Oligonucleotide mediated nucleic acid recombination

• US 8,029,988 B2
• Filed: 11/30/2007
• Issued: 10/04/2011
• Est. Priority Date: 01/19/1999
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis Mayflower Holdings, LLC (Codexis, Inc.)
• Inventors: Patten, Phillip A., Ness, Jon E., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C., Bass, Steven H., Crameri, Andreas, Welch, Mark
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/987,555
• Publication Number:
  US 20080171668A1
• Time in Patent Office: 1,404 Days
• Field of Search: 435/6, 435/440, 536/22.1, 536/23.1, 536/24.3, 536/25.3
• US Class Current: N/A
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

Abstract

Methods of recombining nucleic acids, including homologous nucleic acids, are provided. Families of gene shuffling oligonucleotides and their use in recombination procedures, as well as polymerase and ligase mediated recombination methods are also provided.

Claims

1. A method of recombining homologous nucleic acids, the method comprising:

hybridizing a chemically synthesized set of family gene shuffling oligonucleotides, wherein the set of family shuffling gene oligonucleotides comprises a plurality of homologous oligonucleotide member types, wherein the homologous oligonucleotide member types are present in non-equimolar amounts;

elongating the set of family gene shuffling oligonucleotides, thereby providing a population of recombined nucleic acids; and

selecting one or more recombined nucleic acids from the population of recombined nucleic acids for a desired enzymatic property,

wherein the selected one or more recombined nucleic acids encode industrial enzymes.

2. The method of claim 1, wherein members of the set of family gene shuffling oligonucleotides overlap.

3. The method of claim 1, wherein elongating the set of family gene shuffling oligonucleotides is performed with a polymerase or a ligase.

4. The method of claim 1, wherein the set of family gene shuffling oligonucleotides encodes an evolutionary intermediate nucleic acid.

5. The method of claim 1, the method further comprising, prior to selecting the one or more recombined nucleic acids for the desired property,

denaturing the population of recombined nucleic acids, thereby providing denatured recombined nucleic acids;

reannealing the denatured recombined nucleic acids; and

extending or ligating the resulting reannealed recombined nucleic acids.

6. The method of claim 5, wherein, prior to selecting the one or more recombined nucleic acids for the desired property, the reannealed recombined nucleic acids are recombined.

7. The method of claim 5, further comprising recombining the resulting selected recombined nucleic acids.

8. The method of claim 7, further comprising selecting the resulting multiple recombined nucleic acids for the desired property.

9. The method of claim 1, the method further comprising:

denaturing the population of recombined nucleic acids, thereby providing denatured recombined nucleic acids;

reannealing the denatured nucleic acids;

extending the resulting reannealed recombined nucleic acids; and

repeating the above three steps at least once.

10. The method of claim 1, wherein a plurality of members of the population of recombined nucleic acids are screened for the desired property and are determined to have the desired property, thereby providing first round screened nucleic acids, the method further comprising:

hybridizing a second set of family gene shuffling oligonucleotides, which second set of family gene shuffling oligonucleotides are derived from the first round screened nucleic acids; and

,

elongating the second set of family gene shuffling oligonucleotides, thereby providing a population of further recombined nucleic acids.

11. The method of claim 10, wherein members of the second set of family gene shuffling oligonucleotides overlap.

12. The method of claim 10, further comprising

sequencing the first round screened nucleic acids,

deriving the second set of family gene shuffling oligonucleotides from the first round screened nucleic acids by aligning sequences of the first round screened nucleic acids to identify regions of identity and regions of diversity in the first round screened nucleic acids, and

synthesizing the second set of family gene shuffling oligonucleotides to produce a plurality of oligonucleotides, each of which comprises subsequences corresponding to at least one region of diversity.

13. The method of claim 10, wherein the first round screened nucleic acids encode polypeptides of about 50 amino acids or less.

14. The method of claim 10, wherein the second set of family shuffling gene oligonucleotides comprise a plurality of oligonucleotide member types which comprise consensus region subsequences derived from a plurality of the first round screened nucleic acids.

15. The method of claim 1, wherein the set of family shuffling gene oligonucleotides comprise a plurality of oligonucleotide member types which comprise consensus region subsequences derived from a plurality of homologous target nucleic acids.

16. The method of claim 1, wherein the set of family shuffling gene oligonucleotides comprise at least one module shuffling oligonucleotide(s).

17. The method of claim 1, wherein the set of family shuffling gene oligonucleotides comprise a plurality of module shuffling oligonucleotides, each comprising at least a first subsequence from a first sequence module and a second subsequence from a second sequence module.

18. The method of claim 1, wherein the set of family shuffling gene oligonucleotides comprise a plurality of module shuffling oligonucleotides, wherein one or more of the plurality of oligonucleotides each comprise at least a first subsequence from a first sequence module and a second subsequence from a second sequence module.

19. The method of claim 1, wherein the set of family shuffling oligonucleotides comprise a plurality of codon-varied oligonucleotides.

20. The method of claim 1, the set of family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprises at least 3 member types.

21. The method of claim 1, the set of family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprising at least 5 member types.

22. The method of claim 1, the set of family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprising at least 10 member types.

23. The method of claim 1, the set of family shuffling gene oligonucleotides comprising a plurality of homologous oligonucleotide member types, wherein the homologous oligonucleotide member types are present in approximately equimolar amounts.

24. A method of recombining homologous nucleic acids, the method comprising:

hybridizing a chemically synthesized set of family gene shuffling oligonucleotides;

elongating the set of family gene shuffling oligonucleotides, thereby providing a population of recombined nucleic acids;

selecting one or more recombined nucleic acids from the population of recombined nucleic acids for a desired enzymatic property, wherein the selected one or more recombined nucleic acids encode industrial enzymes, and wherein a plurality of members of the population of recombined nucleic acids are screened for the desired property and are determined to have the desired property, thereby providing first round screened nucleic acids;

sequencing the first round screened nucleic acids,

deriving a second set of family gene shuffling oligonucleotides from the first round screened nucleic acids by aligning sequences of the first round screened nucleic acids to identify regions of identity and regions of diversity in the first round screened nucleic acids;

hybridizing the second set of family gene shuffling oligonucleotides, which second set of family gene shuffling oligonucleotides are derived from the first round screened nucleic acids, wherein members of the second set of family gene shuffling oligonucleotides overlap;

elongating the second set of family gene shuffling oligonucleotides, thereby providing a population of further recombined nucleic acids; and

synthesizing the second set of family gene shuffling oligonucleotides to produce a plurality of oligonucleotides, each of which comprises subsequences corresponding to at least one region of diversity.