Screening for novel bioactivities

• US 6,030,779 A
• Filed: 10/06/1997
• Issued: 02/29/2000
• Est. Priority Date: 07/18/1995
• Status: Expired due to Term

• Current Assignee: BP Corporation North America Incorporated (BP PLC)
• Original Assignee: Diversa Corporation (BASF SE)
• Inventors: Short, Jay M.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/944,795
• Time in Patent Office: 876 Days
• Field of Search: 435/5, 435/6, 435/91.2, 536/22.1
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

Abstract

Disclosed is a process for identifying clones having a specified enzyme activity by screening for the specified enzyme activity in a library of clones prepared by (i) selectively isolating target nucleic acid from nucleic acid derived from at least one microorganism, by use of at least one polynucleotide probe comprising at least a portion of a nucleic acid sequence encoding an enzyme having the specified enzyme activity; and (ii) transforming a host with isolated target nucleic acid to produce a library of clones which are screened for the specified enzyme activity.

Claims

1. A method for identifying a desired activity encoded by a genomic DNA population comprising:

(a) obtaining a single-stranded genomic DNA population;

(b) contacting the single-stranded DNA population of (a) with a DNA probe tunder conditions and for sufficient time to allow hybridization and to produce a double-stranded complex of probe and members of the genomic DNA population which hybridize thereto;

(c) separating the complex from the single-stranded DNA population of (b);

(d) releasing from the probe the members of the genomic population which had been bound to the probe;

(e) forming double-stranded DNA from the members of the genomic population of (d);

(f) introducing the double-stranded DNA of (e) into a suitable host cell to produce an expression library containing a plurality of clones containing the selected DNA; and

(g) screening the expression library for the desired activity.

2. The method of claim 1, wherein the genomic DNA population is derived from uncultivated or cultivated microorganisms.

3. The method of claim 2, wherein the uncultivated or cultivated microorganisms are isolated from an environmental sample.

4. The method of claim 3, wherein the microorganisms isolated from an environmental sample are extremophiles.

5. The method of claim 4, wherein the extremophiles are selected from the group consisting of thermophiles, hyperthermophiles, psychrophiles, halophiles, acidophiles, barophiles and psychrotrophs.

6. The method of claim 1, wherein the genomic DNA, or fragments thereof, comprise one or more operons, or portions thereof.

7. The method of claim 6, wherein the operons, or portions thereof, encodes a complete or partial metabolic pathway.

8. The method of claim 7, wherein the operons or portions thereof encoding a complete or partial metabolic pathway encodes polyketide synthases.

9. The method of claim 1, wherein the expression library containing a plurality of clones is selected from the group consisting of phage, plasmids, phagemids, cosmids, phosmids, viral vectors and artificial chromosomes.

10. The method of claim 1, wherein the suitable host cell is selected from the group consisting of a bacterium, fungus, plant cell, insect cell and animal cell.

11. The method of claim 1, wherein the DNA probe bound to a ligand is comprised of at least a portion of the coding region sequence of DNA for a known bioactivity.

12. The method of claim 1, wherein the ligand is selected from the group consisting of antigens or haptens, biotin or iminobiotin, sugars, enzymes, apoenzymes homopolymeric oligonucleotides and hormones.

13. The method of claim 1, wherein the binding partner for said ligand is selected from the group consisting of antibodies or specific binding fragments thereof, avidin or streptavidin, lectins, enzyme inhibitors, apoenzyme cofactors, homopolymeric oligonucleotides and hormone receptors.

14. The method of claim 1, wherein a solid phase is selected from the group consisting of a glass or polymeric surface, a packed column of polymeric beads or magnetic or paramagnetic particles.

15. The method of claim 1, further comprising producing a cell-free extract of the expression library.

16. The method of claim 15, further comprising combining the expression library extract with a host cell-free protein extract from a metabolically rich host organism.

