Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-2022056431-A1
Application number	US-202117522370-A
Country	US
Kind code	A1
Filing date	Nov 9, 2021
Priority date	Jul 12, 2018
Publication date	Feb 24, 2022
Grant date	—

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. Methods for producing PAL enzymes are also provided. In some embodiments, the engineered PAL polypeptides are optimized to provide enhanced catalytic activities that are useful under industrial process conditions for the production of pharmaceutical compounds.

First claim

We claim:

1 . An engineered polynucleotide encoding an engineered phenylalanine ammonia lyase comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 4, wherein said engineered phenylalanine ammonia lyase comprises a substitution at position 104 and at least one substitution selected from positions 220, 222, and 359 in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8.

2 . An engineered polynucleotide sequence encoding at least one engineered phenylalanine ammonia lyase, wherein said polynucleotide sequence comprises at least 85% sequence identity to SEQ ID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139, wherein the polynucleotide sequence of said engineered phenylalanine ammonia lyase comprises at least one substitution at one or more positions.

3 . The engineered polynucleotide sequence of claim 1 , wherein said polynucleotide sequence comprises at least 85% sequence identity to SEQ ID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139.

4 . The engineered polynucleotide sequence of claim 1 , wherein said polynucleotide sequence comprises SEQ ID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139.

5 . The engineered polynucleotide sequence of claim 1 , wherein said polynucleotide sequence comprises a sequence set forth in the odd-numbered sequences set forth in SEQ ID NOS: 3-1221.

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

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

8 . An expression vector comprising at least one polynucleotide sequence of claim 1 .

9 . A host cell comprising at least one expression vector of claim 8 .

10 . A host cell comprising at least one polynucleotide sequence of claim 1 .

11 . The host cell of claim 10 , wherein said host cell is a eukaryotic host cell.

12 . The host cell of claim 11 , wherein said host cell is Escherichia coli.

13 . A method of producing an engineered phenylalanine ammonia lyase in a host cell, comprising culturing the host cell of claim 10 , in a culture medium under suitable conditions, such that at least one engineered phenylalanine ammonia lyase is produced.

14 . The method of claim 13 , further comprising the step of recovering at least one engineered phenylalanine ammonia lyase from said culture medium and/or said host cell.

15 . The method of claim 13 , further comprising the step of purifying said at least one phenylalanine ammonia lyase.

Assignees

• Codexis Inc

Inventors

• Novick Scott J
• Garcia Ravi David
• Ching Charlene
• Dellas Nikki
• Entwistle David
• Mitchell Vesna

Classifications

• C12Y403/01024

  Phenylalanine ammonia-lyase (4.3.1.24) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12N9/0022

  with oxygen as acceptor (1.4.3) · CPC title