Engineered biocatalysts useful for carbapenem synthesis

Patent metadata

Field	Value
Publication number	US-9540622-B2
Application number	US-201414760963-A
Country	US
Kind code	B2
Filing date	Jan 16, 2014
Priority date	Jan 18, 2013
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

The present disclosure provides engineered pNB esterase polypeptides useful for the synthesis of the carbapenem antibiotic, imipenem. The disclosure also provides polynucleotides encoding the engineered pNB esterases, host cells capable of expressing the engineered pNB esterases, and methods of using the engineered pNB esterases in the production of imipenem.

First claim

What is claimed is:

1. An engineered polypeptide having p-nitrobenzyl esterase (pNB) activity, comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO: 2 at residue positions selected from: 108, 115, 116, 130, 193, 214, 219, 273, 276 and 321, wherein the differences at positions 115 and 273 are 115Q/W and 273A/E/T/V, respectively, and optionally wherein said engineered polypeptide further comprises a different residue at position 362.

2. The engineered polypeptide of claim 1 in which the residue differences as compared to SEQ ID NO: 2 at the residue positions 108, 115, 116, 130, 193, 214, 219, 273, 276, 321, and 362 are selected from 108L/Y, 116S, 130T, 164T, 193A/D/E/V, 214G, 219A/D/L/V, 276A/T/L, 321A, and 362A/D/Q/S/V.

3. The engineered polypeptide of claim 1 in which the amino acid sequence comprises one or more residue differences as compared to SEQ ID NO: 2 selected from: 108L/Y, 193A/D/E/V, 219A/D/L/V, and 362A/D/Q/S/V.

4. The engineered polypeptide of claim 1 in which the amino acid sequence comprises a residue difference as compared to SEQ ID NO: 2 at position 193 selected from: 193A/D/E/V.

5. The engineered polypeptide of claim 4 in which the amino acid residue difference as compared to SEQ ID NO: 2 at position 193 is 193V.

6. The engineered polypeptide of claim 5 in which the amino acid sequence further comprises residue differences as compared to SEQ ID NO: 2 at positions 219 and 273 selected from 219L/V and 273A/V.

7. The engineered polypeptide of claim 6 in which the amino acid sequence further comprises residue differences as compared to SEQ ID NO: 2 at positions 108 and 362 selected from 108L/Y and 362A/D/Q/S/V.

8. The engineered polypeptide of claim 7 in which the amino acid sequence further comprises a residue difference as compared to SEQ ID NO: 2 at position 115 selected from 115Q/W.

9. The engineered polypeptide of claim 2 in which the amino acid sequence comprises a combination of residue differences as compared to SEQ ID NO: 2 selected from:
193V, 219V, and 273A;
108Y, 193D, 219V, 273A, and 362S;
108Y, 193V, 219V, 273A, and 362Q;
108Y, 115Q, 193V, 219L, 273A, and 362Q; and
108Y, 115Q, 193V, 219V, 273A, and 362Q.

10. The engineered polypeptide of claim 1 in which the amino acid sequence further comprises one or more residue differences as compared to SEQ ID NO: 2 selected from: 116S, 130T, 164T, 214G, 276A/T/L, and 321A.

11. The engineered polypeptide of claim 1 in which the amino acid sequence further comprises a residue difference as compared to SEQ ID NO: 2 selected from: 49G, 94G, 96S, 227T, 251V, 267R, 271L, 274L, 313F, 322C/Y, 343V, 356R, 359A, 398L, 412E, 437T, 464A, and 481R.

12. The engineered polypeptide of claim 1 in which the amino acid sequence does not comprise a residue difference as compared to SEQ ID NO: 2 at positions 60, 144, 317, 322, 334, 358, and 370.

13. The engineered polypeptide of claim 2 in which the pNB esterase has at least 1.2 fold, 2 fold, 5 fold, 10 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, or greater increased activity as compared to the polypeptide of SEQ ID NO: 4 in converting compound (2) to compound (1) under suitable reaction conditions.

