Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-11913045-B2
Application number	US-202017009925-A
Country	US
Kind code	B2
Filing date	Sep 2, 2020
Priority date	Apr 18, 2013
Publication date	Feb 27, 2024
Grant date	Feb 27, 2024

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 95% sequence identity to reference sequence SEQ ID NO:24, and wherein the engineered polypeptide comprises an amino acid residue difference at positions 158, 256, and 399, wherein the amino acid 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 difference 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, 521S, 522Y/F/N, 528L, 546R, and 564 G/L/M, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.

3. 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, increased tolerance to acidic pH, as compared to the reference sequence SEQ ID NO:24.

4. The engineered polynucleotide of claim 3 , wherein said encoded engineered polypeptide exhibits an improved property selected from reduced sensitivity to proteolysis and/or increased tolerance to acidic pH.

5. The engineered polynucleotide of claim 4 , wherein said encoded engineered polypeptide is resistant to proteolysis by at least one digestive tract enzyme, and further wherein said engineered polypeptide is resistant to proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/or elastases.

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

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

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

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

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

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

12. The expression vector of claim 11 , wherein said control sequence is a promoter.

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

14. A method of producing an engineered polypeptide in a host cell comprising culturing a host cell comprising at least one expression vector of claim 11 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

15. The method of claim 14 , further comprising recovering said at least one engineered polypeptide from the culture and/or host cells.

16. The method of claim 15 , 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

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y403/01005

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

• C12Y403/01024

  Phenylalanine ammonia-lyase (4.3.1.24) · 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

• A61P25/00

  Drugs for disorders of the nervous system · CPC title