Engineered adenosine kinase variants

Abstract

The present disclosure provides engineered adenosine kinase polypeptides, and recombinantpolynucleotides encoding the engineered adenosine kinases. The present disclosure further providesmethod of using the engineered adenosine kinases.

Description

Docket No. 5244.285WO2ENGINEERED ADENOSINE KINASE VARIANTSCROSS- REFERENCE

[0001] This application claims priority to U. S. Provisional Application No. 63 / 789,681. filed on April 16, 2025 which is incorporated herein by reference in its entirety’.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name 5244_285WO2.xml. created on April 15, 2026, with a file size of 1,343,488 bytes, is part of the specification and is hereby incorporated by reference herein in its entirety.TECHNICAL FIELD

[0003] The present disclosure relates to engineered adenosine kinase variants, polynucleotides encoding the engineered adenosine kinase variants, and uses of the engineered enzymes.BACKGROUND

[0004] Adenosine kinase (AdoK) catalyzes the phosphorylation of adenosine (Ado) to nucleoside monophosphate (5’-AMP) using ATP as a phosphate donor, and is found ubiquitously in eukaryotes. Adenosine kinase is a key step in the purine nucleotide salvage pathway involved in maintaining the proper levels of cellular nucleotides. Phosphorylation by adenosine kinase keeps the intracellular levels of adenosine low and maintains the homeostasis of concentration gradient across the cell membrane, which makes the cytosol a reservoir for extracellular adenosine kinase under physiological conditions.

[0005] In practical applications, adenosine kinase is used in enzyme cascade reactions beginning from nucleoside substrates for ATP regeneration or production of synthetic nucleotides. Although, enzyme cascades offer the opportunity to deal with unstable or toxic intermediates and used to shift the equilibrium of thermodynamically unfavorable reactions to increase the reaction yield, use of enzyme cascades requires adaptation and optimization to circumvent incompatibility' issues between the individual enzy matic reactions.SUMMARY

[0006] The present disclosure provides engineered adenosine kinases for use in transformation of nucleoside substrate to the corresponding nucleoside monophosphate. In some embodiments, the adenosine kinases are engineered to have an improved property’ compared to a reference adenosine kinase.

[0007] In one aspect, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Docket No. 5244.285WO294%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 10-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 10-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 162, or 268. or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

[0008] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 162, or 268, or to a reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 162, or 268, or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

[0009] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or to the reference sequence corresponding to SEQ ID NO: 10, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0010] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0011] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position a substitution at amino acid position 1, 4, 9, 11, 12, 46.88, 131, 138, 139, 140, 158, 175, 189. 193, 199, 200, 201, 203, 223, 267, 270, 277, 278, 280. 281, 289, 293, 302, 315, 321; or substitution 13L / R / T, 23E, 40L / Q, 43A, 53D / H, 54Q, 55Q / S / T / W / Y, 74A / C / G / L / M / Q, 90G / Q, 91G, 94R, 107 / K / L / P, 108S, 109D, 111 / H / S, 112K, 116V, 119K, 120G, 121E, 126V, 128E / L / M, 1291, 133C / E / K / R / S, 134K / Y, 147L / P, 157K / L / Q / R / S, 160A / R / T / W, 163S,Docket No. 5244.285WO2164I / R, 167E / R / S, 168E / G / Q / S / Y, 169W, 170A / C / L, 171Q / T, 172A / R / T / V, 182T, 185L / N,, 190E / L, 191P, 192R, 194F / K, 195C / N, 196G, 206E / H / T, 207S, 210E / V, 21 IS, 222E / P / V, 225K / M / S / T, 233E, 234E, 243A / E / L / N / T, 250L, 25 IP, 254K, 271L / T, 274V, 308K, 312C, 314A / L / M, and 320E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0012] In some embodiments, the engineered adenosine kinase comprises at least a substitution or amino acid residue IV. 4N, 9C / L / R, 11K / P, 12E / P / R / T, 13L / R / T, 23E, 40 / L / Q, 43 A, 46L. 53D / H, 54Q, 55Q / S / T7W / Y, 74A / C / G / L / M / Q, 88R, 90G / Q, 91G, 94R, 107 / K / L / P, 108S, 109D, 111H / S, 112K, 116V, 119K, 120G, 121E, 126V, 128E / L / M, 1291, 13 IS, 133C / E / K / R / S, 134K / Y, 138Q, 139L / S, 140P, 147L / P, 157K / L / Q / R / S, 158S, 160A / R / T / W, 163S, 164I / R, 167E / R / S, 168E / G / Q / S / Y, 169W, 170A / C / L, 171Q / T, 172A / R / T / V, 175L / W, 182T, 185L / N, 189 A / H, 190E / L, 191P, 192R, 193M / T, 194F / K, 195C / N, 196G, 199A / L / M, 200V, 200A, 201S / T, 203E, 206E / H / T, 207S, 210E / V, 21 IS, 222E / P / V, 223G / L, 225K / M / S / T. 233E, 234E, 243A / E / L / N / T, 250L, 251P, 254K. 267G, 270D, 271L / T. 274V, 277K / R, 278A / G / P / Q / T, 280R / V / W, 281S, 289L. 293T, 302H, 308K, 312C, 314A / L / M, 315V, 320E, and 3211, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0013] In some embodiments, the engineered adenosine kinase comprises at least substitution or amino acid residue M1V, H4N, G9C / L / R, G11K / P, M12E / P / R / T, S13L / P / R / T, A21G, D23E, R40H / L / Q, R43A, V46L, M53D / H, R54Q, R55Q / S / T7W / Y, R74A / C / G / L / M / Q / T / V, T80V, Y88R, E90G / Q, H91G, T94M / R, D106G / L / R, D107G / K / L / P. L108S, S109D, P111A / H / S, Q112K, T116V, M119K, D120G, Q121E, A126S / V, H128E / L / M, S129I, A131S, M133C / E / K / L / R / S, R134K / Y, N138Q, H139L / S, V140P, R141S, S147L / P, T147S. T155A / I, E157K / L / Q / R / S, G158S, I160A / R / T / W, A163S. E164I / R / S, A167E / R / S / V, A168E / G / Q / S / Y, M169L / W, G170A / C / L / S.I171Q / T, P172A / R / T / V, F175L / W. P182G / T, S185L / N, P187Q, L189A / H, R190E / L, E191P / S, F192R, I193M / T, E194F / K, Q195C / G / N, A196G. V199A / L / M, A200V, V200A, V201S / T, D203E, S206E / H / T. R207A / L / M / S, Q210E / V, A211I / L / M / S, S222A / E / G / P / V, R223G / L, Q225K / M / S / T, Q231L. S233E, A234E, K243A / E / L / N / R / T. A250I / L, K251P / T, A252V, R254K, A267G, I270D, Y271L / T. Q274V. Y277K / R, D278A / G / P / Q / T, L280R / V / W, T281S. M289L. K293T. Q302H.E308K. N310A. R312C. K314A / L / M, Q315V, A320E, and L321I. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0014] In some aspects, the amino acid sequence of the engineered adenosine kinase further comprises at least a substitution at amino acid position 13, 14, 20, 21. 23, 24, 26, 27, 28. 29, 30, 31, 33, 34, 35, 38. 40, 41, 42, 43. 46, 49, 50, 51, 52. 53, 54, 55, 56. 64, 67, 73, 74, 77. 79, 80, 83, 86. 87,Docket No. 5244.285WO289, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229. 231, 232, 233, 234, 236, 237, 238, 239, 240. 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251. 252, 253, 254, 255, 256, 258, 265, 271, 274. 275, 276, 287, 288, 291, 292, 293, 294, 295, 296, 299. 303, 305, 307, 308, 310, 311, 312, 314, 315. 316, 317, or 320, or combinations thereof, wherein tire amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0015] In some aspects, the ammo acid sequence of the engineered adenosine kinase can further comprises at least a substitution or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 3 I F / II / L / R / W / Y. 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q, 43E / K / R / S / V, 461, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V. 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M. 93 A, 94M / N, 100G, 101F / W, 1021, 103L / Q / R, I ()4A / C / D / G / L / N / P / R / S / T / \V7Y. 106E / G / L / M / P / R / T. 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T, 111A / G / L, 112I / L. 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C. 121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, I30A. 133A / L, 134H, 136F / H, 141 S, 144Q / S.146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G.164A / G / K / Q / S / W. 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V. 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V. 185S / T. 187H / Q / S / V, 188T, 1901, 191A / R / S / W, 192L. 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H. 207A / L / M / R / T / V.211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V. 217A / V, 218D / L / N / R / V. 221 A, 222A / G / M / Q, 225E / G. 229C / H / V, 231L / M / V. 232S. 233A / S / V, 234A / M / N / R / V / Y. 236C / S / V. 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W. 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W. 245F / Q / W, 246A / R / S / T / V, 247L, 248E / G / K / L / R / V. 249A / G / I / L / M / R / S, 250H / I / M / T. 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T. 255A / C / E / G / I / K / S / W. 256C / H / M, 258R / S / V. 265Y, 271W, 274R, 275S, 276D / E / G / S, 287A / S / V / Y, 288V. 291G, 292F / I / K / V, 293M, 294L, 295 A. 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G. 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0016] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 88, 131, 138. 139, 175. 189, 199, 200. 203, 270, 280. or 281; or substitution 53D / H. 54Q, 91G, 111H, 116V, 120G, 126V. 133C / E / K / R / S, 134K / Y. 157L / Q / R,Docket No. 5244.285WO2160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R, 194K, 196G, 206T, 225M / S / T, or 308K; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0017] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 53, 54, 88. 91, 111, 116, 126, 131, 133, 134. 138, 139, 157, 160, 163, 168, 170, 175, 189, 192, 196. 199, 200, 203, 206, 225, 270, 280, 281, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0018] In some embodiments, amino acid sequence of the engineered adenosine kinase comprises at least a substitution selected from the group comprising 53D / H, 54Q, 88R, 91G, 111A / H, 116V. 126V, 131S, 133C / L / R / S. 134K / Y, 138Q, 139L / S, 157L / Q / R. 160A / R / T / W, 163S. 168E / G / Q / S / Y, 170A / C / L / S. 175L / W, 189A / H. 192R, 196G, 199A, 200A, 203E, 206T. 225M / S / T, 270D, 280R / V / W, or 281 S. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0019] In some embodiments, amino acid sequence of the engineered adenosine kinase comprises at least a substitution M53D / H, R54Q. Y88R, H91G. Pl 11A / H. T116V. A126V. A131S, M133C / L / R / S, R134K / Y, N138Q. H139L / S, E157L / Q / R. I160A / R / T7W, A163S. A168E / G / Q / S / Y, G170A / C / L / S, F175L / W, L189A / H. F192R, A196G, V199A. V200A. D203E. S206T, Q225M / S / T. I270D, L280R / V / W, or T281S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0020] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0021] In some embodiments, the adenosine kinase comprises an amino acid sequence having at least 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2. 9.2. 10.2, 11.2. 12.2. and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10Docket No. 5244.285WO2

[0022] In some embodiments, die engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812.

[0023] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0024] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity’ to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or to the reference sequence corresponding to SEQ ID NO: 24, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0025] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 53, 54, 88. 91, 111, 116, 120, 126, 131, 133. 134, 138, 139, 157, 160, 163, 168, 170, 175, 189, 192. 194, 196, 199, 200, 203, 206, 225, 270, 280, 281, or 308, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0026] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 88, 131, 138, 139, 175. 189, 199, 200. 203, 270, 280. 281; or substitution 53D / H. 54Q, 91G, 111H, 116V, 120G, 126V. 133C / E / K / R / S, 134K / Y. 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R, 194K, 196G, 206T, 225M / S / T, or 308K; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.Docket No. 5244.285WO2

[0027] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 53, 53 / 111 / 133, 53 / 111 / 133 / 308, 53 / 111 / 138, 53 / 133, 54, 54 / 116 / 168, 54 / 134 / 168 / 200 / 225, 54 / 134 / 168 / 225 / 280, 54 / 134 / 225, 54 / 168 / 225, 54 / 200 / 280, 111, 111 / 120 / 133, 111 / 133, 111 / 138 / 160, 111 / 138 / 160 / 200, 111 / 160 / 200, 116, 116 / 134 / 168 / 194, 116 / 134 / 168 / 200, 116 / 168 / 225, 133, 133 / 138, 133 / 160 / 170 / 200, 134. 134 / 168, 134 / 168 / 225 / 280, 134 / 225. 138, 138 / 170, 160, 168 / 200 / 280, 168 / 225, 189 / 200 / 225, 200, 200 / 280, 225, 280, 138 / 200. or 168 / 189 / 200, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0028] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 53D / H, 54Q, 88R, 91G, 111H, 116V, 120G, 126V, 131S, 133C / E / K / R / S, 134K / Y, 138Q, 139L / S, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L, 175L / W, 189A / H, 192R, 194K, 196G, 199A, 200A / V, 203E, 206T, 225M / S / T, 270D, 280R / V / W, 28 IS, or 308K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0029] In some embodiments, the amino acid sequence of the engineered adenosine kinase further comprises at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74. 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172. 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191. 192, 194, 195, 196, 197, 202, 206, 207, 208. 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229. 231, 232, 233, 234, 236, 237, 238, 239, 240. 241, 242, 243, 244, 245, 246, 247, 248. 249, 250, 251. 252, 253, 254, 255, 256, 258. 265, 271, 274. 275, 276, 287, 288, 291, 292, 293, 294. 295, 296, 299. 303, 305, 307, 308, 310, 311. 312, 314, 315. 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0030] In some embodiments, the amino acid sequence of the engineered adenosine kinase further comprises at least a substitution or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q, 43E / K / R / S / V, 461, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V. 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M. 93 A, 94M / N, 100G, 101F / W, 1021, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T. 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T, 111A / G / L, 112I / L. 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C.Docket No. 5244.285WO2121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141 S, 144Q / S, 146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 1901, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V. 221A, 222A / G / M / Q, 225E / G, 229C / H / V, 231L / M / V, 232S, 233A / S / V, 234A / M / N / R / V / Y. 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W, 246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S. 250H / I / M / T, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V. 254I / L / M / P / Q / T, 255 A / C7E / G / I / K / S / W. 256C / H / M.258R / S / V, 265Y, 271W, 274R. 275S, 276D / E / G / S, 287A / S / V / Y, 288V, 291G, 292F / I / K / V, 293M.294L. 295 A, 296N, 299G / S, 303D / F / H / M / S / V / W. 305S / T. 307F / G, 308D / F / L / V, 310A / C.311E / F / G / L / M / Q / R / S / T. 312R. 314Q / S / V, 315S, 316A / D / E / I / K / S / Y. 317M / W / Y. or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0031] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 53, 53 / 111 / 133, 53 / 111 / 133 / 308, 53 / 111 / 138, 53 / 133, 54, 54 / 116 / 168, 54 / 134 / 168 / 200 / 225, 54 / 134 / 168 / 225 / 280, 54 / 134 / 225, 54 / 168 / 225, 54 / 200 / 280, 111, 111 / 120 / 133, 111 / 133, 111 / 138 / 160, 111 / 138 / 160 / 200, 111 / 160 / 200.116, 116 / 134 / 168 / 194. 116 / 134 / 168 / 200, 116 / 168 / 225, 133, 133 / 138. 133 / 160 / 170 / 200, 134. 134 / 168, 134 / 168 / 225 / 280, 134 / 225. 138, 138 / 170. 160, 168 / 200 / 280. 168 / 225, 189 / 200 / 225. 200, 200 / 280, 225, 280, or 138 / 200, 168 / 189 / 200. wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0032] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 53H, 53H / 111 A / 133E, 53H / 111A / 133E / 308K.53H / 111A / 138Q, 53H / 133E. 54Q, 54Q / 116V / 168E. 54Q / 134K / 168E / 225T / 280V.54Q / 134K / 168S / 200V / 225T. 54Q / 134K / 225T, 54Q / 168E / 225T, 54Q / 200V / 280V, 111A, 111A / 120G / 133K.. 111A / 133E. 111A / 138Q / 160R, 111A / 138Q / 160R / 200V, 111A / 160R / 200V, 116 V, 116V / 134K / 168E / 194K. 116V / 134K / 168E / 200V. 116V / 168Q / 225T. 133E. 133E / 138Q. 133K, 133K / 160R / 170A / 200V, 133L, 133L / 138Q, 134K, 134K / 168E, 134K / 168E / 225T / 280V, 134K / 225T, 138Q, 138Q / 170A, 160R, 168S / 200V / 280V, 168S / 225T. 189A / 200V / 225T. 200V, 200V / 280V, 225T, 280V. 138Q / 200V. or 168S / 189A / 200V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.Docket No. 5244.285WO2

[0033] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2, 11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0034] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12-321 of SEQ ID NO: 162, or to the reference sequence corresponding to SEQ ID NO: 162, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence comprising residues 12-321 of SEQ ID NO:162, or relative to the reference sequence corresponding to SEQ ID NO: 162,

[0035] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, or more sequence identity' to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 162, or relative to the reference sequence corresponding to SEQ ID NO: 162.

[0036] In some aspects, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position 4 / 126 / 187 / 233, 21, 21 / 129 / 147, 21 / 147, 21 / 147 / 158, 21 / 147 / 158 / 231 / 251, 21 / 147 / 158 / 231 / 251 / 254 / 321, 21 / 147 / 158 / 231 / 293, 21 / 147 / 158 / 234, 21 / 147 / 158 / 234 / 254. 21 / 147 / 158 / 234 / 254 / 293, 21 / 147 / 158 / 251 / 254 / 293, 21 / 147 / 231 / 234, 21 / 147 / 231 / 234 / 251 / 254 / 293, 21 / 147 / 231 / 234 / 251 / 293, 21 / 147 / 234 / 254, 21 / 147 / 234 / 293, 21 / 147 / 251, 21 / 147 / 251 / 254, 21 / 147 / 251 / 293 / 320, 21 / 147 / 254, 21 / 147 / 254 / 293, 21 / 147 / 293, 21 / 158 / 231 / 234 / 251, 21 / 158 / 234, 21 / 158 / 251, 21 / 158 / 251 / 254, 21 / 158 / 293, 21 / 231, 21 / 231 / 234, 21 / 231 / 234 / 251 / 254, 21 / 231 / 234 / 251 / 254 / 293, 21 / 231 / 234 / 251 / 293, 21 / 231 / 251 / 254, 21 / 234 / 251, 21 / 234 / 293, 21 / 251, 21 / 251 / 254, 21 / 254, 21 / 293, 23 / 126 / 157 / 233, 23 / 126 / 157 / 250, 23 / 126 / 187, 23 / 126 / 233, 23 / 126 / 233 / 252 / 289, 23 / 126 / 250 / 289, 23 / 233, 23 / 233 / 252 / 289 / 310, 23 / 250 / 252 / 289, 112 / 126 / 310, 126 / 157 / 187, 126 / 157 / 233 / 250 / 252, 126 / 157 / 233 / 252, 126 / 167 / 310, 126 / 187, 126 / 187 / 233, 126 / 187 / 310, 126 / 233, 126 / 233 / 250 / 252, 126 / 233 / 252 / 289, 126 / 233 / 252 / 302, 126 / 233 / 289, 126 / 250 / 252. 126 / 250 / 289, 126 / 252. 126 / 252 / 289, 126 / 289, 126 / 310, 141 / 187. 147 / 158, 147 / 158 / 182 / 234 / 251, 147 / 158 / 231, 147 / 158 / 231 / 234 / 251. 147 / 158 / 251 / 254, 147 / 158 / 254, 147 / 158 / 254 / 293, 147 / 231 / 234, 147 / 231 / 234 / 251 / 293, 147 / 231 / 234 / 254, 147 / 231 / 293, 147 / 234, 147 / 234 / 251 / 254, 147 / 251. 147 / 251 / 254, 147 / 251 / 293, 147 / 254, 147 / 293, 157 / 187 / 233 / 252,Docket No. 5244.285WO2157 / 233 / 250 / 289, 157 / 252, 157 / 252 / 289, 157 / 289 / 310, 157 / 310, 158, 158 / 231 / 234, 158 / 231 / 234 / 254, 158 / 234, 158 / 234 / 251 / 254, 158 / 234 / 254, 158 / 251 / 254 / 293, 158 / 254, 167 / 252 / 289, 185 / 187 / 233 / 310, 187 / 233, 187 / 233 / 252, 187 / 233 / 252 / 310, 187 / 250, 187 / 250 / 252, 187 / 252, 187 / 289, 187 / 310, 225 / 250 / 252, 231 / 234 / 251, 231 / 234 / 251 / 254, 231 / 254, 233 / 250 / 252, 233 / 250 / 252 / 289, 233 / 250 / 252 / 289 / 310, 233 / 289, 233 / 289 / 310, 233 / 310, 234 / 293, 250 / 252, 250 / 252 / 289, 250 / 252 / 310, 250 / 289, 250 / 289 / 310, 251 / 254, 251 / 254 / 293, 252 / 289, 252 / 310, 254 / 293, 289, 289 / 310, and 293, and 310, or combinations thereof, wherein the amino acid positions are relative to the reference sequence comprising amino acid residues 12-321 of SEQ ID NO: 162, or relative to the reference sequence corresponding to SEQ ID NO: 162.

[0037] In some aspects, the ammo acid sequence of the engineered adenosine kinase comprises a substitution or substitution set selected from 4N / 126V / 187Q / 233E, 21G, 21G / 129I / 147S, 21G / 147S, 21G / 147S / 158S, 21G / 147S / 158S / 231L / 251P, 21G / 147S / 158S / 231L / 251P / 254K / 321I, 21G / 147S / 158S / 231L / 293T, 21G / 147S / 158S / 234E, 21G / 147S / 158S / 234E / 254K, 21G / 147S / 158S / 234E / 254K / 293T, 21G / 147S / 158S / 251P / 254K / 293T, 21G / 147S / 231L / 234E, 21G / 147S / 231L / 234E / 251P / 254K / 293T, 21G / 147S / 231L / 234E / 251T / 293T, 21G / 147S / 234E / 254K, 21G / 147S / 234E / 293T, 21G / 147S / 251P. 21G / 147S / 251P / 254K, 21G / 147S / 251P / 293T / 320E, 21G / 147S / 251T, 21G / 147S / 251T / 254K, 21G / 147S / 254K, 21G / 147S / 254K / 293T, 21G / 147S / 293T, 21G / 158S / 231L / 234E / 251T, 21G / 158S / 234E, 21G / 158S / 251P / 254K. 21G / 158S / 251T, 21G / 158S / 251T / 254K. 21G / 158S / 293T, 21G / 231L, 21G / 231L / 234E. 21G / 231L / 234E / 251P / 254K.21G / 231L / 234E / 251P / 254K / 293T, 21G / 231L / 234E / 251T / 293T, 21G / 231L / 251P / 254K, 21G / 234E / 251P, 21G / 234E / 293T, 21G / 251P, 21G / 251P / 254K, 21G / 251T / 254K, 21G / 254K.21G / 293T, 23E / 126V / 157S / 233E, 23E / 126V / 157S / 250I. 23E / 126V / 187Q, 23E / 126V / 233E, 23E / 126V / 233E / 252V / 289L. 23E / 126V / 250I / 289L, 23E / 233E, 23E / 233E / 252V / 289L / 310A.23E / 250I / 252V / 289L, 112K / 126V / 310A, 126S / 167V / 310A, 126V, 126V / 157S / 187Q, 126V / 157S / 233E / 250L / 252V. 126V / 157S / 233E / 252V. 126V / 187Q, 126V / 187Q / 233E, 126V / 187Q / 310A, 126V / 233E, 126V / 233E / 250L / 252V, 126V / 233E / 252V / 289L, 126V / 233E / 252V / 302H, 126V / 233E / 289L, 126V / 2501 / 252 V. 126V / 250I / 289L, 126V / 250L / 252V, 126V / 252V, 126V / 252V / 289L, 126V / 289L, 126V / 310A. 141S / 187Q. 147S / 158S, 147S / 158S / 182T / 234E / 251P, 147S / 158S / 231L, 147S / 158S / 231L / 234E / 251T, 147S / 158S / 251P / 254K, 147S / 158S / 254K, 147S / 158S / 254K / 293T. 147S / 231L / 234E, 147S / 231L / 234E / 251P / 293T, 147S / 231L / 234E / 254K, 147S / 231L / 293T. 147S / 234E, 147S / 234E / 251T / 254K, 147S / 251P, 147S / 251P / 254K, 147S / 251P / 293T, 147S / 251T, 147S / 251T / 254K, 147S / 251T / 293T, 147S / 254K, 147S / 293T, 157K / 310A, 157S, 157S / 187Q / 233E / 252V, 157S / 233E / 250I / 289L, 157S / 252V, 157S / 252V / 289L, 157S / 289L / 310A, 158S, 158S / 231L / 234E, 158S / 231L / 234E / 254K, 158S / 234E, 158S / 234E / 251P / 254K, 158S / 234E / 254K, 158S / 251T / 254K / 293T, 158S / 254K, 167S / 252V / 289L, 185N / 187Q / 233E / 310A, 187Q, 187Q / 233E, 187Q / 233E / 252V, 187Q / 233E / 252V / 310A,Docket No. 5244.285WO2187Q / 250I / 252V, 187Q / 250L, 187Q / 252V, 187Q / 289L, 187Q / 310A, 225K / 250I / 252V, 231L / 234E / 251T, 231L / 234E / 251T / 254K, 231L / 254K, 233E, 233E / 250I / 252V, 233E / 250I / 252V / 289L, 233E / 250I / 252V / 289L / 310A, 233E / 289L, 233E / 289L / 310A, 233E / 310A, 234E, 234E / 293T, 2501, 250I / 252V, 250I / 252V / 289L, 250I / 289L / 310A, 250L / 252V, 250L / 252V / 310A, 250L / 289L, 251P / 254K, 251P / 254K / 293T, 251T / 254K, 252V / 289L, 252V / 310A, 254K, 254K / 293T, 289L, 289L / 310A, and 293T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 162, or relative to the reference sequence corresponding to SEQ ID NO: 162.

