Combinatorial screening of mixed populations of organisms

• US 7,018,793 B1
• Filed: 09/15/2000
• Issued: 03/28/2006
• Est. Priority Date: 12/07/1995
• Status: Expired due to Fees

• 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:
  US09/663,620
• Time in Patent Office: 2,020 Days
• Field of Search: 435/6, 435/183, 435/200, 536/23.2, 536/23.1, 536/23.5, 536/23.6, 536/23.7, 536/23.74
• US Class Current: 506/14
• 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 having an activity of interest, comprising:

(a) incubating nucleic acids obtained directly without selection from a mixed population of organisms from an environmental source, said nucleic acid has at least a portion of a nucleic acid sequence encoding a molecule of interest, with at least one oligonucleotide probe labeled with a detectable label under such conditions and such time to allow interaction of complementary sequences;

(b) identifying nucleic acid sequences having a complement to the oligonucleotide probe using an analyzer that detects the detectable label;

(c) generating a library from the identified nucleic acid sequences;

(d) screening the library for a specified activity;

(e) mutating a nucleic acid sequence contained in a clone from the library having the specified activity; and

(d) comparing the activity of an expression product of the clone from (e) following mutation with the specified activity of an expression product of the clone without mutation, wherein a difference in the activity is indicative of an effect of introducing at least one sequence mutation, thereby identifying a protein having an activity of interest.

2. The method of claim 1, wherein the activity of interest is an enzymatic activity.

3. The method of claim 2, wherein the enzymatic activity is provided by a glycosidase.

4. The method of claim 1, wherein the nucleic acids are obtained from extremophiles.

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

6. The method of claim 4, wherein the extremophiles comprise hyperthermophiles.

7. The method of claim 4, wherein the extremophiles comprise psychrophiles.

8. The method of claim 4, wherein the extremophiles comprise halophiles.

9. The method of claim 4, wherein the extremophiles comprise psychrotophs.

10. The method of claim 4, wherein the extremophiles comprise alkalophiles.

11. The method of claim 4, wherein the extremophiles comprise acidophiles.

12. The method of claim 1, wherein the step of (a) comprises hybridization screening.

13. The method of claim 1, wherein the step of (a) comprises polymerase chain reaction (PCR) screening.

14. The method of claim 1, wherein the step of (a) comprises biopanning.

15. The method of claim 1, wherein the mutagenesis is via nucleic acid shuffling.

16. The method of claim 1, wherein the library is generated in a prokaryotic cell.

17. The method of claim 1, wherein the library is generated in a Streptomyces sp.

18. The method of claim 17, wherein the Streptomyces is Streptomyces venezuelae.

19. The method of claim 16, wherein the prokaryotic cell is gram negative.

20. The method of claim 16, wherein the prokaryotic cell is a Bacillus sp.

21. The method of claim 16, wherein the prokaryotic cell is a Pseudomonas sp.

22. The method of claim 1, wherein prior to step (a), the method comprises the steps of

(1) isolating organisms from environmental libraries;

(2) extracting nucleic acids from the isolated organisms;

(3) pooling the extracted nucleic acids; and

(4) generating gene libraries from the pooled nucleic acids;

whereby the nucleic acids from the generated gene libraries are subject to step (a).

23. The method of claim 1, wherein the library comprises cDNA sequences.

24. The method of claim 1, wherein the library comprises genomic sequences.

25. The method of claim 1, furtber comprising comparing the mutated nucleic acid sequence of interest to the non-mutated nucleic acid sequence to identify the nucleotide sequence mutation.

26. The method of claim 25, wherein the comparison is performed using a sequence comparison algorithm.

27. The method of claim 1, wherein, prior to (e), the nucleic acid sequences are further screened for a desired characteristic.

28. The method of claim 1, wherein the library is normalized before screening the library.

29. The method of claim 1, wherein the nucleic acid of (a) comprises one or more open reading frames.

30. The method of claim 2, wherein the improved activity of interest comprises an enhanced enzymatic activity compared to that of wild-type.