Methods for generating and screening novel metabolic pathways

• US 5,824,485 A
• Filed: 04/24/1996
• Issued: 10/20/1998
• Est. Priority Date: 04/24/1995
• Status: Expired due to Term

• Current Assignee: Cubist Pharmaceuticals, Inc. (Merck & Co., Inc.)
• Original Assignee: Chromaxome Corporation (Terragen Diversity, Inc.)
• Inventors: Thompson, Katie A., Nasby, Nicole Marie, Peterson, Todd C., Foster, Lyndon M., Brian, Paul
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Brusca, John S.
• Application Number:
  US08/639,255
• Time in Patent Office: 909 Days
• Field of Search: 536/23.1, 435/6, 435/69.1, 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 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 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.

Claims

1. 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.

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 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.

7. 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.

8. The gene expression library of claim 1, 2 or 3 in which the cDNA or genomic DNA fragments are derived from an environmental sample.

9. 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.

10. The gene expression library of claim 9 in which the operon or portions thereof encodes a complete or partial metabolic pathway.

11. The gene expression library of claim 1, 2 or 3 in which at least one expression construct is contained in a host cell.

12. 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.

13. 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.

14. The gene expression library of claim 11 in which the host cell is a bacterium, fungus, plant cell, insect cell, or animal cell.

15. 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.

16. 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.

17. 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.

18. 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.

36. 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.

37. 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.

38. 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.

39. 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.

40. 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.

41. 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.

42. The gene expression library of claim 11 in which the host cells are in a matrix.

43. 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.

44. The gene expression library of claim 4 in which the DNA vector integrates into the genome of a host organism.

2. 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.

3. 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.

19. 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.

22. 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.

23. The method of claim 22 in which the DNA vector is a shuttle vector that replicates in different host cell species or strains.

24. 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.

25. 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.

26. The method of claim 19, 20, or 21 in which the cDNA or genomic DNA fragments are derived from an environmental sample.

27. 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.

28. The method of claim 27 in which the operon encodes a complete or partial metabolic pathway.

29. The method of claim 19, 20, or 21 further comprising introducing the library of expression constructs into a host cell.

30. The method of claim 29 in which the host cell is a bacterium, fungus, plant cell, insect cell, or animal cell.

31. 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.

32. 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.

33. 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.

34. 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.

35. 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.

45. The method of claim 22 in which the DNA vector integrates into the genome of a host organism.

20. 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.

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 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.