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 tyrosine ammonia lyase, wherein said recombinant tyrosine ammonia lyase comprises an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO:10, and wherein said recombinant tyrosine ammonia lyase comprises a mutation in at least one position wherein said position is selected from position 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, wherein the positions correspond to SEQ ID NO:10. 
     
     
         2 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said tyrosine ammonia lyase comprises at least one mutation in at least one position selected from S73, 177, A88, V91, S93, E95, A97, L108, L219, M222, D253, Y304, G307, S315, L364, Y367, Q389, A394, P396, N400, G401, 1423, A447, N453, A462, R490, A500, A550, and/or P564, and wherein the positions correspond to SEQ ID NO:10. 
     
     
         3 . The recombinant tyrosine ammonia lyase of  claim 2 , wherein said tyrosine ammonia lyase further comprises at least one mutation selected from S731, 177M, A88S, V91R, S93L/P/R/T/W, E95D, A97T, L108C, L219M, M222T, D253G, Y304F, G307P/H, S315A, L364H/M/Y, Y367F, Q389T, A394V, P396Q, N400M/T, G401C/L, I423F, A447T, N453C, A462T, R490T, A500S, A550V, P564S, and wherein the positions correspond to SEQ ID NO:10. 
     
     
         4 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said tyrosine ammonia lyase further comprises a substitution set selected from F18H/L47A/T54K/G59R/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/Q521K; F18H/L47A/G59R/177M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q/Q521K; F18H/T54K/S73K/I77M/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K/V554I; F18H/L47A/T54K/G59R/S73K/I77M/S315A/L364M/Q389T/N400M/N453C/Q521K/C565P; F18H/L47A/T54K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C565P; F18H/L47A/G59R/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C; F18H/L47A/S73K/I77M/L214Q/S315A/N400M/N453C/Q521K; F18H/L47A/S73K/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q; F18H/G59R/I77M/S315A/L364M/Q389T/N400M/N453C/Q521K; F18H/I77M/S315A/L364M/Q389T/N400M/N453C/C503Q/C565P; F18H/G59R/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/L47A/I77M/L214Q/S315A/N400M/N453C/C503Q/Q521K; F18H/G59R/S73K/I77M/L214Q/S315A/N400M/N453C; F18H/L47A/G59R/S73K/177M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q; F18H/L47A/S491/T54K/S73K/177M/A97T/L214Q/S315A/Q389T/N400M/N453C; F18H/S73K/177M/S315A/L364M/Q389T/N400M/N453C; F18H/T54K/G59R/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/Q521K; F18H/S73K/I77M/N193D/R305M/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/I77M/L214Q/S315A/L364M/N400M/N453C/Q521K; F18H/L47A/I77M/S315A/N400M/N453C/Q521K; F18H/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q; F18H/I77M/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/L47A/S73K/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C565P; F18H/L47A/T54K/S73K/177M/L214Q/S315A/L364M/N400M/N453C/C503Q; F18H/L47A/S73K/177M/L214Q/R305M/S315A/L364M/N400M/N453C; F18H/L47A/G59R/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C565P; F18H/L47A/T54K/G59R/C64S/S73K/177M/L214Q/S315A/L364M/N400M/N453C/C503Q; F18H/L47A/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/T54K/G59R/S73K/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C565P; F18H/S73K/177M/S315A/L364M/Q389T/N400M/N453C/C503Q/C565P; F18H/S73K/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q/Q521K; F18H/L47A/I77M/L214Q/S315A/N400M/N453C/C503Q; F18H/177M/S315A/L364M/Q389T/N400M/N453C; F18H/L47A/G59R/S73K/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/C565P; F18H/L47A/S73K/I77M/L214Q/S315A/Q389T/N400M/N453C/Q521K/C565P; F18H/L47A/G59R/S73K/177M/S315A/Q389T/N400M/N453C; F18H/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/L47A/T54K/G59R/S73K/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/I77M/S315A/L364M/N400M/N453C/Q521K; F18H/T54K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/L47A/I77M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/S73K/177M/R305M/S315A/L364M/N400M/N453C/C503Q; F18H/L47A/I77M/S315A/Q389T/N400M/N453C/Q521K; F18H/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C; F18H/G59R/I77M/S315A/Q389T/N400M/N453C/Q521K; F18H/L47A/T54K/G59R/S73K/I77M/L214Q/H250N/S315A/L364M/N400M/N453C; F18H/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/G59R/S73K/I77M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/T54K/G59R/S73K/177M/S315A/L364M/Q389T/N400M/N453C; F18H/L47A/I77M/A97T/L214Q/S315A/Q389T/N400M/N453C/Q521K; F18H/L47A/S73K/I77M/R305M/S315A/L364M/Q389T/N400M/N453C/Q521K/C565P; F18H/L47A/S73K/I77M/L214Q/S315A/Q389T/N400M/N453C/C503Q/Q521K; F18H/S73K/