IP Library › Granted Patent US 12,421,509
Granted Patent B2
US 12,421,509 · App. 18/467,259 · Granted Sep 23, 2025

Engineered phosphopentomutase variant enzymes

Inventors: Scott J. Novick (Palo Alto, CA); Xiang Yi (San Carlos, CA); Nikki Dellas (San Carlos, CA); Oscar Alvizo (Fremont, CA); Jovana Nazor (Milpitas, CA); Da Duan (Foster City, CA); Vesna Mitchell (Santa Clara, CA); Jonathan Vroom (South San Francisco, CA); Santhosh Sivaramakrishnan (Redwood City, CA); Nandhitha Subramanian (Milton Keynes, GB); Jeffrey C. Moore (Westfield, NJ); Mark Huffman (Rahway, NJ); Agustina Rodriguez-Granillo (Rahway, NJ); Deeptak Verma (Rahway, NJ); Grant S. Murphy (Rahway, NJ); Nicholas Marshall (Rahway, NJ); Jay Russell (Rahway, NJ); Keith A. Canada (Freehold, NJ)
Assignee: Codexis, Inc.
C12N9/90C12N15/52C12N15/74C12N15/815C12Y504/02007
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Quick Facts
Patent No.
US 12,421,509
App. No.
18/467,259
Granted
Sep 23, 2025
Kind
B2
Abstract

The present invention provides engineered phosphopentomutase (PPM) enzymes, polypeptides having PPM activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing PPM enzymes are also provided. The present invention further provides compositions comprising the PPM enzymes and methods of using the engineered PPM enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

Claims (19)

1. An engineered phosphopentomutase comprising a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 2, wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 2 at one or more positions in said polypeptide sequence selected from 98, 64/98/99/146/179/231/261/357, 98/99/146/231/357, 98/99/146/179/261/357/397, 98/99/146/153/231/261/357, 98/99/146/231, 98/99/146/179/231/261/357, 98/99/231/261/357/397, 98/99/146/261/357/397, 98/99/146/153/231/357, 98/99/146/153/231/357, 98/99/146/179/231/261/357/397, 98/99/146/231/261/357/397, 98/99/146/231/261/357, 98/99/146/179/231/261/357/397, 98/99/146/261/357/397, 98/99/146/179/231/357, 98/99/146/231/357, 98/99/146/153/179/231/357, and 98/99/146/153/231/261/357/397.

2. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 118, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 118 at one or more positions selected from 64/99/114/231/357, 65, 67, 87/225, 99/114, 99/114/147/231/261/397, 99/114/147/231/357, 99/114/147/257, 99/114/147/261, 99/114/153, 99/114/153/231/357, 99/114/153/261, 99/114/153/261/357, 99/114/231, 99/114/231/261, 99/114/231/357, 99/114/257/261/357, 99/114/357, 99/231/257/261, 99/257, 114/231/257/261, 114/231/357/397, 114/257/357, 114/392, 154, 211, 211/316, 225, 234/310, 257, 257/261, 257/357, 263, 350, 357, and 359.

3. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85%, sequence identity to SEQ ID NO: 266, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 266 at one or more positions selected from 65/225, 65/225/263, 97, 118, 143/194, 147, 156, 160, 172, 176, 188, 192, 194, 207, 211, 217, 225, 227, 236, 238, 257, 257/261, 261, 263, 306, and 397.

4. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 266, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution relative to SEQ ID NO: 266 set at one or more positions selected from 116/238, 118/147/192/238, 118/147/225/257, 118/147/238/257/357, 118/156/192/194/231/257, 118/194/238, 118/231/257/357, 118/238, 118/257/357, 147/194/217/225/236/257, 147/225/238/357, 147/236/238/257, 156/192/217/236/257, 156/217/236/257/357, 156/236/238, 192/238, 192/238/357, 225/238/357, 227, 231/257, 236, 236/238/257, 238/257, and 257.

5. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 420, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 420 at one or more positions selected from 12, 94, 118/147/227/238/257/357, 118/151, 118/236/357, 121, 142, 148, 150, 151, 172, 181, 185, 186, 189, 193, 199, 206, 218, 235, 237, 239, 256, 266, 267, 281, 300, 308, and 383.

6. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85%, sequence identity to SEQ ID NO: 420, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 420 at one or more positions in said polypeptide sequence selected from 98/151/172/235/256/266/267, 98/256,108/235/267, 148/150/172/235, 148/151/172/235/256, 148/151/235/256/301, 148/151/235/267, 150/151/172/235/260, 150/151/235, 150/172/235/267/301, 150/235, 150/267, 151, 151/172/235, 151/172/235/256/301, 151/235/260, 151/256, 151/267/301, 172, 172/235, 172/235/237/256/267, 172/267/301/312, 235, 235/256/267, 235/256/267/307, 235/260, 235/267, 235/267/301, 256, 260, 267, 267/301, 301, 307, and 312.

7. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 562, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 562 at one or more positions in said polypeptide sequence selected from 21, 118, 120, 151, 155, 220, 225, and 357.

8. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 562, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions relative to SEQ ID NO: 562 in said polypeptide sequence selected from 21/66/120/155, 21/117/120/139, 21/117/120/151/155/225, 21/117/120/222, 21/117/222/225, 21/120, 21/120/139, 21/120/151, 21/120/151/155/222/225, 21/120/151/222/225, 21/120/151/225/316, 21/120/155, 21/120/222/357, 21/139/151/357, 21/139/357, 21/151/155/222/225, 21/225/357, 21/357, 50/357,117/120/151/155, 117/120/151/222, 117/120/151/222/225, 117/120/225, 117/139/151/225, 117/151/155/222/357, 117/155/222/357, 117/220/225, 120, 120/139, 120/139/151, 120/139/151/222, 120/139/222, 120/139/357, 120/151/155, 120/151/155/222/225, 120/151/155/225, 120/151/222, 120/151/222/225, 120/151/225, 120/155, 120/155/222, 120/155/225, 120/155/357, 120/220, 120/220/222/357, 120/220/225, 120/222, 120/225, 120/225/357, 120/225/401, 120/357, 139/357, 151/155, 155/222/225/357, 220/357, 222/357, 298/357, and 357.

9. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 656, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 656 at one or more positions in said polypeptide sequence selected from 6, 8, 39, 46, 53, 108, 133, 151, 160, 192, 196, 200, 217, 225, 236, 239, 251, 257, 272, 284, 335, 341, 368, 369, 391, and 397.

10. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85%, sequence identity to SEQ ID NO: 790, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 790 at one or more positions in said polypeptide sequence selected from 8, 8/53, 8/53/120/225/257/272, 8/53/146/151/225/227/257, 8/53/151/192/227/357/369/397, 8/53/151/225, 8/53/151/225/227, 8/53/151/225/227/257/272/369, 8/53/151/257/272/357, 8/53/151/272/369, 8/53/151/357, 8/53/257/272/357, 8/53/369, 8/120/151, 8/151, 8/151/192/227/257/369, 8/151/192/227/357/397, 8/151/225/257/357/397, 8/151/225/357, 8/151/227/257/272, 8/151/257/272/397, 8/225/257/272, 8/225/257/357/369, 8/272, 8/357, 9, 53/151, 53/151/272, 53/151/357, 53/151/357/369, 53/192, 53/257/272/357, 53/257/357, 53/272, 53/397, 99, 118, 120, 151/192/257, 151/225/357, 151/227/257, 151/257, 151/357/369, 172, 227/272/357/369, 235, 238, 256, 257, 257/272, 257/357, 355, 357, and 357/397.

