Polynucleotides encoding engineered tyrosine ammonia lyase variants

Patent metadata

Field	Value
Publication number	US-10287567-B2
Application number	US-201715432429-A
Country	US
Kind code	B2
Filing date	Feb 14, 2017
Priority date	Apr 16, 2014
Publication date	May 14, 2019
Grant date	May 14, 2019

Abstract

The present invention provides engineered tyrosine ammonia-lyase (TAL) polypeptides and compositions thereof. In some embodiments, the engineered TAL polypeptides have been optimized to provide enhanced catalytic activity while reducing sensitivity to proteolysis and increasing tolerance to acidic pH levels. The invention also provides methods for utilization of the compositions comprising the engineered TAL polypeptides for therapeutic and industrial purposes.

First claim

What is claimed is:

1. A polynucleotide encoding a recombinant tyrosine ammonia lyase, wherein said recombinant tyrosine ammonia lyase comprises an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO:14, and wherein said recombinant tyrosine ammonia lyase comprises a mutation at position 107.

2. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 1 , wherein said tyrosine ammonia lyase further comprises at least one additional mutation at position 18, 47, 54, 59, 64, 73, 77, 88, 91, 93, 95, 97, 108, 214, 219, 222, 253, 304, 307, 315, 364, 367, 389, 394, 396, 400, 401, 423, 447, 453, 462, 490, 500, 503, 521, 550, 554, 564, and/or 565, and wherein the positions correspond to SEQ ID NO: 14.

3. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 1 , wherein said recombinant tyrosine ammonia lyase is thermostable.

4. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 1 , wherein said recombinant tyrosine ammonia lyase is resistant to proteolysis.

5. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 4 , wherein said recombinant tyrosine ammonia lyase is resistant to at least one digestive tract protease.

6. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 5 , wherein said digestive tract protease is selected from chymotrypsin, trypsin, carboxypeptidases, and elastases.

7. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 1 , wherein said recombinant tyrosine ammonia lyase is acid stable.

8. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 1 , wherein said recombinant tyrosine ammonia lyase is a deimmunized tyrosine ammonia lyase.

9. The polynucleotide encoding the recombinant tyrosine ammonia lyase of claim 1 , wherein said recombinant tyrosine ammonia lyase is purified.

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

11. An expression vector comprising the recombinant polynucleotide sequence of claim 1 .

12. The expression vector of claim 11 , wherein said recombinant polynucleotide sequence is operably linked to a control sequence.

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

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

15. A host cell comprising the expression vector of claim 11 .

16. The host cell of claim 15 , wherein said host cell is prokaryotic or eukaryotic.

17. The host cell of claim 16 , wherein said host cell is Escherichia coli.

18. A method of producing a tyrosine ammonia lyase variant, comprising culturing said host cell of claim 15 , under conditions that said tyrosine ammonia lyase encoded by said recombinant polynucleotide is produced.

19. The method of claim 18 , further comprising the step of recovering said tyrosine ammonia lyase.

20. The method of claim 19 , further comprising the step of purifying said tyrosine ammonia lyase.

Assignees

• Codexis Inc

Inventors

• Huisman Gjalt W
• Agard Nicholas J
• Elgart David
• Zhang Xiyun

Classifications

• A61K31/122

  having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin · CPC title

• A61K38/51

  Lyases (4) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y403/01023

  Tyrosine ammonia-lyase (4.3.1.23) · CPC title

• C12P13/225

  Tyrosine; 3,4-Dihydroxyphenylalanine · CPC title