Combinatorial screening of mixed populations of organisms
View Patent ↗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.
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.