11. The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 846, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set relative to SEQ ID NO: 846 at one or more positions in said polypeptide sequence selected from 6, 7, 9/108/118/172/192/238/355, 9/108/118/172/235/238/256/257/355, 9/108/118/192/256/257/355, 9/108/192/256/257/355, 9/118, 9/118/172/192/257/355, 9/118/172/192/355, 9/118/172/235/238/257, 9/118/172/256/355, 9/118/235/256/355, 9/118/355, 9/172/192/235/238/256/355, 9/172/192/235/256, 9/172/192/355, 9/192/235/256, 9/192/235/355, 9/192/355, 9/235/238/256/355, 9/235/256/257/355, 9/256/355, 9/355, 17, 22, 22/62, 52, 58, 62, 65, 68, 76, 82, 87, 92, 108/118/172/235/238/256/257/355, 108/118/172/256/257/355, 108/172/192/235/238/256/355, 108/172/192/238/256/355, 114, 118, 118/172/235, 118/172/256/257, 118/235/256/257/355, 118/256, 118/355, 126, 133, 137, 139, 151, 155, 172/192/355, 172/235/238/256/257/355, 172/256/257/355, 172/257/355, 180, 188, 189, 192/235/256/355, 192/355, 200, 217, 219, 221, 225, 231, 235, 235/256/257/355, 251, 257, 266, 286, 287, 288, 296, 308, 324, 327, 332, 333, 334, 335, 337, 338, 341, 355, 363, 389, 391, and 393.

12. The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises a polypeptide sequence that is at least 85% more identical to the sequence of at least one engineered phosphopentomutase variant set forth in Table 6.1, 7.1, 8.1, 9.1, 10.1, 12.1, 12.2, 13.1, 14.1, and/or 15.1.

13. The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises a polypeptide sequence that is at least 85% identical to SEQ ID NO: 76, 118, 266, 420, 562, 656, 790, and/or 846.

14. The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises a variant engineered phosphopentomutase set forth in SEQ ID NO: 76, 118, 266, 420, 562, 656, 790, and/or 846.

15. The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises a polypeptide sequence that is at least 85% identical to the sequence of at least one engineered phosphopentomutase variant set forth in the even numbered sequences of SEQ ID NOS: 76, 108, 110, 116, 118, 120, 122, 124, 126, 130, 132, 136, 138, 142, 144, 146, 152, 154, 160, 164, 168, 170, 172, 174, 176, 186, 190, 198, 200, 204, 206, 212, 214, 216, 230, 240, 244, 248, 252, and 266-1152.

16. The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises a polypeptide sequence forth in at least one of the even numbered sequences of SEQ ID NOS: 76, 108, 110, 116, 118, 120, 122, 124, 126, 130, 132, 136, 138, 142, 144, 146, 152, 154, 160, 164, 168, 170, 172, 174, 176, 186, 190, 198, 200, 204, 206, 212, 214, 216, 230, 240, 244, 248, 252, and 266-1152.

17. The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises at least one improved property compared to wild-type E. coli phosphopentomutase.

18. The engineered phosphopentomutase of claim 17 , wherein said improved property comprises improved production of compound 1

and/or compound 3

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 Feb 8, 2024
From: NOVICK, SCOTT J.; YI, XIANG; DELLAS, NIKKI; ALVIZO, OSCAR; NAZOR, JOVANA; DUAN, DA; MITCHELL, VESNA; VROOM, JONATHAN; SIVARAMAKRISHNAN, SANTHOSH; SUBRAMANIAN, NANDHITHA; MOORE, JEFFREY C.; HUFFMAN, MARK A.; VERMA, DEEPTAK; MURPHY, GRANT S.; MARSHALL, NICHOLAS M.; RUSSELL, JAY H.; CANADA, KEITH A.; RODRIGUEZ-GRANILLO, AGUSTINA; MERCK SHARP & DOHME; NOVICK, SCOTT J.; YI, XIANG; DELLAS, NIKKI; ALVIZO, OSCAR; NAZOR, JOVANA; DUAN, DA; MITCHELL, VESNA
To: CODEXIS, INC.
Reel/Frame 066529/0216 →
Continuity (4)
Continuation 17316165 · May 10, 2021
Division 16460062 · Jul 2, 2019
Provisional Application 62695491 · Jul 9, 2018
Related Publication 20240060064A1 · Feb 22, 2024
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