Method for screening for enzyme activity

• US 6,054,267 A
• Filed: 08/02/1996
• Issued: 04/25/2000
• 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): Campbell, Eggerton A.
• Application Number:
  US08/692,002
• Time in Patent Office: 1,362 Days
• Field of Search: 435/6, 435/77, 435/78, 435/7.1, 435/69.1
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• 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 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 301/03001 Alkaline phosphatase (3.1.3.1)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)

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 DNA from DNA derived from at least one microorganism, by use of at least one probe DNA comprising at least a portion of a DNA sequence encoding an enzyme having the specified enzyme activity; and (ii) transforming a host with isolated target DNA to produce a library of clones which are screened for the specified enzyme activity.

Claims

1. A method for enriching for DNA sequences containing at least a partial coding region for at least one specified enzyme activity in a DNA sample comprising selecting and recovering a mixture of target DNA from a mixture of organisms, by use of a mixture of DNA probes comprising at least a portion of a DNA sequence encoding at least one enzyme having a specified enzyme activity and transforming host cells with recovered target DNA to produce an expression library of a plurality of clones.

2. The method of claim 1, wherein the organisms are microorganisms.

3. The method of claim 2, wherein the microorganisms are uncultured microorganisms.

4. The method of claim 1, further comprising screening the expression library for the specified enzyme activity.

5. The method of claim 1, wherein the target DNA obtained from the DNA population is selected by:

a) converting double stranded DNA into single stranded DNA;

b) recovering from the converted single stranded DNA, single stranded target DNA which hybridizes to probe DNA;

c) converting recovered single stranded target DNA to double stranded DNA; and

d) transforming a host cell with the double stranded DNA of c).

6. The method of claim 5, wherein the probe is directly or indirectly bound to a solid phase by which it is separated from single stranded DNA which is not so hybridized.

7. The method of claim 6, which further comprises:

a) releasing single stranded DNA from said probe; and

b) amplifying the released single stranded DNA prior to converting to double stranded DNA.

8. The method of claim 1, wherein said target DNA is gene cluster DNA.

9. The method of claim 3, wherein the uncultured microorganisms are derived from an environmental sample.

10. The method of claim 3, wherein the uncultured microorganisms comprise a mixture of terrestrial microorganisms or marine microorganisms or airborne microorganisms, or a mixture of terrestrial microorganisms, marine microorganisms and airborne microorganisms.

11. The method of claim 1, wherein the clones comprise a construct selected from the group consisting of phage, plasmids, phagemids, cosmids, phosmids, viral vectors, and artificial chromosomes.

12. The method of claim 1, wherein the target DNA comprises one or more operons, or portions thereof, of the DNA population.

13. The method of claim 12, wherein the operon or portions thereof encodes a complete or partial metabolic pathway.

14. The method of claim 3, wherein the uncultured microorganisms comprise extremophiles.

15. The method of claim 14, wherein the extremophiles are selected from the group consisting of thermophiles, hyperthermophiles, psychrophiles, barophiles, and psychrotrophs.

16. The method of claim 4, wherein the host cell is selected from the group consisting of a bacterium, fungus, plant cell, insect cell and animal cell.

17. The method of claim 1, wherein the target DNA encodes a protein.

18. The method of claim 17, wherein the protein is an enzyme.

19. The method of claim 18 wherein the enzyme is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

20. A method for making a gene library, comprising randomly concatenating cDNA or genomic DNA fragments obtained from one or more species of uncultured organisms, and ligating the concatenated DNA fragments to a DNA vector to generate a library of constructs in which genes are contained in the cDNA or genomic DNA fragments.

21. The method of claim 20, wherein the uncultured organisms comprise a mixture of terrestrial microorganisms or marine microorganisms, or a mixture of terrestrial microorganisms and marine microorganisms.

22. The method of claim 20, wherein the uncultured organisms are derived from an environmental sample.

23. The method of claim 20, wherein the cDNA or genomic fragments comprise at least an operon, or portions thereof, of the donor microorganisms.

24. The method of claim 23, wherein the operon encodes a complete or partial metabolic pathway.