14. The engineered polypeptide of claim 1 in which the amino acid sequence comprises a sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120.

15. The engineered polypeptide of claim 1 in which the polypeptide is immobilized on a solid support.

16. The engineered polypeptide of claim 15 in which the solid support is a bead or resin comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene/DVB copolymer or polymethacrylate with octadecyl functional groups.

17. A process for preparing compound (1), or a salt or hydrate of compound (1)

wherein the method comprises contacting a substrate compound (2), or a salt or hydrate of compound (2)

with the engineered pNB esterase polypeptide of claim 1 under suitable reaction conditions.

18. The process of claim 17 in which the substrate of compound (2) is at a loading of from about 1 to about 200 g/L, from about 2 to about 100 g/L, or about 5 to about 50 g/L.

19. The process of claim 18 in which the substrate of compound (2) is at a loading of at least about 0.5 g/L, 1 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 100 g/L, 150 g/L, or 200 g/L.

20. The process of claim 17 in which the suitable reaction conditions comprise a buffer selected from borate, phosphate, carbonate, triethanolamine (TEA), Tris, and 2-(N-morpholino)ethanesulfonic acid (MES).

21. The process of claim 20 in which the buffer comprises MES, wherein the MES concentration is about 0.01 to about 0.4 M, about 0.05 to about 0.4 M, about 0.1 to about 0.3 M, or about 0.1 to about 0.2 M.

22. The process of claim 21 in which the MES concentration is about 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.07 M, 0.1 M, 0.12 M, 0.14 M, 0.16 M, 0.18 M, 0.2 M, 0.3 M, or 0.4 M.

23. The process of claim 17 in which the suitable reaction conditions comprise a pH from about 5 to about 10, pH from about 6 to about 9, or pH from about 7 to about 8.

24. The process of claim 23 in which the suitable reaction conditions comprise a pH of about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

25. The process of claim 17 in which the suitable reaction conditions comprise a temperature of about 5° C. to about 65° C., about 10° C. to about 60° C., about 15° C. to about 55° C., about 15° C. to about 45° C., about 15° C. to about 35° C., about 20° C. to about 55° C., or about 30° C. to about 60° C.

26. The process of claim 25 in which the suitable reaction conditions comprise a temperature of about 5° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C.

27. The process of claim 17 in which the suitable reaction conditions comprise dimethylformamide (DMF) as a co-solvent present at a concentration of from about 2% to about 30% v/v; or from about 5% to about 20% v/v.

28. The process of claim 27 in which the DMF is present at a concentration of about 2% v/v, 5% v/v, 10% v/v, 15% v/v, 20% v/v, 25% v/v, or 30% v/v.

29. The process of claim 17 in which the pNB esterase polypeptide is at a concentration of about 0.01 to about 50 g/L, about 0.05 to about 50 g/L, about 0.1 to about 40 g/L, about 1 to about 40 g/L, about 2 to about 40 g/L, about 5 to about 40 g/L, about 5 to about 30 g/L, about 0.1 to about 10 g/L, about 0.5 to about 10 g/L, about 1 to about 10 g/L, about 0.1 to about 5 g/L, about 0.5 to about 5 g/L, or about 0.1 to about 2 g/L.

30. The process of claim 29 in which the pNB esterase polypeptide is at a concentration of about 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.5 g/L, 1 g/L, 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, or 50 g/L.

31

Assignees

• Codexis Inc

Inventors

• Sukumaran Joly
• Smith Derek
• Yang Hong
• Yeo Wan Lin
• Moore Jeffrey C

Classifications

• C12Y301/01

  Carboxylic ester hydrolases (3.1.1) · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12P17/184

  containing a beta-lactam ring, e.g. thienamycin · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P11/00

  Preparation of sulfur-containing organic compounds · CPC title