# US 2019/0376097 A1 · Engineered tyrosine ammonia lyase > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0376097 A1 - Document ID: US20190376097A1 - Status: Published application - Kind: A1 - Application number: 16/436,176 - Filed: 2019-06-10 - Published: 2019-12-12 - Earliest claimed: 2018-06-12 - Assignees: Codexis, Inc. - Applicants: Codexis, Inc. - Inventors: Gjalt W. Huisman; Joyce Liu; Nikki Dellas; Chinping Chng; William Casey Hallows - Web page: https://patentgrep.com/patent/US20190376097A1 ## CPC - C12P 7/42: Hydroxy-carboxylic acids - A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)} - A61K 9/0053: {Mouth and digestive tract, i.e. intraoral and peroral administration} - A61K 31/122: having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin - C12N 9/88: Lyases (4.) - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - C12P 13/225: {Tyrosine; 3,4-Dihydroxyphenylalanine} - C12Y 403/01023: Tyrosine ammonia-lyase (4.3.1.23) - C12Y 403/01024: Phenylalanine ammonia-lyase (4.3.1.24) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## 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 ### Claim 1 A recombinant tyrosine ammonia lyase and/or biologically active recombinant tyrosine ammonia lyase fragment comprising an amino acid sequence comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 2, 6, 28, 92, 308, 338, 504, and/or 736. ### Claim 2 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 3 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 4 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 5 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 6 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 7 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 8 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 9 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyase 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. ### Claim 10 (depends on 1) The recombinant tyrosine lyase variant of claim 1, comprising any variant provided in Tables 2-1, 3-1, 4-1, 5-1, 6-1, 6-2, 7-1, and/or 8-1. ### Claim 11 (depends on 1) The recombinant tyrosine lyase variant of claim 1, wherein said variant comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. ### Claim 12 A recombinant tyrosine lyase variant comprising at least 90% sequence identity to at least one of the even numbered sequences in SEQ ID NOS: 4-780. ### Claim 13 (depends on 8) The recombinant tyrosine lyase variant of claim 8, wherein said polypeptide of said recombinant tyrosine ammonia lyase comprises one of the even-numbered sequences of SEQ ID NOS: 4-780. ### Claim 14 A recombinant tyrosine ammonia lyase comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 6, 28, 92, 308, 338, 504, and/or 736. ### Claim 15 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said recombinant tyrosine ammonia lyase is thermostable. ### Claim 16 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said recombinant tyrosine ammonia lyase is resistant to proteolysis. ### Claim 17 (depends on 16) The recombinant tyrosine ammonia lyase of claim 16, wherein said recombinant tyrosine ammonia lyase is resistant to at least one digestive tract protease. ### Claim 18 (depends on 17) The recombinant tyrosine ammonia lyase of claim 17, wherein said digestive tract protease is selected from chymotrypsin, trypsin, carboxypeptidases, and elastases. ### Claim 19 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said recombinant tyrosine ammonia lyase is acid stable. ### Claim 20 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said recombinant tyrosine ammonia lyase is a deimmunized tyrosine ammonia lyase. ### Claim 21 (depends on 1) The recombinant tyrosine ammonia lyase of claim 1, wherein said recombinant tyrosine ammonia lyase is purified. ### Claim 22 (depends on 1) 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. ### Claim 23 (depends on 22) The recombinant tyrosine ammonia lyase of claim 22, wherein said reference sequence is selected from SEQ ID NO: 6, 28, 92, 308, 338, 504, and/or 736. ### Claim 24 (depends on 1) A composition comprising at least one recombinant tyrosine ammonia lyase of claim 1. ### Claim 25 (depends on 1) A recombinant polynucleotide sequence encoding at least one recombinant tyrosine ammonia lyase set forth in claim 1. ### Claim 26 (depends on 25) The recombinant polynucleotide sequence of claim 25, wherein said polynucleotide sequence is codon-optimized. ### Claim 27 (depends on 25) The recombinant polynucleotide sequence of claim 25, wherein said polynucleotide comprises at least 90% sequence identity to at least one of the odd-numbered sequences of SEQ ID NOS: 3-779. ### Claim 28 (depends on 26) The recombinant polynucleotide sequence of claim 26, wherein said polynucleotide comprises at least 90% sequence identity to at least one of the odd-numbered sequences of SEQ ID NOS: 3-779. ### Claim 29 (depends on 27) The recombinant polynucleotide sequence of claim 27, wherein said