US2022389464A1PendingUtilityA1

Engineered tyrosine ammonia lyase

75
Assignee: CODEXIS INCPriority: Jun 12, 2018Filed: Aug 5, 2022Published: Dec 8, 2022
Est. expiryJun 12, 2038(~11.9 yrs left)· nominal 20-yr term from priority
Inventors:Gjalt W. HuismanJoyce LiuNikki DellasChinping ChngWilliam Casey Hallows
C12P 13/225A61K 9/0019C12P 7/42C12N 9/88C12Y 403/01024C12Y 403/01023A61K 31/122A61K 9/0053C12N 15/63
75
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Claims

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 and enhanced acid stability, 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.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A recombinant polynucleotide sequence encoding a tyrosine ammonia lyase comprising an amino acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6, 28, 92, 308, 338, 504, and/or 736. 
     
     
         2 . The recombinant polynucleotide sequence encoding a 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 16/18/39/47/54/59/73/91/209/214/285/290/305/307/364/407/503/521/524/565, 16/18/39/47/54/59/73/91/209/214/285/290/307/364/407/450/470/503/521/524/565, 16/18/39/47/54/59/73/91/209/214/285/290/407/503/521/524/565, 16/18/39/47/54/59/73/91/209/214/290/305/307/364/407/470/503/521/524/565, 16/18/39/47/54/59/73/91/209/285/290/307/364/407/450/470/503/521/524/565, 16/18/39/47/54/59/73/91/214/285/290/364/403/407/450/470/503/521/524, 16/18/39/47/54/59/73/91/285/290/305/307/364/407/470/503/521/524/565, 16/18/39/54/73/91/209/210/285/290/307/364/407/450/470/503/521/524, 16/39/47/54/59/73/91/98/209/214/285/290/307/407/450/470/503, 16/39/47/54/59/73/91/209/214/285/290/305/307/364/407/450/470/521/524/565, 16/39/47/54/59/73/91/209/214/285/290/305/307/364/407/450/503/521/565, 16/39/47/54/59/73/91/209/214/285/290/305/307/364/407/470/503/521, 16/39/47/54/59/73/91/209/214/285/290/305/307/364/407/470/503/521/524/565, 16/39/47/54/59/73/91/209/214/285/290/305/364/503/524/565, 16/39/47/54/59/73/91/209/214/285/290/307/364/407/470/503/521/524/565, 16/39/47/54/59/73/91/209/285/290/305/307/364/407/470/503/521/524/565, 16/39/47/54/59/73/209/214/285/290/307/364/470/503/521/524/565, 16/39/54/59/73/91/209/214/285/290/364/450/470/503/521/524, 16/54/59/73/91/209/214/285/290/307/364/407/450/503/521/524/565, 18/39/47/54/59/73/91/209/214/285/290/305/307/364/407/503/521/524, 18/39/47/54/59/73/91/209/214/285/290/305/307/364/407/503/521/524/565, 18/39/47/54/59/73/91/285/290/305/307/364/407/470/521/524/565, 18/39/47/54/59/91/209/214/285/290/307/364/407/450/470/503/521/524/565, 18/39/47/54/73/91/98/209/214/285/290/305/307/364/407/470/503/521/524, 18/39/47/54/73/91/209/214/285/290/305/364/407/470/503/521/524, 18/39/47/54/73/91/209/285/290/305/307/364/407/470/503/521/524, 18/39/54/59/73/91/209/214/285/290/307/364/407/470/503/521/524/565, 18/47/54/59/73/91/285/290/307/364/470/503/521/524/565, 39/47/54/59/73/91/209/210/285/290/307/364/407/450/470/503/521/524/565, 39/47/54/73/91/209/214/285/290/364/407/450/470/503/521/524/565, 39/47/59/73/91/209/214/285/290/305/307/364/407/470/503/521/524/565, 39/54/59/73/91/209/214/285/290/364/407/470/503/524/565, and 39/54/59/73/91/285/290/307/450/470/503/524/565, wherein the positions are numbered with reference to SEQ ID NO: 6. 
     
     
         3 . 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 18/222, 18/222/364/400, 18/222/400/490, 18/364/400, 18/364/453, 18/389/400/490, 18/389/490, 18/400, 77/222/389, 222/364/389, 222/453, 315, 315/364, 315/389/453, 389/400, 400, and 400/453, wherein the positions are numbered with reference to SEQ ID NO: 28. 
     
     
         4 . 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 43/410/413, 49/114/204/425, 61, 72, 72/306/307, 72/306/309, 73, 98, 98/404/410, 104, 106, 110, 112, 114, 114/145/204/258/275, 114/145/204/275/424/435, 114/145/247, 114/145/275, 114/145/275/344/424/435, 114/176, 114/176/518, 114/204, 114/204/247/258, 114/204/258/424/435, 114/204/266/425, 114/204/275/425, 114/204/275/518, 114/204/344, 114/204/435, 114/204/435/518, 114/204/518, 114/247/275/344/518, 114/258/275, 114/258/424/425/518, 114/518, 119/404/410, 145/204/275/435, 145/258/275/518, 145/424/435, 145/435, 175, 176/204, 176/204/518, 176/247, 176/247/258, 176/258/518, 176/275, 176/518, 204, 204/247/258/275, 204/247/435, 204/247/518, 204/258/275, 204/275/344, 204/518, 214, 247, 247/258, 247/258/518, 247/275/344/518, 247/424/435, 247/518, 258/344, 258/435, 275, 275/344, 306, 306/307/309, 306/307/394, 306/307/394/404/410, 306/410, 307, 344, 344/424/435/518, 345, 378/407, 394/410, 394/413, 395, 407, 410, 410/517, 413, 413/557, 418, 424, 424/435, 435, 435/518, 450, 464, and 518, wherein the positions are numbered with reference to SEQ ID NO: 92. 
     