[0038] In some aspects, the ammo acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set selected from H4N / A126V / P187Q / S233E, A21G, A21G / S129I / T147S, A21G / T147S, A21G / T147S / G158S, A21G / T147S / G158S / Q231L / K251P, A21G / T147S / G158S / Q231L / K251P / R254K / L321I, A21G / T147S / G158S / Q231L / K293T, A21G / T147S / G158S / A234E, A21G / T147S / G158S / A234E / R254K, A21G / T147S / G158S / A234E / R254K / K293T, A21G / T147S / G158S / K251P / R254K / K293T, A21G / T147S / Q231L / A234E, A21G / T147S / Q231L / A234E / K251P / R254K / K293T,A21 G / T 147S / Q231L / A234E / K251T / K293T, A21 G / Tl 47S / A234E / R254K, A21G / T147S / A234E / K293T, A21G / T147S / K251P, A21G / T147S / K251P / R254K.A21G / T147S / K251P / K293T / A320E, A21G / T147S / K251T, A21G / T147S / K251T / R254K, A21G / T147S / R254K, A21G / T147S / R254K / K293T. A21G / T147S / K293T, A21G / G158S / Q231L / A234E / K251T. A21G / G158S / A234E, A21G / G158S / K251P / R254K, A21G / G158S / K251T, A21G / G158S / K251T / R254K, A21G / G158S / K293T. A21G / Q231L, A21G / Q231L / A234E. A21G / Q231L / A234E / K251P / R254K.A21 G / Q231 L / A234E / K251 P / R254K / K293T. A21 G / Q231 L / A234E / K251 T / K293T.A21G / Q231L / K251P / R254K, A21G / A234E / K251P. A21G / A234E / K293T, A21G / K251P.A21G / K251P / R254K, A21G / K251T / R254K, A21G / R254K, A21G / K293T,D23E / A126V / E 157S / S233E, D23E / A126V / E 157S / A250I, D23E / A126V / P 187Q, D23E / A126V / S233E, D23E / A126V / S233E / A252V / M289L, D23E / A126V / A250I / M289L.D23E / S233E, D23E / S233E / A252V / M289L / N310A, D23E / A250I / A252V / M289L, Q112K / A126V / N310A, A126S / A167V / N310A, A126V. A126V / E157S / P187Q.A126V / E157S / S233E / A250L / A252V, A126V / E157S / S233E / A252V. A126V / P187Q, A126V / P187Q / S233E, A126V / P187Q / N310A, A126V / S233E, A126V / S233E / A250L / A252V. A126V / S233E / A252V / M289L, A126V / S233E / A252V / Q302H, A126V / S233E / M289L, A126V / A250I / A252V, A126V / A250I / M289L, A126V / A250L / A252V, A126V / A252V, A126V / A252V / M289L, A126V / M289L, A126V / N310A, R141S / P187Q, T147S / G158S, T147S / G158S / P182T / A234E / K251P, T147S / G158S / Q231L, T147S / G158S / Q231L / A234E / K251T, T147S / G158S / K251P / R254K, T147S / G158S / R254K, T147S / G158S / R254K / K293T,Docket No. 5244.285WO2T147S / Q231L / A234E, T147S / Q231L / A234E / K251P / K293T, T147S / Q231L / A234E / R254K, T147S / Q231L / K293T, T147S / A234E, T147S / A234E / K251T / R254K, T147S / K251P, T147S / K251P / R254K, T147S / K251P / K293T, T147S / K251T, T147S / K251T / R254K, T147S / K251T / K293T, T147S / R254K, T147S / K293T, E157K / N310A, E157S, E157S / P187Q / S233E / A252V, E157S / S233E / A250I / M289L, E157S / A252V, E157S / A252V / M289L, E157S / M289L / N310A, G158S, G158S / Q231L / A234E, G158S / Q231L / A234E / R254K, G158S / A234E, G158S / A234E / K251P / R254K, G158S / A234E / R254K, G158S / K251T / R254K / K293T, G158S / R254K, A167S / A252V / M289L. S185N / P187Q / S233E / N310A, P187Q, P187Q / S233E, P187Q / S233E / A252V. P187Q / S233E / A252V / N310A. P187Q / A250I / A252V. P187Q / A250L, P187Q / A252V, P187Q / M289L, P187Q / N310A. Q225K / A250I / A252V, Q231L / A234E / K251T, Q231L / A234E / K251T / R254K. Q231L / R254K, S233E, S233E / A250I / A252V, S233E / A2501 / A252V / M289L, S233E / A250I / A252V / M289L / N310A, S233E / M289L.S233E / M289L / N310A, S233E / N310A. A234E. A234E / K293T, A250I. A250I / A252V, A250I / A252V / M289L, A250I / M289L / N310A, A250L / A252V, A250L / A252V / N310A.A250L / M289L, K251P / R254K. K251P / R254K / K293T, K251T / R254K, A252V / M289L, A252V / N310A, R254K, R254K / K293T. M289L, M289L / N310A, and K293T. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 162. or relative to the reference sequence corresponding to SEQ ID NO: 162.

[0039] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%. 84%. 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identify to the reference sequence comprising residues 12-321 of SEQ ID NO: 268, or to the reference sequence corresponding to SEQ ID NO: 268, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence comprising residues 12-321 of SEQ ID NO: 268. or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0040] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identify' to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0041] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 9. 11, 12, 40 / 193, 46, 199, 201. 223, 267, T1, 278, andDocket No. 5244.285WO2315; a substitution of 13L / R / T, 40L / Q, 55Q / S / T / W / Y, 74A / C / G / L / M / Q, 90G / Q, 94R, 107 / K / L / P, 108S, 109D, 111 / H / S, 119K, 121E, 128E / L / M, 147L / P, 164I / R, 167E / R / S, 169W, 171Q / T, 172A / R / T / V, 182T, 185L / N, 190E / L, 191P, 194F / K, 195C / N, 206E / H / T, 207S, 210E / V, 21 IS, 222E / P / V, 243A / E / L / N / T, 271L / T, 274V, 312C, 314A / L / M or combinations thereof, wherein the amino acid positions are relative to the reference sequence comprising amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0042] In some aspects, the ammo acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 9C, 9L, 9R, UK, IIP, 12E, 12P, 12R, 12T, 13L, 13P, 13R, 13T, 40H / 193T, 40L, 40Q, 43A, 46L, 55Q, 55S, 55T, 55W, 55Y. 74A, 74C, 74G, 74L, 74M, 74Q, 74T, 74V, 80V, 90G, 90Q, 94M, 94R, 106G, 106L, 106R, 107G, 107K, 107L, 107P, 108S, 109D, 111S, 119K, 121E, 128E, 128L, 128M, 140P, 147L, 147P, 155A, 1551, 1641, 164R, 164S, 167E, 167R, 167V, 169L, 169W, 171Q, 171T, 172A, 172R, 172T, 172V, 182G, 185L, 190E, 190L, 191P, 191S, 193M, 194F, 195C, 195G, 195N, 199L, 199M, 201S, 201T, 206E, 206H, 207A, 207L, 207M, 207S, 210E, 210V, 2111, 21 IL. 21 IM, 211S, 222A, 222E, 222G, 222P, 222V, 223G, 223L, 243A, 243E, 243L. 243N, 243R, 243T, 267G, 271L, 271T, 274V, 277K, 277R, 278A, 278G, 278P, 278Q, 278T, 312C, 314A, 314L, 314M, and 315V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0043] In some aspects, the ammo acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue G9C, G9L, G9R, G1 IK, G1 IP, M12E, M12P, M12R, M12T, S13L, S13P, S13R, S13T, R40H / I193T, R40L, R40Q, R43A, V46L, R55Q, R55S, R55T, R55W, R55Y, R74A, R74C, R74G, R74L, R74M. R74Q, R74T, R74V, T80V, E90G, E90Q, T94M, T94R, D106G, D106L, D106R, D107G, D107K, D107L, D107P, L108S, S109D, PHIS, M119K, Q121E, H128E, H128L, H128M, V140P, S147L, S147P, T155A, T155I, E164I, E164R. E164S, A167E, A167R, A167V, M169L. M169W, I171Q, I171T, P172A, P172R, P172T, P172V, P182G, S185L, R190E, R190L, E191P, E191S, I193M, E194F, Q195C, Q195G, Q195N, V199L, V199M, V201S, V201T, S206E, S206H, R207A, R207L, R207M, R207S, Q210E, Q210V, A211I, A211L, A211M, A211S. S222A, S222E, S222G, S222P, S222V. R223G, R223L. K243A. K243E. K243L, K243N. K243R. K243T, A267G. Y271L. Y271T, Q274V, Y277K. Y277R. D278A. D278G, D278P. D278Q. D278T. R312C, K314A. K314L, K314M, and Q315V, or combinations thereof, wherein tire amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0044] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least one substitution position or substitution position set selected from 1 / 250 / 289, 126 / 157, 126 / 157 / 250, 126 / 157 / 250 / 289, 126 / 157 / 289, 126 / 250, 126 / 250 / 289, 126 / 289, 157 / 250, 157 / 250 / 289, 157 / 289, 250 / 289, and 289; the substitution 126V, 157S, 250L,or combinations thereof, wherein theDocket No. 5244.285WO2amino acid positions arc relative to the reference sequence comprising amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0045] In some aspects, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set selected from 1V / 250I / 289L, 126V, 126V / 157K, 126V / 157S / 250I, 126V / 157S / 250I / 289L, 126V / 157S / 289L, 126V / 250I. 126V / 250I / 289L, 126V / 289L, 157S, 157S / 250I, 157S / 250I / 289L, 157S / 289L, 250I / 289L, and 289L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0046] In some aspects, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set selected from M1V / A250I / M289L, A 126V, A126V / E157K, A126V / E157S / A250I, A126V / E157S / A250I / M289L, A126V / E157S / M289L, A126V / A250I, A126V / A250I / M289L. A126V / M289L, E157S, E157S / A250I, E157S / A250I / M289L, E157S / M289L, A250I / M289L, and M289L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

[0047] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence comprising residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812. or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812.

[0048] In some embodiments, the engineered adenosine kinase has adenosine kinase activity and at least one improved property as compared to a reference adenosine kinase. In some embodiments, the engineered adenosine kinase has an improved property selected from i) increased activity on unmodified nucleoside, ii) increased thermostability, iii) increased activity on cytidine, iv) increased activity on 2-fluoro modified nucleosides, and v) increased activity on 2-O-methyl modified nucleosides, or any combinations of i). ii). iii). iv), and v), compared to a reference adenosine kinase.

[0049] In some embodiments, the reference adenosine kinase has an amino acid sequence corresponding to residue 12-321 of SEQ ID NO: 10. 24, 162, or 268, or an amino acid sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

[0050] In some embodiments, the engineered adenosine kinase comprises a fusion polypeptide.

[0051] In some embodiments, the engineered adenosine kinase is purified or provided as a purified preparation. In some embodiments, the engineered adenosine kinase is immobilized on a substrate or support medium.

[0052] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenosine kinase described herein.Docket No. 5244.285WO2

[0053] In some embodiments, die recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, wherein the recombinant polynucleotide encodes an engineered adenosine kinase.

[0054] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or comprises an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811.

[0055] In some aspects, the recombinant polynucleotide has preferred codons or is codon optimized.

[0056] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding an engineered adenosine kinase described herein. In some embodiments, the expression vector comprises a recombinant polynucleotide operably linked to a control sequence. In some aspects, the control sequence comprises a promoter.

[0057] In a further aspect, the present disclosure provides a host cell comprising an expression vector for expression of an engineered adenosine kinase. In some aspects, the host cell is selected from a bacterial cell, fungal cell, insect cell or mammalian cell.

[0058] In a further aspect, the present disclosure provides a method of producing an engineered adenosine kinase polypeptide, comprising culturing a host cell under suitable culture conditions such that the engineered adenosine kinase poly peptide is produced. In some embodiments, the method further comprises recovering or isolating the engineered adenosine kinase polypeptide from the culture and / or host cells. In some embodiments, the method further comprises purifying the engineered adenosine kinase polypeptide.

[0059] In another aspect, the present disclosure provides a composition comprising an engineered adenosine kinase described herein. In some embodiments, the composition is a solution or a lyophilizate. In some embodiments, the composition further comprises one or more of buffer, a nucleoside substrate, and a phosphate donor. In some embodiments, the nucleoside substrate comprises a nucleoside substrate. In some aspects, the nucleoside substrate is a modified nucleoside substrate. In some embodiments, the composition further comprises another enzy me, for example, an adenylate kinase, acetate kinase, 3’-O-kinase, and / or pyruvate oxidase.

[0060] In another aspect, the engineered adenosine kinases are used in the conversion of a nucleoside substrate to the corresponding nucleoside monophosphate. In some embodiments, a method of converting a nucleoside to corresponding nucleoside monophosphate (NMP) comprises contacting aDocket No. 5244.285WO2nucleoside with an engineered adenosine kinase described herein in the presence of a suitable phosphate donor under suitable reaction conditions to convert the nucleoside to the corresponding nucleoside monophosphate (NMP).

[0061] In some embodiments, the nucleoside substrate comprises a modified nucleoside substrate. In some embodiments, the modified nucleoside substrate comprises a modified sugar moiety or a modified nucleobase or a combination thereof. In some aspects, the modified nucleoside comprises a modified sugar moiety, wherein the sugar moiety has a modified 2’-position or 3’-position. In some aspects, the modified sugar moiety has a modified 2 ’-position of the sugar moiety. In some aspects, the 2’-position is a 2'-0-alkyl or 2-halo. In some aspects, the 2’-position is a 2’-O-alkyl, and wherein the 2’-O-alkyl is 2’-O-CH3or 2’-O-CH2CH3. In some aspects, the 2’-position is 2-halo. In some aspects, the 2’-position is 2’-F or 2‘-Br. In some aspects, the modified sugar moiety is modified at the 3 ’-position. In some aspects, the 3 ’-position is modified with a blocking group, wherein the blocking group is formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONH2), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl, O-(2 -nitrobenzyl), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl. phosphate, or triphosphate.

[0062] In some aspects, the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine. In some aspects, the nucleoside has at the 2’ -position of the sugar moiety a H or OH. In some aspects of the methods, the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine, and the nucleoside has at the 2’-position of the sugar moiety an OH, thereby by resulting in the corresponding product rAMP, rGMP, rUMP, rCMP, or rTMP, respectively. In some aspects of the method, the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine, and the nucleoside has at the 2’-position of the sugar moiety an H, thereby by resulting in the corresponding product dAMP, dGMP, dUMP, dCMP, or dTMP, respectively.

[0063] In some embodiments, the nucleoside is a modified nucleoside comprising a modified nucleobase. In some aspects, the modified nucleobase is 5 -bromo-uracil, 5 -iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.

[0064] In some embodiments, the phosphate donor is a nucleoside triphosphate, also referred to as phosphate donor NTP. In some aspects, the phosphate donor NTP is rATP or dATP. In some embodiments, the phosphate donor NTP is adenosine-5’ -gamma-thiotriphosphate (ATPyS) or deoxy adenosine-5’ -gamma-thiotriphosphate (dATPyS), thereby resulting in product nucleoside 5’-O-thiophosphate (NMP-S). In some embodiments, the phosphate donor NTP has the same nucleoside structure of the nucleoside substrate.

[0065] In some embodiments, the method further comprises regenerating the NTP using a NTP regeneration system. In various aspects, the NTP regenerating system comprises creatine kinase,Docket No. 5244.285WO2pyruvate kinase, polyphosphate kinase, and / or acetate-kinase. In some aspects, the NTP regenerating system comprises pyruvate kinase and substrate phosphoenolpyruvate. In some aspects, the NTP regenerating system comprises creatine kinase and substrate creatine phosphate. In some aspects, the NTP regenerating system comprises polyphosphate kinase and substrate polyphosphate. In some aspects, the NTP regenerating system is acetate kinase and substrate acetyl phosphate. In some aspects, the methods further comprise pyruvate oxidase and substrate pyruvate and inorganic phosphate. In some aspects, the methods further comprise including a catalase.

[0066] In an embodiment, an NTP with a phosphate group at the 3’ position of the sugar (NQP) produced from an NMP produced by an engineered adenosine kinase of the application is provided. In sonic aspects, the NQP further comprises a modification of the nucleoside, NTP and / or NQP at the 2’ position of the sugar. In some aspects, the NQP further comprises a modification of the nucleoside, NTP and / or NQP at the nucleobase comprising a noncanonical nucleobase known to those skilled in the art, a removable tag, a cleavable linker, or a radio, a photo, and / or a chemical sensor; a modification of the nucleoside, NTP and / or NQP at the phosphate chain, optionally comprising an a-phosphothioate linkage or other non-phosphodiester linkage; or a combination of a modification at the nucleobase and a modification at the phosphate chain.DETAILED DESCRIPTION

[0067] The present disclosure provides engineered adenosine kinase enzyme, recombinant polynucleotide encoding the engineered adenosine kinases, and methods of using the engineered adenosine kinases. In some embodiments, are engineered to have an improved property compared to a reference adenosine kinase.Abbreviations and Definitions

[0068] In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary' skill in the art, unless specifically defined otherw ise. Accordingly, the following tenns are intended to have the following meanings.

[0069] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherw ise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide.

[0070] Similarly, “comprise.” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Thus, as used herein, the term “comprising” and its cognates are used in their inclusive sense (z.e., equivalent to the term “including” and its corresponding cognates).Docket No. 5244.285WO2

[0071] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.”

[0072] “ About” means an acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.

[0073] “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0074] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0075] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0076] “ Adenosine kinase,” “AdoK,” or “Adk,” refers to an enzyme that catalyzes the phosphorylation of adenosine (A or ADO) to adenosine-5 ’-monophosphate (AMP). In some embodiments, adenosine kinase is classified in EC 2.7.1.20. Although the primary substrate is adenosine, adenosine kinases as used herein include enzymes that act on other nucleosides.

[0077] “Creatine kinase” refers to an enzyme that catalyzes the reversible interconversion of creatine: ATP to creatine phosphate: ADP.

[0078] “Polyphosphate kinase” refers to an enzyme that catalyzes the transfer of phosphate group(s) from high-energy, phosphate-donating molecules, such as polyphosphate (PolyPn), to specific substrates / molecules. Two families of polyphosphate kinases, PPKK1 and PPK2. are known. PPKls preferentially synthesize polyphosphate from NTP and the corresponding reverse reaction, and PPK2s preferentially consume polyphosphate to phosphorylate nucleoside mono- or diphosphates, and the corresponding reverse reactions. In some embodiments, polyphosphate kinase includes enzymes classified in EC 2.7.4.1.

[0079] “Acetate kinase (“AcK”) refers to enzymes that are capable of catalyzing the phosphorylation of nucleoside diphosphates or analogues thereof, to nucleoside triphosphates or the corresponding analogues, using acetyl phosphate or another phosphoryl group donor. Acetate kinases as used herein includes naturally occurring, wild-type enzymes or engineered enzymes. In some embodiments, acetate kinases are naturally occurring, wild-type basic metabolic enzymes found primarily in prokaryotes that catalyze the following reaction: acetate + ATP <->acetyl phosphate + ADP. Acet lDocket No. 5244.285WO2phosphate is an intermediate in the formation of acetyl-CoA. In some embodiments, acetate kinases are derived from the naturally occurring, wild-ty pe enzymes.

[0080] “Adenylate kinase,” “AdyK,” or “ Ayk” refers to an enzyme that catalyzes the interconversion of ATP, ADP, and AMP through transfer of phosphoryl groups. In some embodiments, adenylate kinase also includes enzymes capable of catalyzing the interconversion of NTP, NDP and NMP. In some embodiments, adenylate kinase are enzymes classified in EC 2.7.3.4.

[0081] “Pyruvate oxidase” or “Pox” refers to an enzyme that catalyzes the catalyzes the reaction between pyruvate, inorganic phosphate, and oxygen to generate acetyl phosphate and H2O2. In some embodiments, pyruvate oxidase include enzymes classified in EC 1.2. 3.3.

[0082] “Catalase” refers to an enzyme that converts hydrogen peroxide (H2O2) to H2O and O2. Catalase can be used to remove residual hydrogen peroxide in applications where hydrogen peroxide is present or is a product in a process. In some embodiments, catalases includes enzymes classified hi EC 1.11.1.6.

[0083] “ Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g.. glycosylation or phosphory lation). Included yvithin this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids.

[0084] “ Fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology’ techniques known in the art).

[0085] “ Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gin or Q), histidine (His or H), isoleucine (lie or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phc or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), try ptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which tire abbreviation is used, the amino acid may be in either the L- or D -configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon. “D-Ala” and “L-Ala” designate D-alanine and L-alanine. respectively. When the one-letter abbreviations are used, upper case letters designateDocket No. 5244.285WO2amino acids in the L-configuration about the oc-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0086] “Polynucleotide" and “nucleic acid' refer to two or more nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2’ deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo- and 2’ deoxyribonucleotides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double -stranded, or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e.. adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0087] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C): thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2’-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’-deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated.

[0088] “Nucleobase” or “base" refers to those naturally occurring and synthetic heterocyclic moieties commonly known to those who utilize nucleic acid or polynucleotide technology to thereby generate polymers that can hybridize to polynucleotides in a sequence-specific manner. Non-limiting examples of nucleobases include, among others, adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil. 5-methylcytosine, pseudoisocytosine, pseudoisouridine, 2-thiouracil and 2-thiothymine. 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine. N9-(7-deaza-guanine). N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).

[0089] “Nucleoside" refers to glycosylamines comprising a nucleobase, and a 5-carbon sugar (e.g., ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In contrast, the term “nucleotide" refers to the glycosylamines comprising a nucleobase, a 5-carbon sugar, and one or more phosphate groups, as further described herein. In some embodiments, nucleosides can be phosphory lated by kinases to produce nucleotides.Docket No. 5244.285WO2

[0090] “Nucleoside monophosphate" or “NMP” refers to glycosylamines comprising a nucleobase, a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose), and a phosphate moiety at the 5’-position. In some embodiments herein, “nucleoside monophosphate" is abbreviated as “NMP". Non-limiting examples of nucleoside monophosphates include cytidine monophosphate (CMP), uridine monophosphate (UMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), and inosine monophosphate (IMP). In some embodiments, “nucleoside monophosphate" may refer to a non-natural nucleoside monophosphate. The terms “nucleoside" and “nucleotide” may be used interchangeably in some contexts.

[0091] “Nucleoside diphosphate” or “NDP" refers to glycosylamines comprising a nucleobase, a 5-carbon sugar (e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate) moiety at the 5’-position. In some embodiments herein, “nucleoside diphosphate" is abbreviated as “NDP". Nonlimiting examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP). In some embodiments, “nucleoside diphosphate" may refer to a non-natural nucleoside diphosphate. The terms “nucleoside” and “nucleotide” may be used interchangeably in some contexts.

[0092] “Nucleoside triphosphate” or “NTP" refers to glycosylamines comprising a nucleobase, a 5-carbon sugar (e.g., ribose or deoxy ribose), and a triphosphate moiety at the 5 ’-position. In some embodiments herein, “nucleoside triphosphate” is abbreviated as “NTP”. Non-limiting examples of nucleoside triphosphates include cytidine triphosphate (CTP), uridine triphosphate (UTP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), and inosine triphosphate (ITP). In some embodiments, “nucleoside triphosphate” may refer to a non-natural nucleoside triphosphate. Tire terms “nucleoside” and “nucleotide” may be used interchangeably in some contexts.

[0093] “ Modified” or “non-natural” in context of a nucleoside or nucleotide refers to a nucleoside or nucleotide that has been altered to a non-naturally occurring nucleoside or nucleotide. In some embodiments, modifications to nucleoside or nucleotides include modifications to the base, sugar moiety’, and / or phosphate. In some embodiments, the common modifications of the 2‘-position of the sugar moiety’ with fluoro (F) or -O-CH3is denoted by “fN” and “mN", respectively, where N denotes the nucleobase on the nucleoside or nucleotide. In some embodiments, the presence of a 5’-thiophosphate is denoted by “*N".

[0094] “ Locked nucleoside” or “locked nucleotide” refers to nucleoside or nucleotide, respectively, in which the ribose moiety’ is modified with a bridge connecting the 2’ oxygen and 4’ carbon (see, e.g., Obika et al., Tetrahedron Letters, 1997. 38(50):8735-8738; (Drum etal., Current Pharmaceutical Design, 2008, 14(11): 1138-1142). In some embodiments, the bridge is a methylene or ethyleneDocket No. 5244.285WO2bridge. In some embodiments, the ribose moiety of the locked nucleoside or locked nucleotide is in the C3’-endo (beta-D) or C2‘-endo (alpha-L) conformation.