polynucleotide comprises an odd-numbered sequence of SEQ ID NOS: 3-779. ### Claim 30 (depends on 28) The recombinant polynucleotide sequence of claim 28, wherein said polynucleotide comprises an odd-numbered sequence of SEQ ID NOS: 3-779. ### Claim 31 (depends on 25) An expression vector comprising the recombinant polynucleotide sequence of claim 25. ### Claim 32 (depends on 31) The expression vector of claim 31, wherein said recombinant polynucleotide sequence is operably linked to a control sequence. ### Claim 33 (depends on 32) The expression vector of claim 32, wherein said control sequence is a promoter. ### Claim 34 (depends on 33) The expression vector of claim 33, wherein said promoter is a heterologous promoter. ### Claim 35 (depends on 31) A host cell comprising the expression vector of claim 31. ### Claim 36 (depends on 35) The host cell of claim 35, wherein said host cell is prokaryotic. ### Claim 37 (depends on 35) The host cell of claim 35, wherein said host cell is eukaryotic. ### Claim 38 (depends on 35) A method of producing a tyrosine ammonia lyase variant, comprising culturing said host cell of claim 35, under conditions that said tyrosine ammonia lyase encoded by said recombinant polynucleotide is produced. ### Claim 39 (depends on 38) The method of claim 38, further comprising the step of recovering said tyrosine ammonia lyase. ### Claim 40 (depends on 38) The method of claim 38, further comprising the step of purifying said tyrosine ammonia lyase. ### Claim 41 (depends on 24) A pharmaceutical composition for the treatment of tyrosinemia, comprising the composition of claim 24. ### Claim 42 (depends on 41) The pharmaceutical composition of claim 41, further comprising a pharmaceutically acceptable carrier and/or excipient. ### Claim 43 (depends on 41) The pharmaceutical composition of claim 41, wherein said composition is suitable for oral administration to a human. ### Claim 44 (depends on 41) The pharmaceutical composition of any of claim 41, wherein said composition comprises nitisinone. ### Claim 45 (depends on 41) The pharmaceutical composition of any of claim 41, wherein said composition comprises at least one phenylalanine ammonia lyase. ### Claim 46 (depends on 41) The pharmaceutical composition of any of claims 41, wherein said composition comprises nitisinone and at least one phenylalanine ammonia lyase. ### Claim 47 (depends on 41) The pharmaceutical composition of claim 41, wherein said composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion. ### Claim 48 (depends on 47) The pharmaceutical composition of claim 47, wherein said pill, tablet, capsule, or gelcap further comprises an enteric coating. ### Claim 49 (depends on 41) The pharmaceutical composition of claim 41, wherein said composition is suitable for parenteral injection into a human. ### Claim 50 (depends on 49) The pharmaceutical composition of claim 49, wherein said composition is coadministered with nitisinone. ### Claim 51 (depends on 49) The pharmaceutical composition of claim 49, wherein said composition further comprises nitisinone. ### Claim 52 (depends on 49) The pharmaceutical composition of claim 49, wherein said composition further comprises at least one phenylalanine ammonia lyase. ### Claim 53 (depends on 49) The pharmaceutical composition of claim 49, wherein said composition further comprises nitisinone and at least one phenylalanine ammonia lyase. ### Claim 54 (depends on 41) 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 41 to said subject. ### Claim 55 (depends on 54) The method of claim 54, wherein said symptoms of tyrosinemia or alkaptonuria are ameliorated. ### Claim 56 (depends on 54) The method of claim 54, wherein said subject is able to eat a diet that is less restricted in its phenylalanine and/or tyrosine content than diets required by subjects exhibiting the symptoms of tyrosinemia or alkaptonuria. ### Claim 57 (depends on 54) The method of claim 54, wherein said subject is an infant or child. ### Claim 58 (depends on 54) The method of claim 54, wherein said subject is an adult or young adult. ### Claim 59 (depends on 1) A method for the production of L-tyrosine and/or L-tyrosine derivatives comprising the steps of providing at least one recombinant TAL variant of claim 1, and a suitable substrate, and combining said TAL variant(s) and said substrate under conditions such that L-tyrosine and/or at least one L-tyrosine derivative is produced. ### Claim 60 (depends on 1) A method for the production of coumaric acid and/or at least one coumaric acid derivative, comprising the steps of providing at least one recombinant TAL variant of claim 1 and a suitable substrate, and combining said TAL variant(s) and said substrate under conditions such that coumaric acid and/or at least one coumaric acid derivative is produced. ### Claim 61 (depends on 24) Use of the composition provided in claim 24. ### Claim 62 (depends on 41) Use of the composition provided in claim 41.