Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-10870843-B2
Application number	US-201916601993-A
Country	US
Kind code	B2
Filing date	Oct 15, 2019
Priority date	Apr 18, 2013
Publication date	Dec 22, 2020
Grant date	Dec 22, 2020

Abstract

The present invention provides engineered phenylalanine ammonia-lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.

First claim

What is claimed is:

1. An engineered polynucleotide encoding a polypeptide comprising an amino acid sequence with at least 95% sequence identity to reference sequence SEQ ID NO:24, wherein said polypeptide comprises a mutation at position 509, wherein said positions are numbered with reference to SEQ ID NO: 24.

2. The engineered polynucleotide of claim 1 , wherein said encoded engineered polypeptide further comprises at least one amino acid residue differences as compared to SEQ ID NO:24, wherein the amino acid residue differences are at one or more amino acid positions selected from 20, 24, 27, 39, 43, 45, 47, 54, 58, 59, 62, 70, 73, 80, 82, 91, 94, 98, 104, 105, 110, 112, 115, 117, 118, 119, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 134, 135, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 153, 154, 156, 157, 158, 159, 172, 174, 175, 176, 177, 178, 180, 187, 191, 195, 199, 205, 206, 210, 212, 213, 214, 232, 240, 243, 245, 247, 248, 250, 256, 257, 266, 270, 275, 278, 279, 285, 286, 289, 290, 292, 304, 305, 307, 308, 309, 319, 321, 326, 331, 332, 334, 349, 355, 364, 365, 369, 370, 371, 372, 374, 375, 377, 378, 379, 381, 382, 383, 384, 385, 387, 389, 394, 396, 399, 400, 403, 404, 407, 417, 418, 425, 431, 432, 433, 434, 435, 436, 437, 438, 439, 443, 446, 447, 453, 456, 459, 460, 461, 463, 471, 472, 473, 474, 475, 476, 477, 478, 479, 482, 483, 503, 507, 521, 522, 524, 525, 528, 538, 546, 547, 551, 558, 560, 564, 565, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.

3. The engineered polynucleotide of claim 2 , wherein said encoded polypeptide further comprises at least one amino acid residue difference as compared to SEQ ID NO:24, selected from 112C, 134Q, 158H, 180A, 195E, 240R/W, 243I/L, 245L, 256G, 257W/A, 270K, 304H, 307Q/M, 308Q, 326F, 349M, 353A/N, 364Q, 394V, 399N, 400K, 404A, 443H, 453G, 459F, 460G, 463N, 474Q, 521S, 522Y/F/N, 528L, 546R, and 564 G/L/M, wherein said positions are numbered with reference to SEQ ID NO:24.

4. The engineered polynucleotide of claim 1 , wherein said encoded engineered polypeptide exhibits at least one improved property selected from enhanced catalytic activity, reduced sensitivity to proteolysis, and increased tolerance to acidic pH, as compared to the reference sequence SEQ ID NO:24.

5. The engineered polynucleotide of claim 4 , wherein said encoded polypeptide exhibits at least one improved property selected from reduced sensitivity to proteolysis, and increased tolerance to acidic pH.

6. The engineered polynucleotide of claim 5 , wherein said encoded polypeptide is resistant to proteolysis by at least one digestive tract enzyme, wherein said engineered polypeptide is resistant to proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/or elastases.

7. The engineered polynucleotide of claim 1 , wherein said encoded polypeptide is deimmunized.

8. The engineered polynucleotide of claim 1 , wherein said encoded polypeptide has phenylalanine ammonia lyase activity.

9. The engineered polynucleotide of claim 1 , wherein said polynucleotide is purified.

10. The engineered polynucleotide of claim 1 , wherein said polynucleotide is operably linked to at least one control sequence.

11. The engineered polynucleotide of claim 1 , wherein said polynucleotide is codon-optimized.

12. An expression vector comprising at least one polynucleotide of claim 10 .

13. An expression vector comprising at least one polynucleotide of claim 11 .

14. The expression vector of claim 12 , wherein said control sequence is a promoter.

15. The expression vector of claim 11 , further comprising at least one control sequence, wherein said control sequence is a promoter.

16. A host cell transformed with at least one expression vector of claim 12 .

17. A host cell transformed with at least one expression vector of claim 13 .

18. A host cell transformed with at least one expression vector of claim 14 .

19. A host cell transformed with at least one expression vector of claim 15 .

20. A method of producing an engineered polypeptide in a host cell comprising culturing a host cell comprising at least one expression vector of claim 12 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

21. The method of claim 20 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

22. The method of claim 21 , further comprising the step of purifying said at least one engineered polypeptide.

23. A composition comprising at least one engineered polynucleotide of claim 1 .

Assignees

• Codexis Inc

Inventors

• Huisman Gjalt W
• Agard Nicholas J
• Mijts Benjamin
• Vroom Jonathan
• Zhang Xiyun

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12Y403/01005

  Phenylalanine ammonia-lyase (4.3.1.5) (C12Y403/01023 - C12Y403/01025 takes precedence) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y403/01024

  Phenylalanine ammonia-lyase (4.3.1.24) · CPC title