[0095] “Biocatalysis,” “biocatalytic,” “biotransformation,” and “biosynthesis” refer to the use of enzymes to perform chemical reactions on organic compounds.

[0096] " Wild-type” and “naturally occurring” refer to the form found in nature. For example, a wildtype polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0097] “Recombinant,” “engineered.” “variant,” “non-natural.” and “non-naturally occurring" when used with reference to a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, which has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid or polypeptide is identical a naturally occurring cell, nucleic acid or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0098] “ Percent (%) sequence identity” is used herein to refer to comparisons among polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted by any suitable method, including, but not limited to the local homology algorithm of Smith and Waterman (Smith and Waterman. Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443). by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA.. 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT,Docket No. 5244.285WO2FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucl. Acids Res., 1977, 25(17):3389-3402, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (See, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty’ score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10. and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary’ determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0099] "‘Substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity, at least between 89 to 95 percent sequence identity, or more usually, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison, In some specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percentDocket No. 5244.285WO2sequence identity or more (c.g., 99 percent sequence identity). In some embodiments, residue positions that are not identical in sequences being compared differ by conservative amino acid substitutions.

[0100] “ Reference sequence” refers to a defined sequence used as a basis for a sequence and / or activity comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.

[0101] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues w herein a sequence may be compared to a reference sequence. In some embodiments, the comparison w indow’ is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100. or longer windows.

[0102] “Corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered adenosine kinase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.Docket No. 5244.285WO2

[0103] “ Amino acid difference”, “residue difference” and “substitution” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to die amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X53 as compared to SEQ ID NO: 10” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 20 of SEQ ID NO: 10. Thus, if the reference polypeptide of SEQ ID NO: 10 has a methionine at position 53, then a “residue difference at position X53 as compared to SEQ ID NO: 2” refers to an amino acid substitution of any residue other than methionine at the position of the polypeptide corresponding to position 53 of SEQ ID NO: 10. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “ Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g.. in the Tables presented in the Examples), the present invention also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by aIn some instances, a polypeptide of the present invention can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB,” without the identifier for the residue in die reference sequence. In some embodiments, the phrase “an amino acid residue nB” denotes the presence of the amino residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.

[0104] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence, In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “ / ” (e.g., X53D / X53H, X53D / H, or 53D / H).

[0105] “Amino acid substitution set” or “substitution set” refers to a group of amino acid substitutions in a polypeptide sequence, as compared to a reference sequence. A substitution set can have 2, 3, 4, 5. 6. 7, 8, 9, 10, 11. 12. 13, 14, 15, ormore amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any ofDocket No. 5244.285WO2the variant adenosine kinases listed in the Tables provided in the Examples. In these substitution sets, the individual substitutions are separated by a semicolon (“;”; e.g., M53H; M133E) or slash (“ / ”; e.g., M53H / M133E or 53H / 133E).

[0106] “ Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with an hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0107] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary' non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0108] “ Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered adenosine kinase enzyme. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. Deletions are typically indicated by in amino acid sequences.

[0109] “ Insertion” refers to modification to the polypeptide by addition of one or more amino acids to the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art.Docket No. 5244.285WO2The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0110] “Functional fragment” and “biologically active fragment” are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered adenosine kinase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0111] “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g.. protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or via in vitro synthesis). The recombinant adenosine kinase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant adenosine kinase polypeptides can be an isolated polypeptide.

[0112] “Substantially pure polypeptide” or “purified protein” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. However, in some embodiments, the composition comprising adenosine kinase comprises adenosine kinase that is less than 50% pure (e.g., about 10%, about 20%. about 30%, about 40%, or about 50%). Generally, a substantially pure adenosine kinase composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e.. contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant adenosine kinase polypeptides are substantially pure polypeptide compositions.

[0113] “Improved enzyme property” refers to at least one improved property7of an enzyme. In some embodiments, the present invention provides engineered adenosine kinase polypeptides that exhibit an improvement in any enzyme property' as compared to a reference adenosine kinase polypeptide and / or a wild-type adenosine kinase polypeptide, and / or another engineered adenosine kinase polypeptide. Thus, the level of “improvement” can be determined and compared between various adenosine kinase polypeptides, including wild-type, as well as engineered adenosine kinases. Improved propertiesDocket No. 5244.285WO2include, but are not limited, to such properties as increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and altered temperature profile. In additional embodiments, the term is used in reference to the at least one improved property of adenosine kinase enzymes. In some embodiments, the present invention provides adenosine kinase polypeptides that exhibit an improvement in any enzyme property' as compared to a reference adenosine kinase polypeptide and / or a wild-type adenosine kinase polypeptide, and / or another engineered adenosine kinase polypeptide. Thus, the level of “improvement” can be determined and compared between various adenosine kinase polypeptides, including wild-type, as well as engineered adenosine kinases.

[0114] “ hicreased enzymatic activity” and ’‘enhanced catalytic activity” refer to an improved property of the engineered polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of enzyme) as compared to the reference enzyme. In some embodiments, the terms refer to an improved property of engineered adenosine kinase polypeptides provided herein, which can be represented by an increase in specific activity (e.g.. product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of adenosine kinase) as compared to the reference adenosine kinase enzyme. In some embodiments, the terms are used in reference to improved adenosine kinase enzymes provided herein. Exemplary methods to determine enzyme activity of the engineered adenosine kinases of the present invention are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km. Vmaxor kcat, changes of which can lead to increased enzy matic activity. For example, improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to as much as 2-fold. 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzymatic activity than the naturally occurring adenosine kinase or another engineered adenosine kinase from which the adenosine kinase polypeptides were derived.

[0115] “Conversion” refers to the enzymatic conversion (or biotransformation) of a substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymaticDocket No. 5244.285WO2activity" or “activity ” of an adenosine kinase polypeptide can be expressed as “percent conversion” of the substrate to the product in a specific period of time.

[0116] “ Stringent hybridization conditions” is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of a hybrid is a function of ion strength, temperature, G / C content, and the presence of chaotropic agents. The Tmvalues for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 1989, 168:761-777; Bolton et al., Proc. Natl. Acad. Sci. USA., 1962, 48:1390; Bresslauer et al., Proc. Natl. Acad. Sci. USA, 1986, 83:8893-8897; Freier et al., Proc. Natl. Acad. Sci. USA., 1986, 83:9373-9377; Kierzek et al., Biochem., 1986, 25:7840-7846; Rychlik et al., Nucl. Acids Res., 1990, 18:6409-6412 (erratum, Nucl. Acids Res., 1991, 19:698); Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, (2001); Suggs et al., in “Developmental Biology Using Purified Genes,” Brown et al. [eds.], pp. 683-693, Academic Press. Cambridge, MA (1981); and Wetmur, Crit. Rev. Biochem. Mol. Biol., 1991, 26:227-259). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered adenosine kinase enzyme of the present invention.

[0117] “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42 °C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tmas determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it w ill not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution. 5xSSPE, 0.2% SDS at 42 °C, followed by washing in O.lxSSPE, and 0.1% SDS at 65 °C. AnotherDocket No. 5244.285WO2high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w / v) SDS at 65 °C and washing in 0.1x SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0118] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0119] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the adenosine kinase enzymes may be codon optimized for optimal production in the host organism selected for expression.

[0120] “Preferred.” “optimal.” and “high codon usage bias” codons when used alone or in combination refer(s) interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 1998, 14:372-73; Stenico et al., Nucl. Acids Res., 1994, 22:2437-46; and Wright, Gene, 1990, 87:23-29). Codon usage tables are available for many different organisms (See e.g., Wada et al., Nucl. Acids Res., 1992, 20:2111-2118; Nakamura et al., Nucl. Acids Res., 2000, 28:292; Duret, et al., supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D. C., p. 2047-2066 (1996)). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g.,Docket No. 5244.285WO2Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); Uberbacher, Meth. Enzymol., 1996, 266:259-281; and Tiwari et al., Comput. Appl. Biosci., 1997, 13:263-270).

[0121] “ Control sequence” refers herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter sequence, signal peptide sequence, initiation sequence and transcription terminator. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals.

[0122] "‘Operably linked” or “operatively linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide, and where appropriate the encoded polypeptide of interest.

[0123] “Promoter sequence” refers to a nucleic acid sequence that defines and / or initiates expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0124] “Heterologous” refers to the relationship between two or more nucleic acid or protein sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

[0125] “ Vector” refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide of interest and where appropriate the encoded polypeptide.

[0126] " Expression" includes any step involved in the production of the poly peptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0127] “ Produces” refers to the production of proteins and / or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translationalDocket No. 5244.285WO2modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0128] “Culturing” refers to the growing of a population of microbial cells under any suitable conditions (e.g., using a liquid, gel or solid medium).

[0129] " Host cell” and “host strain” refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., the polynucleotides encoding the adenosine kinase variants). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

[0130] “pH stable” refers to an adenosine kinase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hr) compared to the untreated enzyme.

[0131] “ Thermostable” refers to an adenosine kinase poly peptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures (e.g., 40-80 °C) for a period of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposed to the same elevated temperature.

[0132] “ Solvent stable” refers to an adenosine kinase polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to vary ing concentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl adenosine, methyl tert-but l ether, etc.) for a period of time (e.g., 0.5-24 h) compared to the wild-ty pe enzyme exposed to the same concentration of the same solvent.

[0133] “Thermo- and solvent stable” refers to an adenosine kinase polypeptide that is both thermostable and solvent stable.

[0134] “Stereoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one stereoisomer over another. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or it may be complete where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly alternatively reported in the art (typically as a percentage) as the enantiomeric excess (“e.e.”) calculated therefrom according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. Where the stereoisomers are diastereoisomers, the stereoselectivity is referred to as diastereoselectivity. the fraction (typically reported as a percentage) of one diastereomer in a mixture of tw o diastereomers, commonly alternatively reported as the diastereomeric excess (“d.e.”). Enantiomeric excess and diastereomeric excess are types of stereomeric excess.Docket No. 5244.285WO2

[0135] “Rcgiosclcctivity” and “regioselective reaction” refer to a reaction in which one direction of bond making or breaking occurs preferentially over all other possible directions. Reactions can completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%), or partially regioselective (x%, wherein the percentage is set dependent upon the reaction of interest), if the product of reaction at one site predominates over the product of reaction at other sites.

[0136] “Chemoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one product over another.

[0137] “Suitable reaction conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature. pH. buffers, co-solvents, etc.) under which an adenosine kinase polypeptide of the present invention is capable of converting a substrate to the desired product compound. Some exemplary “suitable reaction conditions” are provided herein.

[0138] " Loading.” such as in “compound loading” or "enzyme loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0139] “ Substrate” in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the engineered enzymes provided herein (e.g., engineered adenosine kinase polypeptides).

[0140] “ Product” in the context of an enzy matic conversion process refers to the compound or molecule resulting from the action of an enzymatic polypeptide on a substrate.

[0141] “Increasing” yield of a product (e.g.. a nucleoside triphosphate or analogue) from a reaction occurs when a particular component present during the reaction (e.g., an adenosine kinase enzyme) causes more product to be produced, compared with a reaction conducted under the same conditions with the same substrate and other substituents, but in the absence of the component of interest.

[0142] " Substantially free” of a particular enzyme if the amount of that enzyme compared with other enzymes that participate in catalyzing the reaction is less than about 2%, about 1%, or about 0.1% (wt / wt).

[0143] “Fractionating” a liquid (e.g.. a culture broth) means applying a separation process (e.g., salt precipitation, column chromatography, size exclusion, and filtration) or a combination of such processes to provide a solution in which a desired protein comprises a greater percentage of total protein in the solution than in the initial liquid product.

[0144] “Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, andDocket No. 5244.285WO2preferably 1 to 6 carbon atoms inclusively. An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (Ci-C4)alkyl refers to an alkyl of 1 to 4 carbon atoms).

[0145] “Alkenyl” refers to groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.

[0146] " Alkynyl” refers to groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.

[0147] “Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR’-, -PH-, -S(O)-, -S(O)2-, -S(O) NR’-, -S(O)2NR’-, and the like, including combinations thereof, where each Ra is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0148] “Alkoxy” refers to the group -OR’ wherein R’ is an alkyl group is as defined above including optionally substituted alkyl groups as also defined herein.

[0149] “ Amino” refers to the group -NHi. Substituted amino refers to the group -NHR’, NR’R’, and NR’R’R’, where each R’ is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, ary l, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfony l, and the like. Typical amino groups include, but arc limited to, dimcthylamino, dicthylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy -sulfamino, and the like.

[0150] “Oxo” refers to =0.

[0151] “Oxy” refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0152] “Carboxy” refers to -COOH.

[0153] “Carbonyl” refers to -C(O)-, which may have a variety7of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0154] “Alkyloxycarbonyl” refers to -C(O)OR’, where R’ is an alkyl group as defined herein, which can be optionally substituted.

[0155] As used herein, “aminocarbonyl” refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NR’R’, where the amino group NR’R’ is as defined herein.

[0156] “Halogen” and “halo” refer to fluoro (F), chloro (C), bromo (B), and iodo (I).

[0157] “Hy droxy” refers to -OH.Docket No. 5244.285WO2

[0158] “Cyano” refers to -CN.

[0159] “Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 12 carbon atoms inclusively having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, pyridyl, naphthyl and the like.

[0160] " Heterocycle,” “heterocyclic,” and interchangeably “heterocycloalkyl,” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, from 2 to 10 carbon ring atoms inclusively and from 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline. cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine. imidazoline, piperidine, piperazine, pyrrolidine, indoline and the like.

[0161] “Heteroaryl” refers to an aromatic heterocyclic group of from 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusively selected from oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).

[0162] “Heteroarylalkyl” refers to an alkyl substituted with a heteroary l (i.e.. heteroaryl-alkyl-groups). preferably having from 1 to 6 carbon atoms inclusively in the alkyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groups are exemplified by pyridylmethyl and the like.

[0163] “ Heteroarylalkenyl” refers to an alkenyl substituted with a heteroary l (i.e., heteroaryl-alkenyl-groups). preferably having from 2 to 6 carbon atoms inclusively in the alkenyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety.

[0164] “Heteroarylalkynyl” refers to an alkynyl substituted with a heteroaryl (i.e., heteroary 1-alkynyl- groups), preferably having from 2 to 6 carbon atoms inclusively in the alky ny l moiety and from 5 to 12 ring atoms inclusively in the heteroary l moiety’.

[0165] “Membered ring” is meant to embrace any cyclic structure. The number preceding the term “membered” denotes the number of skeletal atoms that constitute the ring. Thus, for example, cyclohexy l, py ridine, pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.Docket No. 5244.285WO2

[0166] “Phosphate’’ as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxy gen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups. A phosphate group or chain may be modified, as further described herein.

[0167] “Phosphorylated” as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).

[0168] “Thiophosphate” refers to an instance where a non-bridging oxygen in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP is replaced with a sulfur. In some embodiments, nucleoside 5 ’-thiomonophosphate is referred to as NMP-S. In some embodiments, nucleoside-5’-l-thio(diphosphate) and nucleoside-5’-l-thio(triphosphate) are referred to as NDPaS and NTPaS, respectively. In some embodiments, nucleoside-5’-2-thio(diphosphate) and nucleoside-5’-2-thio(triphosphate) are referred to as NDP|3S and NTPpS, respectively.

[0169] “Dithiophosphate” refers to an instance where two non-bridging oxygens in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP are replaced with tw o sulfurs.

[0170] Unless otherwise specified, positions occupied by hydrogen in the foregoing groups can be further substituted with substituents exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxy alky 1, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamide, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted ar l, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyd, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalk l, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; and preferred heteroatoms are oxygen, nitrogen, and sulfur. It is understood that where open valences exist on these substituents they can be further substituted with alkyl, cycloalkyl, ary l, heteroaryl, and / or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur -bonded substituents, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct formation of a bond or by fomiation of bonds to a new' heteroatom, preferably oxygen, nitrogen, or sulfur. It is further understood that the above substitutions can be made provided that replacing the hydrogen with the substituent does not introduce unacceptable instability to the molecules of the present invention, and is otherwise chemically reasonable.Docket No. 5244.285WO2

[0171] “Optional" and “optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. One of ordinary skill in the art would understand that with respect to any molecule described as containing one or more optional substituents, only sterically practical and / or synthetically feasible compounds are meant to be included.

[0172] “Optionally substituted” refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl, the “alkyl” portion and the “aryl” portion of the molecule may or may not be substituted, and for the series “optionally substituted alkyl, cycloalkyl, ary l and heteroaryl,” the alkyl, cycloalkyl, ary l, and heteroaryl groups, independently of the others, may or may not be substituted.Engineered Adenosine Kinase Polypeptides

[0173] In one aspect, the present disclosure provides adenosine kinase (AdoK) enzymes engineered to have improved properties compared to the parent adenosine kinase enzyme. In some embodiments, the engineered adenosine kinase enzymes exhibit increased activity on nucleoside substrates, increased thermostability, and / or increased activity on modified nucleoside substrates. The engineered adenosine kinases are useful in the conversion of nucleosides to their corresponding nucleoside monophosphate products, particularly in enzyme cascade systems.

[0174] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 10-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 10-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10.24, 162, or 268, or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

[0175] In some embodiments, an engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. 24, 162. or 268, or to a reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 162, or 268. or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.Docket No. 5244.285WO2

[0176] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or to the reference sequence corresponding to SEQ ID NO: 10, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0177] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or to the reference sequence corresponding to SEQ ID NO: 24, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0178] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity’ to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0179] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 1, 4, 9, 11, 12, 13, 21, 23, 40, 43, 46, 53, 54. 55, 74, 80, 88, 90, 91, 94, 106, 107, 108, 109, 111, 112. 116, 119, 120, 121, 126, 128, 129, 131, 133, 134, 138.139, 140, 141, 147, 155, 157, 158, 160, 163. 164, 167, 168, 169, 170, 171, 172, 175, 182, 185, 187, 189, 190, 191, 192, 193, 194, 195, 196, 199, 200, 201, 203, 206, 207, 210, 211, 222, 223, 225, 231, 233, 234, 243, 250, 251, 252, 254, 267, 270, 271, 274, 277, 278, 280, 281, 289, 293, 302, 308, 310, 312, 314, 315, 320, and 321, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0180] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue IV, 4N, 9C / L / R. 11K / P, 12E / P / R / T, 13L / P / R / T, 21G, 23E, 40H / L / Q, 43A, 46L, 53D / H. 54Q, 55Q / S / T / W / Y, 74A / C / G / L / M / Q / T / V, 80V, 88R, 90G / Q, 91G, 94M / R, 106G / L / R, 107G / K / L / P, 108S, 109D, 111A / H / S, 112K, 116V, 119K, 120G. 121E, 126S / V,Docket No. 5244.285WO2128E / L / M, 1291, 131S, 133C / E / K / L / R / S, 134K / Y, 138Q, 139L / S, 140P, 141S, 147L / P, 147S, 155A / I, 157K / L / Q / R / S, 158S, 160A / R / T / W, 163S, 164I / R / S, 167E / R / S / V, 168E / G / Q / S / Y, 169L / W, 170A / C / L / S, 171Q / T, 172A / R / T / V, 175L / W, 182G / T, 185L / N, 187Q, 189A / H, 190E / L, 191P / S, 192R. 193M / T, 194F / K, 195C / G / N, 196G, 199A / L / M, 200V, 200A, 201S / T, 203E, 206E / H / T, 207A / L / M / S, 210E / V, 211I / L / M / S, 222A / E / G / P / V, 223G / L, 225K / M / S / T, 231L, 233E, 234E, 243A / E / L / N / R / T, 250I / L, 251P / T, 252V, 254K, 267G, 270D, 271L / T, 274V, 277K / R, 278A / G / P / Q / T, 280R / V / W, 281S, 289L, 293T, 302H, 308K, 310A, 312C, 314A / L / M, 315V, 320E, and 3211, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0181] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue M1V, H4N, G9C / L / R, G11K / P, M12E / P / R / T, S13L / P / R / T, A21G, D23E, R40H / L / Q, R43A. V46L, M53D / H, R54Q, R55Q / S / T / W / Y, R74A / C / G / L / M / Q / T / V, T80V, Y88R, E90G / Q, H91G, T94M / R, D106G / L / R, D107G / K / L / P, L108S, S109D, P111A / H / S, Q112K, T116V, M119K, D120G, Q121E, A126S / V, H128E / L / M, S129I, A131S, M133C / E / K / L / R / S, R134K / Y, N138Q, H139L / S, V140P, R141S, S147L / P. T147S, T155A / I, E157K / L / Q / R / S, G158S. I160A / R / T7W, A163S, E164I / R / S, A167E / R / S / V, A168E / G / Q / S / Y, M169L / W, G170A / C / L / S, I171Q / T, P172A / R / T / V, F175L / W, P182G / T, S185L / N, P187Q, L189A / H, R190E / L, E191P / S, F192R, I193M / T, E194F / K, Q195C / G / N, A196G, V199A / L / M, A200V, V200A, V201S / T. D203E, S206E / H / T, R207A / L / M / S, Q210E / V, A211I / L / M / S, S222A / E / G / P / V, R223G / L, Q225K / M / S / T, Q231L, S233E. A234E. K243A / E / L / N / R / T, A2501 / L, K251P / T, A252V, R254K, A267G. I270D, Y271L / T, Q274V, Y277K / R. D278A / G / P / Q / T, L280R / V / W. T281S, M289L, K293T, Q302H. E308K. N310A. R312C. K314A / L / M, Q315V, A320E, and L321I. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0182] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position a substitution at amino acid position 1, 4, 9. 11, 12, 46, 88, 131. 138, 139, 140, 158, 175. 189, 193, 199. 200, 201, 203. 223, 267, 270, 277, 278. 280, 281, 289, 293, 302. 315, 321; or substitution 13L / R / T, 23E. 40L / Q, 43 A, 53D / H, 54Q, 55Q / S / T / W / Y. 74A / C / G / L / M / Q, 90G / Q, 91G. 94R, 107 / K / L / P. 108S. 109D, 111 / H / S. 112K. 116V, 119K, 120G, 121 E, 126 V, 128E / L / M, 1291, 133C / E / K / R / S, 134K / Y, 147L / P, 157K / L / Q / R / S, 160A / R / T / W, 163S, 164I / R, 167E / R / S, 168E / G / Q / S / Y, 169W, 170A / C / L / , 171Q / T, 172A / R / T / V, 182T, 185L / N,, 190E / L, 191 P, 192R, 194F / K. 195C / N, 196G. 206E / H / T, 207S, 210E / V, 21 IS, 222E / P / V.225K / M / S / T, 233E, 234E, 243A / E / L / N / T, 250L, 251P, 254K, 271L / T, 274V. 308K. 312C, 314A / L / M, and 320E, or combinations thereof, wherein the amino acid positions are relative to theDocket No. 5244.285WO2reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0183] In some embodiments, the engineered adenosine kinase comprises at least a substitution or amino acid residue IV. 4N. 9C / L / R. 11K / P, 12E / P / R / T, 13L / R / T, 23E, 40 / L / Q, 43A, 46L. 53D / H, 54Q, 55Q / S / T7W / Y, 74A / C / G / L / M / Q, 88R, 90G / Q, 91G, 94R, 107 / K / L / P, 108S, 109D, 111H / S, 112K, 116V, 119K, 120G, 121E, 126V, 128E / L / M, 1291, 13 IS, 133C / E / K / R / S, 134K / Y. 138Q, 139L / S, MOP, 147L / P, 157K / L / Q / R / S, 158S, 160A / R / T / W, 163S, 164I / R, 167E / R / S, 168E / G / Q / S / Y.169W, 170A / C / L, 171Q / T, 172A / R / T / V, 175L / W, 182T, 185L / N, 189 A / H, 190E / L, 191P, 192R, 193M / T, 194F / K, 195C / N, 196G, 199A / L / M, 200V, 200A, 201S / T, 203E, 206E / H / T, 207S, 210E / V, 21 IS, 222E / P / V, 223G / L, 225K / M / S / T, 233E, 234E, 243A / E / L / N / T, 250L, 251P, 254K, 267G, 270D, 271L / T, 274V, 277K / R, 278A / G / P / Q / T, 280R / V / W, 281S, 289L, 293T, 302H, 308K, 312C, 314A / L / M, 315V, 320E, and 3211, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0184] In some embodiments, the engineered adenosine kinase comprises at least substitution or amino acid residue M1V, H4N, G9C / L / R, G11K / P, M12E / P / R / T, S13L / P / R / T, A21G, D23E, R40H / L / Q, R43A, V46L, M53D / H. R54Q, R55Q / S / T / W / Y, R74A / C / G / L / M / Q / T / V, T80V, Y88R, E90G / Q, H91G, T94M / R, D106G / L / R, D107G / K / L / P, L108S, S109D, P111A / H / S, Q112K, T116V, M119K, D120G, Q121E, A126S / V, H128E / L / M, S129I, A131S, M133C / E / K / L / R / S, R134K / Y, N138Q, H139L / S, V140P, R141S, S147L / P, T147S, T155A / I, E157K / L / Q / R / S, G158S, I160A / R / T / W, A163S, E164I / R / S, A167E / R / S / V, A168E / G / Q / S / Y, M169L / W, G170A / C / L / S, I171Q / T, P172A / R / T / V, F175L / W, P182G / T, S185L / N, P187Q, L189A / H, R190E / L, E191P / S, F192R, I193M / T, E194F / K, Q195C / G / N, A196G, V199A / L / M, A200V, V200A, V201S / T, D203E, S206E / H / T, R207A / L / M / S, Q210E / V, A211I / L / M / S, S222A / E / G / P / V, R223G / L, Q225K / M / S / T, Q231L, S233E, A234E, K243A / E / L / N / R / T. A250I / L, K251P / T, A252V, R254K, A267G, I270D, Y271L / T, Q274V. Y277K / R, D278A / G / P / Q / T, L280R / V / W, T281S, M289L. K293T. Q302H.E308K, N310A. R312C, K314A / L / M, Q315V, A320E, and L321I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0185] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 88, 131, 138, 139, 175, 189, 199, 200, 203, 270, 280, or 281; or substitution 53D / H, 54Q, 91G, 111H, 116V, 120G. 126V, 133C / E / K / R / S, 134K / Y, 157L / Q / R, 160A / R / T7W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R, 194K, 196G, 206T, 225M / S / T, or308K; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.Docket No. 5244.285WO2