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q; F18H/L47A/T54K/177M/S315A/L364M/Q389T/N400M/N453C; F18H/T54K/177M/S315A/L364M/Q389T/N400M/N453C/C503Q/C565P; F18H/L47A/C64S/S73K/177M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q; F18H/S73K/I77M/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/I77M/S315A/L364M/Q389T/N400M/N453C/Q521K/C565P; F18H/L47A/G59R/I77M/L214Q/S315A/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/I77M/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/G59R/I77M/L214Q/R305M/S315A/L364M/N400M/N453C/C503Q/Q521K; F18H/L47A/G59R/S73K/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q; F18H/L47A/G59R/I77M/S315A/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/G59R/I77M/S315A/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/T54K/G59R/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/S73K/177M/A97T/L214Q/R305M/S315A/Q389T/N400M/N453C/Q521K/C565P; F18H/S73K/I77M/S315A/Q389T/N400M/N453C/C565P; F18H/L47A/I77M/S315A/L364M/Q389T/N400M/N453C; F18H/L47A/C64S/S73K/I77M/L214Q/S315A/Q389T/N400M/N453C/C503Q/C565P; F18H/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/Q521K/C565P; F18H/L47A/T54K/S73K/177M/L214Q/S315A/L364M/Q389T/L392Q/N400M/N453C/C503Q/Q521K; F18H/L47A/T54K/G59R/S73K/I77M/L214Q/S315A/N400M/N453C/C503Q/Q521K; F18H/L47A/T54K/G59R/I77M/L214Q/S315A/Q389T/N400M/N453C/C503Q/C565P; F18H/L47A/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/T54K/S73K/177M/A97T/L214Q/S315A/N400M/N453C/C503Q; F18H/L47A/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/T54K/177M/S315A/L364M/N400M/N453C; F18H/L47A/T54K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K; F18H/T54K/G59R/177M/S315A/L364M/Q389T/N400M/N453C; F18H/L47A/G59R/I77M/L214Q/S315A/Q389T/N400M/N453C/C503Q/C565P; F18H/L47A/I77M/S315A/N400M/N453C/C565P; F18H/T54K/G59R/I77M/L214Q/S315A/L364M/N400M/N453C/Q521K; F18H/L47A/S73K/I77M/L214Q/S315A/L364M/N400M/N453C; F18H/L47A/T54K/177M/L214Q/S315A/L364M/Q389T/N400M/N453C; F18H/L47A/T54K/G59R/S73K/I77M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q; F18H/L47A/G59R/177M/L214Q/S315A/L364M/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/G59R/177M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/G59R/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/S73K/177M/S315A/L364M/Q389T/N400M/N453C/Q521K/C565P; F18H/L47A/T54K/177M/A97T/L214Q/S315A/N400M/N453C/C503Q; F18H/L47A/I77M/A97T/L214Q/S315A/N400M/N453C/Q521K/C565P; F18H/L47A/G59R/S73K/I77M/S315A/L364M/N400M/N453C/C565P; F18H/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/Q521K; F18H/L47A/G59R/S73K/I77M/L214Q/S315A/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/C64S/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/Q521K; F18H/L47A/G59R/S73K/I77M/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/G59R/S73K/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q; F18H/L47A/I77M/S315A/L364M/N400M/N453C/C503Q; F18H/T54K/G59R/S73K/177M/L214Q/S315A/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/S73K/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q/Q521K; F18H/I77M/L214Q/S315A/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/G59R/177M/A97T/L214Q/S315A/L364M/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/I77M/S315A/N400M/N453C/C503Q/Q521K/C565P; F18H/G59R/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/Q521K; F18H/L47A/T54K/S73K/177M/L214Q/S315A/Q389T/N400M/N453C/C503Q/C565P; F18H/G59R/S73K/I77M/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/T46N/L47A/S73K/I77M/L214Q/S315A/L364M/M370Q/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q/Q521K; F18H/L47A/177M/S315A/L364M/Q389T/N400M/N453C/C503Q; F18H/L47A/T54K/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/I77M/A97T/S315A/Q389T/N400M/N453C/Q521K; F18H/L47A/S73K/I77M/A97T/L214Q/S315A/L364M/N400M/N453C/C503Q/Q521K; F18H/G59R/S73K/I77M/L214Q/S315A/Q389T/N400M/N453C; F18H/L47A/C64S/S73K/177M/S315A/L364M/Q389T/N400M/N453C/C503Q/C565P; F18H/L47A/G59R/I77M/L214Q/S315A/Q389T/N400M/N453C; F18H/L47A/I77M/L214Q/S315A/Q389T/N400M/N453C/Q521K/C565P; F18H/G59R/S73K/177M/S315A/Q389T/N400M/N453C/C565P; F18H/S73K/I77M/L214Q/S315A/L364M/Q389T/N400M/N453C/Q521K; F18H/L47A/C64S/S73K/I77M/L214Q/S315A/L364M/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/T54K/177M/A97T/S315A/Q389T/N400M/N453C/C503Q; F18H/L47A/G59R/I77M/L214Q/S315A/N400M/N453C/C503Q/Q521K/C565P; F18H/L47A/S73K/177M/A97T/S315A/Q389T/N400M/N453C/C503Q/Q521K; F18H/L47A/S73K/177M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q; F18H/L47A/T54K/G59R/I77M/L214Q/S315A/Q389T/N400M/N453C/C503Q; and F18H/L47A/I77M/A97T/L214Q/S315A/Q389T/N400M/N453C/C503Q, and wherein the positions correspond to SEQ ID NO:10. 
     
