Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-10294468-B2
Application number	US-201816176896-A
Country	US
Kind code	B2
Filing date	Oct 31, 2018
Priority date	Apr 18, 2013
Publication date	May 21, 2019
Grant date	May 21, 2019

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 an engineered polypeptide comprising an amino acid sequence with at least 90% sequence identity to reference sequence SEQ ID NO:24, wherein said amino acid sequence comprises an amino acid residue difference at position 353.

2. The engineered polynucleotide encoding an engineered polypeptide of claim 1 , wherein said engineered polypeptide further comprises at least one additional substitution at a position in SEQ ID NO:24, and wherein said positions are numbered with reference to SEQ ID NO:24.

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

4. The engineered polynucleotide encoding an engineered polypeptide of claim 1 wherein the engineered polypeptide 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, 509, 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.

5. The engineered polynucleotide encoding an engineered polynucleotide encoding an engineered polypeptide of claim 4 , wherein the further amino acid residue difference as compared to SEQ ID NO:24 is selected from one or more of the following substitutions 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, 509L, 521S, 522Y/F/N, 528L, 546R, and 564 G/L/M, when optimally aligned with the polypeptide of SEQ ID NO:24.

6. The engineered polynucleotide encoding an engineered polypeptide of claim 3 , wherein the improved property is selected from reduced sensitivity to proteolysis and/or increased tolerance to acidic pH.

7. The engineered polynucleotide encoding an engineered polypeptide of claim 6 , wherein the engineered 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.

8. The engineered polynucleotide encoding an engineered polypeptide of claim 1 , wherein said engineered polypeptide is deimmunized.

9. The engineered polynucleotide encoding an engineered polypeptide of claim 1 , wherein said engineered polypeptide has phenylalanine ammonia lyase activity.

10. The engineered polynucleotide encoding an engineered polypeptide of claim 1 , wherein said polypeptide is purified.

11. The engineered polynucleotide sequence of claim 1 , wherein said polynucleotide sequence is operably linked to a control sequence.

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

13. An expression vector comprising at least one engineered polynucleotide sequence of claim 1 , and at least one control sequence.

14. The expression vector of claim 13 , wherein said control sequence is a promoter.

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

16. A method of producing an engineered polypeptide in a host cell comprising culturing a host cell comprising at least one expression vector of claim 13 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

17. The method of claim 16 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

18. The method of claim 16 , further comprising the step of purifying said at least one engineered polypeptide.

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

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title

• C12Y403/01024

  Phenylalanine ammonia-lyase (4.3.1.24) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title