[0186] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 53D / H, 54Q, 88R, 91G, 111H, 116V, 120G, 126V, 13 IS, 133C / E / K / R / S, 134K / Y, 138Q, 139L / S, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L, 175L / W, 189A / H, 192R, 194K, 196G, 199A, 200A / V, 203E, 206T, 225M / S / T, 270D, 280R / V / W, 28 IS, or 308K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0187] In some embodiments, where the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position at least a substitution at amino acid position 88, 131, 138, 139, 175, 189, 199, 200, 203, 270, 280, or 281; or substitution 53D / H, 54Q, 91G, 111H, 116V, 120G, 126V, 133C / E / K / R / S, 134K / Y, 157L / Q / R, 160A / R / T7W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R, 194K, 196G, 206T, 225M / S / T, or 308K; or combinations thereof, the amino acid sequence can further comprise at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128. 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152. 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 179, 180, 181, 182, 183, 185. 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 212, 215, 216, 217, 218. 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247. 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 274, 275, 276, 287, 288, 291, 292, 293. 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 315, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0188] In some embodiments, the amino acid sequence of the engineered adenosine kinase can further comprises at least a substitution or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31 F / H / L / R / W / Y. 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q. 43E / K / R / S / V, 461, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R. 54V, 55H, 56F / G / S / Y. 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T. 90L / R / S / T / V, 91Y, 92M, 93 A, 94M / N, 100G, 101F / W, 1021, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E. 109E / G / L / P / R / S / T, 111A / G / L. 112I / L, 113G / S. 116M,119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V. 172S. 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V. 185S / T. 187H / Q / S / V, 188T, 1901, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H. 207A / L / M / R / T / V.Docket No. 5244.285WO2211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V, 221A, 222A / G / M / Q, 225E / G, 229C / H / V, 231L / M / V, 232S, 233A / S / V, 234A / M / N / R / V / Y. 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W, 246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M, 258R / S / V, 265Y, 271W, 274R. 275S, 276D / E / G / S, 287A / S / V / Y, 288V, 291G, 292F / I / K / V, 293M.294L, 295 A, 296N, 299G / S, 303D / F / H / M / S / V / W. 305S / T. 307F / G, 308D / F / L / V, 310A / C.311E / F / G / L / M / Q / R / S / T, 312R. 314Q / S / V, 315S, 316A / D / E / I / K / S / Y. 317M / W / Y. or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0189] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 53, 54, 88, 91, 111, 116, 126. 131, 133, 134, 138, 139, 157, 160, 163, 168, 170, 175. 189, 192, 196, 199, 200, 203, 206, 225, 270, 280, 281. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0190] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 53D / H, 54Q, 88R, 91G, 111A / H, 116V, 126V, 13 IS, 133C / L / R / S, 134K / Y, 138Q, 139L / S, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L / S, 175L / W, 189A / H, 192R, 196G, 199 A, 200V, 203E, 206T, 225M / S / T, 270D, 280R / V / W, or 281S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0191] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) M53D / H, R54Q. Y88R, H91G, P111A / H, T116V, A126V, A131S, M133C / L / R / S, R134K / Y, N138Q, H139L / S, E157L / Q / R, I160A / R / T / W, A163S, A168E / G / Q / S / Y, G170A / C / L / S, F175L / W, L189A / H, F192R, A196G, V199A, A200V, D203E, S206T, Q225M / S / T, I270D, L280R / V / W, or T281S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0192] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at an amino acid position set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to aminoDocket No. 5244.285WO2acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0193] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least one substitution as set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0194] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.2, 8.2, 9.2, 10.2. 11.2. 12.2. and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0195] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2. 8.2. 9.2, 10.2. 11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0196] In some embodiments, the adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%. 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2. 12.2. and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0197] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812.

[0198] In some embodiments, die engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutionsDocket No. 5244.285WO2relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0199] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or to the reference sequence corresponding to SEQ ID NO: 24, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0200] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 53, 54, 88. 91, 1 11. 116, 120, 126, 131, 133. 134, 138, 139. 157, 160, 163, 168, 170, 175, 189, 192. 194, 196, 199. 200, 203, 206, 225, 270, 280, 281, or 308, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0201] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 53D / H. 54Q, 88R, 91G. 111A / H, 116V, 120G, 126V. 131S, 133C / E / K / L / R / S. 134K / Y, 138Q, 139L / S. 157L / Q / R, 160A / R / T / W. 163S. 168E / G / Q / S / Y, 170A / C / L / S. 175L / W, 189A / H. 192R. 194K, 196G, 199 A, 200A / V. 203E, 206T, 225M / S / T, 270D.280R / V / W. 281 S, or 308K. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0202] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 88. 131. 138, 139. 175, 189, 199. 200, 203, 270. 280, 281; or substitution 53D / H. 54Q.91G, 111H, 116V, 120G, 126V. 133C / E / K / R / S, 134K / Y. 157L / Q / R.160A / R / T / W, 163S, 168E / G / Q / S / Y. 170A / C / L, 192R, 194K, 196G. 206T. 225M / S / T, or 308K; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0203] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 53D / H, 54Q. 88R, 91G, 111H, 116V, 120G. 126V, 131S, 133C / E / K / R / S, 134K / Y. 138Q. 139L / S. 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y. 170A / C / L, 175L / W, 189A / H. 192R. 194K, 196G, 199A. 200A / V. 203E, 206T, 225M / S / T, 270D. 280R / V / W, 281 S, or 308K, or combinations thereof, wherein the amino acid positions are relative to the referenceDocket No. 5244.285WO2sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0204] In some embodiments, where the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position In some embodiments, where the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position at least a substitution at amino acid position 88, 131, 138, 139, 175, 189, 199, 200, 203, 270, 280. or 281; or substitution 53D / H. 54Q, 91G, 111H, 116V, 120G, 126V, 133C / E / K / R / S, 134K / Y. 157L / Q / R, 160A / R / T7W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R, 194K, 196G, 206T, 225M / S / T, or 308K; or combinations thereof, the amino acid sequence can further comprise at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155. 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171. 172, 177, 179, 180, 181, 182, 183, 185, 187. 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207. 208, 210, 211, 212, 215, 216, 217, 218, 221. 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239. 240, 241, 242, 243, 244, 245. 246, 247, 248. 249, 250, 251, 252, 253, 254, 255, 256. 258, 265, 271. 274, 275, 276, 287, 288, 291. 292, 293, 294. 295, 296, 299, 303, 305, 307, 308, 310. 311, 312, 314. 315, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24. or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0205] In some embodiments, the amino acid sequence of the engineered adenosine kinase can further comprise at least a substitution or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27 A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31 F / H / L / R / W / Y. 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q. 43E / K / R / S / V. 461, 49M. 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R. 54V, 55H, 56F / G / S / Y. 64M / T. 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T. 90L / R / S / T / V, 91Y, 92M, 93A. 94M / N.100G, 101F / W, 1021, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E. 109E / G / L / P / R / S / T, 111A / G / L, 112I / L, 113G / S. 116M,119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q. 126L / S, 128F / V / W. 129A / G / S, BOA, 133A / L, 134H.136F / H. 141S, 144Q / S, 146C / 1, 147A / N / S, 151L, 152A / C. 155A / 1 / N / T / V / W. 156Y. 157P. 160L, 162F. 163G, 164A / G / K / Q / S / W, 167F / T / V / W. 168R / V, 169A / C / D / I / L / M / S. 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 1901, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H. 207A / L / M / R / T / V. 211A / C / F / H / I / K / L / M / R / T / W. 212A, 215L, 216V, 217A / V. 218D / L / N / R / V, 221A. 222A / G / M / Q. 225E / G. 229C / H / V, 231L / M / V. 232S. 233A / S / V, 234A / M / N / R / V / Y. 236C / S / V. 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W. 245F / Q / W,Docket No. 5244.285WO2246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M, 258R / S / V, 265Y, 271W, 274R, 275S, 276D / E / G / S, 287A / S / V / Y. 288V, 291G, 292F / I / K / V, 293M, 294L, 295 A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T. 307F / G, 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R. 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0206] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 53, 53 / 111 / 133, 53 / 111 / 133 / 308, 53 / 111 / 138, 53 / 133, 54, 54 / 116 / 168, 54 / 134 / 168 / 200 / 225, 54 / 134 / 168 / 225 / 280, 54 / 134 / 225, 54 / 168 / 225, 54 / 200 / 280, 111, 111 / 120 / 133, 111 / 133, 111 / 138 / 160, 111 / 138 / 160 / 200, 111 / 160 / 200, 116, 116 / 134 / 168 / 194, 116 / 134 / 168 / 200, 116 / 168 / 225, 133, 133 / 138, 133 / 160 / 170 / 200, 134, 134 / 168, 134 / 168 / 225 / 280, 134 / 225. 138, 138 / 170, 160, 168 / 200 / 280, 168 / 225, 189 / 200 / 225, 200, 200 / 280, 225, 280, or 138 / 200, 168 / 189 / 200, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0207] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 53H, 53H / 111 A / 133E, 53H / 111A / 133E / 308K, 53H / 111A / 138Q, 53H / 133E, 54Q, 54Q / 116V / 168E, 54Q / 134K / 168E / 225T / 280V, 54Q / 134K / 168S / 200V / 225T, 54Q / 134K / 225T, 54Q / 168E / 225T, 54Q / 200V / 280V, 111A, 111 A / 120G / 133K, 111A / 133E, 111A / 138Q / 160R, 111A / 138Q / 160R / 200V, 111A / 160R / 200V, 116V, 116V / 134K / 168E / 194K, 116V / 134K / 168E / 200V. 116V / 168Q / 225T. 133E, 133E / 138Q, 133K, 133K / 160R / 170A / 200V, 133L, 133L / 138Q, 134K, 134K / 168E, 134K / 168E / 225T / 280V, 134K / 225T, 138Q. 138Q / 170A, 160R, 168S / 200V / 280V, 168S / 225T.189A / 200V / 225T, 200V, 200V / 280V, 225T, 280V, 138Q / 200V, or 168S / 189A / 200V. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0208] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set M53H, M53H / P111A / M133E, M53H / P111A / M133E / E308K, M53H / P111A / N138Q, M53H / M133E, R54Q, R54Q / T116V / A168E, R54Q / R134K / A168E / Q225T / L280V. R54Q / R134K / A168S / A200V / Q225T, R54Q / R134K / Q225T, R54Q / A168E / Q225T, R54Q / A200V / L280V, P111A, Pll 1A / D120G / M133K, P111A / M133E, P 111 A / N 138Q / 1160R, P 111 A / N 138Q / 1160R / A200 V, P 111 A / 1160R / A200 V, T 116 V, T116V / R134K / A168E / E194K, T116V / R134K / A168E / A200V. T116V / A168Q / Q225T, M133E, M133E / N138Q, M133K, M133K / I160R / G170A / A200V, M133L, M133L / N138Q, R134K,Docket No. 5244.285WO2R134K / A168E, R134K / A168E / Q225T / L280V, R134K / Q225T, N138Q, N138Q / G170A, I160R, A168S / A200V / L280V, A168S / Q225T, L189A / A200V / Q225T, A200V, A200V / L280V, Q225T, L280V, N138Q / A200V, or A168S / L189A / A200V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0209] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at an amino acid position set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0210] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least one substitution set forth in Tables 7.2, 8.2, 9.2, 10.2. 11.2, 12.2, and 13.2. wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0211] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.2, 8.2, 9.2, 10.2. 11.2. 12.2, and 13.2. wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0212] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2. 9.2, 10.2, 11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0213] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0214] In some embodiments, die engineered adenosine kinase comprises an amino acid sequence comprising residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally included 1, 2, 3,Docket No. 5244.285WO24, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenosine kinase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally includes 1, 2, 3, 4. or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenosine optionally includes 1, 2, 3. 4, or 5 substitutions.

[0215] In some embodiments, the engineered adenosine kinase has adenosine kinase activity and at least one improved property’ as compared to a reference adenosine kinase.

[0216] In some embodiments, the engineered adenosine kinase has increased activity on a nucleoside substrate as compared to the reference adenosine kinase. In some embodiments, the engineered adenosine kinase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2. 3, 4, 5, 10, 15. or 20 fold or more activity compared to the reference adenosine kinase. In some embodiments, the increased activity is on guanosine, adenosine, cytidine, uridine, or thymidine. Examples of increased activity on nucleosides is presented in the Examples.

[0217] In some embodiments, the engineered adenosine kinase has increased activity (e.g., product formation) on cytidine or guanosine. In some embodiments, the engineered adenosine kinase has at least 1.1. 1.2, 1.3, 1.4, 1.5, 2, 3, 4. 5. or 10 fold ormore activity on cytidine and / or guanosine compared to the reference adenosine kinase.

[0218] In some embodiments, the engineered adenosine kinase is capable of converting nucleoside substrate to NMP product at greater than 30%, 40%. 50%, 60%, 70%, 80%. 85%, 90%, 91%, 92%.93%, 94%, 95%, 96%. 97%, 98%, or 99%. In some embodiments, the engineered adenosine kinase is capable of converting substrate adenosine, guanosine, cytidine, uridine, or thymidine to product AMP, GMP. CMP, UMP, or TMP, respectively, at greater than 30%. 40%, 50%, 60%. 70%. 80%, 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%. 97%, 98%, or 99%.

[0219] In some embodiments, the engineered kinase has increased activity (e.g., product formation) on 2’-fluoro modified nucleosides. In some embodiments, the 2’-F modified nucleosides are 2’-F-2’-deoxyadenosine. 2 ’-F-2’ -deoxy cytidine, 2 ’-F-2’ -deoxy guanosine, and / or 2’-F-2’-deoxyuridine. In some embodiments, the engineered adenosine kinase has at least 1.1, 1.2, 1.3, 1.4. 1.5. 2, 3, 4, 5, or 10 fold or more activity on 2’-F modified nucleosides as compared to the reference adenosine kinase.

[0220] In some embodiments, the engineered kinase has increased activity (e.g., product formation) on 2’-O-CH3 modified nucleosides. In some embodiments, the 2’-O-CH3 modified nucleosides are 2’-O-CH3adenosine, 2’-O-CH3cytidine, 2’-O-CH3 guanosine, and / or 2’-O-CH3uridine. In some embodiments, the engineered adenosine kinase has at least 1.1, 1.2. 1.3, 1.4, 1.5, 2, 3, 4, 5, or 10 fold or more activity on 2’-O-CH3modified nucleosides as compared to the reference adenosine kinase.Docket No. 5244.285WO2

[0221] In some embodiments, the engineered adenosine kinase is capable of converting substrate 2’-fluoro-nucleoside to product 2’-fluoro-NMP at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenosine kinase is capable of converting substrate 2’ -fluoro-adenosine, 2 ’-fluoro-guanosine, 2’-fluoro-cytidine, 2’ -fluoro-uridine, or 2 ’-fluoro-thymidine to product 2 ’-fluoro- AMP, 2’-fluoro-GMP, 2’-fluoro-CMP, 2‘-fluoro-UMP, or 2‘-fluoro-TMP, respectively, at greater than 30%, 40%, 50%. 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99%.

[0222] In some embodiments, the engineered adenosine kinase is capable of converting 2’-O-methyl-nucleoside substrate to product 2’-O-methyl-NMP at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenosine kinase is capable of converting substrate 2‘-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2‘-O-methyl-thymidine to product 2’-O-methyl-AMP, 2'-O-methyl-GMP, 2’-O-methyl-CMP, 2’-O-methyl-UMP, or 2’-O-methyl-TMP, respectively, at greater than 30%. 40%, 50%, 60%, 70%. 80%, 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0223] Exemplary conditions for the conversion of a nucleoside to the corresponding NMP, including the conversion of 2 ’-fluoro-nucleoside to 2’-fluoro NMP and the conversion of 2’-O-methyl nucleoside to 2’-O-methyl NMP are provided in the Examples.

[0224] In some embodiments, the engineered adenosine kinase has increased thermostability as compared to the reference adenosine kinase. In some embodiments, increased thermal stability is at condition of 45 °C for 60 min.

[0225] In some embodiments, the engineered adenosine kinase has an improved property selected from i) increased activity on unmodified nucleoside, ii) increased thermostability, iii) increased activity on cytidine and / or guanosine, iv) increased activity on 2’-F modified nucleosides, and v) increased activity on 2’-O-CHs modified nucleosides, or any combinations of i), ii), iii), iv), and v). compared to a reference adenosine kinase.

[0226] In some embodiments, for comparing the improved property, the reference adenosine kinase has an amino acid sequence corresponding to residues 12-321 of SEQ ID NO: 10, 24, 162. or 268, or an amino acid sequence corresponding to SEQ ID NO: 10, 24, 162. or 268. In some embodiments, the reference adenosine kinase has an amino acid sequence corresponding to residues 12-321 of SEQ ID NO: 10, or an amino acid sequence corresponding to SEQ ID NO: 10.

[0227] In some embodiments, the engineered adenosine kinase is provided in the form of a fusion polypeptide. In some embodiments, the engineered adenosine kinase is fused to variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and / or purification, hi some embodiments, the fusion protein or polypeptide of theDocket No. 5244.285WO2engineered adenosine kinase comprises a glycinc-histidinc or histidine -tag (His-tag). In some embodiments, the fusion protein or polypeptide of the engineered adenosine kinase comprises one or more lysine residues, for example, 1-12 lysine units, such as for conjugation to a support medium. In some embodiments, the fusion protein of the engineered adenosine kinase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered adenosine kinase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N-) tenninus of engineered adenosine kinase polypeptide. In some embodiments, the fusion is to the carboxy (C-) tenninus of the adenosine kinase polypeptide.

[0228] In some embodiments, the present disclosure further provides functional fragments or biologically active fragments of engineered adenosine kinase polypeptides described herein. Thus, for each and every embodiment herein of an engineered adenosine kinase, a functional fragment or biologically active fragment of the engineered adenosine kinase is provided herewith. In some embodiments, a functional fragment or biologically active fragments of an engineered adenosine kinase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the adenosine kinase polypeptide from which it was derived (i.e.. the parent adenosine kinase). In some embodiments, functional fragments or biologically active fragments comprise at least about 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, or 99% of the parent sequence of the adenosine kinase. In some embodiments the functional fragment will be truncated by less than 5, less than 10. less than 15, less than 10, less than 25, less than 30, less than 35, less than 40. less than 45, and less than 50 amino acids.

[0229] In some embodiments, a functional fragment of an engineered adenosine kinase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered adenosine kinase, hi some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60. less than 65, or less than 70 amino acids.

[0230] In some embodiments, the functional fragments or biologically active fragments of the engineered adenosine kinase polypeptide described herein include at least a mutation or mutation set in the amino acid sequence of an engineered adenosine kinase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the engineered adenosine kinase displays the enhanced or unproved property associated with the mutation or mutation set in the parent adenosine kinase.

[0231] In some embodiments, the engineered adenosine kinase is purified, as described herein. In some embodiments, the purified preparation has the engineered adenosine kinase at least 60%, 70%, 80%, 85%, 90%, or 95% greater of the protein content of the preparation.Docket No. 5244.285WO2

[0232] In some embodiments, an engineered adenosine kinase described herein is provided immobilized on a substrate or support medium, such as a solid substrate, a porous substrate, a membrane, or particles. The polypeptide can be entrapped in matrixes or membranes. In some embodiments, matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacryla ide, and collagen. In some embodiments, the solid matrices, includes, among others, activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.

[0233] In some embodiments, the engineered adenosine kinase is immobilized on the surface of a support material. In some embodiments, the polypeptide is adsorbed on the support material. In some embodiments, the polypeptide is immobilized on the support material by covalent attachment.Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polymethacrylate, polyacrylate, polystyrene, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.

[0234] As further described below, solid supports useful for immobilizing the engineered polypeptides of the present disclosure include, but are not limited to, beads or resins comprising polymethacrylate or polyacry late with epoxide functional groups, polymethacrylate or polyacrylate with amino epoxide functional groups, styrene / DVB copolymer or polymethacrylate or polyacry late with octadecyl functional groups. In some embodiments, exemplary solid supports useful for immobilizing the engineered polypeptides of the present invention include, but are not limited to, EnginZyme (including, EziG-1, EziG-1, and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP. EC-HFA / S, EXA252, EXE119 and EXE120).Polynucleotides Encoding Engineered Polypeptides, Expression Vectors and Host Cells

[0235] In another aspect, the present disclosure provides recombinant polynucleotides encoding the engineered adenosine kinases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the engineered adenosine kinase. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding the engineered adenosine kinase polypeptide(s) is introduced into appropriate host cells to express the corresponding adenosine kinase polypeptide(s).

[0236] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremelyDocket No. 5244.285WO2large number of nucleic acids to be made, all of which encode an engineered adenosine kinase of the present disclosure. Thus, the present disclosure provides methods and compositions for the production of each and every possible variation of polynucleotides that could be made that encode the engineered adenosine kinase polypeptides described herein by selecting combinations based on the possible codon choices, and all such polynucleotide variants are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., Tables 7.2, 8.2, 9.2, 10.2. 11.2, 12.2, and 13.2) and in the Sequence Listing.

[0237] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungal cells arc used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, codon optimized polynucleotides encoding an engineered adenosine kinase polypeptide described herein contain preferred codons at about 40%. 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

[0238] Accordingly, in some embodiments, a recombinant polynucleotide of the present disclosure comprises a polynucleotide sequence encoding an engineered adenosine kinase polypeptide described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide has preferred codons for expression (e.g., codon optimized). In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized for expression in eukaryotic or prokaryotic cells, as further discussed below.

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ IDNOs: 10-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 10-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 162, or 268, or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

[0240] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. 24, 162, or 268, or to a reference sequence corresponding to SEQDocket No. 5244.285WO2ID NO: 10, 24, 162, or 268, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 162, or 268, or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

[0241] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or to the reference sequence corresponding to SEQ ID NO: 10, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or to the reference sequence corresponding to SEQ ID NO: 24, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0243] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ IDNOs: 12-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 1, 4, 9, 11, 12, 13, 21, 23, 40, 43, 46. 53, 54, 55, 74, 80, 88, 90, 91, 94, 106, 107, 108, 109, 111, 112, 116. 119, 120, 121, 126, 128, 129, 131, 133, 134, 138, 139. 140, 141, 147, 155, 157, 158, 160, 163, 164, 167. 168, 169, 170, 171, 172, 175, 182, 185, 187, 189, 190.191, 192, 193, 194, 195, 196, 199, 200, 201. 203, 206, 207, 210, 211, 222, 223, 225, 231, 233, 234,Docket No. 5244.285WO2243, 250, 251, 252, 254, 267, 270, 271, 274, 277, 278, 280, 281, 289, 293, 302, 308, 310, 312, 314, 315, 320, and 321, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0245] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 88, 131, 138, 139, 175, 189, 199, 200, 203. 270, 280, or 281; or substitution 53D / H, 54Q. 91G, 111H, 116V, 120G, 126V, 133C / E / K / R / S. 134K / Y, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R, 194K, 196G, 206T, 225M / S / T, or 308K; or combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence further comprising at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26. 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55. 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101. 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116. 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 274, 275, 276, 287. 288, 291, 292, 293, 294, 295, 296, 299, 303, 305, 307. 308, 310, 311, 312, 314, 315, 316, 317, or 320, or combinations thereof, wherein tire amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position 53, 54, 88, 91, 111, 116, 126, 131, 133, 134, 138, 139, 157, 160, 163, 168, 170, 175, 189, 192, 196, 199, 200, 203, 206, 225, 270, 280, 281, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2,Docket No. 5244.285WO29.2, 10.2, 11.2, 12.2, and 13.2, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%.75%, 80%, 81%, 82%. 83%. 84%, 85%, 86%. 87%. 88%, 89%, 90%. 91%. 92%, 93%, 94%. 95%.96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%.75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%. 92%, 93%, 94%. 95%.96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812.Docket No. 5244.285WO2wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or to the reference sequence corresponding to SEQ ID NO: 24, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0255] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 53. 54, 88, 91, 111, 116, 120, 126, 131, 133, 134, 138, 139, 157.160, 163, 168, 170, 175, 189, 192, 194, 196. 199, 200, 203, 206, 225, 270, 280, 281, or 308, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0256] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 88. 131, 138, 139. 175, 189, 199, 200, 203. 270, 280, 281; or substitution 53D / H, 54Q. 91G, 111H, 116V. 120G, 126V, 133C / E / K / R / S. 134K / Y, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R, 194K, 196G, 206T, 225M / S / T, or 308K; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence that can further comprise at least a substitution or substitution set at amino acid position 13, 14, 20, 21. 23, 24, 26, 27, 28, 29, 30. 31, 33, 34, 35. 38, 40, 41, 42, 43. 46, 49, 50, 51. 52, 53, 54, 55. 56, 64, 67, 73, 74. 77, 79, 80, 83, 86. 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107. 108, 109, 111. 112, 113, 116, 119. 120, 121, 126, 128, 129, 130, 133, 134. 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160.162, 163, 164, 167, 168, 169, 170, 171, 172. 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191.192, 194, 195, 196, 197, 202, 206, 207, 208. 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229.231, 232, 233, 234, 236, 237, 238, 239, 240. 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,Docket No. 5244.285WO2252, 253, 254, 255, 256, 258, 265, 271, 274, 275, 276, 287, 288, 291, 292, 293, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 315, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0258] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2.10.2.11.2, 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%. 81%. 82%, 83%, 84%. 85%. 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%. 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%. 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%.93%, 94%. 95%. 96%, 97%, 98%. 99%. or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, wherein the recombinant polynucleotide encodes an engineered adenosine kinase.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or comprises an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811.Docket No. 5244.285WO2

[0263] In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to a reference polynucleotide encoding an engineered adenosine kinase polypeptide described herein, e.g., a recombinant polynucleotide provided in Tables 7.2, 8.2, 9.2. 10.2, 11.2, 12.2. and 13.2, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reference polynucleotide corresponding to nucleotide residues 34-963 of SEQ ID NO: 23 or to a reference polynucleotide sequence corresponding to SEQ ID NO: 23 or a reverse complement thereof. In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reference polynucleotide corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811. or a reverse complement thereof.

