Methods of evolving a polynucleotides by mutagenesis and recombination

• US 6,365,408 B1
• Filed: 01/04/2000
• Issued: 04/02/2002
• Est. Priority Date: 06/19/1998
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/477,763
• Time in Patent Office: 819 Days
• Field of Search: 435/440, 435/6, 435/91.2, 536/23.2, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 506/1
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

A method of mutating a polynucleotide such that it has a desired or improved functional property is disclosed.

Claims

1. A method of evolving a polynucleotide toward acquisition of a desired functional property, comprising:

mutagenizing the polynucleotide to generate a population of mutant forms of the polynucleotide;

introducing a plurality of the mutant forms as components of a vector into host cells, and culturing the host cells, wherein mutant forms recombine with each other to form recombinant sequences;

selecting or screening the recombinant sequences for the desired functional property.

2. The method of claim 1, further comprising ligating a plurality of the mutant forms to form ligated mutant forms and introducing ligated mutant forms as components of a vector into the host cells.

3. The method of claim 1, wherein the mutagenizing is by random mutagenesis.

4. The method of claim 1, wherein the polynucleotide is a naturally occurring polynucleotide.

5. The method of claim 1, wherein the mutagenizing is performed by error-prone PCR.

6. The method of claim 1, wherein the mutagenizing is performed by oligonucleotide cassette mutagenesis.

7. The method of claim 6, wherein the oligonucleotide cassette mutagenesis is performed using oligonucleotides having codon positions encoding variant amino acids selected from a defined subset of amino acid residues and codon positions encoding invariant amino acids.

8. The method of claim 7, wherein codon positions encoding the variant amino acids comprises the sequence NNK.

9. The method of claim 1, wherein the mutagenic forms are of unknown sequence.

10. The method of claim 1, further comprising recombining the selected or screened recombinant sequences, and screening the resulting sequences for further enhancement of the desired functional property.

11. The method of claim 1, wherein the desired functional property is a increased drug resistance, or capacity to bind to a receptor.

12. The method of claim 1, wherein the desired functional property is an enzymatic activity.

13. The method of claim 1, wherein the mutant forms differ from the polynucleotide by point mutations.

14. The method of claim 1, wherein the mutant forms encode a set of random peptides.

15. The method of claim 1, wherein the host cells are yeast or mammalian cells.

16. The method of claim 1, wherein the host cells are bacterial.

17. The method of claim 1, wherein the host cells are eukaryotic.

18. The method of claim 1, wherein the mutant forms are introduced into the host cells by electroporation.

19. The method of claim 1, wherein the vector further comprises a marker gene.

20. The method of claim 10, wherein the second recombining step is performed in vitro.

21. The method of claim 10, wherein the second recombining step is performed in vivo.

22. The method of claim 21, wherein the second recombining step comprises introducing a plurality of the selected or screened recombinant sequences as components of a vector into host cells and culturing the host cells, wherein the recombinant forms recombine with each other to produce further recombined sequences.

23. The method of claim 10, wherein the second recombining step comprises conducting multiple cycles of denaturation, renaturation and incubation in the presence of a polymerase on overlapping segments from the selected or screened recombinant sequences under conditions where by one segment serves as a template for another segment thereby producing further recombined sequences.

24. The method of claim 23, wherein the overlapping segments are random.

25. The method of claim 23, wherein the overlapping segments are non-random.

26. The method of claim 23, wherein the overlapping segments are produced by cleavage of the selected or screened recombinant sequences.

27. The method of claim 26, wherein the selected or screened recombinant sequences are DNA and cleavage is by DNaseI digestion.

28. The method of claim 23, wherein the overlapping segments are produced by chemical synthesis.

29. A method of identifying a recombined polynucleotide with a desired functional property comprising:

introducing point mutations into a polynucleotide by cassette oligonucleotide mutagenesis in order to generate a population of mutant forms of the polynucleotide; and

shuffling a plurality of the mutant forms in order to produce recombinant sequences; and

selecting or screening the recombinant sequences for the desired functional property.

30. The method of claim 29, wherein the shuffling step is performed in vitro.

31. The method of claim 30, wherein the shuffling step is performed in vivo.

32. The method of claim 29, wherein the shuffling step comprises conducting multiple cycles of denaturation, renaturation and incubation in the presence of a polymerase on overlapping segments from the mutant forms under conditions whereby one segment serves as a template for another segment thereby producing further recombined sequences.

33. The method of claim 32, wherein the overlapping segments are random.

34. The method of claim 32, wherein the overlapping segments are non-random.

35. The method of claim 32, wherein the overlapping segments are produced by cleavage of the mutant forms.

36. The method of claim 32, wherein the mutant forms are DNA and cleavage is by DNaseI digestion.

37. The method of claim 32, wherein the overlapping segments are produced by chemical synthesis.

38. The method of claim 32, further comprising recombining the selected or screened recombinant sequences, and screening the resulting sequences for further enhancement of the desired functional property.

39. The method of claim 29, wherein the oligonucleotide cassette mutagenesis is performed by using oligonucleotides having codon positions encoding variant amino acids selected from a defined subset of amino acid residues and codon positions encoding variant amino acids.

40. The method of claim 39, wherein the codon positions encoding the variant amino acids comprise the sequence NNK.