17. The method of claim 16, wherein the host organism is Streptomyces.

18. The method of claim 16, wherein the host organism is Bacillus.

19. A method for preselecting a desired DNA from a genomic DNA population comprising:

(a) obtaining a single-stranded genomic DNA population;

(b) contacting the single-stranded DNA population of (a) with a ligand-bound oligonucleotide probe that is complementary to a secretion signal sequence unique to a given class of proteins under conditions permissive of hybridization to form a double-stranded complex;

(c) contacting the double-stranded complex of (a) with a solid phase specific binding partner for said ligand so as to produce a solid phase complex;

(d) separating the solid phase complex from the single-stranded DNA population of (a);

(e) releasing the members of the genomic population which had bound to said solid phase bound probe;

(f) separating the solid phase bound probe from the members of the genomic population which had bound thereto;

(g) forming double-stranded DNA from the members of the genomic population of (e);

(h) introducing the double-stranded DNA of (g) into a suitable host cell to form an expression library containing a plurality of clones containing the selected DNA; and

(i) screening the expression library for the desired activity.

20. The method of claim 19, wherein the genomic DNA population is derived from uncultivated or cultivated microorganisms.

21. The method of claim 20, wherein the uncultivated or cultivated microorganisms are isolated from an environmental sample.

22. The method of claim 21, wherein the microorganisms isolated from an environmental sample are extremophiles.

23. The method of claim 22, wherein the extremophiles are selected from the group consisting of thermophiles, hyperthermophiles, psychrophiles, halophiles, acidophiles, barophiles and psychrotrophs.

24. The method of claim 19, wherein the genomic DNA, or fragments thereof, comprise one or more operons, or portions thereof.

25. The method of claim 24, wherein the operons, or portions thereof, encodes a complete or partial metabolic pathway.

26. The method of claim 25, wherein the operons or portions thereof encoding a complete or partial metabolic pathway encodes polyketide synthases.

27. The method of claim 19, wherein the expression library containing a plurality of clones is selected from the group consisting of phage, plasmids, phagemids, cosmids, phosmids, viral vectors and artificial chromosomes.

28. The method of claim 19 wherein the a suitable host cell is selected from the group consisting of a bacterium, fungus, plant cell, insect cell and animal cell.

29. The method of claim 19, wherein the DNA probe bound to a ligand is comprised of at least a portion of the coding region sequence of DNA for a known bioactivity.

30. The method of claim 19, wherein the ligand is selected from the group consisting of antigens or haptens, biotin or iminobiotin, sugars, enzymes, apoenzymes homopolymeric oligonucleotides and hormones.

31. The method of claim 19, wherein the binding partner for said ligand is selected from the group consisting of antibodies or specific binding fragments thereof, avidin or streptavidin, lectins, enzyme inhibitors, apoenzyme cofactors, homopolymeric oligonucleotides and hormone receptors.

32. The method of claim 19, wherein a solid phase is selected from the group consisting of a glass or polymeric surface, a packed column of polymeric beads or magnetic or paramagnetic particles.

33. The method of claim 19, further comprising producing a cell-free extract of the expression library.

34. The method of claim 33, firther comprising combining the expression library extract with a cell-free protein extract from a metabolically rich host organism.

35. The method of claim 34, wherein the host organism is Streptomyces.

36. The method of claim 34, wherein the host organism is Bacillus.

37. A method for identifying a desired bioactivity or biomolecule comprising:

(a) producing one or more expression libraries derived from nucleic acid directly isolated from the environment;

(b) combining a cell-free extract from the expression library or libraries with crude or partially purified extracts, or pure proteins from a metabolically rich cell line to form an extract mixture free from the library and cell line cells; and

(c) screening said mixture to identify an activity or molecule produced by the extract mixture.

38. A method for identifying a desired bioactivity or biomolecule comprising:

(a) producing one or more expression libraries containing clones having nucleic acid inserts derived from nucleic acid directly isolated from the environment;

(b) transferring said clones into a metabolically rich cell line; and

(c) screening said cell line to identify clones having a bioactivity or biomolecule of interest.