     
         5 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is thermostable. 
     
     
         6 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is resistant to proteolysis. 
     
     
         7 . The recombinant tyrosine ammonia lyase of  claim 6 , wherein said recombinant tyrosine ammonia lyase is resistant to at least one digestive tract protease. 
     
     
         8 . The recombinant tyrosine ammonia lyase of  claim 7 , wherein said digestive tract protease is selected from chymotrypsin, trypsin, carboxypeptidases, and elastases. 
     
     
         9 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is acid stable. 
     
     
         10 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is a deimmunized tyrosine ammonia lyase. 
     
     
         11 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is purified. 
     
     
         12 . The recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) reduced sensitivity to proteolysis; iii) increased tolerance to acidic pH; iv) reduced aggregation; v) decreased Km for tyrosine; vi) decreased immunogenicity; or a combination of any of i), ii), iii), iv), v), and/or vi), as compared to a reference sequence. 
     
     
         13 . The recombinant tyrosine ammonia lyase of  claim 12 , wherein said reference sequence is SEQ ID NO:6, 8, 10, 12, or 14. 
     
     
         14 . A composition comprising at least one recombinant tyrosine ammonia lyase of  claim 1 . 
     
     
         15 . A recombinant polynucleotide sequence encoding at least one recombinant tyrosine ammonia lyase set forth in  claim 1 . 
     
     
         16 . The recombinant polynucleotide sequence of  claim 15 , wherein said polynucleotide sequence is codon-optimized. 
     
     
         17 . An expression vector comprising the recombinant polynucleotide sequence of  claim 16 . 
     
     
         18 . The expression vector of  claim 17 , wherein said recombinant polynucleotide sequence is operably linked to a control sequence. 
     
     
         19 . The expression vector of  claim 18 , wherein said control sequence is a promoter. 
     
     
         20 . The expression vector of  claim 19 , wherein said promoter is a heterologous promoter. 
     
     
         21 . A host cell comprising the expression vector of  claim 20 . 
     
     
         22 . The host cell of  claim 21 , wherein said host cell is selected from prokaryotic and eukaryotic cells. 
     
     
         23 . A method of producing a tyrosine ammonia lyase variant, comprising culturing said host cell of  claim 21 , under conditions that said tyrosine ammonia lyase encoded by said recombinant polynucleotide is produced. 
     
     
         24 . The method of  claim 23 , further comprising the step of recovering said tyrosine ammonia lyase. 
     
     
         25 . The method of  claim 24 , further comprising the step of purifying said tyrosine ammonia lyase. 
     
     
         26 . A pharmaceutical composition for the treatment of tyrosinemia, comprising the enzyme composition of  claim 14 . 
     
     
         27 . The pharmaceutical composition of  claim 26 , further comprising a pharmaceutically acceptable carrier and/or excipient. 
     
     
         28 . The pharmaceutical composition of  claim 26 , wherein said composition is suitable for oral administration to a human. 
     
     
         29 . The pharmaceutical composition of  claim 26 , wherein said composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion. 
     
     
         30 . The pharmaceutical composition of  claim 26 , wherein said composition is coadministered with nitisinone 
     
     
         31 . The pharmaceutical composition of  claim 30 , wherein said composition comprises nitisinone. 
     
     
         32 . The pharmaceutical composition of  claim 29 , wherein said pill, tablet, capsule, or gelcap further comprises an enteric coating. 
     
     
         33 . The pharmaceutical composition of  claim 26 , wherein said composition is suitable for parenteral injection into a human. 
     
     
         34 . A method for treating and/or preventing the symptoms of tyrosinemia or alkaptonuria in a subject, comprising providing a subject having tyrosinemia or alkaptonuria, and providing the pharmaceutical composition of  claim 26  to said subject. 
     
     
         35 . The method of  claim 34 , wherein said symptoms of tyrosinemia or alkaptonuria are ameliorated. 
     
     
         36 . The method of  claim 34 , wherein said subject is able to eat a diet that is less restricted in its methionine, phenylalanine and/or tyrosine content than diets required by subjects exhibiting the symptoms of tyrosinemia or alkaptonuria. 
     
     
         37 . The method of  claim 34 , wherein said subject is an infant, child, young adult, or adult. 
     
     
         38 . A method for the production of L-tyrosine and/or L-tyrosine derivatives comprising the steps of providing at least one recombinant tyrosine ammonia lyase variant of  claim 1  and a suitable substrate, and combining said recombinant tyrosine ammonia lyase variant(s) and said substrate under conditions such that L-tyrosine and/or at least one L-tyrosine derivative is produced. 
     
     
         39 . A method for the production of coumaric acid, comprising the steps of providing at least one recombinant TAL variant of  claim 1  and a suitable substrate, and combining said recombinant tyrosine ammonia lyase variant(s) and said substrate under conditions such that coumaric acid is produced.