IP Library › Granted Patent US 11,060,075
Granted Patent B2
US 11,060,075 · App. 16/666,280 · Granted Jul 13, 2021

Engineered DNA polymerase variants

Inventors: Mathew G. Miller (San Carlos, CA); Vesna Mitchell (Santa Clara, CA); Jovana Nazor (Milpitas, CA); Donald S. Baskerville (Louisville, CO); Nikki Dellas (Mountain View, CA); David Elgart (San Mateo, CA); Jonathan Vroom (South San Francisco, CA); Sandy M. Gomes (Redwood City, CA); Nandhitha Subramanian (San Francisco, CA); Ericka Bermudez (Aptos, CA)
Assignee: Codexis, Inc.
C12N9/1252
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Quick Facts
Patent No.
US 11,060,075
App. No.
16/666,280
Granted
Jul 13, 2021
Kind
B2
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 (27)

1. An engineered DNA polymerase comprising a polypeptide sequence having at least 85% or more sequence identity to the reference sequence of SEQ ID NO: 6, wherein the engineered DNA polymerase comprises a mutation at position 515 in its polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 6.

2. The engineered DNA polymerase of claim 1 , further comprising at least one substitution or substitution set at amino acid positions 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 , further comprising at least one substitution or substitution set 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, 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 , further comprising at least one substitution or substitution set 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 further 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 , further comprising at least one substitution or substitution set 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 , further comprising at least one substitution or substitution set 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 , further comprising at least one substitution or substitution set 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 , 240/579, further comprising at least one substitution or substitution set at amino acid positions selected from 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 , further comprising at least one substitution at amino acid positions selected from 257, 420, 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 , further comprising at least one substitution or substitution set 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 , further comprising at least one substitution or substitution set 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 , further comprising at least one substitution or substitution set 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 , further comprising at least one substitution or substitution set 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 , further comprising at least one substitution or substitution set 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 , further comprising at least one substitution or substitution set 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 has DNA polymerase activity.

18. 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.

19. The engineered DNA polymerase of claim 18 , 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.

20. 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.

21. 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 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 thermostability 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.

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

24. A composition comprising the engineered DNA polymerase of claim 1 .

25. The engineered DNA polymerase of claim 1 , wherein the polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%—or more sequence identity to the reference sequence of SEQ ID NO: 6.

26. The engineered DNA polymerase of claim 1 , wherein the polypeptide sequence has at least 95%, 96%, 97%, 98%,—or more sequence identity to the reference sequence of SEQ ID NO: 6.

27. The engineered DNA polymerase of claim 1 , wherein the polypeptide sequence has at least 99%—or more sequence identity to the reference sequence of SEQ ID NO: 6.

Assignments (2)
SECURITY INTEREST Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 25, 2019
From: MILLER, MATHEW G.; MITCHELL, VESNA; NAZOR, JOVANA; BASKERVILLE, DONALD S.; DELLAS, NIKKI; ELGART, DAVID; VROOM, JONATHAN; GOMES, SANDY M.; SUBRAMANIAN, NANDHITHA; BERMUDEZ, ERICKA
To: CODEXIS, INC.
Reel/Frame 051101/0895 →
Continuity (2)
Provisional Application 62752215 · Oct 29, 2018
Related Publication 20200131485A1 · Apr 30, 2020