[0264] In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to a reverse complement of a reference polynucleotide encoding an engineered adenosine kinase polypeptide described herein, wherein the recombinant polynucleotide hybridizing under stringent conditions encodes an adenosine kinase polypeptide comprising an amino acid sequence having one or more amino acid differences as compared to SEQ ID NO: 10, 24. 162, or 268. at residue positions selected from any positions as set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2. In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions to a reverse complement of a reference polynucleotide encoding an engineered adenosine kinase polypeptide described herein encodes an adenosine kinase polypeptide having one or more amino acid differences present in an engineered adenosine kinase having an amino acid sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or an amino acid sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid differences are relative to SEQ ID NO: 10, 24, 162, or 268.

[0265] In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of SEQ ID NO: 23 or to a reference polynucleotide sequence corresponding to SEQ ID NO: 23 or a reverse complement thereof. In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811,Docket No. 5244.285WO2or an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or a reverse complement thereof.

[0266] In some additional embodiments, the polynucleotide hybridizing under highly stringent conditions comprises a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% or more sequence identity to a reverse complement of a polynucleotide reference sequence corresponding to nucleotide residues 34-963 of SEQ ID NO: 23, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 23 encodes an engineered adenosine kinase polypeptide. In some additional embodiments, the polynucleotide hybridizing under highly stringent conditions comprises a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reverse complement of a polynucleotide reference sequence corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811 encodes an engineered adenosine kinase polypeptide.

[0267] In some embodiments, a recombinant polynucleotide encoding any of the adenosine kinase provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the recombinant polynucleotide encoding the polypeptides are provided as expression vectors where one or more control sequences is operably linked to the recombinant polynucleotide to regulate the expression of the polynucleotide and / or encoded polypeptide. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are known in the art.

[0268] In some embodiments, the control sequences include, among others, promoter sequences, Kozak sequence, leader sequences, polyadenylation sequences, pro-peptide sequences, signal peptide sequences, regulatory elements, and transcription terminators. As know n in the art, suitable promoters can be selected based on the host cells used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrasc gene (sacB), Bacillus lichcniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus lichenifonnis penicillinase gene (penP). Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g.. Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA. 1978, 75:3727-3731), as well as the lac promoter (See e.g., DeBoer et al.. Proc. Natl Acad. Sci. USA. 1983. 80: 21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase. Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha- amylase. Aspergillus niger or Aspergillus awamori glucoamylase (glaA),Docket No. 5244.285WO2Rhizomucor michci lipase, Aspergillus oryzac alkaline protease, Aspergillus or zac triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI). Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3 -phosphoglycerate kinase. In some embodiments, promoters effective in Pichia cells are used. Other useful promoters for yeast host cells are known in the art (See e.g.. Romanos et al., Yeast. 1992. 8:423-488). In some embodiments, for insect host cells, suitable promoters include, among others, baculovirus promoters (e.g., PIO and polyhedron promoters), OpIE2 promoter, and Nephotettix cincticeps actin promoters. In some embodiments, promoters effective in Pichia cells are used. In some embodiments, for insect host cells, suitable promoters include, among others, baculovirus promoters (e.g., PIO and polyhedron promoters), OpIE2 promoter, and Nephotettix cincticeps actin promoters. In some embodiments, for mammalian host cells, suitable promoters include, among others, promoters of cytomegalovirus (CMV), chicken P-actin promoter fused with the CMV enhancer, simian virus 40 (SV40), human phosphoglycerate kinase, beta actin, elongation factor-la or glyceraldehyde-3 -phosphate dehydrogenase, or Gallus P-actin.

[0269] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e.. a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the leucine decarboxylase polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. For bacterial expression, the transcription terminators can be a Rho-dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3 -phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to those from cytomegalovirus (CMV). Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.Docket No. 5244.285WO2

[0270] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a nontranslated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the adenosine kinase polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3 -phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include but are not limited to the 5' -UTR element present in orthopoxvirus mRNA.

[0271] In some embodiments, the control sequence comprises a 3‘ untranslated nucleic acid region and polyadeny lation tail nucleic acid sequence, sequences operably linked to the 3' terminus of the protein coding nucleic acid sequence which mediate binding to proteins involved in mRNA trafficking and translation and mRNA half-life. Any polyadenylation sequence and 3’ UTR which is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (See e.g., Guo and Sherman. Mol. Cell. Biol.. 1995. 15:5983-5990). Useful polyadenylation and 3’ UTR sequences for mammalian host cells include, but are not limited to, the 3 -UTRs of a- and (3-globin mRNAs. mouse kappa light chain, chicken ovalbumin, and SV40 that harbor several sequence elements that increase the stability and translation of mRNA.

[0272] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide (s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NCIB 11837 inaltogenic amylase, Bacillus stearothermophilus alphaDocket No. 5244.285WO2amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase. Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include but are not limited to, those from the genes for immunoglobulin gamma (IgG) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide.

[0273] In some embodiments, the control sequence is a regulatory sequence that facilitates the regulation of the expression of the recombinant polynucleotide and / or encoded polypeptide.Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter. Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter. In mammalian cells, suitable regulatory systems include, among others, zinc-inducible sheep metallothionine (MT) promoter, dexamethasone (Dex) -inducible promoter, mouse mammary tumor virus (MMTV) promoter; ecdysone insect promoter, tetracyclineinducible promoter system, RU486-inducible promoter system, and the rapamycin-inducible promoter system.

[0274] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding an adenosine kinase polypeptide, where the recombinant polynucleotide is operably or operatively linked to a control sequence, such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the adenosine kinase polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0275] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent ofDocket No. 5244.285WO2chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0276] In some embodiment, recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0277] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Selectable marker for mammalian cells include, among others, chloramphenicol acetyl transferase (CAT), nourseothricin N-acetyl transferase, blasticidin-S deaminase, blastcidin S acetyltransferase, Sh ble (Zeocin® resistance), aminoglycoside 3 '-phosphotransferase (neomycin resistance), hph (hygromycin resistance), thymidine kinase, and puromycin N-acetyl-transferase.

[0278] In another aspect, the present disclosure provides a host cell comprising at least one recombinant polynucleotide encoding an adenosine kinase polypeptide of the present disclosure, the recombinant polynucleotide(s) being operatively linked to one or more control sequences for expression of the adenosine kinase polypeptide. In some embodiments, the host cells suitable for use in expressing the polypeptides encoded by the expression vectors is a prokaryotic cell or eukaryotic cell known in the art. In some embodiments, the host cell is a bacterial cell, including, among others, E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cell. Exemplary bacterial host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21). In some embodiments, the host cell is a fungal cell, such as filamentous fungal cell or yeast cell, In some embodiments, suitable fungal host cells include, among others. Pichia, Saccharomyces, Yarrowia,Docket No. 5244.285WO2Kluyveromyces, Aspergillus, Trichoderma, Neurospora, Mucor, Penicillium T. Trichoderma, or Myceliophthora fungal cell. Exemplary fungal host cell includes, among others, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus Trichoderma reesei. Neurospora crassa, Mucor circinelloides, Penicillium chrysogenum T. reesei, Trichoderma harzianum, Saccharomyces cerevisiae, or Myceliophthora thermophile. In some embodiments, the host cell is an insect cell. In some embodiments, a suitable insect host cell is a lepidopteran or dipteran insect cell. Exemplary insect host cell includes, among others, Sf9 cell, Sf21 cell, Schneider 2 cell, and BTI-TN-5B1-4 (High Five) cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell or rodent cell. Exemplary mammalian cells include, among others, Expi293. HeLa. U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, Hek 293, 293F, 293E, 293T, COS, Vero, NSO. Sp2 / 0 cell, DUKX-X11, MCF-7. Y79, SO-Rb50, Hep G2, J558L, and CHO cell.

[0279] In another aspect, the present disclosure provides a method of producing the adenosine kinase polypeptides, where the method comprises culturing a host cell comprising an expression vector capable of expressing or producing the adenosine kinase polypeptide under suitable culture conditions such that the adenosine kinase polypeptide is expressed or produced. In some embodiments, the method comprises a step of isolating the adenosine kinase from the culture medium and / or host cell, as described herein. In some further embodiments, the method further comprises purifying the expressed adenosine kinase polypeptide.

[0280] In some embodiments, the adenosine kinase polypeptide expressed in a host cell is recovered from the cells and / or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography, such as described herein.

[0281] Chromatographic techniques for isolation / purification of the adenosine kinase polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying the adenosine kinase depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the adenosine kinase. For affinity chromatography purification, an antibody that specifically binds adenosine kinase polypeptide may be used. In some embodiments, an affinity tag. e g., His-tag, can be introduced into the adenosine kinase polypeptide for purposes of isolation / purification.

[0282] Appropriate culture media and growth conditions for the above-described host cells are well known in the art. Polynucleotides for expression of the adenosine kinases may be introduced intoDocket No. 5244.285WO2cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

[0283] In some embodiments, the polynucleotides encoding the adenosine kinase polypeptide can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, polynucleotide fragments can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al., Tetra. Lett., 1981, 22: 1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as it is typically practiced in automated synthetic methods.

[0284] In some embodiments, a method for preparing the adenosine kinase can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of an adenosine kinase, such as described in the Tables of the Examples, and (b) expressing the engineered adenosine kinase encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and / or substitutions, In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions. The expressed polypeptide can be assessed for the desired property, e.g., adenosine kinase activity on one or more nucleosides.Compositions

[0285] In a further aspect, the present disclosure provides compositions of the engineered adenosine kinases disclosed herein. In some embodiments, the engineered adenosine kinase polypeptide in the composition is isolated or purified. In some embodiments, the adenosine kinase is combined with other components and compounds to provide compositions and formulations comprising the engineered adenosine kinase polypeptide as appropriate for different applications and uses.

[0286] In some embodiments, the composition comprises at least one adenosine kinase described herein. For example, a composition comprises at least one engineered adenosine kinase exemplifiedDocket No. 5244.285WO2in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2, and the Sequence Listing. In some embodiments, the composition comprising an engineered adenosine kinase is an aqueous solution. In some embodiments, the composition comprising an engineered adenosine kinase is a lyophilizate.

[0287] In some embodiments, the composition further comprises at least a buffer. Exemplary buffers are provided in the Examples, e.g., MOPS, triethanolamine, etc. In some embodiments, the buffer is present at a concentration of about 1 mM-500 mM, 5 mM-450 mM, 10 mM-400 mM, 20 mM-350 mM, 30 mM-300 mM, 40 mM-250 mM, or 50 mM-200 mM. In some embodiments, the buffer is present at a concentration of about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM.

[0288] In some embodiments, the compositions comprises further comprises an additional enzyme, including, among others, an adenylate kinase, acetate kinase, or 3’-O-kinase, or combinations thereof.

[0289] In some embodiments, the composition further comprises a nucleoside substrate. In some embodiments, the nucleoside substrate is an unmodified nucleoside, e.g., adenosine, guanosine, cytidine, thymidine, or uridine. In some embodiments, the concentration of nucleoside in the composition is greater than that found in cells, e.g., bacterial cells or mammalian cells. In some embodiments, the nucleoside substrate concentration is about 0.1 mM to 200 mM, 0.5 mM to 180 mM. 1 mM to 150 mM. 2 mM to 120 mM. 5 mM to 100 mM. 10 mM to 80 mM. or 20 mM to 60 mM. as further described herein. In some embodiments, the nucleoside substrate concentration is about 100 mM-200 mM. In some embodiments, the nucleoside substrate concentration is about 0.1 mM, 0.5 mM, 1 mM. 2 mM, 5 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM. 120 mM, 150 mM, 180 mM, or 200 mM.

[0290] In some embodiments, the concentration of adenosine in the composition is at least 2 mM. 3 mM, 4 mM. 5 mM, 7 mM. or 10 mM, or greater. In some embodiments, the concentration of adenosine in the composition is about 0.1 mM to 200 mM. 0.5 mM to 180 mM. 1 mM to 150 mM. 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the adenosine substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of adenosine in the composition is at least 0.1 mM, 0.5 mM. 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM. 10 mM. 20 mM, 60 mM, 80 mM, 100 mM. 120 mM, 150 mM. 180 mM, or 200 mM, or greater.

[0291] In some embodiments, the concentration of cytosine in the composition is at least 0.5 mM. 1 mM, 2 mM. 3 mM, 4 mM. 5 mM, 7 mM, or 10 mM. or greater. In some embodiments, the concentration of cytosine in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM. 1 mM to 150 mM. 2 mM to 120 mM. 5 mM to 100 mM. 10 mM to 80 mM. or 20 mM to 60 mM. In some embodiments, the cytosine substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of cytosine in the composition is at least 0.1 mM, 0.5 mM. 1 mM, 2 mM. 3 mM, 4Docket No. 5244.285WO2mM, 5 mM, 7 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.

[0292] In some embodiments, the concentration of guanosine in the composition is greater than 1 mM, 2 mM, 3 mM, 4 mM, 5 mM. 7 mM, or 10 mM, or greater. In some embodiments, the concentration of guanosine in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the guanosine substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of guanosine in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM. 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.

[0293] In some embodiments, the concentration of uridine in the composition is at least 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM. or 10 mM or greater. In some embodiments, the concentration of uridine in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the uridine substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of uridine in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.

[0294] In some embodiments, the concentration of thymidine in the composition is at least 0.1 mM, 0.5 mM. 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, or 10 mM. or greater. In some embodiments, the concentration of thymidine in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the thymidine substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of thymidine in the composition is at least 0.1 mM, 0.5 mM. 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM. 10 mM. 20 mM. 60 mM, 80 mM, 100 mM, 120 mM, 150 mM.180 mM, or 200 mM, or greater.

[0295] In some embodiments, the nucleoside substrate is a modified nucleoside, such as a modified sugar moiety and / or nucleobase. In some embodiments, the nucleoside is modified on the 2’-or 3’-position of the sugar moiety (e.g.. 2’-halo. 2’-O-CH3). In some embodiments, the modified nucleoside comprises a 2’-fluoro modified nucleoside. In some embodiments, the 2’-fluoro modified nucleoside is 2’-fluoro-deoxyadenosine. 2’-fluoro-deoxycytidine. 2’-fluoro-deoxyguanosine, 2’-fluoro-deoxyuridine, and / or 2 ’-fluoro-deoxy thymidine. In some embodiments, the modified nucleoside comprises a 2’-O-CH3modified nucleoside. In some embodiments, the 2’-O-CH3modified nucleoside is 2’-O-CH3adenosine. 2’-O-CH3cytidine, 2’-O-CH3guanosine, and / or 2’-O-CH3uridine. In some embodiments, the modified nucleoside concentration is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM,Docket No. 5244.285WO2or 20 mM to 60 mM, as further described herein. In some embodiments, the modified nucleoside concentration is about 100 mM-200 mM. In some embodiments, the modified nucleoside concentration is about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM.

[0296] In some embodiments, the modified nucleoside is modified at the 3 ’-position of the sugar moiety. In some embodiments, the 3 ’-position of the sugar moiety is modified with a blocking group, preferably a reversible blocking group. In some embodiments, the blocking group is a formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONH2), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl. O-(2 -nitrobenzy l), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl, phosphate, diphosphate, or triphosphate. Reversible 3’-blockcd nucleoside / nucleotides are described in Chen et al., Genomics, Proteomics & Bioinformatics, 2013, 11(1):34-40, Metzker et al., Nucleic Acids Res., 1994, 22 (20): 4259-4267; Sabat et al., Front Chem.2023; 11: 1161462; and patent publications US5763594, US9650406, US20200216891;W02004 / 018497; and WO 2014 / 139596; all of which are incorporated by reference) In some embodiments, the modified sugar moiety is a 3’-O-phosphate.

[0297] In some embodiments, the modified nucleoside comprises modified 2’ - and 3 ’-positions of the sugar moiety, such as described herein. Exemplary modified NDP substrates with modified 2’-and 3 ’-positions include, among others, 2’-fluoro or 2‘-O-methyl, and 3 ’-O-allyl, 3'-O-(2-nitrobenzyl), 3 ’-O-azidomethyl (O-CH2N3), 3 ’ -O-tert-buty Idithiomethy 1, or 3 ‘-phosphate.

[0298] In some embodiments, the modified sugar moiety of the nucleoside in the compositions comprises a “locked” nucleoside. In some embodiments, the locked NDP is a locked adenosine, locked guanosine, locked cytidine, locked thymidine, or locked uridine.

[0299] In some embodiments, the modified nucleoside comprises a modified nucleobase. In some embodiments, the modified nucleobases is 5-bromo-uraciL 5 -iodo-uracil. 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole. Other nucleobases are described herein.

[0300] In some embodiments, the composition further comprises a phosphate donor for the adenosine kinase, for example, NTP. In some embodiments, the phosphate donor NTP (also referred to as phosphate donor NTP) has the same nucleoside structure as the nucleoside substrate. By way of example and not limitation, if the nucleoside substrate is modified at the 2’ -position of the sugar moiety, the donor NTP has the same modified 2’ -position of the sugar moiety. Additionally, the phosphate donor NTP has the same nucleobase as the NMP substrate.

[0301] In some embodiments, the composition comprises an engineered adenosine kinase and one or more components of a NTP regenerating system. In some embodiments, the components of the NTP regeneration system includes, among others, pyruvate kinase, creatine kinase, adenosine kinase,Docket No. 5244.285WO2and / or polyphosphate kinase. In some embodiments, the components of the NTP regeneration system includes a substrate (i.e., phosphate donor) for the enzyme in the NTP regenerating system, for example, phosphoenolpyruvate, creatine phosphate, and polyphosphate, respectively.

[0302] In some embodiments, where the NTP regenerating system uses an acetate kinase, the NTP regenerating system further comprises a pyruvate oxidase, pyruvate, and phosphate, for converting the acetate to acetyl phosphate.

[0303] In some embodiments, the composition comprises an immobilized engineered adenosine kinase, as described herein. In some embodiments, the immobilized adenosine kinase is immobilized with other enzymes, e.g., an adenylate kinase, acetate kinase, and / or 3’-O-kinase.Uses and Methods

[0304] In another aspect, the present disclosure provides uses of the engineered adenosine kinase enzymes, either alone or in combination with other enzymes. In some embodiments, the engineered adenosine kinase is used in the production of nucleoside monophosphate (NMP) from a nucleoside. In some embodiments, a method of converting a nucleoside to nucleoside monophosphate comprises contacting a nucleoside with an engineered adenosine kinase described herein in the presence of a suitable phosphate donor NTP under suitable reaction conditions to convert the nucleoside to the corresponding product nucleoside monophosphate.

[0305] In some embodiments, the nucleoside substrate is a naturally occurring or unmodified nucleoside. In some embodiments, the nucleoside substrate is a nucleoside found on mRNA or DNA.

[0306] In some embodiments, the nucleoside has at the 2’ -position of the sugar moiety a H or OH.

[0307] In some embodiments, the nucleoside comprises a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine, 6,8-diaminopurine, 5 -methylcytosine (m5C), 2-thiouridine, pseudouridine, dihydrouridine, inosine, and 7-methylguanosine (m7G). In some embodiments, the nucleoside has at the 2’ -position of the sugar moiety a H or OH.

[0308] In some embodiments, the unmodified nucleoside is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2 '-position of the sugar moiety an OH, thereby resulting in corresponding product rAMP, rGMP, rUMP, rCMP, or rTMP, respectively.

[0309] In some embodiments, the unmodified nucleoside is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2 '-position of the sugar moiety an H, thereby by resulting in corresponding product dAMP, dGMP, dUMP, dCMP, or dTMP, respectively.Docket No. 5244.285WO2

[0310] In some embodiments of the method, the nucleoside substrate is a modified nucleoside, thereby resulting in production of the corresponding modified nucleoside monophosphate. In some embodiments, the modified nucleoside is modified in the sugar moiety or the nucleobase, or combination of modified sugar moiety and modified nucleobase.

[0311] In some embodiments of the method, the modified nucleoside comprises a modified sugar moiety. In some embodiments, the modified sugar moiety is modified at the 2 ’-position or 3 ’-position of the sugar moiety.

[0312] In some embodiments, the modified sugar moiety is a modified at the 2’-position of the sugar moiety. In some embodiments, the 2’-position of the sugar moiety is halo. 2’-O-R’, or 2’-O-COR’. where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a Ci-Cialkyl. In some embodiments, the modified 2 ’-position is a 2’-O-R’, wherein in R’ is alkyloxy alkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some embodiments, the 2’-position of the sugar moiety of the nucleoside substrate is -O-R’. wherein R’ is -CH3or -CH2CH3or -CH2CH2OCH3. In some embodiments, the modified 2’-position is 2’-O-(2 -methoxyethyl). 2’-O-allyl, 2’-O-propargyl, 2’-O-ethylamine, 2’-O-cyanoethyl. or 2’-O-acetalester.

[0313] In some embodiments of the method, the modified 2’-position is 2’-halo. In some embodiments, the modified 2’-position is F (i.e., 2’-F) or Br (i.e., 2’-Br).

[0314] In some embodiments, the modified nucleoside substrate is a locked nucleoside. In some embodiments, the locked nucleoside is a locked adenosine, locked guanosine, locked cytidine, locked thymidine, or locked uridine. In some embodiments, the ribose moiety of the locked nucleoside is in the C3’-endo (beta-D-LNA) or C2’-endo (alpha-L-LNA) conformation. In some embodiments, the locked nucleoside has a methylene bridge. In some embodiments, the locked nucleoside has an ethylene bridge. Various locked nucleotides are described in, for example, WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202. WO 2008 / 154401. WO 2009 / 067647, WO 2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 2002, 12. 73-76, Seth et al. J. Org. Chem. 2010. Vol 75(5) pp. 1569-81, and Mitsuoka et al.. Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016. 59. 9645-9667.

[0315] In some embodiments of the method, the modified sugar moiety on the nucleoside substrate is modified at the 3 ’-position of the sugar moiety. In some embodiments, the 3 ’-position of the sugar moiety is modified with a blocking group, preferably a reversible blocking group. In some embodiments, the blocking group is a formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONH2), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl, O-(2-nitrobenzyl), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl. phosphate, diphosphate, orDocket No. 5244.285WO2triphosphate. Reversible 3’-blockcd nuclcosidc / nuclcotidcs arc described in Chen et al., Genomics, Proteomics & Bioinformatics, 2013, 11(1):34-40, Metzker et al., Nucleic Acids Res., 1994, 22 (20): 4259-4267; Sabat et al., Front Chem. 2023; 11: 1161462; and patent publications US5763594, US9650406, US20200216891; W02004 / 018497; and WO 2014 / 139596; all of which are incorporated by reference) In some embodiments, the modified sugar moiety is a 3’-O-phosphate.

[0316] In some embodiments of the method, the modified nucleoside comprises a modified nucleobase. In some embodiments, the modified nucleobase is 5 -bromo-uracil, 5 -iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.

