Altered thermostability of enzymes

• US 6,632,600 B1
• Filed: 11/15/2000
• Issued: 10/14/2003
• Est. Priority Date: 12/07/1995
• 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): Nashed, Nashaat T.
• Application Number:
  US09/714,780
• Time in Patent Office: 1,063 Days
• Field of Search: 435/6, 435/4, 435/14, 435/15, 435/16, 435/18, 435/19, 435/21, 435/22, 435/23, 435/24, 435/25, 435/26, 435/27, 435/28
• US Class Current: 435/4
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• 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 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)

Abstract

Provided is a method of screening gene libraries derived from a mixed population of organisms for a bioactivity or biomolecule of interest. The mixed population of organisms can be a cultured population or an uncultured population from, for example, the environment. Also provided are methods of screening isolates or enriched populations of organisms, which isolates include a population that is spatially, temporally, or hierarchical, for example, of a particular species, genus, family, or class of organisms. Identified clones containing a biomolecule or bioactivity of interest can be further variegated or the DNA contained in the clone can be variegated to create novel biomolecules or bioactivities of interest.

Claims

1. A method for identifying a protein or polypeptide having an altered thermostability compared with a thermostability of a wild-type protein or polypeptide, the method comprising:

a) constructing a library of clones, wherein the clones are constructed from nucleic acids obtained from an unselected mixed population of organisms from an environmental sample;

(b) determining thermostability of one or more wild-type proteins or polypeptides expressed from one or more clones of the library;

(c) subjecting one or more of the clones of (b) to mutagenesis;

(d) determining thermostability of modified proteins or polypeptides expressed from the clones of (c);

(e) comparing the thermostability of the one or more wild-type proteins or polypeptides of (b) with the thermostability of at least one corresponding modified protein or polypeptide of (d), thereby identifying one or more clones expressing a modified proteins or polypeptides having an altered thermostability, and;

(f) selecting at least one protein or polypeptide having an altered thermostability.

2. The method of claim 1, further comprising comparing the mutated nucleic acid sequence encoding the modified protein polypeptide to the nucleic acid sequence encoding the corresponding wild-type protein, thereby identifying the nucleotide sequence mutation.

