Engineered tyrosine ammonia lyase

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.

What is claimed is: 
     
         1 . A recombinant polynucleotide encoding a recombinant tyrosine ammonia lyase comprising an amino acid sequence comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2114, wherein said recombinant tyrosine ammonia lyase further comprises a substitution at position 305, and wherein the positions correspond to SEQ ID NO: 2114, further wherein said recombinant tyrosine ammonia lyase is suitable for use in treatment of tyrosinemia. 
     
     
         2 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said tyrosine ammonia lyase further comprises at least one substitution in at least one position or set of positions selected from 2, 4, 4/8/11/13/22/111/504, 4/8/11/17/324/428/504, 4/8/11/22/111, 4/8/22/111/504, 4/17/241/519/547, 4/324, 4/428, 6, 7, 8/11/13/324, 8/11/17/22/324/504/519, 8/11/111/504, 10, 11/13/504, 11/17/22/324, 14, 16, 39, 57, 228, 228/259, 236, 305/307/425, 316, 385, 425, 430, 437, 478, and 505, wherein the positions are numbered with reference to SEQ ID NO: 2114. 
     
     
         3 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant polynucleotide comprises a nucleic acid sequence comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2113. 
     
     
         4 . The recombinant polynucleotide sequence of  claim 1 , wherein said polynucleotide sequence is codon-optimized. 
     
     
         5 . The recombinant polynucleotide sequence of  claim 2 , wherein said polynucleotide sequence is codon-optimized. 
     
     
         6 . The recombinant polynucleotide sequence of  claim 3 , wherein said polynucleotide sequence is codon-optimized. 
     
     
         7 . An expression vector comprising a recombinant polynucleotide sequence of  claim 1 . 
     
     
         8 . An expression vector comprising a recombinant polynucleotide sequence of  claim 2 . 
     
     
         9 . An expression vector comprising a recombinant polynucleotide sequence of  claim 3 . 
     
     
         10 . The expression vector of  claim 7 , wherein said recombinant polynucleotide sequence is operably linked to at least one control sequence. 
     
     
         11 . The expression vector of  claim 8 , wherein said recombinant polynucleotide sequence is operably linked to at least one control sequence. 
     
     
         12 . The expression vector of  claim 9 , wherein said recombinant polynucleotide sequence is operably linked to at least one control sequence. 
     
     
         13 . The expression vector of  claim 10 , wherein said control sequence is a promoter. 
     
     
         14 . The expression vector of  claim 11 , wherein said control sequence is a promoter. 
     
     
         15 . The expression vector of  claim 12 , wherein said control sequence is a promoter. 
     
     
         16 . The expression vector of  claim 13 , wherein said promoter is a heterologous promoter. 
     
     
         17 . The expression vector of  claim 14 , wherein said promoter is a heterologous promoter. 
     
     
         18 . The expression vector of  claim 15 , wherein said promoter is a heterologous promoter. 
     
     
         19 . A host cell comprising the expression vector of  claim 7 . 
     
     
         20 . A host cell comprising the expression vector of  claim 8 . 
     
     
         21 . A host cell comprising the expression vector of  claim 9 . 
     
     
         22 . The host cell of  claim 19 , wherein said host cell is selected from prokaryotic and eukaryotic cells. 
     
     
         23 . The host cell of  claim 20 , wherein said host cell is selected from prokaryotic and eukaryotic cells. 
     
     
         24 . The host cell of  claim 21 , wherein said host cell is selected from prokaryotic and eukaryotic cells. 
     
     
         25 . A method of producing a recombinant tyrosine ammonia lyase, comprising culturing said host cell of  claim 19 , under conditions that said recombinant tyrosine ammonia lyase encoded by said recombinant polynucleotide is produced. 
     
     
         26 . The method of  claim 25 , further comprising the step of recovering said recombinant tyrosine ammonia lyase. 
     
     
         27 . The method of  claim 26 , further comprising the step of purifying said recombinant tyrosine ammonia lyase. 
     
     
         28 . A method of producing a recombinant tyrosine ammonia lyase, comprising culturing said host cell of  claim 20 , under conditions that said recombinant tyrosine ammonia lyase encoded by said recombinant polynucleotide is produced. 
     
     
         29 . The method of  claim 28 , further comprising the step of recovering said recombinant tyrosine ammonia lyase. 
     
     
         30 . The method of  claim 29 , further comprising the step of purifying said recombinant tyrosine ammonia lyase. 
     
     
         31 . A method of producing a recombinant tyrosine ammonia lyase, comprising culturing said host cell of  claim 21 , under conditions that said recombinant tyrosine ammonia lyase encoded by said recombinant polynucleotide is produced. 
     
     
         32 . The method of  claim 31 , further comprising the step of recovering said recombinant tyrosine ammonia lyase. 
     
     
         33 . The method of  claim 32 , further comprising the step of purifying said recombinant tyrosine ammonia lyase.