words
The gene expression library of claim 1, 2 or 3 in which the expression construct comprises a plasmid vector, a phage vector, a viral vector, a cosmid vector, or an artificial chromosome.
Claim 8
Dependent Claim
24 words
The gene expression library of claim 4 in which the vector is a shuttle vector that replicates in different host cell species or strains.
Claim 9
Dependent Claim
39 words
The gene expression library of claim 1, 2 or 3 in which the cDNA or genomic DNA fragments are derived from bacteria, fungi, algae, lichens, plants, protozoans, metazoans, coelenterates, insects, mollusca, sponges, worms, amphibians, reptiles, tunicates, birds or mammals.
Claim 10
Dependent Claim
30 words
The gene expression library of claim 1, 2 or 3 in which the donor organisms comprise a mixture of terrestrial microorganisms, freshwater microorganisms, or marine microorganisms, or a combination thereof.
Claim 11
Dependent Claim
24 words
The gene expression library of claim 1, 2 or 3 in which the cDNA or genomic DNA fragments are derived from an environmental sample.
Claim 12
Dependent Claim
27 words
The gene expression library of claim 7 in which the cDNA or genomic DNA fragments comprise one or more operons, or portions thereof, of the donor microorganism.
Claim 13
Dependent Claim
21 words
The gene expression library of claim 9 in which the operon or portions thereof encodes a complete or partial metabolic pathway.
Claim 14
Dependent Claim
23 words
The gene expression library of claim 1, 2 or 3 in which at least one expression construct is contained in a host cell.
Claim 15
Dependent Claim
31 words
The gene expression library of claim 11 in which the host cells have been modified by the introduction, induction or overproduction of active efflux systems prior to containing the expression constructs.
Claim 16
Dependent Claim
37 words
The gene expression library of claim 11 in which the host cells have been modified by the introduction, induction or overproduction of a known metabolic pathway of interest or portion thereof prior to containing the expression constructs.
Claim 17
Dependent Claim
23 words
The gene expression library of claim 11 in which the host cell is a bacterium, fungus, plant cell, insect cell, or animal cell.
Claim 18
Dependent Claim
45 words
The gene expression library of claim 14 in which the host cell is Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Myxococcus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Spodoptera frugiperda, Aspergillus nidulans, Arabidopsis thaliana, Nicotiana tabacum, COS cells, 293 cells, VERO cells, NIH/3T3 cells or CHO cells.
Claim 19
Dependent Claim
34 words
The gene expression library of claim 11 in which the host cells further contain a reporter regimen tailored to identify clones in the library that are expressing desirable metabolic pathways, gene products, or compounds.
Claim 20
Dependent Claim
41 words
The gene expression library of claim 16 in which the reporter regimen comprises DNA encoding a reporter gene operably-associated with a regulatory region that is inducible or modulated by desirable metabolic pathways, gene products, or compounds expressed by the host cell.
Claim 21
Dependent Claim
35 words
The gene expression library of claim 11 in which the host cells are in a matrix containing a reporter regimen tailored to identify clones in the library that are expressing desirable metabolic pathways or compounds.
Claim 22
Independent Claim
84 words
A method for making a combinatorial gene expression library, comprising ligating a DNA vector to one or more cDNA or genomic DNA fragments to generate a library of expression constructs, wherein the cDNA or genomic DNA fragments in the library of expression constructs are obtained from a plurality of species of donor organisms, and wherein genes contained in the cDNA or genomic DNA fragments are operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 23
Independent Claim
84 words
A method for making a combinatorial gene expression library, comprising ligating a DNA vector to one or more cDNA or genomic DNA fragments to generate a library of expression constructs, wherein the cDNA or genomic DNA fragments in the library of expression constructs are obtained from a plurality of species of donor organisms, and wherein genes contained in the cDNA or genomic DNA fragments are operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 24
Independent Claim
75 words
A method for making a chimeric pathway gene expression library, comprising randomly concatenating cDNA or genomic DNA fragments obtained from one or more species of donor organisms, and ligating the concatenated DNA fragments to a DNA vector to generate a library of expression constructs in which genes contained in the cDNA or genomic DNA fragments are operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 25
Independent Claim
75 words
A method for making a chimeric pathway gene expression library, comprising randomly concatenating cDNA or genomic DNA fragments obtained from one or more species of donor organisms, and ligating the concatenated DNA fragments to a DNA vector to generate a library of expression constructs in which genes contained in the cDNA or genomic DNA fragments are operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 26
Independent Claim
86 words
A method for making a biased combinatorial gene expression library, comprising ligating a DNA vector to one or more cDNA or genomic DNA fragments to generate a library of expression constructs, wherein the cDNA or genomic DNA fragments are obtained from a plurality of species of donor organisms and are selected for a specific property, and wherein genes contained in the cDNA or genomic DNA fragments are operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 27
Independent Claim
86 words
A method for making a biased combinatorial gene expression library, comprising ligating a DNA vector to one or more cDNA or genomic DNA fragments to generate a library of expression constructs, wherein the cDNA or genomic DNA fragments are obtained from a plurality of species of donor organisms and are selected for a specific property, and wherein genes contained in the cDNA or genomic DNA fragments are operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 28
Dependent Claim
30 words
The method of claim 19, 20, or 21 in which the DNA vector is a plasmid vector, a phage vector, a viral vector, a cosmid vector, or an artificial chromosome.