3. The method of claim 2, wherein the comparison is performed using a sequence comparison algorithm.

4. The method of claim 1, wherein the protein or polypeptide is an enzyme.

5. The method of claim 4, wherein the enzyme is a lipase.

6. The method of claim 4, wherein the enzyme is an esterase.

7. The method of claim 4, wherein the enzyme is a protease.

8. The method of claim 4, wherein the enzyme is a glycosidase.

9. The method of claim 4, wherein the enzyme is a glycosyl transferase.

10. The method of claim 4, wherein the enzyme is a phosphatase.

11. The method of claim 4, wherein the enzyme is a dehydrogenase.

12. The method of claim 4, wherein the enzyme is a kinase.

13. The method of claim 4, wherein the enzyme is a mono-oxygenate.

14. The method of claim 4, wherein the enzyme is a dioxygenase.

15. The method of claim 4, wherein the enzyme is a haloperoxidase.

16. The method of claim 4, wherein the enzyme is a lignin peroxidase.

17. The method of claim 4, wherein the enzyme is a diarylpropane peroxidase.

18. The method of claim 4, wherein the enzyme is an epoxide hydrolase.

19. The method of claim 4, wherein the enzyme is a nitrile hydratase.

20. The method of claim 4, wherein the enzyme is a nitrilase.

21. The method of claim 4, wherein the enzyme is a transaminase.

22. The method of claim 4, wherein the enzyme is an amidase.

23. The method of claim 4, wherein the enzyme is an acylase.

24. The method of claim 4, wherein the library comprises nucleic acids obtained from extremophiles.

25. The method of claim 4, wherein the extremophiles comprise thermophiles.

26. The method of claim 24, wherein the extremophiles comprise hyperthermophiles.

27. The method of claim 24, wherein the extremophiles comprise psychrophiles.

28. The method of claim 24, wherein the extremophiles comprise halophiles.

29. The method of claim 24, wherein the extremophiles comprise psychrotrophs.

30. The method of claim 24, wherein the extremophiles comprise alkalophiles.

31. The method of claim 24, wherein the extremophiles comprise acidophiles.

32. The method of claim 1, wherein the mutagenesis comprises a method selected from the group consisting of error-prone PCR, nucleic acid shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, ligation reassembly, site saturation mutagenesis and any combination thereof.

33. The method of claim 1, wherein the mutagenesis comprises error-prone PCR.

34. The method of claim 1, wherein the mutagenesis comprises nucleic acid shuffling.

35. The method of claim 1, wherein the mutagenesis comprises oligonucleotide-directed mutagenesis.

36. The method of claim 1, wherein the mutagenesis comprises assembly PCR.

37. The method of claim 1, wherein the mutagenesis comprises sexual polymerase chain reaction (PCR) mutagenesis.

38. The method of claim 1, wherein the mutagenesis comprises in vivo mutagenesis.

39. The method of claim 1, wherein the mutagenesis comprises cassette mutagenesis.

40. The method of claim 1, wherein the mutagenesis comprises recursive ensemble mutagenesis.

41. The method of claim 1, wherein the mutagenesis comprises exponential ensemble mutagenesis.

42. The method of claim 1, wherein the mutagenesis comprises site-specific mutagenesis.

43. The method of claim 1, wherein the library is constructed using prokaryotic cells.

44. The method of claim 1, wherein the library is constructed using Strepromyces sp.

45. The method of claim 44, wherein the Strepromyces comprises Streptomyces venezuelae.

46. The method of claim 43, wherein the prokaryotic cells comprise gram negative cells.

47. The method of claim 43, wherein the prokaryotic cells comprise Bacillus sp.

48. The method of claim 43, wherein the prokaryotic cells comprise Pseudomonas sp.

49. The method of claim 1, further comprising obtaining nucleic acid from the clone identified in (e).

50. The method of claim 49, wherein obtaining nucleic acid from the clone comprises contacting the clone with a complementary nucleic acid, or fragment thereof, thereby allowing hybridization of the nucleic acids of the clone with the complementary nucleic acid. and isolation thereof.

51. The method of claim 50, wherein the complementary nucleic acid or fragment thereof comprises a solid phase bound hybridization probe.

52. The method of claim 1, further comprising screening the clones of the library for a protein or enzymatic activity prior to (c).

53. The method of claim 52, wherein the screening comprises contacting or encapsulating one or more clones of the library with a substrate, wherein a change in the substrate after contact or encapsulation indicates the presence of a protein or enzymatic activity.

54. The method of claim 53, wherein the substrate comprises a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, wherein modulation of the interaction of the first test protein linked to the DNA binding moiety with the second test protein linked to the transcription activation moiety results in a change in the expression of a detectable protein.

55. The method of claim 52, wherein the library is normalized before screening.

56. The method of claim 1, wherein the one or more clones of the library contain one or more open reading frames.

57. The method of claim 56, wherein the one or more open reading frames encode one or more polypeptide(s) of a metabolic pathway.

58. The method of claim 1, wherein the thermostability of the one or more wild-type proteins or polypeptides of (b) is lower than the thermostability of at least one corresponding modified protein or polypeptide of (d).

59. The method of claim 1, wherein the thermostability of the one or more wild-type proteins or polypeptides of (b) is higher than the thermostability of at least one corresponding modified protein or polypeptide of (d).

60. The method of claim 1, further comprising comparing an activity of a modified protein or polypeptide after mutation of its encoding nucleic acid with an activity of the wild-type protein or polypeptide.

61. The method of claim 1, further comprising producing the protein or polypeptide identified in (e).