High throughput screening for novel enzymes

• US 6,174,673 B1
• Filed: 06/16/1998
• Issued: 01/16/2001
• Est. Priority Date: 06/16/1997
• Status: Expired due to Term

• Current Assignee: BASF Enzymes LLC (BASF SE)
• Original Assignee: Diversa Corporation (BASF SE)
• Inventors: Keller, Martin, Short, Jay M.
• Primary Examiner(s): Yucel, Remy
• Application Number:
  US09/098,206
• Time in Patent Office: 945 Days
• Field of Search: 435/6, 435/69.1, 435/440, 435/320.1, 435/471, 435/476
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nuclei acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell sorter to identify said clones. More particularly, this is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; (ii) exposing said libraries to a particular substrate or substrates of interest; and (iii) screening said exposed libraries utilizing a fluorescence activated cell sorter to identify clones which react with the substrate or substrates. Also provided is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; and (ii) screening said exposed libraries utilizing an assay requiring co-encapsulation, a binding event or the covalent modification of a target, and a fluorescence activated cell sorter to identify positive clones.

Claims

1. A method for identifying bioactivities or biomolecules using high throughput screening of genomic DNA comprising:

a) providing an expression library containing a plurality of clones, wherein the DNA for generating the library is obtained from a mixed population of organisms;

b) encapsulating a bioactive fluorescent substrate and at least one clone of the library in a gel microdroplet, wherein the substrate is fluorescent in the presence of a bioactivity or biomolecule;

c) screening the microdroplet with a fluorescent analyzer that detects bioactive fluorescence; and

d) identifying clones detected as positive for bioactive fluorescence, wherein fluorescence is indicative of DNA that encodes a bioactivity or biomolecule.

2. The method of claim 1, wherein the bioactivity is an enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.

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

4. The method of claim 3, wherein the prokaryotic cell is gram negative.

5. The method of claim 4, wherein the prokaryotic cell is E. coli.

6. The method of claim 5, wherein prior to step b), the E. coli is transferred to a Streptomyces sp.

7. The method of claim 6, wherein the Streptomyces sp. is Streptomyces venezuelae.

8. The method of claim 1, wherein the expression library contains DNA obtained from extremophiles.

9. The method of claim 8, wherein the extremophiles are thermophiles.

10. The method of claim 9 wherein the extremeophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.

11. The method of claim 1 wherein the bioactive substrate comprises C12FDG.

12. The method of claim 1, wherein the bioactive substrate comprises a lipophilic tail.

13. The method of claim 1, wherein the samples are heated before step b).

14. The method of claim 11, wherein the heating is at about 70°

C.

15. The method of claim 14, wherein the heating occurs at about 30 minutes.

16. The method of claim 1, wherein the fluorescent analyzer comprises a FACS apparatus.

17. The method of claim 1, wherein the expression library is biopanned before step b).

18. The method of claim 1, including the additional steps of:

subjecting an enzyme encoded by the DNA identified in step d) to directed evolution comprising the steps of;

a) subjecting the enzyme to non-directed mutagenesis; and

b) screening mutant enzymes produced in step a) for a mutant enzyme.

19. The method of claim 1, wherein the prokaryotic expression library is normalized before step b).

20. The method of claim 1, further comprising co-encapsulating an indicator cell in step b).

21. The method of claim 1, wherein the library is a prokaryotic expression library.

22. A method for identifying bioactivities or biomolecules using high throughput screening of prokaryotic genomic DNA in a culture-independent system comprising:

a) generating a prokaryotic expression library containing a plurality of clones, wherein the DNA for generating the library is obtained from a mixed population of organisms;

b) inserting a bioactive fluorescent substrate into the clones of the library, wherein the substrate is fluorescent in the presence of a bioactivity or biomolecule;

c) screening the clones with a fluorescent analyzer that detects bioactive fluorescence; and

d) identifying clones detected as positive for bioactive fluorescence, wherein fluorescence is indicative of DNA that encodes a bioactivity or biomolecule.

23. The method of claim 22, further comprising encapsulation the clone and the bioactive substrate prior to screening.