Claim 29
Dependent Claim
23 words
The method of claim 22 in which the DNA vector is a shuttle vector that replicates in different host cell species or strains.
Claim 30
Dependent Claim
37 words
The method of claim 19, 20, or 21 in which the cDNA or genomic DNA fragments are derived from bacteria, fungi, algae, lichens, plants, protozoans, metazoans, coelenterates, insects, mollusca, sponges, worms, amphibians, reptiles, tunicates, birds, or mammals.
Claim 31
Dependent Claim
28 words
The method of claim 19, 20, or 21 in which the donor organisms comprise a mixture of terrestrial microorganisms, freshwater microorganisms or marine microorganisms, or a combination thereof.
Claim 32
Dependent Claim
22 words
The method of claim 19, 20, or 21 in which the cDNA or genomic DNA fragments are derived from an environmental sample.
Claim 33
Dependent Claim
25 words
The method of claim 25 in which the cDNA or genomic DNA fragments comprise at least an operon, or portions thereof, of the donor microorganisms.
Claim 34
Dependent Claim
16 words
The method of claim 27 in which the operon encodes a complete or partial metabolic pathway.
Claim 35
Dependent Claim
20 words
The method of claim 19, 20, or 21 further comprising introducing the library of expression constructs into a host cell.
Claim 36
Dependent Claim
21 words
The method of claim 29 in which the host cell is a bacterium, fungus, plant cell, insect cell, or animal cell.
Claim 37
Dependent Claim
43 words
The method of claim 30 in which the host cell is Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Myxococcus. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Spodoptera frugiperda, Aspergillus nidulans, Arabidopsis thaliana, Nicotiana tabacum, COS cells, 293 cells, VERO cells, NIH/3T3 cells, or CHO cells.
Claim 38
Dependent Claim
32 words
The method of claim 29 in which the host cells further contain a reporter regimen tailored to identify clones in the library that are expressing desirable metabolic pathways, gene products, or compounds.
Claim 39
Dependent Claim
40 words
The method of claim 32 in which the reporter regimen comprises DNA encoding a reporter gene operably-associated with a regulatory region that is inducible or modulated by the desirable metabolic pathways, gene products, or compounds expressed by the host cell.
Claim 40
Dependent Claim
36 words
The method of claim 29 in which the host cells in which the host cells have been modified by the introduction, induction or overproduction of active efflux systems prior to introducing the library of expression constructs.
Claim 41
Dependent Claim
37 words
The method of claim 29 in which the host cells have been modified by the introduction, induction or overproduction of a known metabolic pathway of interest or portion thereof prior to introducing the library of expression constructs.
Claim 42
Dependent Claim
37 words
A method for identifying a compound of interest in a gene expression library, comprising: (a) culturing the gene expression library of claim 11; and (b) screening the gene expression library for a clone which produces the compound.
Claim 43
Dependent Claim
41 words
A method for screening a gene expression library or a compound of interest, comprising: (a) culturing the gene expression library of claim 16; and (b) detecting a signal generated by the reporter regimen; thereby identifying a clone which produces the compound.
Claim 44
Dependent Claim
41 words
A method for screening a gene expression library or a compound of interest, comprising: (a) culturing the gene expression library of claim 18; and (b) detecting a signal generated by the reporter regimen; thereby identifying a clone which produces the compound.
Claim 45
Dependent Claim
29 words
A method for producing a compound of interest, comprising: (a) culturing the clone identified in claim 36; and (b) recovering the compound from the culture of the identified clone.
Claim 46
Dependent Claim
29 words
A method for producing a compound of interest, comprising: (a) culturing the clone identified in claim 37; and (b) recovering the compound from the culture of the identified clone.
Claim 47
Dependent Claim
29 words
A method for producing a compound of interest, comprising: (a) culturing the clone identified in claim 38; and (b) recovering the compound from the culture of the identified clone.
Claim 48
Dependent Claim
16 words
The gene expression library of claim 11 in which the host cells are in a matrix.
Claim 49
Dependent Claim
37 words
A method for identifying a compound of interest in a gene expression library, comprising: (a) culturing the gene expression library of claim 42; and (b) screening the gene expression library for a clone which produces the compound.
Claim 50
Dependent Claim
20 words
The gene expression library of claim 4 in which the DNA vector integrates into the genome of a host organism.
Claim 51
Dependent Claim
18 words
The method of claim 22 in which the DNA vector integrates into the genome of a host organism.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-10-19 | 2002-04-20 | 2002-10-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-10-19 | 2006-04-20 | 2006-10-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-10-19 | 2010-04-20 | 2010-10-20 | $7700.00 | Window Closed / Lapsed |