[0317] In some embodiments, the nucleobase of the nucleoside substrate is. among others. 5-methylcytosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil. 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines, 5-alkyluridines, 5-halouridines, 6-azapyrimidines, 6-alkylpyrimidines, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, quesosine, 2-thiouridine, 4-thiouridine, 4-acetyltidine, 5- / carboxy hydroxy methyl (uridine. 5-carboxyniethy laminomethy 1-2-thiouridine, 5-carboxymethylaminomethyluridine. -D-galactosylqueosine, 1 -methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7-methylguanosine. 5-methoxyaminomethy 1-2- thiouridine, 5 -methy laminomethy luridine, 5-methylcarbon ylmethyluridine, 5- methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, -D- mannosylqueosine, uridinc-5-ox acctic acid, 2 -thiocytidine, Nl-methyl-adenine, N6-methyl- adenine, 8'-azido-adenine, N, N-dimethy 1-adenosine, aminoallyladenosine, 5'-methyl-urdine, pseudouridine, Nl- methyl -pseudouridine, 5 '-hydroxy -methyl -uridine, 2'-thio-uridine, 4'-thio- uridine, hypoxanthine, xanthine, 5'-methyl-cytidine, 5 '-hydroxy -methylcytidine, 6'-thio- guanine, or N7-methyl-guanine.

[0318] In some embodiments of the method, the phosphate donor is a nucleotide triphosphate (NTP), also referred to herein as phosphate donor NTP. In some embodiments, the phosphate donor NTP is rATP or dATP. In some embodiments, the phosphate donor NTP has the same nucleoside structure as the nucleoside substrate. By way of example and not limitation, if the nucleoside substrate is unmodified, the phosphate donor NTP has an unmodified nucleoside, c.g., unmodified in the sugar moiety and nucleobase. If the nucleoside substrate is modified at the 2‘ -position of the sugar moiety, the phosphate donor NTP has the same modification in the sugar moiety.

[0319] In some embodiments of the method, the phosphate donor is a nucleotide -5 ’-gammathiotriphosphate (NTPyS), thereby resulting in product NMPocS. In some embodiments, the phosphate donor NTP is rATPyS or dATPyS. In some embodiments, the phosphate donor NTPyS has the same nucleoside structure as the nucleoside substrate.Docket No. 5244.285WO2

[0320] In some embodiments, the phosphate donor NTP is present at a concentration of about 0.001 mM to 1 mM, 0.005 mM to 0.5 mM, 0.01 mM to 0.4 mM, 0.05 mM to 0.3 mM, or 0.1 mM to 0.2 mM. In some embodiments, the phosphate donor NTP is present at a concentration of about 0.001 mM, 0.005 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, or 1 mM.

[0321] In some embodiments, the method further comprises a NTP regenerating or recycling system for regenerating NTP from the NDP produced by the adenosine kinase reaction. In some embodiments of the method, the NTP regenerating system comprises a creatine kinase, a pyruvate kinase, a polyphosphate kinase, and / or an acetate-kinase. In the description of enzymes herein, the database accession numbers (e.g.. UniProt, Genbank, NCBI, etc.) disclosing the amino acid sequence arc provided in parentheses.

[0322] In some embodiments, the NTP regenerating system is pyruvate kinase and substrate phosphoenolpyruvate. Various suitable pyruvate kinases are known in the art. In some embodiments, the pyruvate kinase for use in the NTP regenerating system is the pyruvate kinase of. among others, Escherichia coli str. K-12 substr. MG1655 (A0A3L1NNV5), Geobacillus stearothermophilus ATCC 7953 (Q02499), Schizosaccharomyces pombe (QI 0208), Saccharomyces cerevisiae S288C (D6VPH8), Gallus gallus liver (F1NW43), Solanum tuberosum (P22200), Oryctolagus cuniculus Ml / 2 (018919), Mus musculus muscle (P52480), Rattus norvegicus Ml / 2 (A0A8L2Q7B9), or Homo sapien muscle (A0A804F729).

[0323] In some embodiments, the NTP regenerating system is creatine kinase and substrate creatine phosphate. Various suitable creatine kinases are known in the art. In some embodiments, the creatine kinase for use in the NTP regenerating system is tire creatine kinase of, among others, Danio rerio (A8E5L0), Gallus gallus mitochondrial (P11009), Mus musculus (P07310), Bos taurus (Q5E9Y4), Oryctolagus cuniculus (P00563), or Homo sapien M-type (P06732).

[0324] In some embodiments, the NTP regenerating system is polyphosphate kinase and substrate polyphosphate. Various suitable polyphosphate kinases are known in the art. In a preferred embodiment, the polyphosphate kinase is PPK1. In some embodiments, the PPK1 for use in the NTP regenerating system is the PPK1 of, among others, Pseudomonas putida DOT-TIE (AFO50238.1), Escherichia coli K-12 P0A7B1 (AAC75554.1), Clostridium acetobutylicum ATCC 824(NP 347259.1), Thermosynechococcus elongatus (WP 011056068), Acidithiobacillus ferrooxidans (WP 064219446), Acidithiobacillus thiooxidans (WP 031572361). Bacillus acidicola (WP_066264350), Acetobacter aceti (GAN58028), Acetobacter aceti (WP_077811826.1), Thioalkalivibrio denitrificans (WP 077277945.1), or Psychromonas ingrahamii (WP 041766473.1).

[0325] In some embodiments, the NTP regenerating system is an acetate kinase and its substrate acetyl phosphate. Various suitable acetates kinases are known in the art and also describe herein. In some embodiments, the acetate kinase for use in the NTP regeneration system is an acetate kinase ofDocket No. 5244.285WO2Escherichia coli str. K-12 substr. MG1655 (NP 416799.1), Coryncbactcrium jcikciuni K411 (WP_011272972.1), Lactococcus cremoris subsp. cremoris KW2 (WP 011835968.1), Lactococcus lactis (WP 004254593.1), Marinitoga sp. 38H-ov (WP_165147355.1), Thermotoga sp. KOL6 (WP 101510533.1), Thermosipho melaniensis (WP 012057479.1), Thermotoga sp. RQ7 (WP_041844042.1), or Thermosipho africanus (WP_004102380.1).

[0326] In some embodiments, where the NTP regenerating system is an acetate kinase, the method of further comprises a pyruvate oxidase and substrate pyruvate and inorganic phosphate. The pyruvate oxidase converts acetate to acetyl phosphate, which is then used as a phosphate donor by the acetate kinase in the NTP regenerating system. Various pyruvate oxidases are known in the art. In some embodiments, the pyruvate oxidases arc homologs of pyruvate oxidases. In some embodiments, the pyruvate oxidase is a pyruvate oxidase of Bifidobacterium mongoliense (A0A087C4V4), Alkalibacterium subtropicum (A0A1I1KLE2), Pisciglobus halotolerans (A0A1I3CCM7), Jeotgalibaca sp PTS2502 (A0A1U7E9W7), Vagococcus fluvialis (A0A8I2AXT4), Candidatus Gracilibacteria bacterium (A0A2M7FGE0), Bavariicoccus seileri A0A3D4S346), Bifidobacterium aquikefiri (A0A261G4D1), Aerococcus urinae (F2I8Y6), or Aerococcus suis (A0A1W1YA59).

[0327] In some embodiments, the method further comprises a catalase, which converts H₂O₂ produced by the pyruvate oxidase to H2O. Various suitable catalases are known in the art. In some embodiments, the catalase can be the catalase of Archaeoglobus fulgidus, Bacillus stearothermophilus, E. coli, Mycobacterium intracellulare, Synechococcus sp PCC7942, Arabidopsis thaliana, Pisum sativum, or Saccharomyces cerevisiae (see, e.g., Zamocky. M., Progress in Biophysics and Molecular Biology, 1999, 72(l):19-66); see also WO1992017571). In some embodiments, the catalase is a catalase of Bos taurus (P00432), Aspergillus niger (A0A254TZH3), Helicobacter pylori (J0N6C6), Drosophila melanogaster (P17336) or Rattus norvegicus (P04762).

[0328] In the embodiments provided herein and illustrated in the Examples, various ranges of suitable reaction conditions that can be used in the processes, including but not limited to, substrate loading, co-substrate loading. pH, temperature, buffer, solvent system, cofactor, polypeptide loading, and reaction time. Further suitable reaction conditions for carry ing out the process for biocatalytic conversion of substrate compounds to product compounds using the enzymes described herein can be supplemented in view of the guidance provided herein to the skilled artisan, including, but not limited to, contacting the enzymes and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound. The present disclosure contemplates any suitable reaction conditions that may find use in the methods described herein.

[0329] In some embodiments, the substrate compound in the reaction mixtures can be varied, taking into consideration, for example, the desired amount of product compound, the effect of each substrateDocket No. 5244.285WO2concentration on enzy me activity, stability' of enzy me under reaction conditions, and the percent conversion of each substrate to product. In some embodiments, the suitable reaction conditions comprise a nucleoside substrate compound loading of about 0.1 uM to 1 uM, 1 uM to 2 uM, 2 uM to 3 uM, 3 uM to 5 uM, 5 uM to 10 uM, or 10 uM to 100 uM, or greater. In some embodiments, the suitable reaction conditions comprise a nucleoside substrate compound loading of about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the suitable reactions conditions comprise a nucleoside substrate compound loading of about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM. 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM. or 200 mM.

[0330] In some embodiments, the suitable reaction conditions comprise a nucleoside substrate compound loading of at least about 0.5 to about 25 g / L, 1 to about 25 g / L, 5 to about 25 g / L, about 10 to about 25 g / L, about 20 to about 25 g / L, or about 30 to about 60 g / L. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.5 g / L. at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L. at least about 20 g / L, at least about 30 g / L, at least about 40 g / L, at least about 50 g / L, or at least about 60 g / L, or even greater.

[0331] In carry ing out the processes described herein, the enzymes may be added to the reaction mixture in the fonn of a purified enzy me, partially purified enzy me, whole cells transformed with gene(s) encoding the enzy me, as cell extracts and / or ly sates of such cells, and / or as an enzy me immobilized on a solid support or other support medium. Whole cells transformed with gene(s) encoding the enzyme(s) or cell extracts, lysates thereof, and isolated enzymes may be employed in a variety' of different forms, including solid (e.g., lyophilized, spray -dried, and the like) or semisolid (e.g., a crude paste). The cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or the like, followed by a desalting procedure prior to lyophilization (e g., ultrafiltration, dialysis, etc.). Any of the enzyme preparations (including whole cell preparations) may be stabilized by crosslinking using known crosslinking agents, such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like).

[0332] In some embodiments, the gene(s) encoding the poly peptides can be transformed into host cell separately or together into the same host cell. For example, in some embodiments one set of host cells can be transformed with gene(s) encoding one polypeptide and another set can be transformed with gene(s) encoding another polypeptide. Both sets of transformed cells can be utilized together in the reaction mixture in the form of whole cells, or in the form of lysates or extracts derived therefrom. In other embodiments, a host cell can be transformed with gene(s) encoding multiple polypeptides. In some embodiments the polypeptides can be expressed in the form of secreted polypeptides and the culture medium containing the secreted polypeptides can be used for the synthesis reaction.Docket No. 5244.285WO2

[0333] In some embodiments, the improved activity of the engineered adenosine kinase polypeptides disclosed herein provides for processes wherein higher percentage conversion can be achieved with lower concentrations of the engineered polypeptide. In some embodiments of the process, the suitable reaction conditions comprise an engineered poly peptide amount of about 0.1% w / w, 0.2% (w / w), 0.5% (w / w). 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 75% (w / w), 100% (w / w) or more of substrate compound loading.

[0334] In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate of about 50 to 1, 25 to 1, 10 to 1, 5 to 1, 1 to 1, 1 to 5. 1 to 10, 1 to 25 or 1 to 50. In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate from a range of about 50 to 1 to a range of about 1 to 50 or 1 to 100.

[0335] In some embodiments, the engineered polypeptide is present at about 0.01 g / L to about 50 g / L; about 0.01 to about 0.1 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0.1 g / L to about 2 g / L. In some embodiments, the adenosine kinase polypeptide is present at about 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or 50 g / L.

[0336] In the embodiments of the process, the reaction conditions comprise a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and / or during the course of the reaction. In some embodiments, the suitable reaction conditions comprise a solution pH from about 4 to about 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, or pH from about 6 to about 8. In some embodiments, the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6. 6.5. 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0337] In some embodiments, in the course of the reaction, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range, for example, among others, by the addition of an acid or a base, before and / or during the course of the reaction. Alternatively, the pH may be controlled by using a buffer. Accordingly, in some embodiments, the reaction condition comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, by way of example and not limitation, borate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine, and 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris), and the like. In some embodiments, the buffer is present in a concentration of 1 mM-500 mM, 5 mM to 450 mM, 10 mM to 400 mM, 20 mM to 350 mM, 30 mM to 300 mM, 40 mM to 200 mM, or 50 mM toDocket No. 5244.285WO2100 mM. In some embodiments, the buffer is present in the composition at about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM or 500 mM. In some embodiments, the reaction conditions comprise water as a suitable solvent with no buffer present.

[0338] In the embodiments of the processes herein, a suitable temperature is used for the reaction conditions, for example, taking into consideration the increase in reaction rate at higher temperatures, and the activity of the enzyme during the reaction tune period. In some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C to about 95 °C, about 10 °C to about 75 °C, about 15 °C to about 95 °C. about 20 °C to about 95 °C, about 20 °C to about 65 °C, about 25 °C to about 70 °C, or about 50 °C to about 70 °C. In some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C, 15 °C, 20 °C, 25 °C, 30 °C. 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C or 95 °C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout die course of the reaction, hi some embodiments, the temperature during the enzymatic reaction can be adjusted over a temperature profile during the course of the reaction.

[0339] In some embodiments, the processes are carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and / or co-solvent systems, which generally comprise aqueous solvents, organic solvents and / or polymeric solvents. The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the processes using the engineered adenosine kinase polypeptides can be carried out in an aqueous co-solvent system comprising an organic solvent (e.g., ethanol, isopropanol (IP A), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) ethyl acetate, buty l acetate, 1 -octanol, heptane, octane, methyl t butyl ether (MTBE). toluene, and the like), ionic or polar solvents (e.g., 1-ethyl 4 methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl 3 methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, and the like). In some embodiments, the co-solvent can be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol. The non-aqueous co- solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases. Exemplary aqueous co-solvent systems can comprise water and one or more co-solvents selected from an organic solvent, polar solvent, and polyol solvent. In general, the co-solvent component of an aqueous co-solvent system is chosen such that it does not adversely inactivate the enzymes under the reaction conditions.Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified enzymes with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.Docket No. 5244.285WO2

[0340] In some embodiments of the process, the suitable reaction conditions comprise an aqueous co-solvent, where the co-solvent comprises DMSO at about 1% to about 50% (v / v), about 1 to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 10% to about 30% (v / v), or about 10% to about 20% (v / v). In some embodiments of the process, the suitable reaction conditions can comprise an aqueous co-solvent comprising ethanol at about 1% (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v). about 45% (v / v), or about 50% (v / v).

[0341] In some embodiments, the reaction conditions comprise a surfactant for stabilizing or enhancing the reaction. Surfactants can comprise non-ionic, cationic, anionic and / or amphiphilic surfactants. Exemplary surfactants, include by way of example and not limitation, nonyl phenoxypolyethoxylethanol (NP40), TRITON™ X-100 polyethylene glycol ter / -octylphenyl ether, polyoxyethy Icnc-stcarv lamine, cetyltrimethylammonium bromide, sodium oleylamidosulfate, polyoxyethylene-sorbitamnonostearate, hexadecyldimethylamine, etc. Any surfactant that may stabilize or enhance the reaction may be employed. The concentration of the surfactant to be employed in the reaction may be generally from 0.1 to 50 nig / mL, particularly from 1 to 20 mg / niL.

[0342] In some embodiments, the reaction conditions include an antifoam agent, which aids in reducing or preventing formation of foam in the reaction solution, such as when the reaction solutions are mixed or sparged. Anti-foam agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alky l amides, alkyl sulfates, etc ), and hydrophobic (e.g., treated silica, polypropy lene, etc.), some of which also function as surfactants. Exemplary anti-foam agents include Y-30® (Dow Coming), poly-glycol copolymers, oxy / ethoxylated alcohols, andpoly dimethylsiloxanes. In some embodiments, the anti-foam can be present at about 0.001% (v / v) to about 5% (v / v), about 0.01% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the anti-foam agent can be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v). about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v) or more as desirable to promote the reaction.

[0343] In some embodiments, the quantities of reactants used in the sy nthesis reaction will generally vary' depending on the quantities of product desired, and concomitantly die amount of substrates employed. Those having ordinary' skill in the art will readily understand how to vary' these quantities to tailor them to the desired level of productivity' and scale of production.

[0344] In some embodiments, the order of addition of reactants is not critical. The reactants may be added together at the same time to a solvent (e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like), or alternatively, some of the reactants may be added separately, and some together at different time points. For example, where applicable, the cofactor, co-substrate and substrate may be added first to the solvent.Docket No. 5244.285WO2

[0345] In some embodiments, die reaction components (c.g., enzy me, salts, etc.) may be provided to the reaction in a variety' of different forms, including powder (e.g., ly ophilized, spray dried, and the like), solution, emulsion, suspension, and the like. The reactants can be readily lyophilized or spray dried using methods and equipment that are known to those having ordinary' skill in the art. For example, the protein solution can be frozen at -80 °C in small aliquots, then added to a pre-chilled lyophilization chamber, followed by die application of a vacuum.

[0346] For improved mixing efficiency when an aqueous co-solvent system is used, the polypeptide(s), and co-substrate may be added and mixed into the aqueous phase first. The substrate may be added and mixed in, followed by the organic phase or the substrate may be dissolved in the organic phase and mixed in. Alternatively, the substrate may be premixed in the organic phase, prior to addition to the aqueous phase.

[0347] The processes of the present invention are generally allowed to proceed until further conversion of substrate to product does not change significantly with reaction time (e.g., less than 10% of substrate being converted, or less than 5% of substrate being converted). In some embodiments, the reaction is allowed to proceed until there is complete or near complete conversion of substrate to product. Transformation of substrate to product can be monitored using known methods by detecting substrate and / or product, with or without derivatization. Suitable analytical methods include gas chromatography, HPLC, MS, and the like. In some embodiments, after suitable conversion to product, the reactants are separated from the product and additional reactants are added.

[0348] Any of the processes disclosed herein using the polypeptides for the preparation of products can be carried out under a range of suitable reaction conditions, including but not limited to ranges of substrates, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time. In one example, the suitable reaction conditions for the conversion of a nucleoside to aNMP comprise: (a) substrate loading of about 1-200 mM nucleoside; (b) about 0.01 g / L to 5 g / L engineered adenosine kinase poly peptide; (c) 1-100 mM MgCF; (e) 5 to 100 mM of buffer, e.g., Tris-HC1; (1) 0.01-100 mM NTP (e.g., ATP); (g) pH at 5-9; and (h) temperature of about 15 °C to 70 °C. In some embodiments, additional reaction components or additional techniques carried out to supplement the reaction conditions. These can include taking measures to stabilize or prevent inactivation of the enzy me, reduce product inhibition, shift reaction equilibrium to formation of the desired product.

[0349] As described herein, the methods of using the polypeptides described herein can be carried out using the enzymes bound or immobilized on a solid support. Methods of poly peptide immobilization are known in the art. In some embodiments, the engineered polypeptides can be bound non-covalently or covalently. Various methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are described in, for example,Docket No. 5244.285WO2Yi ct al., Proc. Bioclicm., 2007, 42(5): 895-898; Martin ct al., Appl. Microbiol. Biotcchnol., 2007, 76(4): 843-851; Koszelewski et al., J. Mol. Cat. B: Enzymatic, 2010, 63: 39-44; Truppo et al., Org. Proc. Res. Dev., published online: dx.doi.org / 10.1021 / op200157c; Hermanson, “Bioconjugate Techniques,” 2nd Ed., Academic Press, Cambridge, MA (2008); Mateo et al., Biotechnol. Prog., 2002, 18(3):629-34; and “Bioconjugation Protocols: Strategies and Methods," In “Methods in Molecular Biology,” Niemeyer (ed.), Humana Press, New York, NY (2004); the disclosures of each which are incorporated by reference herein). Solid supports useful for immobilizing the engineered polypeptides of the present disclosure include, but are not limited to, beads or resins comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene / DVB copolymer or polymethacrylate with octadecyl functional groups. In some embodiments, exemplary solid supports useful for immobilizing the engineered polypeptides of the present invention include, but are not limited to. EnginZyme (including, EziG-1, EziG-1, and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC -EP, EC-HFA / S. EXA252. EXE119 and EXE120).

[0350] In further embodiments, any of the above described processes for the conversion of one or more substrate compounds to product compound can further comprise one or more steps selected from: extraction; isolation; purification; and crystallization of product compound. Methods, techniques, and protocols for extracting, isolating, purifying, and / or crystallizing the product from biocatalytic reaction mixtures produced by the above disclosed processes are known to the ordinary artisan. Additionally, illustrative methods are provided in the Examples below.EXAMPLES

[0351] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

[0352] In the experimental disclosure below, the following abbreviations apply where applicable: ppm (parts per million); M (molar); mM (millimolar), uM and pM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and mL (milliliter); ul, pl, uL and pL (microliter); cm (centimeters); mm (millimeters); um and pm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); D (daltons); rpm (rotations per minute); °C (degrees Celsius); LB (Luria Broth); IPTG (isopropyl-P-D-thiogalactoside); LiK (lithium-potassium); DNA (deoxyribonucleic acid); and RNA (ribonucleic acid).Nucleotide Modifications and OligonucleotidesDocket No. 5244.285WO2Example 1Adenosine kinase (AdoK) selection, plasmid construction, and directed evolution

[0353] The adenosine kinase (AdoK) enzyme (SEQ ID NO: 10) is a carbohydrate kinase family protein encoded by the genome of Xcmthemonas campestris (UniProt ID: A0A3E1LD95). A synthetic gene (SEQ ID NO: 9) encoding an A-term inal 6-histidine tagged version of the WT AdoK was designed with codon optimization for E. coli expression, synthesized, and subcloned into the E. coli expression vector pCK100900i (See, e.g., US Pat. No. 7,629,157 and US Pat. Appln. Publn.2016 / 0244787) all of which are hereby incorporated by reference). This plasmid construct wasDocket No. 5244.285WO2transformed into an E. colt strain derived from W3110. Directed evolution techniques were used to generate libraries of gene variants from these plasmids (see, e.g., WO2024 / 129232). The substitutions in the enzy me variants described herein are indicated with reference to the N-terminal 6-histidine tagged version of the AdoK enzyme (i.e., SEQ ID NO: 10) or variants thereof, as indicated.Example 2AdoK expression and lysate processing for high throughput (HTP) screening High Throughput (HTP) Growth of AdoK Enzyme and Variants

[0354] Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose and 30 pg / mL chloramphenicol. After overnight incubation at 37 °C, colonies were placed into the wells of 96-well shallow flat bottom NUNC™ (Thermo-Scientific) plates filled with 180 pl / well LB medium supplemented with 1% glucose and 30 pg / mL chloramphenicol. The cultures were allowed to grow overnight for 18-20 horns in a shaker (200 rpm, 30 °C, and 85% relative humidity; Kulmer). Overnight growth samples (20 pL) were transferred into Costar 96-well deep plates filled with 380 pL of Terrific Broth supplemented with 30 pg / mL chloramphenicol. The plates were incubated for 120 minutes in a shaker (250 rpm, 30 °C, and 85% relative humidity; Kuhner) until the ODeoo reached between 0.4-0.8. The cells were then induced with 40 pL of 10 mM IPTG in sterile water and incubated overnight for 18-20 hours in a shaker (250 rpm, 30 °C, and 85% relative humidity;Kuhner). The cells were pelleted (4,000 rpm for 20 min), the supernatants were discarded, and the cells were frozen at -80 °C prior to analysis.Thermal lysis of HTP Cell Pellets with Lysozyme (Examples 13-25)

[0355] For lysis, 400 pL lysis buffer containing 50 mM triethanolamine buffer, pH 7.5, and 0.1 g / L lysozyme were added to the cell pellet in each well. The cells were shaken vigorously at room temperature for 5 minutes on a bench top shaker. An aliquot of the re-suspended cells (5-100 uL) was transferred to a 96-well format 200 pL BioRad PCR plate, diluting to 100 uL in lysis buffer if necessary, then briefly spun-down prior to 1 h heat treatment at the temperature indicated, typically 45-60 °C. Following heat-treatment, the cell debris was pelleted by centrifugation (4,000 rpm at 4 °C for 10 min), and clear supernatants were then used in biocatalytic reactions to determine their activity levels.Example 3Shake Flask Expression and Purification of AdoKShake Flask Expression

[0356] Selected HTP cultures grown as described above were plated onto LB agar plates with 1% glucose and 30 pg / mL chloramphenicol and grown overnight at 37 °C. A single colony from eachDocket No. 5244.285WO2culture was transferred to 5 mL of LB broth with 1% glucose and 30 pg / mL chloramphenicol. The cultures were grown for 20 h at 30 °C, 250 rpm, and subcultured at a dilution of approximately 1:50 into 250 mL of Terrific Broth with 30 pg / mL of chloramphenicol, to a final ODeoo of about 0.05. The cultures were incubated for approximately 195 min at 30 °C, 250 rpm, to an ODeoo of about 0.6, and then induced with the addition of IPTG at a final concentration of 1 mM. The induced cultures were incubated for 20 h at 30 °C, 250 rpm. Following this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the pellets were resuspended in 35 mL of 20 mM triethanolamine, pH 7.5. This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-l 10L). The crude lysate was pelleted by centrifugation (10.000 rpm for 60 min at 4 °C), and the supernatant was then filtered through a 0.2 pm PES membrane to further clarify the lysate.Purification of AdoK from Shake Flask Lysates

[0357] AdoK lysates were supplemented with l / 50th volume of SF elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.02% v / v Triton X-100 reagent) per well. Lysates were then purified using an AKTA Start purification system and a 5 mL HisTrap FF column (GE Healthcare). The SF wash buffer comprised 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v / v Triton X-100 reagent.

