Methods for generating and screening novel metabolic pathways

• US 5,783,431 A
• Filed: 10/24/1996
• Issued: 07/21/1998
• Est. Priority Date: 04/24/1996
• Status: Expired due to Term

• Current Assignee: Cubist Pharmaceuticals, LLC (Merck & Co., Inc.)
• Original Assignee: Chromaxome Corporation (Merck & Co., Inc.)
• Inventors: Peterson, Todd C., Foster, Lyndon M., Brian, Paul
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Brusca, John S.
• Application Number:
  US08/738,944
• Time in Patent Office: 635 Days
• Field of Search: 536/23.1, 435/172.3, 435/320.1
• US Class Current: 506/10
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

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 provides mobilizable combinatorial gene expression libraries that can be transferred from one species of host organism to another for expression. Also provided are specialized cloning vectors for making mobilizable gene expression libraries. 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.

Claims

1. A mobilizable combinatorial gene expression library, comprising a pool of expression constructs, each expression construct comprising a shuttle vector that replicates in different species or strains of host cell, said shuttle vector 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 are each operably-associated with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism.

2. The gene expression library of claim 1 wherein the cDNA or genomic DNA fragments contained in the expression constructs are randomly concatenated, and are derived from one or more species of donor organisms.

3. The gene expression library of claim 1 wherein the cDNA or genomic DNA fragments contained in the expression constructs are preselected for a specific property.

4. 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.

5. The gene expression library of claim 4 in which the vector is a shuttle vector that replicates in different host cell species or strains.

6. The gene expression library of claim 1, 2, or 3 in which the donor organisms comprise a mixture of microorganisms.

7. The gene expression library of claim 1, 2, or 3 in which each expression construct is contained in a host cell.

8. The gene expression library of claim 7 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.

9. The gene expression library of claim 7 in which the host cell is Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Pseudomonas aeruginosa, Myxococcus xanthus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Spodoptera frugiperda, Aspergillus nidulans, Arabidopsis thaliana, Nicotiana tabacum, COS cells, 293 cells, VERO cells, NIH/3T3 cells, or CHO cells.

10. The gene expression library of claim 7 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.

11. The gene expression library of claim 7 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.

12. The gene expression library of claim 7 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, gene products, or compounds.

22. The gene expression library of claim 3 wherein the cDNA or genomic DNA fragments are preselected for homology to nucleic acid sequences encoding proteins in a metabolic pathway.

24. The gene expression library of claim 1, 2, or 3 in which each expression construct is contained in a host cell that contains proteins that mediate transfer of the expression construct by conjugation.

13. A method for making a mobilizable combinatorial gene expression library, comprising ligating a shuttle vector that replicates in different species or strains of host cell, to one or more cDNA or genomic DNA fragments to form a pool of expression constructs, wherein said cDNA or genomic DNA fragments in the pool of expression constructs are obtained from a plurality of species of donor organisms, and wherein the genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.

14. The method of claim 13 wherein the cDNA or genomic DNA fragments contained in the expression constructs are randomly concatenated, and are derived from one or more species of donor organisms.

15. The method of claim 13 wherein the cDNA or genomic DNA fragments contained in the expression constructs are preselected for a specific property.

16. The method of claim 13, 14, or 15 in which the DNA vector is a plasmid vector, a phage vector, a viral vector, a cosmid vector, or an artificial chromosome.

17. The method of claim 13, 14, or 15 in which the shuttle vector further comprises an origin of transfer.

23. The method of claim 15 wherein the cDNA or genomic DNA fragments are preselected for homology to nucleic acid sequences encoding proteins in a metabolic pathway.

18. A method for making a combinatorial gene expression library comprising transferring a pool of expression constructs in a species of host organism to another species or strain of host organism, said expression construct comprising a shuttle vector that replicates in different species or strains of host cell, said shuttle vector comprising one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are obtained from a plurality of species of donor organisms, and wherein the genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.

19. The method of claim 18 wherein the pool of expression constructs is transferred by conjugation.

20. The method of claim 18 wherein the pool of expression constructs is transferred by isolating the expression constructs from a first species of host organism, and introducing the expression constructs into a second species or strain of host organism.

25. The method of claim 18 wherein the pool of expression constructs is contained in host cells that contain proteins that mediate transfer of the expression constructs by conjugation.

21. 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 in the library 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 each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.