Protein activity screening of clones having DNA from uncultivated microorganisms

• US 5,958,672 A
• Filed: 06/03/1996
• Issued: 09/28/1999
• 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): Wax, Robert A.
• Assistant Examiner(s): Nashed, Nashaat T.
• Application Number:
  US08/657,409
• Time in Patent Office: 1,212 Days
• Field of Search: 435/4, 435/183, 435/69.1
• US Class Current: 435/4
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions

Abstract

Disclosed is a process of screening clones having DNA from an uncultivated microorganism for a specified protein, e.g. enzyme, activity by screening for a specified protein, e.g. enzyme, activity in a library of clones prepared by (i) recovering DNA from a DNA population derived from at least one uncultivated microorganism; and (ii) transforming a host with recovered DNA to produce a library of clones which is screened for the specified protein, e.g. enzyme, activity.

Claims

1. A method for identifying a protein activity of interest comprising:

culturing a gene expression library comprising a pool of expression constructs, each expression construct comprising a vector containing one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are derived from a plurality of species of donor organisms, and wherein the cDNA or genomic DNA fragments are each operably-associated with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism; and

detecting the protein activity encoded by the cDNA or genomic DNA fragments.

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

3. The method of claim 2, wherein the enzymatic activity is selected from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase activity.

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

5. The method of claim 4, wherein the microorganisms are derived from an environmental sample.

6. The method of claim 4, wherein the microorganisms are a mixed population of uncultured organisms.

7. The method of claim 1, wherein the DNA fragment comprises one or more operons, or portions thereof.

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

9. A method for identifying a protein activity of interest comprising:

culturing a gene expression library, comprising a pool of expression constructs, each expression construct comprising a vector containing one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are derived from a plurality of species of donor microorganisms, and wherein the cDNA or genomic DNA fragments are each operably-associated with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism; and

detecting the protein activity encoded by the cDNA or genomic DNA fragments.

10. The method of claim 9, wherein the protein activity is an enzymatic activity.

11. The method of claim 10, wherein the enzymatic activity is selected from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase activity.

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

13. The method of claim 9, wherein the microorganisms are a mixed population of uncultured organisms.

14. The method of claim 9, wherein the DNA fragment comprises one or more operons, or portions thereof.

15. The method of claim 14, wherein the operon or portions thereof encodes a complete or partial metabolic pathway.