[0358] Elution fractions containing protein were identified by UV absorption (A280) and pooled, then dialyzed overnight in dialysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1, 0.1 mM EDTA. and 50% glycerol) in a 3.5K Slide-A-Lyzer™ dialysis cassette (Thermo Fisher) for buffer exchange. AdoK concentrations in the preparations were measured by absorption at 280 nrn.Example 4Biosynthetic cascade reactions for production of nucleotide triphosphates (NTPs) NTP biosynthetic reaction setupDocket No. 5244.285WO2

[0359] Reactions were performed in 384-wcll format 40 pL BioRad PCR plates. AdoK variants were assayed in the presence of adenylate kinase (AdyK) and acetate kinase (AcK) enzy me variants to provide for direct conversion of nucleosides to the corresponding triphosphate. The reactions were set up as follows: (i) all reaction components, except for the nucleoside substrate and the AdoK lysate, were premixed in a single solution and were aliquoted into each well of the 384-well plates, (ii) AdoK lysate solution was then added into the wells, and (iii) an aliquot of the substrate nucleoside in DMSO was added to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thennocycler at the indicated temperature and reaction time, then held at 10 °C prior to analysis.Example 5High-performance liquid chromatography (HPLC) analysis of phosphorylated products Sample preparation and reaction analysis using HPLC:

[0360] The nucleoside substrates, along with their respective 5 ’-monophosphate (NMP), 5’-diphosphate (NDP), and 5 ‘-triphosphate (NTP) products produced using reactions set up as described in Example 4 were analyzed using HPLC. Mobile phases consisted of 50 mM potassium phosphate (pH 7) with 2 mM tetrabutylammonium hydrogen sulfate (Solvent A), acetonitrile (solvent B), and water (Solvent C). Products were detected by UV absorption at 254 mn. In some instances, a Zorbax RR StableBond Aq, 3.0 x 150 mm, 3.5 uni (Agilent, #863954-314) column was used. In other instances, a Zorbax RR StableBond Aq. 3.0 x 100 mm, 3.5 pm (Agilent, #861954-314) column was used, while in other instances, a Zorbax RR StableBond Aq, 2.1 x 50 mm, 3.5 pm (Agilent, #871700-914) column was used.Example 6Analysis of NTP production in biosynthetic cascade using capillary electrophoresis Coupling of NTP biosynthetic reactions to TdT for CE:

[0361] For high-throughput (HTP) determination of NTP yield, NTP biosynthetic cascade reactions were terminated with either a heatkill at 95 °C for 2 minutes or by dilution with 75% methanol. Samples were then diluted into a coupling reaction. The reaction contained 20 mM triethanolamine (TEoA), 0.25 mM CoCL, 0.001 units of inorganic pyrophosphatase (New England Biolabs), 4 pM SEQ ID NO: 8 (terminal nucleotidyl transferase), 12.375 pM unlabeled oligonucleotide, and 0.125 pM 5 ’-6-F AM -labeled oligonucleotide. Reactions were carried out at 50 °C for 60 minutes, followed by 2 minutes at 95 °C.Sample preparation for reaction analysis using CE:

[0362] For analysis of reaction samples, capillary electrophoresis was performed using either an ABI 3500XL Genetic Analyzer (ThermoFisher) or a SeqStudio™ Flex Genetic Analyzer (ThermoFisher).Docket No. 5244.285WO2Reactions (1 pL) were quenched by the additions of 19 pL of I mM aqueous ethylenediaminetetraacetic acid (EDTA). Quenched reactions were diluted to 1.25 nM oligonucleotide, and a 2-pL aliquot of this solutions was transferred to a new 96-well MicroAmp Optical PCR plate or a 384-well MicroAinp Optical PCR plate containing 18 pL Hi-Di™ Formamide (ThermoFisher) containing the Alexa633 size standard. The ABI 3500XL and SeqStudio™ Flex were configured with POP6 polymer, 50 cm capillaries, and a 55 °C oven temperature. Pre-run settings were 18 kV for 50 sec. Injection was 10 kV for 2 sec, and the run settings were 19 kV for 620-640 sec. FAM-labeled oligo substrates and products were identified by their sizes relative to the sizing ladder.Example 7Improvements over SEQ ID NO: 10 in the conversion of nucleosides to nucleotidesHTP Screening for Improved AdoK Variants

[0363] SEQ ID NO: 10 was selected as the parent AdoK enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and / or recombination of previously identified beneficial mutations). The poly peptides encoded by each gene were produced in HTP and prepared as described in Table 7.1.

[0364] Reactions were performed as described in Example 4 using conditions summarized in Table 7.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.

[0365] Activity relative to SEQ ID NO: 10 (Activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 10 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 7.2.Docket No. 5244.285WO2Example 8Improvements over SEQ ID NO: 10 in the conversion of nucleosides to nucleotides HTP Screening for Improved AdoK Variants

[0366] The AdoK of SEQ ID NO: 10 was selected as the parent AdoK enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis andDocket No. 5244.285WO2recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 11.1.

[0367] Reactions were performed as described in Example 4 using conditions summarized in Table 11.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.

[0368] Stability relative to SEQ ID NO: 10 (Stability FIOP) was calculated based on the percentage of extension products obser ed for the variant compared with the percentage obser ed with SEQ ID NO: 10 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 8.2.Docket No. 5244.285WO2Example 9Improvements over SEQ ID NO: 24 in the conversion of nucleosides to nucleotidesHTP Screening for Improved AdoK Variants

[0369] The AdoK of SEQ ID NO: 24 was selected as the parent AdoK enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1.

[0370] Reactions were performed as described in Example 4 using conditions summarized in Table 9.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.Docket No. 5244.285WO2

[0371] Activity relative to SEQ ID NO: 24 (Activity' FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 24 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 9.2.Docket No. 5244.285WO2Example 10Improvements over SEQ ID NO: 24 in the conversion of nucleosides to nucleotidesHTP Screening for Improved AdoK Variants

[0372] The AdoK of SEQ ID NO: 24 was selected as the parent AdoK enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 10.1.

[0373] Reactions were performed as described in Example 4 using conditions summarized in Table 10.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.

[0374] Activity relative to SEQ ID NO: 24 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 24 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 10.2.Docket No. 5244.285WO2Example 11Improvements over SEQ ID NO: 162 in the conversion of nucleosides to nucleotides HTP Screening for Improved AdoK Variants

[0375] The AdoK of SEQ ID NO: 162 was selected as the parent AdoK enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 11.1.

[0376] Reactions were performed as described in Example 4 using conditions summarized in Table 11.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.Docket No. 5244.285WO2

[0377] Activity relative to SEQ ID NO: 162 (Activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 162 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 11.2.Docket No. 5244.285WO2Docket No. 5244.285WO2Docket No. 5244.285WO2Docket No. 5244.285WO2Example 12Improvements over SEQ ID NO: 268 in the conversion of nucleosides to nucleotides HTP Screening for Improved AdoK Variants

[0378] The AdoK of SEQ ID NO: 268 was selected as the parent AdoK enzy me. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 12.1.

[0379] Reactions were performed as described in Example 4 using conditions summarized in Table 12.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.Docket No. 5244.285WO2

[0380] Activity relative to SEQ ID NO: 268 (Activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 268 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 12.2.Docket No. 5244.285WO2Docket No. 5244.285WO2Docket No. 5244.285WO2Example 13Improvements over SEQ ID NO: 268 in the conversion of nucleosides to nucleotides HTP Screening for Improved AdoK Variants

[0381] The AdoK of SEQ ID NO: 268 was selected as the parent AdoK enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 13.1.

[0382] Reactions were performed as described in Example 4 using conditions summarized in Table 13.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.Docket No. 5244.285WO2

[0383] Activity relative to SEQ ID NO: 268 (Activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 268 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 13.2.Docket No. 5244.285WO2

[0384] While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, without departing from the spirit and scope of the invention.

[0385] For all purposes, each and every' publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

Claims

Docket No. 5244.285WO2CLAIMSWhat is claimed is:

1. An engineered adenosine kinase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 10-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 10-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24. 162, or 268. or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

2. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%.95%, 96%, 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 162, or 268, or to a reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, 24, 1 2. or 268, or relative to the reference sequence corresponding to SEQ ID NO: 10, 24, 162, or 268.

3. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

4. The engineered adenosine kinase of any one of claims 1-3, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 1, 4, 9, 11, 12, 46, 88, 131, 138, 139, 140, 158. 175, 189, 193, 199, 200, 201, 203, 223, 267, 270, Til. K, 280, 281, 289, 293, 302, 315, 321; or substitution 13L / R / T, 23E, 40L / Q, 43A, 53D / H, 54Q, 55Q / S / T / W / Y, 74A / C / G / L / M / Q, 90G / Q, 91G, 94R, 107 / K / L / P, 108S, 109D, 111 / H / S, 112K, 116V, 119K, 120G, 121E, 126V, 128E / L / M, 1291, 133C / E / K / R / S, 134K / Y, 147L / P, 157K / L / Q / R / S, 160A / R / T / W, 163S, 164I / R, 167E / R / S, 168E / G / Q / S / Y, 169W, 170A / C / L / , 171Q / T, 172A / R / T / V, 182T, 185L / N,, 190E / L, 191P, 192R, 194F / K, 195C / N, 196G, 206E / H / T, 207S, 210E / V, 21 IS,Docket No. 5244.285WO2222E / P / V, 225K / M / S / T, 233E, 234E, 243A / E / L / N / T, 250L, 25 IP, 254K, 271L / T, 274V, 308K, 312C, 314A / L / M, and 320E, or combinations thereof, wherein die amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

5. The engineered adenosine kinase of any one of claims 1-3, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue IV, 4N, 9C / L / R, 11K / P, 12E / P / R / T, 13L / R / T, 23E, 40 / L / Q, 43 A, 46L, 53D / H, 54Q, 55Q / S / T / W / Y, 74A / C / G / L / M / Q, 88R, 90G / Q, 91G, 94R, 107 / K / L / P, 108S, 109D, 111H / S, 112K. 116V, 119K, 120G, 121E. 126V. 128E / L / M, 1291, 13 IS, 133C / E / K / R / S. 134K / Y, 138Q, 139L / S, 140P, 147L / P, 157K / L / Q / R / S, 158S, 160A / R / T / W. 163S. 164I / R. 167E / R / S. 168E / G / Q / S / Y, 169W, 170A / C / L, 171Q / T. 172A / R / T / V, 175L / W. 182T. 185L / N, 189A / H, 190E / L. 191P. 192R. 193M / T, 194F / K, 195C / N. 196G, 199A / L / M, 200V. 200A, 201S / T, 203E, 206E / H / T, 207S, 210E / V. 21 IS. 222E / P / V, 223G / L. 225K / M / S / T, 233E, 234E, 243A / E / L / N / T, 250L, 2 IP, 254K, 267G, 270D. 271L / T, 274V, 277K / R. 278A / G / P / Q / T, 280R / V / W, 281S, 289L, 293T, 302H, 308K. 312C, 314A / L / M, 315V, 320E, and 3211, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

6. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue M1V. H4N, G9C / L / R, G11K / P, M12E / P / R / T. S13L / P / R / T, A21G, D23E, R40H / L / Q, R43A, V46L, M53D / H, R54Q, R55Q / S / T / W / Y, R74A / C / G / L / M / Q / T / V, T80V, Y88R, E90G / Q. H91G, T94M / R, D106G / L / R, D107G / K / L / P, L108S, S109D. P111A / H / S. Q112K, T116V. M119K, D120G, Q121E, A126S / V. H128E / L / M, S129I, A131S, M133C / E / K / L / R / S, R134K / Y, N138Q, H139L / S, V140P, R141S. S147L / P, T147S, T155A / I. E157K / L / Q / R / S, G158S. I160A / R / T / W, A163S. E164I / R / S. A167E / R / S / V, A168E / G / Q / S / Y. M169L / W. G170A / C / L / S, I171Q / T, P172A / R / T / V. F175L / W, P182G / T, S185L / N, P187Q, L189A / H, R190E / L, E191P / S. F192R. I193M / T, E194F / K, Q195C / G / N. A196G, V199A / L / M. A200V, V200A, V201S / T, D203E. S206E / H / T. R207A / L / M / S, Q210E / V, A211I / L / M / S, S222A / E / G / P / V. R223G / L. Q225K / M / S / T, Q231L, S233E. A234E.K243A / E / L / N / R / T, A250I / L, K251P / T, A252V, R254K, A267G, I270D, Y271L / T, Q274V, Y277K / R, D278A / G / P / Q / T, L280R / V / W, T281S, M289L, K293T, Q302H, E308K, N310A, R312C, K314A / L / M, Q315V, A320E, and L321I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

7. The engineered adenosine kinase of any one of claims 1-6, wherein the amino acid sequence of the engineered adenosine kinase further comprises at least a substitution at amino acidDocket No. 5244.285WO2position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40. 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156. 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172. 177, 179, 180, 181, 182, 183, 185, 187, 188. 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208. 210, 211, 212, 215, 216, 217, 218, 221, 222. 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240. 241, 242, 243, 244, 245, 246, 247, 248, 249. 250, 251, 252, 253, 254, 255, 256, 258. 265, 271, 274. 275, 276, 287, 288, 291, 292. 293, 294, 295. 296, 299, 303, 305, 307, 308, 310, 311. 312, 314, 315. 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO:

10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

8. The engineered adenosine kinase of claim 7, wherein the amino acid sequence of the engineered adenosine kinase further comprises at least a substitution or amino acid residue 13A / E / P, 14S / T / V, 20F / V. 21G, 23N, 24I / N / V, 26F, 27 A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y. 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q.43E / K / R / S / V, 461, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V. 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79 A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M, 93 A, 94M / N, 100G, 101F / W, 1021, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T, 111A / G / L, 112I / L, 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, BOA, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 15 IL, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 1901, 191A / R / S / W, 192L. 194A / R, 195G / M / T / V, 196S, 197R. 202F. 206A / H, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V. 221A, 222A / G / M / Q, 225E / G, 229C / H / V. 231L / M / V, 232S, 233A / S / V. 234A / M / N / R / V / Y, 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V. 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W. 246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T. 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M. 258R / S / V, 265Y, 271W. 274R. 275S, 276D / E / G / S, 287A / S / V / Y. 288V, 291G, 292F / I / K / V, 293M. 294L. 295 A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G, 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V. 315S. 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-Docket No. 5244.285WO29. The engineered adenosine kinase of any one of claims 1-8, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 53, 54, 88, 91, 111, 116, 126, 131, 133, 134, 138, 139, 157, 160, 163, 168, 170, 175, 189, 192, 196, 199, 200, 203, 206, 225, 270. 280, 281, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO:

10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

10. The engineered adenosine kinase of any one of claims 1-9, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution 53D / H, 54Q, 88R, 91G, 111A / H. 116V, 126V, 131S, 133C / L / R / S, 134K / Y. 138Q, 139L / S, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L / S, 175L / W. 189A / H, 192R, 196G. 199 A. 200A, 203E, 206T, 225M / S / T.270D, 280R / V / W, or 281 S. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

11. The engineered adenosine kinase of any one of claims 1-10, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution M53D / H, R54Q, Y88R, H91G, P111A / H, T116V, A126V, A131S, M133C / L / R / S, R134K / Y, N138Q, H139L / S, E157L / Q / R, I160A / R / T / W, A163S. A168E / G / Q / S / Y, G170A / C / L / S, F175L / W, L189A / H. F192R, A196G, V199A. V200A. D203E. S206T, Q225M / S / T. I270D, L280R / V / W, or T281S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

12. The engineered adenosine kinase of any one of claims 1-11. wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.

2. 11.2, 12.2, and 13.

2. wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO:

10. or relative to the reference sequence corresponding to SEQ ID NO: 10.

13. The engineered adenosine kinase of any one of claims 1-4, wherein the adenosine kinase comprises an amino acid sequence having at least 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.

2. 10.2, 11.2, 12.

2. and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO:

10. or relative to the reference sequence corresponding to SEQ ID NO: 10.Docket No. 5244.285WO214. The engineered adenosine kinase of any one of claims 1-4, comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812.

15. The engineered adenosine kinase of claim 1. comprising an amino acid sequence having at least 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO:

24. or to the reference sequence corresponding to SEQ ID NO:

24. wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

16. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

17. The engineered adenosine kinase of claim 15 or 16. wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 53, 54, 88.91, 111, 116, 120, 126, 131, 133, 134. 138, 139, 157. 160, 163, 168, 170, 175, 189, 192, 194. 196, 199. 200, 203, 206, 225, 270, 280, 281, or 308, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

18. The engineered adenosine kinase of any one of claims 15-17, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 53D / H, 54Q, 88R, 91G, 111A / H, 116V, 120G, 126V, 131S, 133C / E / K / L / R / S, 134K / Y, 138Q, 139L / S. 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L / S, 175L / W, 189A / H, 192R, 194K, 196G, 199 A, 200A / V, 203E, 206T. 225M / S / T, 270D, 280R / V / W, 281 S, or 308K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.Docket No. 5244.285WO219. The engineered adenosine kinase of claim 15 or 16, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 88, 131, 138, 139, 175, 189, 199, 200, 203, 270, 280, 281; or substitution 53D / H, 54Q, 91G, 111H, 116V, 120G, 126V, 133C / E / K / R / S, 134K / Y, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y, 170A / C / L, 192R. 194K, 196G, 206T, 225M / S / T, or 308K; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

20. The engineered adenosine kinase of claim 19, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 53D / H. 54Q, 88R, 91G, 111H, 116V, 120G. 126V. 131S. 133C / E / K / R / S, 134K / Y. 138Q, 139L / S, 157L / Q / R, 160A / R / T / W, 163S, 168E / G / Q / S / Y. 17 A / C / L, 175L / W. 189A / H, 192R, 194K, 196G. 199 A, 200 A / V. 203E, 206T, 225M / S / T, 270D, 280R / V / W, 28 IS, or 308K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

21. The engineered adenosine kinase of claim 19 and 20, wherein die ammo acid sequence of the engineered adenosine kinase further comprises at least a substitution at amino acid position 13, 14, 20, 21. 23, 24, 26, 27, 28, 29, 30, 31, 33. 34, 35, 38, 40, 41, 42, 43, 46, 49. 50, 51, 52, 53, 54, 55, 56. 64, 67, 73, 74. 77, 79, 80, 83, 86, 87, 89, 90, 91. 92, 93, 94, 100, 101. 102, 103, 104, 106, 107, 108. 109, 111, 112. 113, 116, 119, 120, 121, 126, 128, 129. 130, 133, 134. 136, 141, 144, 146, 147, 151. 152, 155, 156. 157, 160, 162, 163, 164, 167, 168, 169. 170, 171, 172. 177, 179, 180, 181, 182, 183. 185, 187, 188. 190, 191, 192, 194, 195. 196, 197, 202. 206, 207, 208. 210, 211, 212, 215, 216, 217. 218, 221, 222. 225, 229. 231, 232, 233. 234, 236, 237. 238, 239, 240. 241, 242. 243, 244, 245, 246. 247, 248, 249. 250, 251. 252, 253, 254. 255, 256, 258. 265, 271, 274. 275, 276. 287, 288, 291, 292. 293, 294.

295. 296, 299. 303, 305, 307. 308, 310, 311. 312, 314.

315. 316, 317. or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

22. The engineered adenosine kinase of claim 21, wherein the amino acid sequence of the engineered adenosine kinase further comprises at least a substitution or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y. 38C / R / V, 40H / M / R / W, 4 IL, 42D / G / N / Q, 43E / K / R / S / V, 461, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V, 55H, 56F / G / S / Y. 64M / T, 67G, 73A / C / E / M. 74F / I / K / R / T / V, 77V, 79 A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M, 93 A, 94M / N, 100G. 101F / W, 1021, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T. 108A / C / E, 109E / G / L / P / R / S / T,Docket No. 5244.285WO2111A / G / L, 112I / L, 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 15 IL, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 1901, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R. 202F, 206A / H, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V. 221A, 222A / G / M / Q, 225E / G, 229C / H / V. 231L / M / V, 232S, 233A / S / V. 234A / M / N / R / V / Y, 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V. 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W. 246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T. 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M. 258R / S / V, 265Y, 271W. 274R, 275S, 276D / E / G / S, 287A / S / V / Y. 288V, 291G, 292F / 1 / K / V, 293M. 294L. 295 A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G, 308D / F / L / V. 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y. or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO:

24. or relative to the reference sequence corresponding to SEQ ID NO: 24.

23. The engineered adenosine kinase of claim 15 or 16, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 53, 53 / 111 / 133, 53 / 111 / 133 / 308, 53 / 111 / 138, 53 / 133, 54, 54 / 116 / 168, 54 / 134 / 168 / 200 / 225, 54 / 134 / 168 / 225 / 280, 54 / 134 / 225, 54 / 168 / 225, 54 / 200 / 280, 111, 111 / 120 / 133, 111 / 133, 111 / 138 / 160, 111 / 138 / 160 / 200, 111 / 160 / 200, 116, 116 / 134 / 168 / 194, 116 / 134 / 168 / 200, 116 / 168 / 225, 133, 133 / 138, 133 / 160 / 170 / 200, 134, 134 / 168, 134 / 168 / 225 / 280, 134 / 225, 138, 138 / 170, 160, 168 / 200 / 280, 168 / 225, 189 / 200 / 225, 200, 200 / 280, 225, 280, or 138 / 200, 168 / 189 / 200, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

24. The engineered adenosine kinase of any one of claim 15, 16, or 23, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 53H, 53H / 111A / 133E. 53H / 111A / 133E / 308K, 53H / 111A / 138Q. 53H / 133E, 54Q. 54Q / 116V / 168E. 54Q / 134K / 168E / 225T / 280V, 54Q / 134K / 168S / 200V / 225T, 54Q / 134K / 225T, 54Q / 168E / 225T. 54Q / 200V / 280V, 111A, 111A / 120G / 133K, 111A / 133E, 111A / 138Q / 160R, 111A / 138Q / 160R / 200V, 111A / 160R / 200V, 116V. 116V / 134K / 168E / 194K. 116V / 134K / 168E / 200V, 116V / 168Q / 225T, 133E, 133E / 138Q, 133K, 133K / 160R / 170A / 200V, 133L, 133L / 138Q, 134K, 134K / 168E, 134K / 168E / 225T / 280V, 134K / 225T, 138Q, 138Q / 170A. 160R, 168S / 200 V / 280 V, 168S / 225T, 189A / 200V / 225T, 200V, 200V / 280V, 225T, 280V, 138Q / 200V, or 168S / 189A / 200V,Docket No. 5244.285WO2wherein the amino acid positions arc relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

25. The engineered adenosine kinase of any one of claims 1-4, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.

2. 12.2, and 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 24, or relative to the reference sequence corresponding to SEQ ID NO: 24.

26. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12-321 of SEQ ID NO: 162, or to the reference sequence corresponding to SEQ ID NO: 162, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence comprising residues 12-321 of SEQ ID NO:162, or relative to the reference sequence corresponding to SEQ ID NO: 162.

27. The engineered adenosine kinase of claim 1. comprising an amino acid sequence having at least 80%. 81%, 82%, 83%. 84%. 85%, 86%, 87%. 88%. 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity' to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812. wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO:

162. or relative to the reference sequence corresponding to SEQ ID NO: 162.

