ENGINEERED DNA POLYMERASE VARIANTS

• US 20200131485A1
• Filed: 10/28/2019
• Published: 04/30/2020
• Est. Priority Date: 10/29/2018
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Miller, Mathew G., Mitchell, Vesna, Nazor, Jovana, Baskerville, Donald S., Dellas, Nikki, Elgart, David, Vroom, Jonathan, Gomes, Sandy M., Subramanian, Nandhitha, Bermudez, Ericka
• Granted Patent: US 11,060,075 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2333/9126 DNA-directed DNA polymerase (2.7.7.7)

Abstract

The present invention provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The invention also provides methods for use of the compositions comprising the engineered DNA polymerase polypeptides for diagnostic and other purposes.

Claims

1. An engineered DNA polymerase comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO:

2, 6, 22, 24, 26, 28, and/or 824, or a functional fragment thereof, wherein the engineered DNA polymerase comprises at least one mutation in its polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO;

2, 6, 22, 24, 26, 28, or 824.

2. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set is selected from 21, 21/66/247/282, 247/282/575, 282/575, 283/647/702/743, 339/647/661/664/668/702/712, 372/391/702, 391, 391/647/659/661/668/671/712/716, 391/647/659/661/668/671/716, 391/647/659/664/668/702/728/732, 391/647/659/664/671/702, 391/647/661/664/671/702/716, 391/647/671/728, 391/659/702/716/732/737, 391/661/664/668/671/716/737, 391/671, 391/702/712/716/732/743, 647/659/661/664/668/702, 647/659/664/668/702/712/737, 647/659/668/671/716/728, 647/668, 647/668/671/712, 659/702/743, 661/664/668/671/716, 668/702, 671/702, 671/702/716, 702, and 743, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

3. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18/387, 24/719, 43/528, 48/760, 101/646, 108/679, 223, 257, 282, 359, 360, 361, 362, 376/619, 390, 391, 394, 394/399, 420, 421, 478, 502, 506, 514, 515, 521, 528, 583/730, 603, 619, 631, 646, 655, 662, 666, 668, 685, 691, 702, 721, 738, 754, 760, and 761, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

4. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 174/361/394/666/668/721, 360/391, 361/391/659, 361/394/420/528/646/666/721/743, 361/394/420/528/666, 361/394/420/646/666/702/721/743, 361/528/646/666, 361/528/646/702/721, 361/528/666, 361/646, 394/420, 502/507/695, 528/646/659/668/743, 528/666, 528/668, 528/743, 619, 666, and 685/691/743, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

22.

5. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 100, 277, 280, 281, 283, 339, 401, 468, 479, 480, 482, 489, 490, 491, 496, 497, and 498, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

22.

6. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 15/134/482/490/497/671/685, 234/497/647, 257/390/420, 257/390/420/647, 257/401/420, 257/401/420/482/647/671/685, 257/482/497/647, 257/647, 257/671/685/702, 281, 281/391/478, 281/391/478/685, 281/391/488/492, 281/391/495/561/659/668, 281/391/659/668, 281/391/668, 281/478/659/685/702, 281/478/668, 281/488, 281/488/492/495/659/668, 281/488/492/668/702, 281/488/495, 281/488/495/668, 281/492/495/668, 281/492/495/668/702, 281/668, 390/401/716, 390/420, 390/491/671, 390/497, 390/671/685, 391, 391/478, 391/478/479/668, 391/478/492/668, 391/479/659/668, 391/488/492/659/685, 391/488/492/668, 391/488/495/668/685/702, 391/492/495, 391/492/495/659, 391/492/515/659/685, 391/495/659, 401, 401/482/659/671/702, 401/490, 401/490/659/671, 401/671, 420, 420/482/659/702, 420/490, 420/490/659/661/671, 420/659/702, 420/661/671, 420/685, 478, 478/479, 478/479/668, 478/479/702, 478/488/659, 478/488/668/685/702, 478/515, 479/492, 479/659/678, 482/497/647/716, 482/497/671/685, 482/671/702/716, 488, 488/492, 488/492/495, 488/495, 488/495/685, 490/497/661/671/685/702/716, 492, 492/495/659/668, 492/659/685, 492/668/685/712, 492/668/712, 495, 495/659, 495/659/685, 497/647, 497/647/659/671, 497/659/691/716, 497/661, 497/661/671, 497/671/702, 497/671/716, 497/685, 497/702, 515, 659, 659/691, and 671, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

24.

7. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 55/579, 108, 108/521, 156/451, 236/755, 240, 247, 248, 256, 298, 299, 299/319, 302, 309, 316, 319, 350, 356, 357, 358, 370, 384, 385, 386, 389, 406, 407, 411, 415, 440, 443, 447, 450, 451, 520, 536, 539, 540, 544, 550/575, 566, 568, 575, 579, 579/767, 600, 601, 601/638, 609/648, 624, 634, 648, 656, 672, 758, 765, 767, 772, 777, 778, 779, 780, 782, 784, and 785, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

24.

8. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 248, 281, 281/302, 281/492, 302/401, 339/491/492/579/712, 390/466/539/712, and 661, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

26.

9. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 240/579, 240/579/702, 248/391/539/579/659/702, 248/391/659, 302/391/579, 339/390/420/425/466/490/491/515/702, 391, 391/482, 391/659, 420/515, 579, 579/659/702, 579/702, and 659/702, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

28.

10. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 257, 420, 515, and 521, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

11. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 71/361/702/721/738, 277, 281, 339, 391/491, 401, 479, 480, 482, 488, 490, 491, 492, 495, 497, 528/646/659/668/743, 702/743, and 743, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

22.

12. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 240, 370, 385, 539, 540, 550/575, 634, and 777, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

24.

13. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 390/391, 482, and 515, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

28.

14. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 281, 281/579, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

28.

15. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 13, 15, 19, 26, 52, 55, 61, 80, 81, 82, 95, 111, 118, 141, 148, 152, 156, 162, 163, 179, 181, 187, 189, 191, 196, 208, 221, 229, 231, 242, 258, 274, 297, 313, 314, 317, 325, 326, 333, 349, 377, 387, 394, 395, 411, 447, 450, 451, 453, 469, 482, 496, 502, 520, 521, 537, 563, 564, 564/572, 567, 569, 575, 580, 601, 603, 619, 620, 648, 667, 673, 690, 705, 719, 731, 758, 761, 772, 774, 775, 778, 783, and 784, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

824.

16. The engineered DNA polymerase of claim 1, wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 15/447/569/775/783/784, 82/242/569, 82/450/567/569, 313, 314/447/569/783/784, 537/667, 567/569/667, and 569, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

824.

17. The engineered DNA polymerase of claim 1, wherein said engineered DNA polymerase comprises a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered DNA polymerase variant set forth in Table 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 6.2, and/or 6.3.

18. The engineered DNA polymerase of claim 1, wherein said engineered DNA polymerase has DNA polymerase activity.

19. The engineered DNA polymerase of claim 1, having at least one improved property, as compared to a wild-type DNA polymerase selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus.

20. The engineered DNA polymerase of claim 19, having at least one improved property, as compared to wild-type DNA polymerase, wherein said improved property is selected from producing increased product in polymerase chain reactions, greater fidelity, and greater thermostability.

21. The engineered DNA polymerase of claim 1, wherein said engineered DNA polymerase produces a greater product yield in polymerase chain reactions than wild-type DNA polymerase, wherein said wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus.

22. The engineered DNA polymerase of claim 1, wherein said engineered DNA polymerase exhibits greater fidelity than wild-type DNA polymerase, wherein said wild-type DNA polymerase selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus.

23. The engineered DNA polymerase of claim 1, wherein said engineered DNA polymerase exhibits greater thermostability than wild-type DNA polymerase, wherein said wild-type DNA polymerase selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus.

24. The engineered DNA polymerase of claim 1, wherein said polymerase is purified.

25. A polynucleotide sequence encoding at least one engineered DNA polymerase of claim 1.

27. The polynucleotide sequence of claim 25, wherein said polynucleotide sequence encodes at least one engineered DNA polymerase comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO:

2, 6, 22, 24, 26, 28, and/or 824.

28. The polynucleotide sequence of claim 27, wherein said sequence comprises SEQ ID NO:

1, 5, 21, 23, 25, 27, and/or 823.

29. The polynucleotide sequence of claim 25, wherein said polynucleotide sequence is operably linked to a control sequence.

30. The polynucleotide sequence of claim 25, wherein said polynucleotide sequence is codon-optimized.

31. An expression vector comprising at least one polynucleotide sequence of claim 25.

32. A host cell transformed with at least one expression vector of claim 31.

33. A method of producing an engineered DNA polymerase polypeptide in a host cell comprising culturing a host cell of claim 32, under suitable culture conditions, such that at least one engineered DNA polymerase is produced.

34. The method of claim 33, further comprising recovering at least one engineered DNA polymerase from the culture and/or host cells.

35. The method of claim 34, further comprising the step of purifying said at least one engineered DNA polymerase.

36. A composition comprising at least one engineered DNA polymerase of claim 1.

38. A method for high-throughput fidelity determination of a DNA polymerase, comprising i) providing:

at least one DNA polymerase set forth in claim 1;

a reporter plasmid comprising genes encoding a first reporter protein and a second reporter protein and a selection marker;

an amplification system, including a thermocycler and reagents for conducting a polymerase chain reaction; and

a purification system;

an transformation system, including competent host cells; and

a flow cytometer;

ii) exposing said DNA polymerase and said reporter plasmid to said amplification system, under conditions such that the reporter construct is amplified by said DNA polymerase to produce PCR product;

iii) circularizing said PCR product to provide circularized PCR amplicons;

vi) transforming said PCR amplicons using said transformation system to produce transformed cells; and

vii) analyzing said transformed cells using said flow cytometer; and

viii) determining the fidelity of said DNA polymerase.

39. The method of claim 38, further comprising the step of inducing said transformed cells.

40. The method of claim 38, wherein said first reporter protein comprises green fluorescent protein.

41. The method of claim 38, wherein said second reporter protein comprises dsRed.

42. The method of claim 38, wherein said selection marker comprises chloramphenicol acetyltransferase.

43. The method of claim 38, wherein said circularization of said PCR amplicons is conducted using at least one ligase.

44. The method of claim 38, wherein said PCR amplicons are purified.

45. The method of claim 38, further comprising determining the fold-improvement in polymerase fidelity as compared to a reference DNA polymerase.

46. The method of claim 45, wherein the reference DNA polymerase is a wild-type polymerase.

47. The method of claim 46, wherein said wild-type polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus.

26. A polynucleotide sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO:

1, 5, 21, 23, 25, 27, 823, and/or or a functional fragment thereof, wherein said engineered polypeptide comprises at least one substitution at one or more amino acid positions.

37. A high-throughput assay system for determination of DNA polymerase fidelity.