     
         5 . 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 27, 31, 32/119/237/513/534, 32/119/509/513/534, 36, 54, 56, 58, 61, 61/214/464, 68, 69, 114, 114/119, 114/410/413/418, 118, 119/145/237/331, 119/237/254/513, 119/331/509/513, 119/509/513, 145/237/509/513/534, 158/509/513/534, 158/534, 189, 193, 209, 214, 214/410/450/464, 237, 237/254/331, 237/509/513, 237/513, 237/513/534, 249/339, 266, 270, 270/532, 282, 286, 289, 289/517, 331, 335, 338, 339, 353, 354, 355, 394/410/464, 394/464, 410/418/450/464, 418, 464, 467, 470, 497, 502, 509/513, 509/513/534, 513/534, 517, 522, 524, 525, 547, 551, 554, and 560, wherein the positions are numbered with reference to SEQ ID NO: 308. 
     
     
         6 . 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 27/31/54, 27/31/502/534, 27/282/410, 31/54, 31/54/237/282, 31/54/282/335/502, 31/237/335, 31/237/335/502, 31/282, 31/335, 31/410, 31/502, 31/534/547, 36/114/119/289/554, 54/237, 54/335/355/410/502, 61/114/119/286/289/551/554, 61/119/339/551/554, 114, 114/119/254/289, 114/119/254/289/551, 114/119/339/364, 114/119/554, 114/286/339/418, 114/364/554, 119/254/286/339/364/418/551, 119/254/289/554, 119/339/364/418/551, 119/339/554, 119/418/551/554, 119/551/554, 119/554, 237/282/410/502, 237/410/502, 237/517, 254, 254/286/289/554, 254/289/339/418/554, 254/418, 282/410, 282/410/502/517/547, 282/502, 286/289, 286/289/418/554, 289, 289/551, 335/410, 339/418/551, 410/547, 502, and 554, wherein the positions are numbered with reference to SEQ ID NO: 338. 
     
     
         7 . 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 116, 130, 133, 136, 158, 162, 211, 251, 267, 280, 319, 343, 356, 366, 367, 369, 386, 390, 403, 428, 439, 440, 443, 444, 462, 463, 474, 477, 478, 479, 481, 530, and 541, wherein the positions are numbered with reference to SEQ ID NO: 338. 
     
     
         8 . 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 54, 54/116/119/364, 54/119/162/364/410, 54/364/403, 54/410/463, 54/462, 54/463, 54/530, 116/162/364, 119/162, 119/162/410, 410, 462, and 474, wherein the positions are numbered with reference to SEQ ID NO: 504. 
     
     
         9 . 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 18, 18/52/463, 18/254, 18/267/554, 18/289, 18/289/339/364/554, 18/289/534/554, 18/289/554, 18/339/418/463, 18/339/463, 18/339/534, 254/289/339/418/463/554, 289/315/339/364/418/463/551, 289/534/554, 339/364/463, 534, and 534/551/554, wherein the positions are numbered with reference to SEQ ID NO: 736. 
     
     
         10 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is thermostable. 
     
     
         11 . The recombinant nucleic acid sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is resistant to proteolysis. 
     
     
         12 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is resistant to at least one digestive tract protease. 
     
     
         13 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said digestive tract protease is selected from chymotrypsin, trypsin, carboxypeptidases, and elastases. 
     
     
         14 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is acid stable. 
     
     
         15 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is a deimmunized tyrosine ammonia lyase. 
     
     
         16 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 1 , wherein said recombinant tyrosine ammonia lyase is purified. 
     
     
         17 . The recombinant polynucleotide sequence encoding a 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. 
     
     
         18 . The recombinant polynucleotide sequence encoding a recombinant tyrosine ammonia lyase of  claim 17 , wherein said reference sequence is selected from SEQ ID NO: 6, 28, 92, 308, 338, 504, and/or 736. 
     
     
         19 . The recombinant polynucleotide sequence of  claim 1 , wherein said polynucleotide sequence is codon-optimized. 
     
     
         20 . An expression vector comprising the recombinant polynucleotide sequence of  claim 19 . 
     
     
         21 . The expression vector of  claim 20 , wherein said recombinant polynucleotide sequence is operably linked to a control sequence. 
     
     
         22 . The expression vector of  claim 21 , wherein said control sequence is a promoter. 
     
     
         23 . A host cell comprising the expression vector of  claim 21 . 
     
     
         24 . The host cell of  claim 23 , wherein said host cell is selected from prokaryotic cells and eukaryotic cells. 
     
     
         25 . A method of producing a tyrosine ammonia lyase variant, comprising culturing said host cell of  claim 24 , under conditions that said tyrosine ammonia lyase encoded by said recombinant polynucleotide is produced. 
     
     
         26 . The method of  claim 25 , further comprising the step of recovering said tyrosine ammonia lyase. 
     
     
         27 . The method of  claim 25 , further comprising the step of purifying said tyrosine ammonia lyase.

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