28. The engineered adenosine kinase of claim 26 or 27, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position 4 / 126 / 187 / 233, 21, 21 / 129 / 147, 21 / 147, 21 / 147 / 158, 21 / 147 / 158 / 231 / 251, 21 / 147 / 158 / 231 / 251 / 254 / 321, 21 / 147 / 158 / 231 / 293, 21 / 147 / 158 / 234, 21 / 147 / 158 / 234 / 254, 21 / 147 / 158 / 234 / 254 / 293, 21 / 147 / 158 / 251 / 254 / 293, 21 / 147 / 231 / 234, 21 / 147 / 231 / 234 / 251 / 254 / 293, 21 / 147 / 231 / 234 / 251 / 293, 21 / 147 / 234 / 254, 21 / 147 / 234 / 293, 21 / 147 / 251, 21 / 147 / 251 / 254, 21 / 147 / 251 / 293 / 320. 21 / 147 / 254. 21 / 147 / 254 / 293, 21 / 147 / 293, 21 / 158 / 231 / 234 / 251, 21 / 158 / 234, 21 / 158 / 251. 21 / 158 / 251 / 254. 21 / 158 / 293. 21 / 231, 21 / 231 / 234, 21 / 231 / 234 / 251 / 254.21 / 231 / 234 / 251 / 254 / 293, 21 / 231 / 234 / 251 / 293, 21 / 231 / 251 / 254, 21 / 234 / 251, 21 / 234 / 293, 21 / 251, 21 / 251 / 254. 21 / 254, 21 / 293, 23 / 126 / 157 / 233, 23 / 126 / 157 / 250. 23 / 126 / 187, 23 / 126 / 233, 23 / 126 / 233 / 252 / 289. 23 / 126 / 250 / 289, 23 / 233. 23 / 233 / 252 / 289 / 310, 23 / 250 / 252 / 289, 112 / 126 / 310.Docket No. 5244.285WO2126 / 157 / 187, 126 / 157 / 233 / 250 / 252, 126 / 157 / 233 / 252, 126 / 167 / 310, 126 / 187, 126 / 187 / 233, 126 / 187 / 310, 126 / 233, 126 / 233 / 250 / 252, 126 / 233 / 252 / 289, 126 / 233 / 252 / 302, 126 / 233 / 289, 126 / 250 / 252, 126 / 250 / 289, 126 / 252, 126 / 252 / 289, 126 / 289, 126 / 310, 141 / 187, 147 / 158, 147 / 158 / 182 / 234 / 251, 147 / 158 / 231, 147 / 158 / 231 / 234 / 251, 147 / 158 / 251 / 254, 147 / 158 / 254, 147 / 158 / 254 / 293, 147 / 231 / 234, 147 / 231 / 234 / 251 / 293, 147 / 231 / 234 / 254, 147 / 231 / 293, 147 / 234, 147 / 234 / 251 / 254, 147 / 251. 147 / 251 / 254, 147 / 251 / 293, 147 / 254, 147 / 293, 157 / 187 / 233 / 252, 157 / 233 / 250 / 289, 157 / 252. 157 / 252 / 289, 157 / 289 / 310, 157 / 310, 158. 158 / 231 / 234, 158 / 231 / 234 / 254, 158 / 234, 158 / 234 / 251 / 254. 158 / 234 / 254, 158 / 251 / 254 / 293, 158 / 254, 167 / 252 / 289, 185 / 187 / 233 / 310.187 / 233, 187 / 233 / 252. 187 / 233 / 252 / 310, 187 / 250. 187 / 250 / 252, 187 / 252, 187 / 289, 187 / 310.225 / 250 / 252, 231 / 234 / 251. 231 / 234 / 251 / 254, 231 / 254, 233 / 250 / 252, 233 / 250 / 252 / 289, 233 / 250 / 252 / 289 / 310, 233 / 289, 233 / 289 / 310, 233 / 310, 234 / 293, 250 / 252, 250 / 252 / 289, 250 / 252 / 310, 250 / 289, 250 / 289 / 310. 251 / 254, 251 / 254 / 293. 252 / 289, 252 / 310. 254 / 293, 289, 289 / 310, and 293, and 310, or combinations thereof, wherein the amino acid positions are relative to the reference sequence comprising amino acid residues 12-321 of SEQ ID NO:

162. or relative to the reference sequence corresponding to SEQ ID NO: 162.

29. The engineered adenosine kinase of any one of claims 26-28, wherein the amino acid sequence of the engineered adenosine kinase comprises a substitution or substitution set selected from 4N / 126V / 187Q / 233E, 21G, 21G / 129I / 147S, 21G / 147S, 21G / 147S / 158S, 21G / 147S / 158S / 231L / 251P, 21G / 147S / 158S / 231L / 251P / 254K / 321I, 21G / 147S / 158S / 231L / 293T, 21G / 147S / 158S / 234E, 21G / 147S / 158S / 234E / 254K, 21G / 147S / 158S / 234E / 254K / 293T, 21G / 147S / 158S / 251P / 254K / 293T, 21G / 147S / 231L / 234E, 21G / 147S / 231L / 234E / 251P / 254K / 293T, 21G / 147S / 231L / 234E / 251T / 293T, 21G / 147S / 234E / 254K. 21G / 147S / 234E / 293T. 21G / 147S / 251P, 21G / 147S / 251P / 254K, 21G / 147S / 251P / 293T / 320E, 21G / 147S / 251T, 21G / 147S / 251T / 254K. 21G / 147S / 254K, 21G / 147S / 254K / 293T. 21G / 147S / 293T, 21G / 158S / 231L / 234E / 251T, 21G / 158S / 234E, 21G / 158S / 251P / 254K. 21G / 158S / 251T, 21G / 158S / 251T / 254K, 21G / 158S / 293T, 21G / 231L.21G / 231L / 234E, 21G / 231L / 234E / 251P / 254K, 21G / 231L / 234E / 251P / 254K / 293T.21G / 231L / 234E / 251T / 293T, 21G / 231L / 251P / 254K. 21G / 234E / 251P, 21G / 234E / 293T, 21G / 251P, 21G / 251P / 254K, 21G / 251T / 254K, 21G / 254K, 21G / 293T, 23E / 126V / 157S / 233E, 23E / 126V / 157S / 250I, 23E / 126V / 187Q. 23E / 126V / 233E, 23E / 126V / 233E / 252V / 289L, 23E / 126V / 2501 / 289L, 23E / 233E. 23E / 233E / 252V / 289L / 310A. 23E / 2501 / 252V / 289L.112K / 126V / 310A. 126S / 167V / 310A. 126V, 126V / 157S / 187Q, 126V / 157S / 233E / 250L / 252V, 126V / 157S / 233E / 252V, 126V / 187Q, 126V / 187Q / 233E, 126V / 187Q / 310A, 126V / 233E, 126V / 233E / 250L / 252V, 126V / 233E / 252V / 289L, 126V / 233E / 252V / 302H, 126V / 233E / 289L, 126V / 250I / 252V, 126V / 250I / 289L, 126V / 250L / 252V, 126V / 252V, 126V / 252V / 289L, 126V / 289L, 126V / 310A, 141S / 187Q, 147S / 158S, 147S / 158S / 182T / 234E / 251P, 147S / 158S / 231L, 147S / 158S / 231L / 234E / 251T. 147S / 158S / 251P / 254K, 147S / 158S / 254K. 147S / 158S / 254K / 293T,Docket No. 5244.285WO2147S / 231L / 234E, 147S / 231L / 234E / 251P / 293T, 147S / 231L / 234E / 254K, 147S / 231L / 293T.147S / 234E, 147S / 234E / 251T / 254K, 147S / 25 IP, 147S / 251P / 254K, 147S / 2 1P / 293T, 147S / 251T, 147S / 2 1T / 254K, 147S / 251T / 293T, 147S / 254K, 147S / 293T, 157K / 310A, 157S, 157S / 187Q / 233E / 252V, 157S / 233E / 250I / 289L, 157S / 252V, 157S / 252V / 289L, 157S / 289L / 310A, 158S, 158S / 231L / 234E, 158S / 231L / 234E / 254K, 158S / 234E. 158S / 234E / 2 1P / 254K, 158S / 234E / 254K, 158S / 251T / 254K / 293T, 158S / 254K, 167S / 252V / 289L. 185N / 187Q / 233E / 310A.187Q, 187Q / 233E, 187Q / 233E / 252V, 187Q / 233E / 252V / 310A. 187Q / 250I / 252V, 187Q / 250L, 187Q / 252V. 187Q / 289L, 187Q / 310A, 225K / 250I / 252V, 231L / 234E / 2 1T, 231L / 234E / 251T / 254K, 231L / 254K, 233E. 233E / 250I / 252V, 233E / 250I / 252V / 289L, 233E / 250I / 252V / 289L / 310A, 233E / 289L, 233E / 289L / 310A, 233E / 310A, 234E, 234E / 293T, 2501, 250I / 252V, 250I / 252V / 289L, 250I / 289L / 310A, 250L / 252V, 250L / 252V / 310A, 250L / 289L, 251P / 254K, 251P / 254K / 293T, 251T / 254K. 252V / 289L. 252V / 310A, 254K, 254K / 293T, 289L, 289L / 310A, and 293T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 162, or relative to the reference sequence corresponding to SEQ ID NO: 162.

30. The engineered adenosine kinase of any one of claims 26-29, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set selected from H4N / A126V / P187Q / S233E, A21G, A21G / S129I / T147S, A21G / T147S,A21 G / T 147S / G158S, A21 G / T 147S / G158S / Q231 L / K251 P, A21G / T147S / G158S / Q231L / K251P / R254K / L321I, A21G / T147S / G158S / Q231L / K293T, A21G / T147S / G158S / A234E, A21G / T147S / G158S / A234E / R254K, A21G / T147S / G158S / A234E / R254K / K293T, A21G / T147S / G158S / K251P / R254K / K293T, A21G / T147S / Q231L / A234E, A21G / T147S / Q231L / A234E / K251P / R254K / K293T,A21 G / T 147S / Q231L / A234E / K251T / K293T, A21 G / Tl 47S / A234E / R254K,A21 G / T 147S / A234E / K293T, A21 G / T 147S / K25 IP, A21 G / T 147S / K251 P / R254K.A21G / T147S / K251P / K293T / A320E, A21G / T147S / K251T, A21G / T147S / K251T / R254K, A21G / T147S / R254K, A21G / T147S / R254K / K293T. A21G / T147S / K293T, A21G / G158S / Q231L / A234E / K251T. A21G / G158S / A234E, A21G / G158S / K251P / R254K, A21G / G158S / K251T, A21G / G158S / K251T / R254K, A21G / G158S / K293T. A21G / Q231L, A21G / Q231L / A234E, A21G / Q231L / A234E / K251P / R254K.A21 G / Q231 L / A234E / K251 P / R254K / K293T, A21 G / Q231 L / A234E / K251 T / K293T.A21G / Q231L / K251P / R254K, A21G / A234E / K251P, A21G / A234E / K293T, A21G / K251P, A21G / K251P / R254K, A21G / K251T / R254K, A21G / R254K, A21G / K293T, D23E / A126V / E157S / S233E, D23E / A126V / E157S / A250I, D23E / A126V / P187Q, D23E / A126V / S233E, D23E / A126V / S233E / A252V / M289L, D23E / A126V / A250I / M289L.D23E / S233E, D23E / S233E / A252V / M289L / N310A, D23E / A250I / A252V / M289L,Docket No. 5244.285WO2QI 12K / A126V / N310A, A126S / A167V / N310A, A126V, A126V / E157S / P187Q, A126V / E157S / S233E / A250L / A252V, A126V / E157S / S233E / A252V, A126V / P187Q, A126V / P187Q / S233E, A126V / P187Q / N310A, A126V / S233E, A126V / S233E / A250L / A252V, A126V / S233E / A252V / M289L, A126V / S233E / A252V / Q302H, A126V / S233E / M289L, A126V / A250I / A252V, A126V / A250I / M289L, A126V / A250L / A252V, A126V / A252V, A126V / A252V / M289L, A126V / M289L, A126V / N310A, R141S / P187Q, T147S / G158S, T147S / G158S / P182T / A234E / K251P, T147S / G158S / Q231L. T147S / G158S / Q231L / A234E / K251T, T147S / G158S / K251P / R254K, T147S / G158S / R254K, T147S / G158S / R254K / K293T, T147S / Q231L / A234E. T147S / Q231L / A234E / K251P / K293T, T147S / Q231L / A234E / R254K, T147S / Q231L / K293T. T147S / A234E, T147S / A234E / K251T / R254K, T147S / K251P, T147S / K251P / R254K. T147S / K251P / K293T. T147S / K251T, T147S / K251T / R254K, T147S / K251T / K293T. T147S / R254K, T147S / K293T. E157K / N310A, E157S, E157S / P187Q / S233E / A252V, E157S / S233E / A250I / M289L. E157S / A252V, E157S / A252V / M289L. E157S / M289L / N310A, G158S, G158S / Q231L / A234E, G158S / Q231L / A234E / R254K, G158S / A234E, G158S / A234E / K251P / R254K. G158S / A234E / R254K, G158S / K251T / R254K / K293T, G158S / R254K, A167S / A252V / M289L, S185N / P187Q / S233E / N310A, P187Q, P187Q / S233E, P187Q / S233E / A252V, P187Q / S233E / A252V / N310A, P187Q / A250I / A252V, P187Q / A250L, P187Q / A252V, P187Q / M289L. P187Q / N310A, Q225K / A250I / A252V. Q231L / A234E / K251T, Q231L / A234E / K251T / R254K, Q231L / R254K, S233E, S233E / A250I / A252V.S233E / A250I / A252V / M289L, S233E / A250VA252V / M289L / N310A. S233E / M289L, S233E / M289L / N310A, S233E / N310A, A234E, A234E / K293T, A250I, A250I / A252V, A250I / A252V / M289L. A250I / M289L / N310A, A250L / A252V, A250L / A252V / N310A, A250L / M289L, K251P / R254K, K251P / R254K / K293T, K251T / R254K, A252V / M289L, A252V / N310A, R254K, R254K / K293T, M289L, M289L / N310A, and K293T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 162, or relative to the reference sequence corresponding to SEQ ID NO: 162.

31. The engineered adenosine kinase of claim 1. comprising an amino acid sequence having at least 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%. 88%. 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to the reference sequence comprising residues 12-321 of SEQ ID NO: 268, or to the reference sequence corresponding to SEQ ID NO: 268, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence comprising residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

32. The engineered adenosine kinase of claim 1, comprising an amino acid sequenceDocket No. 5244.285WO2having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

33. The engineered adenosine kinase of claim 31 or 32, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 9, 11, 12, 40 / 193, 46, 199, 201. 223, 267, 277, 278, and 315; a substitution of 13L / R / T, 40L / Q. 55Q / S / T / W / Y, 74A / C / G / L / M / Q, 90G / Q. 94R, 107 / K / L / P. 108S, 109D, 111 / H / S. 119K. 12 IE, 128E / L / M, 147L / P, 164I / R, 167E / R / S, 169W, 171Q / T, 172A / R / T / V. 182T, 185L / N, 190E / L, 191P, 194F / K, 195C / N. 206E / H / T. 207S. 210E / V, 211 S, 222E / P / V, 243A / E / L / N / T, 271L / T, 274V, 312C, 314A / L / M or combinations thereof, wherein the amino acid positions are relative to the reference sequence comprising amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

34. The engineered adenosine kinase of any one of claims 31-32, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 9C, 9L, 9R, UK, IIP, 12E, 12P, 12R, 12T, 13L, 13P, 13R, 13T, 40H / 193T, 40L, 40Q, 43 A, 46L, 55Q, 55S, 55T, 55W, 55Y, 74A, 74C, 74G, 74L, 74M. 74Q, 74T, 74V, 80V, 90G. 90Q, 94M, 94R.106G, 106L, 106R, 107G, 107K, 107L, 107P, 108S, 109D. 111S, 119K, 121E, 128E, 128L, 128M.140P. 147L. 147P. 155A, 1551. 1641, 164R. 164S. 167E. 167R, 167V, 169L, 169W. 171Q, 171T, 172A, 172R, 172T, 172V, 182G. 185L. 190E. 190L. 191P. 191S. 193M, 194F, 195C, 195G. 195N, 199L. 199M, 201S, 201T, 206E, 206H. 207A. 207L, 207M, 207S, 210E. 210V. 2111, 21 IL. 21 IM, 21 IS, 222A. 222E. 222G, 222P, 222V, 223G, 223L, 243 A. 243E. 243L, 243N, 243 R, 243T, 267G, 271L, 271T, 274V. 277K. 277R, 278A, 278G, 278P, 278Q. 278T. 312C, 314A, 314L, 314M. and 315 V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO:

268. or relative to the reference sequence corresponding to SEQ ID NO: 268.

35. The engineered adenosine kinase of claim 34, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue G9C, G9L, G9R, G11K, G11P, M12E, M12P, M12R, M12T, S13L, S13P, S13R, S13T, R40H / I193T. R40L, R40Q, R43A, V46L, R55Q. R55S, R55T, R55W, R55Y, R74A. R74C, R74G, R74L, R74M, R74Q, R74T, R74V, T80V, E90G. E90Q, T94M, T94R, D106G, D106L, D106R, D107G, D107K, D107L, D107P, L108S, S109D, PHIS, M119K, Q121E, H128E, H128L, H128M, V140P, S147L, S147P, T155A,Docket No. 5244.285WO2T155I, E164I, E164R, E164S, A167E, A167R, A167V, M169L, M169W, I171Q, I171T, P172A, P172R, P172T, P172V, P182G, S185L, R190E, R190L, E191P, E191S, I193M, E194F, Q195C, Q195G, Q195N, V199L, V199M, V201S, V201T, S206E, S206H, R207A, R207L, R207M, R207S, Q210E, Q210V, A211I, A211L, A211M, A211S, S222A, S222E, S222G. S222P, S222V, R223G, R223L, K243A, K243E, K243L, K243N, K243R, K243T, A267G, Y271L, Y271T, Q274V, Y277K, Y277R, D278A, D278G. D278P, D278Q, D278T, R312C, K314A, K314L, K314M, and Q315V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

36. The engineered adenosine kinase of claim 31 or 32. wherein the amino acid sequence of the engineered adenosine kinase comprises at least one substitution position or substitution position set selected from 1 / 250 / 289, 126 / 157, 126 / 157 / 250, 126 / 157 / 250 / 289. 126 / 157 / 289, 126 / 250.126 / 250 / 289, 126 / 289, 157 / 250, 157 / 250 / 289, 157 / 289, 250 / 289, and 289; the substitution 126V, 157S, 250L,or combinations thereof, wherein the amino acid positions are relative to the reference sequence comprising amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

37. The engineered adenosine kinase of claim 36, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set selected from 1V / 250I / 289L, 126V, 126V / 157K, 126V / 157S / 250I. 126V / 157S / 250I / 289L, 126V / 157S / 289L, 126V / 250I, 126V / 250I / 289L, 126V / 289L, 157S, 157S / 250I, 157S / 250I / 289L. 157S / 289L. 250I / 289L, and 289L. or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

38. The engineered adenosine kinase of claim 37, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set selected from M1V / A250I / M289L. A126V, A126V / E157K, A126V / E157S / A250I, A126V / E157S / A250I / M289L, A126V / E157S / M289L, A126V / A250I, A126V / A250I / M289L, A126V / M289L, E157S, E157S / A250I, E157S / A250I / M289L, E157S / M289L, A250I / M289L, and M289L, or combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 268, or relative to the reference sequence corresponding to SEQ ID NO: 268.

39. The engineered adenosine kinase of claim 1. wherein the amino acid sequence of the engineered adenosine kinase comprises residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 and 220-812. or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 12-206 andDocket No. 5244.285WO2220-812.

40. The engineered adenosine kinase of any one of claims 1-26, having adenosine kinase activity and at least one improved property as compared to a reference adenosine kinase.

41. The engineered adenosine kinase of claim 40, wherein the improved property is selected from i) increased activity on unmodified nucleoside, ii) increased thermostability, iii) increased activity on cytidine, iv) increased activity on 2-fluoro modified nucleosides, and v) increased activity on 2-O-methyl modified nucleosides, or any combinations of i), ii), iii), iv), and v), compared to the reference adenosine kinase.

42. The engineered adenosine kinase of any one of claims 1-41, comprising a fusion polypeptide.

43. The engineered adenosine kinase of any one of claims 1-42, wherein the engineered adenosine kinase is purified.

44. The engineered adenosine kinase of any one of claims 1-42, wherein the engineered adenosine kinase is immobilized on a substrate or support medium.

45. A recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenosine kinase of any one of claims 1-42.

46. The recombinant polynucleotide of claim 45. comprising a polynucleotide sequence having at least 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, wherein the recombinant polynucleotide encodes an engineered adenosine kinase.

47. The recombinant polynucleotide of claim 45 or 46. wherein the polynucleotide sequence has preferred codons or is codon optimized.

48. The recombinant polynucleotide of claim 45. wherein the polynucleotide sequence comprises nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811, or comprises an odd-numbered SEQ ID NO. of SEQ ID NOs: 11-205 and 219-811.

49. An expression vector comprising a recombinant polynucleotide of any one of claims 45-48.Docket No. 5244.285WO250. The expression vector of claim 49, wherein the recombinant polynucleotide is operably linked to a control sequence.

51. The expression vector of claim 50, wherein the control sequence comprises at least a promoter.

52. A host cell comprising an expression vector of any one of claims 49-51.

53. The host cell of claim 52, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

54. A method of producing an engineered adenosine kinase polypeptide, comprising culturing a host cell of claim 52 or 53 under suitable culture conditions such that the engineered adenosine kinase polypeptide is produced.

55. The method of claim 54, further comprising recovering the engineered adenosine kinase polypeptide from the culture and / or host cells.

56. The method of claim 54 or 55, further comprising purifying the engineered adenosine kinase polypeptide.

57. A composition comprising an engineered adenosine kinase of any one of claims 1-44.

58. The composition of claim 57, further comprising a nucleoside substrate.

59. The composition of claim 58, wherein the nucleoside is a modified nucleoside substrate.

60. A method of converting a nucleoside or a modified nucleoside to corresponding nucleoside monophosphate (NMP), comprising contacting a nucleoside with an engineered adenosine kinase of any one of claims 1-44 in the presence of phosphate donor NTP under suitable reaction conditions to convert the nucleoside to the corresponding nucleoside monophosphate (NMP).

61. The method of claim 60, wherein the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine.

62. The method of claim 61, wherein the nucleoside has at the 2’-position of the sugar moiety a H or OH.

63. The method of claim 62, wherein the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine, and wherein the nucleoside has at the 2 ’-position of the sugar moiety an OH,Docket No. 5244.285WO2thereby by resulting in corresponding product rAMP, rGMP, rUMP, rCMP, or rTMP, respectively.

64. The method of claim 62, wherein the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine, and wherein the nucleoside has at the 2’-position of the sugar moiety an H, thereby by resulting in corresponding product dAMP, dGMP, dUMP. dCMP, or dTMP, respectively.

65. The method of claim 60, wherein the nucleoside is a modified nucleoside, wherein the nucleoside has a modified sugar moiety or modified nucleobase, or a combination thereof.

66. The method of claim 65, wherein the modified nucleoside comprises a modified sugar moiety, wherein the sugar moiety has a modified 2’-position or 3’-position.

67. The method of claim 66, wherein the modified sugar moiety has a modified 2’-position of the sugar moiety.

68. The method of claim 67, wherein the 2’-position is a 2’-O-alkyl or 2-halo.

69. The method of claim 68, wherein the 2’-position is a 2‘-O-alkyl, and wherein the 2’- O-alkyl is 2’-O-CH3or 2’-O-CH2CH3.

70. The method of claim 68, wherein the 2’ -position is 2-halo.

71. The method of claim 70, wherein the 2’-position is 2’-F or 2’-Br.

72. The method of any one of claims 66-71, wherein the modified sugar moiety is modified at the 3 ’-position.

73. The method of claim 72, wherein the 3 ’-position is modified with a blocking group, wherein the blocking group is formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONH2), O-methyl, O-methoxymethyl. O-methylthiomethyl, O-benzyloxymethyl, O-allyl. O-(2-nitrobenzyl). O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl, phosphate, or triphosphate.

74. The method of any one of claims 66-73, wherein the modified nucleoside comprises a modified nucleobase, wherein the modified nucleobase is 5 -bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.

75. The method of any one of claims 60-74. wherein the phosphate donor NTP is rATP or dATP.

76. The method of any one of claims 60-75, wherein the phosphate donor NTP isDocket No. 5244.285WO2nucleoside-5 ’-gamma-thiotriphosphate (NTPyS), thereby resulting in product nucleoside 5’-thiophosphate (NMP-S).

77. The method of any one of claims 60-76. wherein the phosphate donor NTP has the same nucleoside structure of the nucleoside substrate.

78. The method of any one of claims 60-77, further comprising regenerating the NTP using a NTP regeneration system.

79. The method of claim 78, wherein the NTP regenerating system comprises creatine kinase, pyruvate kinase, polyphosphate kinase, and / or acetate-kinase.

80. The method of claim 79, wherein the NTP regenerating system is pyruvate kinase and substrate phosphoenolpyruvate.

81. The method of claim 79, wherein the NTP regenerating system is creatine kinase and substrate creatine phosphate.

82. The method of claim 79, wherein the NTP regenerating system is polyphosphate kinase and substrate polyphosphate.

83. The method of claim 79, wherein the NTP regenerating system is acetate kinase and substrate acetyl phosphate.

84. The method of claim 83, further comprising pyruvate oxidase and substrate pyruvate and inorganic phosphate.

85. The method of claim 84, further comprising including a catalase.

86. An NTP with a phosphate group at the 3’ position of the sugar (NQP) produced from an NMP produced by an engineered adenosine kinase of any one of claims 1-42.

87. The NQP of claim 86, further comprising a modification of the nucleoside, NTP and / or NQP at the 2‘ position of the sugar.

88. The NQP of any one of claims 86-87 further comprising a modification of the nucleoside, NTP and / or NQP at the nucleobase comprising a noncanonical nucleobase known to those skilled in the art, a removable tag. a cleavable linker, or a radio, a photo, and / or a chemical sensor; a modification of the nucleoside, NTP and / or NQP at the phosphate chain, optionally comprising an «-phosphothioate linkage or other non-phosphodiester linkage; or a combination of a modification at the nucleobase and a modification at the phosphate chain.

Images